prostaglandin-d2 and ciglitazone

Upper aerodigestive cancer is an aggressive malignancy with relatively stagnant long-term survival rates over 20 yr. Recent studies have demonstrated that exploitation of PPARγ pathways may be a novel therapy for cancer and its prevention. We tested whether PPARγ is expressed and inducible in aerodigestive carcinoma cells and whether it is present in human upper aerodigestive tumors. Human oral cancer CA-9-22 and NA cell lines were treated with the PPAR activators eicosatetraynoic acid (ETYA), 15-deoxy-δ- 12,14-prostaglandin J2 (PG-J2), and the thiazolidinedione, ciglitazone, and evaluated for their ability to functionally activate PPARγ luciferase reporter gene constructs. Cellular proliferation and clonogenic potential after PPARγ ligand treatment were also evaluated. Aerodigestive cancer specimens and normal tissues were evaluated for PPARγ expression on gene expression profiling and immunoblotting. Functional activation of PPARγ reporter gene constructs and increases in PPARγ protein were confirmed in the nuclear compartment after PPARγ ligand treatment. Significant decreases in cell proliferation and clonogenic potential resulted from treatment. Lipid accumulation was induced by PPARγ activator treatment. 75% of tumor specimens and 100% of normal control tissues expressed PPARγ RNA, and PPARγ protein was confirmed in 66% of tumor specimens analyzed by immunoblotting. We conclude PPARγ can be functionally activated in upper aerodigestive cancer and that its activation downregulates several features of the neoplastic phenotype. PPARγ expression in human upper aerodigestive tract tumors and normal cells potentially legitimizes it as a novel intervention target in this disease. © 2016 Wiley Periodicals, Inc.

Topics: Antineoplastic Agents; Arachidonic Acids; Cell Line; Cell Line, Tumor; Cell Proliferation; Gene Expression Regulation, Neoplastic; Head and Neck Neoplasms; Humans; Lipid Metabolism; Mouth; Mouth Neoplasms; PPAR gamma; Prostaglandin D2; Thiazolidinediones

Monocytes/macrophages link the innate and adaptive immune systems, and in inflammatory disorders their activation leads to tissue damage. 15-Deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural peroxisome proliferator-activated receptor gamma (PPARγ) ligand, has garnered much interest because it possesses anti-inflammatory properties in a number of experimental models. However, whether it regulates monocytes/macrophage pathophysiology is still unknown. This study was designed to examine the effects of 15d-PGJ(2) on the phagocytosis, proliferation and inflammatory cytokines generation in mouse monocyte/macrophage cell line RAW264.7 and J774A.1 cells upon lipopolysaccharide challenge. Our results showed that 15d-PGJ(2) inhibited the phagocytic activity and cell proliferation in a dose-dependent manner, and suppressed proinflammatory cytokines expression, such as tumor necrosis factor-α, transforming growth factor-β1, interleukin-6, and monocyte chemotactic protein-1. These effects were independent of PPARγ, because PPARγ agonist (troglitazone or ciglitazone) and PPARγ antagonist (GW9662) did not affect these activities mentioned above in cells. Treatment of 15d-PGJ(2) also did not modulate expression and distribution of PPARγ. However, these effects of 15d-PGJ(2) were abrogated by antioxidant N-acetylcysteine. Moreover, treatment of 15d-PGJ(2) induced a significant increase in reactive oxygen species production in RAW264.7 and J774A.1 cells. In conclusion, 15d-PGJ(2) attenuates the biological activities of mouse monocyte/macrophage cell line cells involving oxidative stress, independently of PPARγ. These data further underline the anti-inflammation potential of 15d-PGJ(2).

Topics: Acetylcysteine; Anilides; Animals; Cell Line; Cell Proliferation; Chemokine CCL2; Chromans; Interleukin-6; Lipopolysaccharides; Macrophages; Mice; Monocytes; Oxidative Stress; Phagocytosis; PPAR gamma; Prostaglandin D2; Reactive Oxygen Species; Thiazolidinediones; Troglitazone; Tumor Necrosis Factor-alpha

Peroxisome proliferator-activated receptors (PPARs) have been originally thought to be restricted to lipid metabolism or glucose homeostasis. Recently, evidence is growing that PPARγ ligands have inhibitory effects on tumor growth. To shed light on the potential therapeutic effects on melanoma we tested a panel of PPAR agonists on their ability to block tumor proliferation in vitro. Whereas ciglitazone, troglitazone and WY14643 showed moderate effects on proliferation, 15d-PGJ2 displayed profound anti-tumor activity on four different melanoma cell lines tested. Additionally, 15d-PGJ2 inhibited proliferation of tumor-associated fibroblasts and tube formation of endothelial cells. 15d-PGJ2 induced the tumor suppressor gene p21, a G(2)/M arrest and inhibited tumor cell migration. Shot gun proteome analysis in addition to 2D-gel electrophoresis and immunoprecipitation of A375 melanoma cells suggested that 15d-PGJ2 might exert its effects via modification and/or downregulation of Hsp-90 (heat shock protein 90) and several chaperones. Applying the recently established CPL/MUW database with a panel of defined classification signatures, we demonstrated a regulation of proteins involved in metastasis, transport or protein synthesis including paxillin, angio-associated migratory cell protein or matrix metalloproteinase-2 as confirmed by zymography. Our data revealed for the first time a profound effect of the single compound 15d-PGJ2 on melanoma cells in addition to the tumor-associated microenvironment suggesting synergistic therapeutic efficiency.

Topics: Antineoplastic Agents; Cell Cycle; Cell Line, Tumor; Cell Movement; Cell Proliferation; Chromans; Humans; Melanoma; PPAR gamma; Prostaglandin D2; Proteome; Pyrimidines; Thiazolidinediones; Troglitazone

Neural stem cells (NSCs) are a small population of resident cells that can grow, migrate and differentiate into neuro-glial cells in the central nervous system (CNS). Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates cell growth and differentiation. In this study we analyzed the influence of PPARγ agonists on neural stem cell growth and differentiation in culture. We found that in vitro culture of mouse NSCs in neurobasal medium with B27 in the presence of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) induced their growth and expansion as neurospheres. Addition of all-trans retinoic acid (ATRA) and PPARγ agonist ciglitazone or 15-Deoxy-Δ(12,14)-Prostaglandin J(2) (15d-PGJ2) resulted in a dose-dependent inhibition of cell viability and proliferation of NSCs in culture. Interestingly, NSCs cultured with PPARγ agonists, but not ATRA, showed significant increase in oligodendrocyte precursor-specific O4 and NG2 reactivity with a reduction in NSC marker nestin, in 3-7 days. In vitro treatment with PPARγ agonists and ATRA also induced modest increase in the expression of neuronal β-III tubulin and astrocyte-specific GFAP in NSCs in 3-7 days. Further analyses showed that PPARγ agonists and ATRA induced significant alterations in the expression of many stemness and differentiation genes associated with neuro-glial differentiation in NSCs. These findings highlight the influence of PPARγ agonists in promoting neuro-glial differentiation of NSCs and its significance in the treatment of neurodegenerative diseases.

Topics: Animals; Antigens; Cell Differentiation; Cell Proliferation; Cell Survival; Cells, Cultured; Dose-Response Relationship, Drug; Epidermal Growth Factor; Fibroblast Growth Factor 2; Gene Expression; Glial Fibrillary Acidic Protein; Intermediate Filament Proteins; Mice; Nerve Tissue Proteins; Nestin; Neural Stem Cells; Oligodendroglia; PPAR gamma; Prostaglandin D2; Proteoglycans; Thiazolidinediones; Tretinoin; Tubulin

Ovarian cancer accounts for the highest mortality among all gynecological cancers, mainly due to the fast developing chemoresistance. The death ligand TRAIL induces apoptosis and is able to sensitize tumor cells to cytostatic drugs without affecting physiological tissue. Combined treatment of TRAIL and the antidiabetic acting PPARγ ligands was shown to induce apoptosis synergistically in different ovarian cancer cell lines.. To investigate feasible TRAIL-dependent inhibition of proliferation and induction of apoptosis in chemoresistant ovarian cancer cell lines, the drug- and TRAIL-sensitive HEY cell line was utilized to develop subclones with selective resistance against cisplatin, etoposide, docetaxel, paclitaxel, gemcitabine and pemetrexed, as well as against TRAIL as control cell line. Expression of the key factors of the TRAIL signaling pathway, TRAIL receptors 1-4, caspase-8, FLIP and XIAP, was analyzed before and after TRAIL treatment by immunoblotting.. Cell proliferation experiments showed TRAIL-dependent inhibition that was further increased by combination treatment with the PPARγ ligands. Simultaneous exposure of TRAIL and the PPARγ ligands also resulted in enhanced induction of apoptosis even in partial TRAIL-resistant HEY cell lines. In the parental HEY cell line, additional treatment with the PPARγ ligands led to an increased protein expression of DR5 and a further decline of XIAP expression.. Therefore, the combinational treatment with TRAIL and PPARγ ligands might be a promising experimental therapy because the PPARγ ligands, especially d15-PGJ(2), sensitize drug-resistant ovarian cancer cells to TRAIL-induced apoptosis.

Topics: Apoptosis; CASP8 and FADD-Like Apoptosis Regulating Protein; Caspase 8; Cell Line, Tumor; Chromans; Drug Resistance, Neoplasm; Drug Synergism; Female; Humans; Ovarian Neoplasms; PPAR gamma; Prostaglandin D2; Receptors, TNF-Related Apoptosis-Inducing Ligand; Thiazolidinediones; TNF-Related Apoptosis-Inducing Ligand; Troglitazone

Collagenase-3 (matrix metalloproteinase, MMP-13) plays an important role in the degradation of cartilage in pathologic conditions. MMP-13 is elevated in joint tissues in both rheumatoid arthritis (RA) and osteoarthritis (OA). In addition, inflammation-stimulated synovial fibroblasts are able to release MMP-13 and other cytokines in these diseases. The peroxisome proliferator-activated receptor-γ (PPARγ) ligands are recently considered as new anti-inflammatory compounds and these ligands were reported to ameliorate inflammatory arthritis. The aim of this study is to evaluate the mechanisms how PPARγ ligands inhibit the inflammatory response in synovial fibroblasts. Two PPARγ ligands, cyclopentenone prostaglandin 15-deoxy-Δ(12,14) -prostaglandin-J2 (15d-PGJ2) and synthetic thiazolidinedione compound ciglitazone were examined in this study. Here we found that 15d-PGJ2 and ciglitazone markedly inhibited TNF-α-induced MMP-13 production in human synovial fibroblasts. In addition, activation of nuclear factor κB (NF-κB) is strongly associated with MMP-13 induction by TNF-α and the activation of NF-κB was determined by Western blot, reporter assay, and immunofluorescence. It was found that 15d-PGJ2 markedly attenuated the translocation of NF-κB by direct inhibition of the activation of IKK via a PPARγ-independent manner. Ciglitazone also inhibits TNF-α-induced MMP-13 expression by suppressing NF-κB activation mainly via the modulation of p38-MAPK. Collectively, our data demonstrate that 15d-PGJ2 and ciglitazone attenuated TNF-α-induced MMP-13 expression in synovial fibroblasts primarily through the modulation of NF-κB signaling pathways. These compounds may have therapeutic application in inflammatory arthritis.

Topics: Active Transport, Cell Nucleus; Anti-Inflammatory Agents; Blotting, Western; Cells, Cultured; Dose-Response Relationship, Drug; Fibroblasts; Fluorescent Antibody Technique; Genes, Reporter; Humans; I-kappa B Kinase; I-kappa B Proteins; Matrix Metalloproteinase 13; Mutation; NF-kappa B; NF-KappaB Inhibitor alpha; p38 Mitogen-Activated Protein Kinases; PPAR gamma; Prostaglandin D2; Protein Kinase Inhibitors; Signal Transduction; Synovial Membrane; Thiazolidinediones; Transfection; Tumor Necrosis Factor-alpha

Cyclopentenone prostaglandin 15-deoxy-Δ(12,14)-prostaglandin J(2) (15d-PGJ(2)), which is generated from the dehydration of PGD(2), is a natural ligand of peroxisome proliferator-activated receptor gamma (PPARγ) and a potential apoptotic mediator. The synthetic PPARγ ligands, troglitazone and ciglitazone, inhibit tumor progression in many cells by PPARγ activation, but the mechanism of 15d-PGJ(2) is still unclear. In this study, GW9662, an antagonist of PPARγ, and quercetin, a natural antioxidant, were used to study the apoptotic mechanism of 15d-PGJ(2) in A549 cells. Results showed that 15d-PGJ(2) induced apoptosis, which was associated with the production of reactive oxygen species (ROS) and the decrease of GSH levels. Furthermore, quercetin reduced the activity of caspases in 15d-PGJ(2)-induced apoptotic processes. These results suggest that 15d-PGJ(2) induces apoptosis in A549 cells mainly through the formation of ROS; it does not depend on PPARγ activation. Moreover, these findings support the use of quercetin and PPARγ agonists in non-small cell lung carcinoma.

Topics: Apoptosis; Carcinoma, Non-Small-Cell Lung; Caspases; Cell Line, Tumor; Glutathione; Humans; Lung Neoplasms; PPAR gamma; Prostaglandin D2; Quercetin; Reactive Oxygen Species; Thiazolidinediones

Ligands activating the transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) have antiinflammatory effects. Vascular rejection induced by allogeneic T cells can be responsible for acute and chronic graft loss. Studies in rodents suggest that PPARγ agonists may inhibit graft vascular rejection, but human T-cell responses to allogeneic vascular cells differ from those in rodents, and the effects of PPARγ in human transplantation are unknown.. We tested the effects of PPARγ agonists on human vascular graft rejection using a model in which human artery is interposed into the abdominal aorta of immunodeficient mice, followed by adoptive transfer of allogeneic (to the artery donor) human peripheral blood mononuclear cells. Interferon-γ-dependent rejection ensues within 4 weeks, characterized by intimal thickening, T-cell infiltrates, and vascular cell activation, a response resembling clinical intimal arteritis. The PPARγ agonists 15-deoxy-prostaglandin-J(2), ciglitazone, and pioglitazone reduced intimal expansion, intimal infiltration of CD45RO(+) memory T cells, and plasma levels of inflammatory cytokines. The PPARγ antagonist GW9662 reversed the protective effects of PPARγ agonists, confirming the involvement of PPARγ-mediated pathways. In vitro, pioglitazone inhibited both alloantigen-induced proliferation and superantigen-induced transendothelial migration of memory T cells, indicating the potential mechanisms of PPARγ effects.. Our results suggest that PPARγ agonists inhibit allogeneic human memory T cell responses and may be useful for the treatment of vascular graft rejection.

Topics: Adoptive Transfer; Anilides; Animals; Arteries; Cell Movement; Cell Proliferation; Cytokines; Graft Rejection; Humans; Hypoglycemic Agents; Immunologic Memory; Isoantigens; Mice; Mice, SCID; Pioglitazone; PPAR gamma; Prostaglandin D2; Superantigens; T-Lymphocytes; Thiazolidinediones; Transplantation, Heterologous; Transplantation, Homologous

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) has been shown to induce colon cancer cell apoptosis in the presence of interferon-gamma. We hypothesized that co-treatment using TWEAK with other pro-apoptosis agents could sensitize death receptor-resistant colon cancer cells.. The effects of chemopreventive agents and TWEAK on cell death and apoptosis were determined using propidium iodide (PI) exclusion and M30 CytoDEATH.. We found that 15d-PGJ(2) sensitizes colon cancer cells to TWEAK-induced apoptosis. Caspase inhibition reduced 15d-PGJ(2)-, but not 15d-PGJ(2)+TWEAK-induced apoptosis. 15d-PGJ(2) promoted reactive oxygen species (ROS) production and dissipation of mitochondrial potential (DeltaPsi(m)) that were more marked with combined treatment. ROS, DeltaPsi(m) and cell death were partially normalized by the antioxidant N-acetylcysteine. TWEAK induced nuclear factor-kappa B activation, which was attenuated by 15d-PGJ(2). 15d-PGJ(2) reduced the expression of the anti-apoptotic proteins BCL-X(L) and MCL-1, while increasing BAX and translocation of cytochrome c and apoptosis-inducing factor.. 15d-PGJ(2) sensitized cancer cells to TWEAK-induced apoptosis through an ROS-dependent cell death pathway and may have chemotherapeutic utility as an apoptosis-enhancing agent.

Topics: Apoptosis; Butyrates; Caco-2 Cells; Cell Line, Tumor; Colonic Neoplasms; Cytokine TWEAK; Drug Synergism; HT29 Cells; Humans; Interferon-gamma; Leucine; Ligands; Membrane Potential, Mitochondrial; Mitochondria; PPAR gamma; Prostaglandin D2; Thiazolidinediones; Tumor Necrosis Factors

Activators of the peroxisome proliferator-activated receptor gamma (PPARgamma), originally found to be implicated in lipid metabolism and glucose homeostasis, have been shown to modulate inflammatory responses through interference with cytokine and chemokine production. Given the central role of mesangial cell-derived chemokines in glomerular leukocyte recruitment in human and experimental glomerulonephritis, we studied the influence of natural and synthetic PPARgamma activators on INF-gamma-induced expression of the T cell-attracting chemokines IP-10/CXCL10, Mig/CXCL9 and I-TAC/CXCL11 in mouse mesangial cells.. INF-gamma-treated mesangial cells were cultured in the presence or absence of either the naturally occurring PPARgamma ligand 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) or synthetic PPARgamma activators of the glitazone group. Chemokine mRNA and protein expression and activation of the JAK/STAT signalling pathway were analysed.. The 15d-PGJ(2), but not synthetic PPARgamma ligands, dose-dependently inhibited INF-gamma-induced chemokine gene (mRNA and protein) expression. Combined results from EMSA and western blot analysis revealed the inhibitory ability of 15d-PGJ(2), but not of synthetic PPARgamma ligands, on IFN-gamma-induced tyrosine phosphorylation of JAK1, JAK2, STAT1 and nuclear STAT1 translocation and DNA binding.. Our results demonstrate that 15d-PGJ(2) inhibits INF-gamma-induced chemokine expression in mesangial cells by targeting the JAK/STAT signalling pathway. This effect is independent of an interference with PPARgamma.

Topics: Animals; Cells, Cultured; Chemokine CXCL10; Chemokine CXCL11; Chemokine CXCL9; Chromans; Gene Expression; Glomerulonephritis; Interferon-gamma; Janus Kinases; Mesangial Cells; Mice; PPAR gamma; Prostaglandin D2; Recombinant Proteins; RNA, Messenger; Signal Transduction; STAT1 Transcription Factor; Thiazolidinediones; Troglitazone

To investigate the molecular mechanism of fish adipocyte differentiation, the three subtypes of PPAR genes (alpha, beta and gamma) were characterized in a marine teleost red sea bream (Pagrus major). The primary structures of red sea bream PPARs exhibited high degrees of similarities to their mammalian counterparts, and their gene expression was detected in various tissues including adipose tissue, heart and hepatopancreas. During the differentiation of primary cultured red sea bream adipocytes, three PPARs showed distinct expression patterns: The alpha subtype showed a transient increase and the beta gene expression tended to increase during adipocyte differentiation whereas the gene expression level of PPARgamma did not change. These results suggest that they play distinct roles in adipocyte differentiation in red sea bream. In the differentiating red sea bream adipocytes, mammalian PPAR agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2), ciglitazone and fenofibrate did not show clear effects on the adipogenic gene expression. However, 2-bromopalmitate increased the PPARgamma and related adipogenic gene expression levels, suggesting the gamma subtype plays a central role in red sea bream adipocyte differentiation and in addition, fatty acid metabolites can be used as modulators of adipocyte function. Thus our study highlighted the roles of PPARs in fish adipocyte differentiation and provided information on the molecular mechanisms of fish adipocyte development.

Topics: Adipocytes; Animals; Cell Differentiation; Cells, Cultured; Fenofibrate; Hypoglycemic Agents; Peroxisome Proliferator-Activated Receptors; Phylogeny; Prostaglandin D2; Protein Isoforms; Sea Bream; Thiazolidinediones

Side effects of peroxisome proliferator activated receptor gamma (PPARgamma) agonists such as ciglitazone include anemia, which in theory could be due to decreased formation or premature death of erythrocytes. A form of suicidal erythrocyte death is eryptosis, which is characterized by cell shrinkage and by breakdown of phosphatidylserine asymmetry leading to phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing erythrocytes are recognized by macrophages, engulfed, degraded and thus cleared from circulating blood. Triggers of eryptosis include increase in intracellular Ca(2+) concentration. The present study thus explored, whether the PPARgamma agonist ciglitazone or the natural PPARgamma ligand 15deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) are capable to trigger eryptosis. Phosphatidylserine exposure was determined from annexin V binding and cell shrinkage from decrease of forward scatter of human erythrocytes in FACS analysis. Both, ciglitazone (>or= 5 microM) and 15d-PGJ2 (>or= 3 microM), within 24 hours increased phosphatidylserine exposure and at concentrations of 10 microM led to a significant loss of the cell volume. Ciglitazone further stimulated hemolysis, which, however, affected only a fraction of erythrocytes undergoing eryptosis. According to Fluo3 fluorescence of human erythrocytes, 10 microM ciglitazone or 15d-PGJ2 increased intracellular Ca(2+) activity. In conclusion, ciglitazone and 15d-PGJ2 trigger eryptosis at least in part by an increase in the cytosolic Ca(2+) concentration. The effect most likely contributes to the anemia observed following treatment with PPARgamma agonists.

Topics: Aniline Compounds; Annexin A5; Calcium; Cell Death; Erythrocytes; Hemolysis; Humans; Prostaglandin D2; Thiazolidinediones; Xanthenes

The role of PPARgamma in ciglitazone and 15-d PGJ(2)-induced apoptosis and cell cycle arrest of Jurkat (before and after PPARgamma gene silencing), U937 (express high levels of PPARgamma) and HeLa (that express very low levels of PPARgamma) cells was investigated. PPARgamma gene silencing, per se, induced a G2/M cell arrest, loss of membrane integrity and DNA fragmentation of Jurkat cells, indicating that PPARgamma is important for this cell survival and proliferation. Ciglitazone-induced apoptosis was abolished after knockdown of PPARgamma suggesting a PPARgamma-dependent pro-apoptotic effect. However, ciglitazone treatment was toxic for U937 and HeLa cells regardless of the presence of PPARgamma. This treatment did not change the cell cycle distribution corroborating with a PPARgamma-independent mechanism. On the other hand, 15-d PGJ(2) induced apoptosis of the three cancer cell lines regardless of the expression of PPARgamma. These results suggest that PPARgamma plays an important role for death of malignant T lymphocytes (Jurkat cells) and PPARgamma agonists exert their effects through PPARgamma-dependent and -independent mechanisms depending on the drug and the cell type.

Topics: Apoptosis; Cell Cycle; Cell Division; DNA Fragmentation; G2 Phase; Gene Expression Regulation; Gene Silencing; HeLa Cells; Humans; Jurkat Cells; PPAR gamma; Prostaglandin D2; Thiazolidinediones; U937 Cells

Tumor necrosis factor-alpha (TNF-alpha) plays critical roles in bone resorption at the site of inflammatory joints. The aim of this study is to evaluate the effect of peroxisome proliferator-activated receptor gamma (PPAR-gamma) agonists, a new class of anti-inflammatory compounds, on TNF-alpha-mediated osteoclastogenesis in human monocytes. Human monocytes were differentiated into osteoclasts in the presence of TNF-alpha and macrophage colony-stimulating factor. Tartrate-resistant acid phosphatase (TRAP) staining and a pit formation assay using dentin were used for the identification of activated osteoclasts. The protein and gene expressions of transcription factors were determined by immunofluorescence and real-time RT-PCR analysis, respectively. TNF-alpha-induced osteoclast generation from human peripheral monocytes in a dose-dependent manner, and the induction was not inhibited by osteoprotegerin, a decoy receptor for receptor activator of NF-kappaB ligand. The addition of PPAR-gamma agonists, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) or ciglitazone, to the culture resulted in a remarkably reduced number of generated osteoclasts. In addition, both agonists inhibited the protein and gene expressions of nuclear factor of activated T-cell isoform c1 (NFATc1), c-Fos, c-Jun and NF-kappaB p65, which are known to be associated with osteoclastogenesis. GW9662, an antagonist of PPAR-gamma, fully rescued ciglitazone-induced inhibition, but did not affect 15d-PGJ2-induced inhibition. Monocyte chemoattractant protein-1 (MCP-1), a CC chemokine related to osteoclastogenesis, was induced during TNF-alpha-mediated osteoclast differentiation, and the neutralizing antibody to MCP-1 reduced osteoclast formation by about 40%. 15d-PGJ2 and ciglitazone blocked the induction of MCP-1 by TNF-alpha. Moreover, the addition of MCP-1 rescued the inhibition of TRAP-positive multinucleated cell (TRAP-MNCs) formation by 15d-PGJ2 and ciglitazone, although generated TRAP-MNCs had no capacity to resorb dentin slices. Our data demonstrate that 15d-PGJ2 and ciglitazone down-regulate TNF-alpha-mediated osteoclast differentiation in human cells, in part via suppression of the action of MCP-1. These PPAR-gamma agonists may be a promising therapeutic application for rheumatoid arthritis and inflammatory bone-resorbing diseases.

Topics: Cell Differentiation; Cells, Cultured; Chemokine CCL2; Humans; Monocytes; Osteoclasts; PPAR gamma; Prostaglandin D2; RANK Ligand; Thiazolidinediones; Transcription Factors; Tumor Necrosis Factor-alpha

A peroxisome proliferator-activated receptor gamma (PPARgamma) ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), has been reported to possess anti-inflammatory activity in activated monocytes/macrophages. In this study, we investigated the effect of 15d-PGJ(2) on the lipopolysaccharide (LPS)-induced expression of chemokine mRNAs, especially macrophage inhibitory protein (MIP)-2 (CXCL2), in mouse peritoneal macrophages. The inhibitory actions of the natural PPARgamma ligands, 15d-PGJ(2) and prostaglandin A1 (PGA1), on the expression of RANTES (regulated upon activation, normal T expressed and secreted; CCL5), MIP-1beta (CCL4), MIP-1alpha (CCL3), IFN-gamma-inducible protein 10 kilodaltons (IP-10; CXCL10) and monocyte chemoattractant protein-1 (MCP-1; CCL2) mRNA in LPS-treated cells were stronger than those of the synthetic PPARgamma ligands troglitazone and ciglitazone. However, 15d-PGJ(2) enhanced the expression of LPS-induced MIP-2 (CXCL2) mRNA. A specific PPARgamma antagonist (GW9662) had no effect on the inhibitory action of 15d-PGJ(2) and PGA1 in LPS-induced chemokine mRNA expression and on the synergistic action of 15d-PGJ(2) in LPS-induced MIP-2 (CXCL2) expression. Moreover, LPS itself reduced the expression of PPARgamma. Although the synergistic effect of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) mRNA expression was remarkable, the production of MIP-2 (CXCL2) in cells treated with 15d-PGJ(2) and LPS did not increase compared to the production in cells treated with LPS alone. The synergistic action of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) mRNA expression was dependent on the activation of nuclear factor-kappaB (NF-kappaB), and 15d-PGJ(2) increased the phosphorylation of p38 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in cells stimulated with LPS. These results suggest that the synergistic effect of 15d-PGJ(2) on LPS-induced MIP-2 (CXCL2) expression is PPARgamma-independent, and is mediated by the p38 and SAPK/JNK pathway in mitogen-activated protein kinase signaling pathways, which activates NF-kappaB. Our data may give more insights into the different mechanisms contrary to the anti-inflammatory effect of 15d-PGJ(2) on the expression of chemokine genes.

Topics: Animals; Cells, Cultured; Chemokine CXCL2; Chemokines; Chemokines, CXC; Chromans; Dose-Response Relationship, Drug; Immunologic Factors; Ligands; Lipopolysaccharides; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; PPAR gamma; Prostaglandin D2; Prostaglandins A; RNA, Messenger; Thiazolidinediones; Troglitazone; Up-Regulation

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been shown to stimulate progesterone production by bovine luteal cells. We previously reported higher expression of PPARgamma in old compared with new luteal tissue in the rat. The following studies were conducted to determine the role of PPARgamma in rat corpora lutea (CL) and test the hypothesis that PPARgamma plays a role in the metabolism of progesterone and/or luteal lifespan. Ovaries were removed from naturally cycling rats throughout the estrous cycle, and pseudopregnant rats. mRNA for PPARgamma and P450 side-chain cleavage (SCC) was localized in luteal tissue by in situ hybridization, and protein corresponding to PPARgamma and macrophages identified by immunohistochemistry. Luteal tissue was cultured with agonists (ciglitazone, prostaglandin J2) or an antagonist (GW-9662) of PPARgamma. Progesterone was measured in media by RIA and levels of mRNA for 20alpha-hydroxysteriod dehydrogenase (HSD) and bcl-2 were measured in luteal tissue after culture by RT-PCR. An inverse relationship existed between the expression of mRNA for SCC and PPARgamma. There was no effect of PPARgamma agonists or the antagonist on luteal progesterone production in vitro, or levels of mRNA for 20alpha-HSD. PPARgamma protein was localized to the nuclei of luteal cells and did not correspond with the presence of macrophages. In new CL, ciglitazone decreased mRNA for bcl-2 on proestrus, estrus, and metestrus. Interestingly, GW-9662 also decreased mRNA for bcl-2 on proestrus and diestrus in old and new CL, and on metestrus in new CL. These data indicate that PPARgamma is not a major player in luteal progesterone production or metabolism but may be involved in regulating luteal lifespan.

Topics: Anilides; Animals; Cell Nucleus; Cholesterol Side-Chain Cleavage Enzyme; Corpus Luteum; Corpus Luteum Maintenance; Female; Immunohistochemistry; In Situ Hybridization; PPAR gamma; Pregnancy; Prostaglandin D2; Proto-Oncogene Proteins c-bcl-2; Pseudopregnancy; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazolidinediones; Tissue Culture Techniques

Respiratory syncytial virus (RSV) is the major causative agent of severe lower respiratory tract disease and death in infants worldwide. The epithelial cells of the airways are the target cells for RSV infection and the site of the majority of the inflammation associated with the disease. However, despite five decades of intensive RSV research there exist neither an effective active vaccine nor a promising antiviral and anti-inflammatory therapy. Recently, peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a member of the nuclear hormone receptor superfamily, has been shown to possess anti-inflammatory properties. Therefore, we hypothesized whether the detrimental increase of intercellular adhesion molecule-1 (ICAM-1) on RSV-infected lung epithelial cells (A549 and primary normal human bronchial epithelial cells (NHBE)) might be modulated by natural and synthetic PPAR-gamma agonists (15d-PGJ2, ciglitazone, troglitazone, Fmoc-Leu). Our data show that all PPAR-gamma agonists under study significantly down-regulated the RSV-induced expression of ICAM-1 on A549- and NHBE cells in a dose-dependent manner resulting in a reduced beta2 integrin-mediated adhesion of monocytic effector cells (U937) to RSV-infected A549 cell monolayers. In contrast, the PPAR-alpha agonist bezafibrate had no impact on the RSV-induced ICAM-1 expression. The reduced ICAM-1 expression was associated with a diminished ICAM-1 mRNA level and binding activity of nuclear factor-kappaB (p65/p50) in A549 cells. These findings suggest that PPARgamma agonists have beneficial effects in the suppression of the inflammatory response during RSV infection and therefore might have clinical efficacy in the course of severe RSV-infection.

Topics: Amino Acids; Cell Adhesion; Cell Line; Cells, Cultured; Chromans; Dose-Response Relationship, Drug; Down-Regulation; Epithelial Cells; Fluorenes; Humans; Intercellular Adhesion Molecule-1; Lung; NF-kappa B; PPAR gamma; Prostaglandin D2; Respiratory Mucosa; Respiratory Syncytial Virus Infections; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazolidinediones; Troglitazone; U937 Cells

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) is a nuclear receptor that regulates diverse biological functions including inflammation. The PPARgamma ligands have been reported to exert cardioprotective effects and attenuate myocardial reperfusion injury. Here, we examined the molecular mechanisms of their anti-inflammatory effects. Male Wistar rats were subjected to myocardial ischemia and reperfusion and were treated with the PPAR-gamma ligands, 15-deoxy-Delta-prostaglandin J2 (15d-PGJ2) or ciglitazone, or with vehicle only, in the absence or presence of the selective PPAR-gamma antagonist GW-9662. In vehicle-treated rats, myocardial injury was associated with elevated tissue activity of myeloperoxidase, indicating infiltration of neutrophils, and elevated plasma levels of creatine kinase and tumor necrosis factor-alpha. These events were preceded by activation of the nuclear factor-kappaB pathway. The PPAR-gamma DNA binding was also increased in the heart after reperfusion. Treatment with ciglitazone or 15d-PGJ2 reduced myocardial damage and neutrophil infiltration and blunted creatine kinase levels and cytokine production. The beneficial effects of both ligands were associated with enhancement of PPAR-gamma DNA binding and reduction of nuclear factor-kappaB activation. Treatment with 15d-PGJ2, but not ciglitazone, enhanced DNA binding of heat shock factor 1 and upregulated the expression of the cardioprotective heat shock protein 70. Treatment with 15d-PGJ2, but not ciglitazone, also induced a significant increase in nuclear phosphorylation of the prosurvival kinase Akt. The cardioprotection afforded by ciglitazone was attenuated by the PPAR-gamma antagonist GW-9662. In contrast, GW-9662 did not affect the beneficial effects afforded by 15d-PGJ2. Thus, our data suggest that treatment with these chemically unrelated PPAR-gamma ligands results in diverse anti-inflammatory mechanisms.

Topics: Anilides; Animals; Creatine Kinase; DNA-Binding Proteins; Heat Shock Transcription Factors; HSP70 Heat-Shock Proteins; Hypoglycemic Agents; Immunologic Factors; Inflammation; Ligands; Male; Myocardial Reperfusion Injury; Neutrophil Infiltration; NF-kappa B; Peroxidase; PPAR gamma; Prostaglandin D2; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; Signal Transduction; Thiazolidinediones; Transcription Factors; Tumor Necrosis Factor-alpha; Up-Regulation

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a ligand-activated nuclear receptor with effects on inflammation, atherosclerosis, and apoptosis. The endogenous PPARgamma ligand, 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2), and the synthetic ligand, ciglitazone, have anti-inflammatory properties in endothelial cells. In addition to PPARgamma-dependent effects on the anti-inflammatory process, it has been proposed that PPARgamma ligands may also inhibit the nuclear transcription factor kappaB (NFkappaB) pathway in a PPARgamma-independent manner. The purpose of this study was to compare the effects of 15d-PGJ2 and ciglitazone on the cytokine-induced activation of the NFkappaB pathway. Human umbilical vein endothelial cells (HUVECs) were transiently transfected with NFkappaB-luciferase or PPARgamma elements-luciferase reporter constructs for 48 h. The HUVECs were pretreated with 15d-PGJ2 or ciglitazone (30 microM) for 1 h, followed by a 4-h stimulation with tumor necrosis factor alpha (100 U/mL). Luciferase assay was performed to determine reporter activity. Additionally, HUVECs were transiently transfected with a dominant-negative mutant, which retains ligand and DNA binding but exhibits markedly reduced transactivation. Stimulation of HUVEC with tumor necrosis factor alpha increased NFkappaB activation while decreasing PPARgamma activity. Overexpression of a dominant-negative PPARgamma mutant prevented the inhibitory effect of ciglitazone on cytokine-induced NFkappaB activation in transfected human endothelial cells. Conversely, 15d-PGJ2 inhibited the cytokine-induced NFkappaB activation even in the absence of PPARgamma. Our data suggest that 15d-PGJ2 exerts direct inhibitory effects on the NFkappaB pathway through a PPARgamma-independent mechanism. On the contrary, the inhibitory effect of ciglitazone on the NFkappaB pathway seems to require PPARgamma activation.

Topics: Cells, Cultured; Cyclooxygenase 2; Dose-Response Relationship, Drug; E-Selectin; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Humans; Intercellular Adhesion Molecule-1; Luciferases; Mutation; NF-kappa B; PPAR gamma; Prostaglandin D2; Recombinant Fusion Proteins; Signal Transduction; Thiazolidinediones; Transfection; Tumor Necrosis Factor-alpha; Vascular Cell Adhesion Molecule-1

Peroxisome proliferator-activated receptor gamma (PPARgamma), a ligand-activated transcription factor, is considered as an anti-osteoblastic factor associated with adiposity and the elderly osteoporosis due to a defect in osteoblastogenesis. We have found that oral administration of PPARgamma activator rosiglitazone decreased tibia BMD and serum ALP but left serum calcium and osteoclast marker C-terminal telopeptide unaffected. In addition, we examined the inhibitory mechanisms of PPARgamma on the bone formation by using PPARgamma activators ciglitazone and 15-deoxy-Delta(12,14)-prostaglandin-J2 (15d-PGJ2). Our data indicated that PPARgamma ligands decreased both mineralized bone nodules and alkaline phosphatase (ALP) activities in cultured primary osteoblasts. Reverse transcription polymerase chain reaction (RT-PCR) showed that the expression of bone morphogenetic protein-2 (BMP-2) and osteocalcin (OCN) was inhibited by ciglitizone and 15d-PGJ2. Furthermore, PPARgamma ligands inhibited NF-kappaB associated downstream COX-2 and iNOS osteogenic signaling. The ultrasound (US)-induced elevation of COX-2 and iNOS expression and nitric oxide (NO) production were attenuated in the presence of PPARgamma ligands. Furthermore, local administration of PPARgamma ligands into the metaphysis of rat tibia decreased the bone volume in secondary spongiosa. These results suggest that the activation of PPARgamma inhibits osteoblastic differentiation and the expression of several anabolic mediators involved in bone formation. These data may reflect osteoporosis and less bone formation in the aging people and patients treated with thiazolidinediones.

Topics: Aging; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Proteins; Cell Differentiation; Cell Proliferation; Cells, Cultured; Cyclooxygenase 2; Down-Regulation; Gene Expression Regulation, Enzymologic; Ligands; Nitric Oxide; Nitric Oxide Synthase Type II; Osteoblasts; Osteogenesis; PPAR gamma; Prostaglandin D2; Rats; RNA, Messenger; Rosiglitazone; Thiazolidinediones; Time Factors; Transforming Growth Factor beta

The signaling lipid molecule 15-deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) has multiple cellular functions, including anti-inflammatory and antineoplastic activities. Here, we report that 15d-PGJ2 blocks translation through inactivation of translational initiation factor eIF4A. Binding of 15d-PGJ2 to eIF4A blocks the interaction between eIF4A and eIF4G that is essential for translation of many mRNAs. Cysteine 264 in eIF4A is the target site of 15d-PGJ2. The antineoplastic activity of 15d-PGJ2 is likely attributed to inhibition of translation. Moreover, inhibition of translation by 15d-PGJ2 results in stress granule (SG) formation, into which TRAF2 is sequestered. The sequestration of TRAF2 contributes to the anti-inflammatory activity of 15d-PGJ2. These findings reveal a novel cross-talk between translation and inflammatory response, and offer new approaches to develop anticancer and anti-inflammatory drugs that target translation factors including eIF4A.

Topics: Anti-Inflammatory Agents; Arachidonic Acid; Arsenites; Chromans; Cyclopentanes; Cytoplasmic Granules; Dinoprostone; Emetine; Enzyme Inhibitors; Eukaryotic Initiation Factor-2; Eukaryotic Initiation Factor-4A; Gene Expression Regulation; HeLa Cells; Humans; Hypoglycemic Agents; Inflammation; Poly(A)-Binding Proteins; PPAR gamma; Prostaglandin D2; Prostaglandins A; Protein Biosynthesis; Protein Synthesis Inhibitors; Rosiglitazone; Signal Transduction; Sodium Compounds; T-Cell Intracellular Antigen-1; Thiazolidinediones; TNF Receptor-Associated Factor 2; Troglitazone; Tumor Necrosis Factor-alpha

The PPAR-gamma ligands, 15-deoxy-Delta(12,14)-prostaglandin J(2) and ciglitazone, and the PPAR-alpha ligand, WY-14643, were examined for their effects on proliferation and apoptosis of A375 melanoma, DU145 and PC3 prostate cancer, and MB-MDA-231 breast cancer. While 15-deoxy-Delta(12,14)-prostaglandin J(2) inhibited proliferation of A375 melanoma, ciglitazone was inactive against this and the other cell lines. Restriction of specific amino acids known to inhibit proliferation and induce apoptosis sensitized all cell lines to ciglitazone, and the combined effects were greater than the individual effects of either treatment. WY-14643 alone or in combination with amino acid deprivation was inactive. Normal fibroblasts were resistant to the treatments.

Topics: Amino Acids; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Female; Humans; Hypoglycemic Agents; Immunologic Factors; Ligands; Male; Melanoma; Methionine; Phenylalanine; PPAR gamma; Prostaglandin D2; Prostatic Neoplasms; Thiazolidinediones; Tyrosine

The epithelial cells of the airways are the target cells for respiratory syncytial virus (RSV) infection and the site of the majority of the inflammation associated with the disease. Recently, peroxisome-proliferator-activated receptor gamma (PPARgamma), a member of the nuclear hormone receptor superfamily, has been shown to possess anti-inflammatory properties. Therefore, we investigated the role of PPARgamma agonists (15d-PGJ(2), ciglitazone and troglitazone) on the synthesis of RSV-induced cytokine release from RSV-infected human lung epithelial cells (A549). We observed that all PPARgamma ligands inhibited dose-dependently the release of TNF-alpha, GM-CSF, IL-1alpha, IL-6 and the chemokines CXCL8 (IL-8) and CCL5 (RANTES) from RSV-infected A549 cells. Concomitantly, the PPARgamma ligands diminished the cellular amount of mRNA encoding for IL-6, CXCL8 and CCL5 and the RSV-induced binding activity of the transcription factors NF-kappaB (p65/p50) and AP-1 (c-fos), respectively. Our data presented herein suggest a potential application of PPARgamma ligands in the anti-inflammatory treatment of RSV infection.

Topics: Cell Line, Tumor; Chemokine CCL5; Chemokines, CC; Chromans; Cytokines; Epithelial Cells; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-1; Interleukin-6; Interleukin-8; NF-kappa B; PPAR gamma; Prostaglandin D2; Respiratory Syncytial Viruses; RNA, Messenger; Thiazolidinediones; Transcription Factor AP-1; Troglitazone; Tumor Necrosis Factor-alpha

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily. All three PPAR subtypes, PPAR-alpha, PPAR-beta/delta and PPAR-gamma are expressed in human melanocytes. In this study, we investigated the effects of PPAR-gamma activator on melanocyte growth, and apoptosis. The PPAR-gamma activators ciglitazone, troglitazone, and 15-deoxy-prostaglandin J2 inhibited melanocyte growth in a dose-dependent manner. This inhibitory effect of ciglitazone seemed to occur through induction of apoptosis. Apoptosis was increased after ciglitazone treatment, which was observed by the TUNEL method and flow cytometry. We noted a decrease in extracellular signal regulated kinase protein expression under ciglitazone treatment. Western blot analysis revealed an apparent time-dependent reduction in Bcl-2 protein levels in ciglitazone-treated melanocytes. In terms of Bax expression, a difference was not found. The expression of caspase-3 proteins was increased time-dependently with ciglitazone treatment. These results indicate that melanocyte growth and apoptosis may be modulated through PPAR-gamma and that ciglitazone, a PPAR-gamma activator, inhibits growth of human melanocytes by inducing apoptosis.

Topics: Apoptosis; bcl-2-Associated X Protein; Caspase 3; Caspases; Cell Proliferation; Cells, Cultured; Chromans; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Male; Melanocytes; PPAR gamma; Prostaglandin D2; Proto-Oncogene Proteins c-bcl-2; Thiazolidinediones; Troglitazone

Cancer cell invasion and metastasis require the concerted action of several proteases that degrade extracellular matrix proteins and basement membranes. Recent reports suggest the plasminogen activator system plays a critical role in pancreatic cancer biology. In the present study, we determined the contribution of the plasminogen activator system to pancreatic cancer cell invasion in vitro. Moreover, the effect of peroxisome proliferator-activated receptor (PPAR)-gamma ligands, which are currently in clinical use as antidiabetic drugs and interestingly seem to display antitumor activities, on pancreatic cancer cell invasion and the plasminogen activator system was assessed. Expression of components of the plasminogen activator system [i.e., urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1, and uPA receptor] was detected in six human pancreatic cancer cell lines. Inhibition of urokinase activity by specific synthetic compounds reduced baseline pancreatic cancer cell invasion. The PPAR-gamma ligands 15-deoxy-Delta12,14-prostaglandin J2 and ciglitazone also attenuated pancreatic cancer cell invasion. This effect was abrogated by dominant-negative PPAR-gamma receptors and pharmacologic PPAR-gamma inhibitors. Moreover, activation of PPAR-gamma by ligands increased plasminogen activator inhibitor-1 and decreased uPA levels in pancreatic cancer cells, and this was accompanied by a reduction in total urokinase activity. The present study shows that the plasminogen activator system plays an integral role in pancreatic cancer cell invasion in vitro. Activation of the nuclear receptor PPAR-gamma by ligands reduced pancreatic cancer cell invasion, which was largely mediated by modulation of the plasminogen activator system. These findings further underscore the potential role of PPAR-gamma ligands as therapeutic agents in pancreatic cancer.

Topics: Cell Line, Tumor; Down-Regulation; Humans; Ligands; Neoplasm Invasiveness; Pancreatic Neoplasms; Plasminogen Activator Inhibitor 1; PPAR gamma; Prostaglandin D2; Thiazolidinediones; Up-Regulation; Urokinase-Type Plasminogen Activator

2-Arachidonyl glycerol (2-AG) is an endogenous arachidonic acid derivative that binds cannabinoid receptors CB1 and CB2 and is hence termed an endocannabinoid. 2-AG also modulates a variety of immunological responses, including expression of the autocrine/paracrine T cell growth factor interleukin (IL)-2. The objective of the present studies was to determine the mechanism responsible for IL-2 suppression by 2-AG. Because of the labile properties of 2-AG, 2-AG ether, a nonhydrolyzable analog of 2-AG, was also used. Both 2-AG and 2-AG ether suppressed IL-2 expression independently of CB1 and CB2, as demonstrated in leukocytes derived from CB1/CB2-null mice. Moreover, we demonstrated that both 2-AG and 2-AG ether treatment activated peroxisome proliferator-activated receptor gamma (PPARgamma), as evidenced by forced differentiation of 3T3-L1 cells into adipocytes, induction of aP2 mRNA levels, and activation of a PPARgamma-specific luciferase reporter in transiently transfected 3T3-L1 cells. Consequently, the putative role of PPARgamma in IL-2 suppression by 2-AG and 2-AG ether was examined in Jurkat T cells. Concordant with PPARgamma involvement, the PPARgamma-specific antagonist 2-chloro-5-nitro-N-(4-pyridyl)-benzamide (T0070907) blocked 2-AG- and 2-AG ether-mediated IL-2 suppression. Likewise, 2-AG suppressed the transcriptional activity of two transcription factors crucial for IL-2 expression, nuclear factor of activated T cells and nuclear factor kappaB, in the absence but not in the presence of T0070907. 2-AG treatment also induced PPARgamma binding to a PPAR response element in activated Jurkat T cells. Together, the aforementioned studies identify PPARgamma as a novel intracellular target of 2-AG, which mediates the suppression of IL-2 by 2-AG in a manner that is independent of CB1 and/or CB2.

Topics: 3T3-L1 Cells; Adipogenesis; Animals; Arachidonic Acids; Benzamides; Cells, Cultured; Endocannabinoids; Fatty Acid-Binding Proteins; Female; Gene Expression; Glycerides; Humans; Interferon-gamma; Interleukin-2; Interleukin-4; Jurkat Cells; Mice; Mice, Inbred C57BL; Mice, Knockout; NF-kappa B; NFATC Transcription Factors; Peroxisome Proliferator-Activated Receptors; PPAR gamma; Prostaglandin D2; Protein Binding; Pyridines; Receptor, Cannabinoid, CB1; Receptor, Cannabinoid, CB2; Response Elements; T-Lymphocytes; Thiazolidinediones

We have previously shown that peroxisome proliferator-activated receptor-gamma (PPARgamma) agonists inhibited the inflammatory response of RSV-infected human lung epithelial cells. In this study, we supply evidence that specific PPARgamma agonists (15d-PGJ2, ciglitazone, troglitazone, Fmoc-Leu) efficiently blocked the RSV-induced cytotoxicity and development of syncytia in tissue culture (A549, HEp-2). All PPARgamma agonists under study markedly inhibited the cell surface expression of the viral G and F protein on RSV-infected A549 cells. This was paralleled by a reduced cellular amount of N protein-encoding mRNA determined by real-time RT-PCR. Concomitantly, a reduced release of infectious progeny virus into the cell supernatants of human lung epithelial cells (A549, normal human bronchial epithelial cells (NHBE)) was observed. Similar results were obtained regardless whether PPARgamma agonists were added prior to RSV infection or thereafter, suggesting that the agonists inhibited viral gene expression and not the primary adhesion or fusion process.

Topics: Chromans; Humans; Lung; PPAR gamma; Prostaglandin D2; Respiratory Mucosa; Respiratory Syncytial Viruses; Thiazolidinediones; Troglitazone; Virus Replication

To study the effects of peroxisome proliferators-activated receptor gamma (PPARgamma) agonists on transforming growth factor (TGF)-beta1-induced fibrotic responses in renal fibroblasts, so as to investigate its effects in prevention of tubulointerstitial fibrosis.. Rat renal fibroblasts of the line NRK/49F were cultured and divided into groups. In group 1 TGFbeta1 of the concentrations of 0, 1, 2, 5, and 10 ng/ml was added and co-cultured for 24 h. In group 2 TGFbeta1 of the concentration of 5 ng/ml was added and co-cultured for 0, 6, 12, and 24 h respectively. Groups 3, 4, and 5 were pretreated with 10 micromol/L15d-PGJ2, PPARgamma ligand, 10 micromol/L troglitazone, agonist of, and 10 micromol/L ciglitazone, both PPARgamma agonists, respectively for 2 h, then treated with 5 ng/ml TGFbeta1. A blank control group was set up. The cultured cells were collected. RT-PCR was used to detect the mRNA expression of TGF-beta1-indued fibronectin (FN). Western blotting was used to detect the expression of TGF-beta1-induced FN, Smad, and phosphorylated Smad (p-Smad) proteins.. TGF-beta1 enhanced the FN mRNA expression in a dose- and time-dependent manner. The FN mRNA expression of the 5 ng/ml TGF-beta1 group was 3.7 times that of the control group (P < 0.05). The FN mRNA expression of the 15d-PGJ2, troglitazone-, and ciglitazone-pretreated groups were lower than that of the 5 ng/ml TGF-beta1 group by 37.3%, 41.5%, and 22.7% respectively (all P < 0.05). The p-Smad2/3 protein expression levels of the TGF-beta1 group began to increase 15 minutes after stimulation, increased in a time-dependent manner, peaked 1 hour after, and began to decrease 2 hours later. However, the levels of protein expression of total Smad2 and Smad3 did not change significantly in all groups. Both 2 ng/ml TGFbeta1 and 5 ng/ml TGFbeta1 significantly induced the increase of protein expression of p-Smad2/3 (all P < 0.05). The levels of protein expression of p-Smad2 and p-Smad3 of the 5 ng/ml TGFbeta1 group were 3.42 and 0.97 times those of the 2 ng/ml TGFbeta1 (both P < 0.05). The levels of protein expression of p-Smad2/3 of the 15d-PGJ2, troglitazone-, and ciglitazone-pretreated groups were all significantly lower than that of the 5 ng/ml TGFbeta1 group by 61.2%, 53.0%, and 59.5% (all P < 0.05), However, there was no significant difference among different drug-treated groups (all P > 0.05).. Possibly through abrogating TGF-beta1/Smads signaling pathway, PPARgamma agonists inhibit TGF-beta1-induced renal fibroblast extracellular matrix synthesis and may play a potential role in preventing tubulointerstitial fibrosis as a novel approach to prevent the progress of end stage renal dysfunction.

Topics: Animals; Blotting, Far-Western; Cells, Cultured; Chromans; Dose-Response Relationship, Drug; Extracellular Matrix Proteins; Fibroblasts; Fibronectins; Kidney; Peroxisome Proliferator-Activated Receptors; Prostaglandin D2; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Smad Proteins; Thiazolidinediones; Transforming Growth Factor beta; Troglitazone

Inflammatory mechanisms may play an important role in the pathogenesis of cisplatin nephrotoxicity. Agonists of the peroxisome proliferator-activated receptor-gamma (PPARgamma), such as rosiglitazone, have been recently demonstrated to regulate inflammation by modulating the production of inflammatory mediators and adhesion molecules. The purpose of this study was to examine the protective effects of rosiglitazone on cisplatin nephrotoxicity and to explore the mechanism of its renoprotection.. Mice were treated with cisplatin with or without pre-treatment with rosiglitazone. Renal functions, histological findings, aquaporin 2 (AQP2) and adhesion molecule expression, macrophage infiltration and tumour necrosis factor-alpha (TNF-alpha) levels were investigated. The effect of rosiglitazone on nuclear factor (NF)-kappaB activity and on viability was examined using cultured human kidney (HK-2) cells.. Rosiglitazone significantly decreased both the damage to renal function and histological pathology after cisplatin injection. Pre-treatment with rosiglitazone reduced the systemic levels of TNF-alpha and down-regulated adhesion molecule expression in addition to the infiltration of inflammatory cells after cisplatin administration. Rosiglitazone restored the decreased AQP2 expression after cisplatin treatment. Pre-treatment with rosiglitazone blocked the phosphorylation of the p65 subunit of NF-kappaB in cultured HK-2 cells. Rosiglitazone had a protective effect via a PPARgamma-dependent pathway in cisplatin-treated HK-2 cells.. These results showed that pre-treatment with rosiglitazone attenuates cisplatin-induced renal damage through the suppression of TNF-alpha overproduction and NF-kappaB activation.

Topics: Anilides; Animals; Apoptosis; C-Peptide; Cell Line; Chromans; Cisplatin; Drug Evaluation, Preclinical; Glioma; Humans; Hypoglycemic Agents; Inflammation; Insulin; Intercellular Adhesion Molecule-1; Kidney; Kidney Diseases; Kidney Function Tests; Kidney Tubules, Proximal; Macrophages; Male; Mice; Mice, Inbred C57BL; Monocytes; PPAR gamma; Prostaglandin D2; Protein Transport; Rosiglitazone; Thiazolidinediones; Transcription Factor RelA; Troglitazone; Tumor Necrosis Factor-alpha

Peroxisome proliferator-activated receptor gamma (PPARgamma) agonists cause cell death in several types of cancer cells. The aim of this study was to examine the effects of two PPARgamma agonists, ciglitazone and 15-deoxy-delta(12,14)-prostaglandin J2 (15dPGJ2), on the survival of thyroid carcinoma CGTH W-2 cells. Both ciglitazone and 15dPGJ2 decreased cell viability in a time- and dose-dependent manner. Cell death was mainly due to apoptosis, with a minor contribution from necrosis. Increased levels of active caspase 3, cleaved poly (ADP-ribose) polymerase (PARP), and cytosolic cytochrome-c were noted. In addition, ciglitazone and 15dPGJ2 induced detachment of CGTH W-2 cells from the culture substratum. Both the protein levels and immunostaining signals of focal adhesion (FA) proteins, including vinculin, integrin beta1, focal adhesion kinase (FAK), and paxillin were decreased after PPARgamma agonist treatment. Meanwhile, reduced phosphorylation of FAK and paxillin was noted. Furthermore, PPARgamma agonists induced expression of protein tyrosine phosphatase-PEST (PTP-PEST), and of phosphatase and tensin homologue deleted on chromosome ten (PTEN). The upregulation of these phosphatases might contribute to the dephosphorylation of FAK and paxillin, since pre-treatment with orthovanadate prevented PPARgamma agonist-induced dephosphorylation of FAK and paxillin. Perturbation of CGTH W-2 cells with anti-integrin beta1 antibodies induced FA disruption and apoptosis in the same cells, thus the downregulation of integrin beta1 by PPARgamma agonists resulted in FA disassembly and might induce apoptosis via anoikis. Our results suggested the presence of crosstalk between apoptosis and integrin-FA signaling. Moreover, upregulation and activation of PTEN was correlated with reduced phosphorylation of Akt, and this consequence disfavored cell survival. In conclusion, PPARgamma agonists induced apoptosis of thyroid carcinoma cells via the cytochrome-c caspase 3 and PTEN-Akt pathways, and induced necrosis via the PARP pathway.

Topics: Apoptosis; Asian People; Carcinoma; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Focal Adhesions; Humans; Hypoglycemic Agents; Necrosis; Neoplasm Proteins; PPAR gamma; Prostaglandin D2; Signal Transduction; Thiazolidinediones; Thyroid Neoplasms

Peroxisome proliferator-activated receptor-gamma is a nuclear receptor transcription factor that regulates cell growth, differentiation and homeostasis. PPARgamma agonists have been used to treat obesity, diabetes, cancer and inflammation and recent studies have shown the protective effects of PPARgamma agonists on experimental allergic encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disease model of multiple sclerosis (MS). Our studies have further demonstrated that the PPARgamma agonists, 15d-PGJ2 and Ciglitazone, inhibit EAE through blocking IL-12 signaling leading to Th1 differentiation and the PPARgamma deficient heterozygous mice (PPARgamma+/-) or those treated with PPARgamma antagonists develop an exacerbated EAE in association with an augmented Th1 response. In this study, we show that the PPARgamma antagonists, Bisphenol A diglycidyl ether (BADGE) and 2-chloro-5-nitro-N-(4-pyridyl)benzamide (T0070907), reverse the inhibition of EAE by the PPARgamma agonists, Ciglitazone and 15-Deoxy-Delta(12,14)-Prostaglandin J2, in C57BL/6 wild-type and PPARgamma+/- mice. The reversal of EAE by BADGE and T0070907 was associated with restoration of neural antigen-induced T cell proliferation, IFNgamma production and Th1 differentiation inhibited by Ciglitazone and 15d-PGJ2. These results suggest that Ciglitazone and 15d-PGJ2 ameliorate EAE through PPARgamma-dependent mechanisms and further confirm a physiological role for PPARgamma in the regulation of CNS inflammation and demyelination in EAE.

Topics: Animals; Antigens; Benzamides; Benzhydryl Compounds; Cell Differentiation; Cell Proliferation; Demyelinating Diseases; Encephalomyelitis, Autoimmune, Experimental; Enzyme-Linked Immunosorbent Assay; Epoxy Compounds; Female; Immunologic Factors; Inflammation; Interferon-gamma; Mice; Mice, Inbred C57BL; Myelin Proteins; Myelin-Associated Glycoprotein; Myelin-Oligodendrocyte Glycoprotein; PPAR gamma; Prostaglandin D2; Pyridines; Th1 Cells; Thiazolidinediones

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a transcription factor important for adipogenesis and more recently has been shown to be an anticancer target. PPARgamma ligands, including the endogenous ligand 15-deoxy-Delta12,14-PGJ2 (15d-PGJ2) and synthetic ligands like ciglitazone and troglitazone, all induce apoptosis in normal and malignant human B lymphocytes, but the dependency of PPARgamma for apoptosis induction is unknown. In this study, we used a PPARgamma dominant-negative approach and a small molecule irreversible PPARgamma antagonist and found that these inhibitors prevented PPARgamma activation but did not prevent B cell apoptosis induced by 15d-PGJ2 or ciglitazone. In addition, a PPARgamma agonist that is a structural analog of 15d-PGJ2, and lacks the electrophilic carbon of the 15d-PGJ2 cyclopentenone ring, activated PPARgamma but did not kill B lymphocytes, further supporting a non-PPARgamma-mediated mechanism. To further investigate the apoptotic mechanism, the effects of 15d-PGJ2 and ciglitazone on reactive oxygen species were investigated. 15d-PGJ2, but not ciglitazone, potently induced reactive oxygen species in B lymphocytes, implicating the reactive nature of the 15d-PGJ2 structure in the apoptosis mechanism. In addition, 15d-PGJ2 caused an almost complete depletion of intracellular glutathione. Moreover, incubation with glutathione reduced ethyl ester, an antioxidant, prevented apoptosis induced by 15d-PGJ2, but not by ciglitazone. These findings indicate that the expression of PPARgamma may not be predictive of whether a normal or malignant B lineage cell is sensitive to PPARgamma agonists. Furthermore, these new findings support continued investigation into the use of PPARgamma agonists as agents to attenuate normal B cell responses and as anti-B cell lymphoma agents.

Topics: Apoptosis; B-Lymphocytes; Humans; Ligands; Lymphoma, B-Cell; PPAR gamma; Prostaglandin D2; Reactive Oxygen Species; Thiazolidinediones

Brain abscesses arise from a focal parenchymal infection by various pathogens, particularly Staphylococcus aureus. We have shown that astrocytes are activated upon exposure to S. aureus and may contribute to the excessive tissue damage characteristic of brain abscess. Therefore, modulating astrocyte activation may facilitate a reduction in brain abscess severity. Peroxisome proliferator activated receptor-gamma (PPAR-gamma) agonists are potent inhibitors of microglial activation; however, the effects of these compounds on S. aureus-dependent astrocyte activation have not yet been examined. Here, we demonstrate that two chemically distinct PPAR-gamma agonists, 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone, suppress the production of several pro-inflammatory molecules in S. aureus-stimulated astrocytes including interleukin-1beta and nitric oxide (NO). Interestingly, 15d-PGJ2 attenuated Toll-like receptor 2 (TLR2) and inducible nitric oxide synthase expression, but failed to modulate macrophage inflammatory protein-2 (MIP-2/CXCL2) production, suggesting that 15d-PGJ2 is not a global inhibitor of astrocyte activation. Another novel finding of this study was the fact that both 15d-PGJ2 and ciglitazone were capable of attenuating pre-existing astrocyte activation, indicating their potential benefit in a therapeutic setting. Importantly, 15d-PGJ2 and ciglitazone were still capable of inhibiting S. aureus-induced pro-inflammatory mediator release in PPAR-gamma-deficient astrocytes, supporting PPAR-gamma-independent effects of these compounds. Collectively, these results suggest that 15d-PGJ2 and ciglitazone exert their anti-inflammatory actions on astrocytes primarily independent of the PPAR-gamma pathway.

Topics: Animals; Animals, Newborn; Astrocytes; Cells, Cultured; Hypoglycemic Agents; Inflammation Mediators; Mice; Mice, Inbred C57BL; Mice, Knockout; Nitric Oxide Synthase Type II; PPAR gamma; Prostaglandin D2; Staphylococcus aureus; Thiazolidinediones; Toll-Like Receptor 2

Peritoneal mesothelial cells (PMCs) play an important role in peritoneal inflammatory and immune response. It was reported that the peroxisomal proliferator-activated receptor-gamma (PPARgamma) ligand could effectively reduce inflammatory processes. However, the expression and function of PPARgamma in PMCs has not been reported. This study was to investigate the expression of PPARgamma in rat PMCs and the effect of PPARgamma activation on the production of CD40 and ICAM-1 induced by lipopolysaccharide (LPS).. Rat PMCs (RPMCs) were harvested from the peritoneal cavity of Sprague-Dawley rats and maintained under defined in vitro conditions. The cells were treated separately with LPS, 15d-PGJ(2), and ciglitazone at different time points. The mRNA and protein expression of PPARgamma, CD40 and ICAM-1 were detected by RT-PCR and Western blot, respectively. The intracellular distribution of PPARgamma was detected by immunocytochemistry.. RPMCs expressed PPARgamma both at the mRNA and protein level. The specific signals for PPARgamma were mainly localized in the nucleus with weak staining in the cytoplasm. Stimulation of RPMCs with LPS resulted in a time-dependent increase in the expression of PPARgamma with the peak of mRNA at 3 h and protein at 12 h. Thereafter the expression of PPARgamma gradually attenuated. The mRNA expressions for CD40, ICAM-1 and protein expression of ICAM-1 were significantly upregulated following stimulation with LPS. Both 15d-PGJ(2) and ciglitazone decreased the expression of CD40 mRNA and ICAM-1 protein. However, ciglitazone was less effective than 15d-PGJ(2).. There is constitutive expression of PPARgamma in cultured RPMCs and PPARgamma ligands which strongly inhibit LPS-induced CD40 and ICAM-1 production in RPMCs. It suggested that PPARgamma might play a part in the local defense of the peritoneal cavity by downregulating inflammatory mediators, which may play a potential role in preventing peritoneal fibrosis induced by peritonitis. Further in vivo study is needed to demonstrate the long-term effects.

Topics: Animals; CD40 Antigens; Cells, Cultured; Gene Expression; Hypoglycemic Agents; Immunologic Factors; Intercellular Adhesion Molecule-1; Lipopolysaccharides; Male; Peritoneum; PPAR gamma; Prostaglandin D2; Rats; Rats, Sprague-Dawley; Thiazolidinediones

Recently, we demonstrated that the peroxisome proliferator-activated receptor-gamma (PPAR-gamma) ligands, either 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) or ciglitazone, increased endothelial nitric oxide (.NO) release without altering endothelial nitric oxide synthase (eNOS) expression (4). However, the precise molecular mechanisms of PPAR-gamma-stimulated endothelial.NO release remain to be defined. Superoxide anion radical (O2-.) combines with .NO to decrease.NO bioavailability. NADPH oxidase, which produces O2-., and Cu/Zn-superoxide dismutase (Cu/Zn-SOD), which degrades O2-., thereby contribute to regulation of endothelial cell.NO metabolism. Therefore, we examined the ability of PPAR-gamma ligands to modulate endothelial O2-. metabolism through alterations in the expression and activity of NADPH oxidase or Cu/Zn-SOD. Treatment with 10 microM 15d-PGJ2 or ciglitazone for 24 h decreased human umbilical vein endothelial cell (HUVEC) membrane NADPH-dependent O2-. production detected with electron spin resonance spectroscopy. Treatment with 15d-PGJ2 or ciglitazone also reduced relative mRNA levels of the NADPH oxidase subunits, nox-1, gp91phox (nox-2), and nox-4, as measured using real-time PCR analysis. Concordantly, Western blot analysis demonstrated that 15d-PGJ2 or ciglitazone decreased nox-2 and nox-4 protein expression. PPAR-gamma ligands also stimulated both activity and expression of Cu/Zn-SOD in HUVEC. These data suggest that in addition to any direct effects on endothelial.NO production, PPAR-gamma ligands enhance endothelial.NO bioavailability, in part by altering endothelial O2-. metabolism through suppression of NADPH oxidase and induction of Cu/Zn-SOD. These findings further elucidate the molecular mechanisms by which PPAR-gamma ligands directly alter vascular endothelial function.

Topics: Blotting, Western; Cell Membrane; Cells, Cultured; Electron Spin Resonance Spectroscopy; Endothelial Cells; Humans; Hypoglycemic Agents; Ligands; NADPH Oxidases; Nitric Oxide; PPAR gamma; Prostaglandin D2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Superoxide Dismutase; Superoxides; Thiazolidinediones; Umbilical Veins

Pulmonary fibrosis is a progressive life-threatening disease for which no effective therapy exists. Myofibroblasts are one of the key effector cells in pulmonary fibrosis and are the primary source of extracellular matrix production. Drugs that inhibit the differentiation of fibroblasts to myofibroblasts have potential as antifibrotic therapies. Peroxisome proliferator-activated receptor (PPAR)-gamma is a transcription factor that upon ligation with PPARgamma agonists activates target genes containing PPAR response elements. PPARgamma agonists have anti-inflammatory activities and may have potential as antifibrotic agents. In this study, we examined the abilities of PPARgamma agonists to block two of the most important profibrotic activities of TGF-beta on pulmonary fibroblasts: myofibroblast differentiation and production of excess collagen. Both natural (15d-PGJ2) and synthetic (ciglitazone and rosiglitazone) PPARgamma agonists inhibited TGF-beta-driven myofibroblast differentiation, as determined by alpha-smooth muscle actin-specific immunocytochemistry and Western blot analysis. PPARgamma agonists also potently attenuated TGF-beta-driven type I collagen protein production. A dominant-negative PPARgamma partially reversed the inhibition of myofibroblast differentiation by 15d-PGJ2 and rosiglitazone, but the irreversible PPARgamma antagonist GW-9662 did not, suggesting that the antifibrotic effects of the PPARgamma agonists are mediated through both PPARgamma-dependent and independent mechanisms. Thus PPARgamma agonists have novel and potent antifibrotic effects in human lung fibroblasts and may have potential for therapy of fibrotic diseases in the lung and other tissues.

Topics: Actins; Anilides; Cell Differentiation; Collagen Type I; Fibroblasts; Humans; Hypoglycemic Agents; Lung; Muscle, Smooth; PPAR gamma; Prostaglandin D2; Pulmonary Fibrosis; Rosiglitazone; Thiazolidinediones; Transforming Growth Factor beta

Peroxisome proliferator-activated receptor gamma (PPARgamma) plays a critical albeit poorly defined role in the development and progression of several cancer types including those of the breast, colon, and lung. A PPAR response element (PPRE) reporter assay was utilized to evaluate the selective transactivation of PPARgamma in 10 different cell lines including normal mammary epithelial, breast, lung, and colon cancer cells. Cells were treated with one of four compounds including rosglitizone (Ros), ciglitizone (Cig), 15-deoxy-Delta(12,14)-prostaglandin J2 (PGJ2), or GW 9662 (GW). We observed differences in transactivation between cell lines from different tissue origin, across cell lines from a single tissue type, and selective modulation of PPARgamma within a single cell line by different ligands. Interestingly, GW, a PPARgamma antagonist in adipocytes, enhanced PPRE reporter activation in normal mammary epithelial cells while it had virtually no effect in any of the cancer cell lines tested. Within each cancer type, individual cell lines were found to respond differently to distinct PPARgamma ligands. For instance, Ros, Cig, and PGJ2 were all potent agonist of PPARgamma transactivation in lung adenocarcinoma cell lines while these same ligands had no effect in squamous cell or large cell carcinomas of the lung. Message levels of PPARgamma and retinoid X receptor alpha (RXRalpha) in the individual cell lines were quantitated by real time-polymerase chain reaction (RT-PCR). The ratio of PPARgamma to RXRalpha was predictive of how cells responded to co-treatment of Ros and 9-cis-retinoic acid, an RXRalpha agonist, in two out of three cell lines tested. These data indicate that PPARgamma can be selectively modulated and suggests that it may be used as a therapeutic target for individual tumors.

Topics: Alitretinoin; Anilides; Breast Neoplasms; Caco-2 Cells; Cell Line, Tumor; Colonic Neoplasms; Female; Gene Expression Regulation, Neoplastic; Genes, Reporter; HT29 Cells; Humans; Ligands; Lung Neoplasms; PPAR gamma; Prostaglandin D2; Retinoid X Receptor alpha; RNA, Messenger; Rosiglitazone; Thiazolidinediones; Transfection; Tretinoin

15-Deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) was initially identified as a high affinity natural ligand for the peroxisome proliferator-activated receptor (PPAR)-gamma. Recent studies have shown that it has a potent anti-inflammatory effect by attenuating the expression of proinflammatory mediators in activated macrophages, mainly through the inhibition of nuclear factor (NF)-kappaB-dependent transcription of inflammatory genes. In this study, we investigated the synergistic effect of 15d-PGJ(2) on the expression of LPS-induced chemokine KC mRNA in mouse peritoneal macrophages. The time course of KC mRNA expression in cells stimulated with 15d-PGJ(2) plus LPS simultaneously (15d-PGJ(2)/LPS) showed similar patterns to the cells treated with LPS alone, and 15d-PGJ(2) had no effect on the stability of LPS-induced KC mRNA expression. Although NF-kappaB activity in cells treated with LPS was augmented by 15d-PGJ(2), pyrrolidone dithiocarbamate (PDTC) did not block the synergistic effect of 15d-PGJ(2) on LPS-induced KC mRNA expression. However, the synergistic effect of 15d-PGJ(2) was markedly inhibited when the macrophages were treated with a inhibitor of the mitogen-activated protein kinase (MAPK) signalling pathway, 2'-amino-3'-methoxyflavine (PD98059). Therefore, the mechanism of synergistic action of 15d-PGJ(2) on the expression of LPS-induced KC mRNA in mouse peritoneal macrophages is possibly related to the MAPK signalling pathway, not to NF-kappaB activation. These data may contribute to unravelling some of the different mechanisms contrary to the anti-inflammatory effect of 15d-PGJ(2).

Topics: Animals; Blotting, Northern; Chemokine CXCL1; Chemokines, CXC; Cycloheximide; Dactinomycin; Dose-Response Relationship, Drug; Drug Synergism; Enzyme Inhibitors; Flavonoids; Gene Expression; Hypoglycemic Agents; Immunologic Factors; Intercellular Signaling Peptides and Proteins; Lipopolysaccharides; Macrophages, Peritoneal; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; NF-kappa B; PPAR gamma; Prostaglandin D2; Pyrrolidines; RNA Stability; RNA, Messenger; Specific Pathogen-Free Organisms; Thiazolidinediones; Thiocarbamates; Time Factors; Up-Regulation

Arachidonic acid (AA) metabolizing enzymes and peroxisome proliferator-activated receptors (PPARs) have been shown to regulate the growth of epithelial cells. We have previously reported that exposure to the 5-lipoxygenase activating protein-directed inhibitor MK886 but not the cyclooxygenase inhibitor, indomethacin, reduced growth, increased apoptosis, and up-regulated PPARalpha and gamma expression in breast cancer cell lines. In the present study, we explore approaches to maximizing the proapoptotic effects of PPARgamma on lung cancer cell lines. Non-small-cell cancer cell line A549 revealed dose-dependent PPARgamma reporter activity after treatment with MK886. The addition of indomethacin in combination with MK886 further increases reporter activity. We also show increased growth inhibition and up-regulation of apoptosis after exposure to MK886 alone, or in combination with indomethacin and the PPAR ligand, 15-deoxy-Delta12,14-prostaglandin J2 compared with single drug exposures on the adenocarcinoma cell line A549 and small-cell cancer cell lines H345, N417, and H510. Real-time PCR analyses showed increased PPAR mRNA and retinoid X receptor (RXR)alpha mRNA expression after exposure to MK886 and indomethacin in a time-dependent fashion. The results suggest that the principal proapoptotic effect of these drugs may be mediated through the known antiproliferative effects of the PPARgamma-RXR interaction. We therefore explored a three-drug approach to attempt to maximize this effect. The combination of low-dose MK886, ciglitazone, and 13-cis-retinoic acid interacted at least in a superadditive fashion to inhibit the growth of lung cancer cell lines A549 and H1299, suggesting that targeting PPARgamma and AA action is a promising approach to lung cancer growth with a favorable therapeutic index.

Topics: Acetophenones; Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Arachidonic Acid; Caspases; Cell Growth Processes; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Indoles; Isotretinoin; Ligands; Lung Neoplasms; Peroxisome Proliferator-Activated Receptors; Prostaglandin D2; Pyrimidines; Retinoid X Receptor alpha; RNA, Messenger; Tetrazoles; Thiazolidinediones

15-deoxy-delta(12,14)-PGJ(2)(15d-PGJ(2)) is a natural ligand that activates the peroxisome proliferators-activated receptor (PPAR) gamma, a member of nuclear receptor family implicated in regulation of lipid metabolism and adipocyte differentiation. Recent studies have shown that 15d-PGJ(2) is the potent anti-inflammatory agent functioning via PPARgamma-dependent and -independent mechanisms. Most postulated mechanisms for anti-inflammatory action of PPARgamma agonists are involved in inhibiting NF-kappaB signaling pathway. We examined the possibility that IL-6 signaling via the Jak-Stat pathway is modulated by 15d-PGJ(2) in lymphocytes and also examined whether the inhibition of IL-6 signaling is dependent of PPARgamma. 15d-PGJ(2) blocked IL-6 induced Stat1 and Stat3 activation in primary human lymphocytes, Jurkat cells and immortalized rheumatoid arthritis B cells. Inhibition of IL-6 signaling was induced rapidly within 15 min after treatment of 15d-PGJ(2). Other PPARgamma-agonists, such as troglitazone and ciglitazone, did not inhibit IL-6 signaling, indicating that 15d-PGJ(2) affect the IL-6-induced Jak-Stat signaling pathway via PPARgamma-independent mechanism. Although cycloheximide reversed 15d-PGJ(2)-mediated inhibition of Stat3 activation, actinomycin D had no effect on 15d-PGJ(2)-mediated inhibition of IL-6 signaling, indicating that inhibition of IL-6 signaling occur independent of de novo gene expression. These results show that 15d-PGJ(2) specifically inhibit Jak-Stat signaling pathway in lymphocytes, and suggest that 15d-PGJ(2) may regulate inflammatory reactions through the modulation of different signaling pathway other than NF-kappaB in lymphocytes.

Topics: Arthritis, Rheumatoid; Chromans; Cycloheximide; Dactinomycin; DNA-Binding Proteins; Gene Expression Regulation; Humans; Hypoglycemic Agents; Interleukin-6; Jurkat Cells; Lymphocytes; NF-kappa B; Phosphorylation; PPAR gamma; Prostaglandin D2; Protein Synthesis Inhibitors; Signal Transduction; STAT1 Transcription Factor; STAT3 Transcription Factor; Thiazolidinediones; Trans-Activators; Troglitazone

We recently reported that the peroxisome proliferator-activated receptor gamma (PPARgamma) ligands 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) and ciglitazone increased cultured endothelial cell nitric oxide (NO) release without increasing the expression of endothelial nitric oxide synthase (eNOS). The current study was designed to characterize further the molecular mechanisms underlying PPARgamma-ligand-stimulated increases in endothelial cell NO production.. Treating human umbilical vein endothelial cells (HUVEC) with PPARgamma ligands (10 micromol/L 15d-PGJ2, ciglitazone, or rosiglitazone) for 24 hours increased NOS activity and NO release. In selected studies, HUVEC were treated with PPARgamma ligands and with the PPARgamma antagonist GW9662 (2 micromol/L), which fully inhibited stimulation of a luciferase reporter gene, or with small interfering RNA to PPARgamma, which reduced HUVEC PPARgamma expression. Treatment with either small interfering RNA to PPARgamma or GW9662 inhibited 15d-PGJ2-, ciglitazone-, and rosiglitazone-induced increases in endothelial cell NO release. Rosiglitazone and 15d-PGJ2, but not ciglitazone, increased heat shock protein 90-eNOS interaction and eNOS ser1177 phosphorylation. The heat shock protein 90 inhibitor geldanamycin attenuated 15d-PGJ2- and rosiglitazone-stimulated NOS activity and NO production.. These findings further clarify mechanisms involved in PPARgamma-stimulated endothelial cell NO release and emphasize that individual ligands exert their effects through distinct PPARgamma-dependent mechanisms.

Topics: Anilides; Cells, Cultured; Endothelium, Vascular; Genes, Reporter; Humans; Hypoglycemic Agents; Ligands; Nitric Oxide; PPAR gamma; Prostaglandin D2; RNA, Small Interfering; Rosiglitazone; Signal Transduction; Thiazolidinediones; Umbilical Veins

Although peroxisome proliferator activated receptor gamma (PPARgamma) agonists have been implicated in differentiation and growth inhibition of cancer cells, the potential therapeutic and chemopreventive effects on gastric cancer are poorly defined. We examined the in vitro and in vivo effects of PPARgamma ligands on growth of gastric cancer, and the effect of PPARgamma activation on expression of cyclooxygenase 2 (COX-2) and cancer related genes.. Gastric cell lines (MKN28 and MKN45) were treated with two specific PPARgamma ligands: ciglitazone and 15-deoxy-Delta(12,)(14)-prostaglandin J(2). Cell growth was determined by bromodeoxyuridine incorporation assay and apoptosis was measured by DNA fragmentation. Expression of COX-2 was determined by western blot and real time quantitative polymerase chain reaction (PCR). Expression profiles of cancer related genes were screened with cDNA array. In vivo growth of implanted MKN45 cells in nude mice was monitored after oral treatment with rosiglitazone.. PPARgamma ligands suppressed the in vitro growth of MKN45 cells in a dose dependent manner whereas prostacyclin, a PPARdelta agonist, had no growth inhibitory effect. Growth inhibition was more pronounced in MKN45 cells, which was accompanied by DNA fragmentation and downregulation of COX-2. Screening by cDNA microarray showed that PPARgamma ligand treatment was associated with upregulation of bad and p53, and downregulation of bcl-2, bcl-xl, and cyclin E1 in MKN45 cells, which was confirmed by quantitative real time PCR. In contrast, MKN28 cells with lower PPARgamma and COX-2 expression levels had lower growth inhibitory responses to PPARgamma ligands. Microarray experiments only showed induction of the bad gene in MKN28 cells. In vivo growth of MKN45 cells in nude mice was retarded by rosiglitazone. Mean tumour volume in rosiglitazone treated mice was significantly lower than controls at six weeks (p = 0.019) and seven weeks (p = 0.001) after treatment.. PPARgamma ligands suppress both in vitro and in vivo growth of gastric cancer and may play a major role in cancer therapy and prevention.

Topics: Animals; Antineoplastic Agents; Apoptosis; Cell Division; Cyclooxygenase 2; Dose-Response Relationship, Drug; Gene Expression Regulation, Neoplastic; Humans; Intracellular Signaling Peptides and Proteins; Isoenzymes; Ligands; Male; Membrane Proteins; Mice; Mice, Inbred BALB C; Mice, Nude; Neoplasm Proteins; Neoplasm Transplantation; Nuclear Receptor Coactivators; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Rosiglitazone; Stomach Neoplasms; Thiazolidinediones; Transcription Factors; Tumor Cells, Cultured

We have previously shown that nuclear factor-kappaB inhibition by adenovirus expressing mutated IkappaB-alpha or by proteasome inhibitor increases human articular chondrocytes sensibility to apoptosis. Moreover, the nuclear factor-kappaB inhibitor BAY11-7085, a potent anti-inflammatory drug in rat adjuvant arthritis, is itself a proapoptotic agent for chondrocytes. In this work, we show that BAY 11-7085 but not the proteasome inhibitor MG-132 induced a rapid and sustained phosphorylation of extracellular signal-regulated kinases (ERK1/2) in human articular chondrocytes. The level of ERK1/2 phosphorylation correlated with BAY 11-7085 concentration and chondrocyte apoptosis. 15-Deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) and its precursor prostaglandin (PG) D2 but not PGE2 and PGF2alpha rescued chondrocytes from BAY 11-7085-induced apoptosis. 15d-PGJ2 markedly inhibited BAY 11-7085-induced phosphorylation of ERK1/2. BAY 11-7085 also induced ERK1/2 phosphorylation and apoptosis in human synovial fibroblasts, and these reactions were down-regulated by 15d-PGJ2. Further analysis in synovial fibroblasts showed that only molecules that suppressed BAY 11-7085-induced phosphorylation of ERK1/2 (i.e. 15d-PGJ2, PGD2, and to a lesser extent, MEK1/2 inhibitor UO126, but not prostaglandins E2 and F2alpha or peroxisome proliferator-activated receptor-gamma agonist ciglitazone) were able protect cells from apoptosis. These results suggested that the antiapoptotic effect of 15d-PGJ2 on chondrocytes and synovial fibroblasts might involve inhibition of ERK1/2 phosphorylation.

Topics: Annexin A5; Anti-Infective Agents; Apoptosis; Blotting, Western; Cartilage; Cartilage, Articular; Cell Survival; Cells, Cultured; Chondrocytes; Coloring Agents; Cysteine Endopeptidases; Dinoprost; Dinoprostone; Down-Regulation; Fibroblasts; Humans; I-kappa B Proteins; Immunologic Factors; Leupeptins; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Mutation; NF-kappa B; NF-KappaB Inhibitor alpha; Nitriles; Phosphorylation; Prostaglandin D2; Proteasome Endopeptidase Complex; Receptors, Cytoplasmic and Nuclear; Sulfones; Synovial Membrane; Thiazolidinediones; Transcription Factors

15-Deoxy-delta(12,14)-prostaglandin J(2) (15dPGJ(2) has been recently proposed as a potent anti-inflammatory agent. However, the mechanisms by which 15dPGJ(2) mediates its therapeutic effects in vivo are unclear. We demonstrate that 15dPGJ(2) at micromolar (2.5-10 microm) concentrations induces the expression of heme oxygenase-1 (HO-1), an anti-inflammatory enzyme, at both mRNA and protein levels in human lymphocytes. In contrast, troglitazone and ciglitazone, two thiazolidinediones that mimic several effects of 15dPGJ(2) through their binding to the peroxisome proliferator-activated receptor (PPAR)-gamma, did not affect HO-1 expression, and the positive effect of 15dPGJ(2) on this process was mimicked instead by other cyclopentenone prostaglandins (PG), such as PGD(2) (the precursor of 15dPGJ(2)) and PGA(1) and PGA(2) which do not interact with PPAR-gamma. Also, 15dPGJ(2) enhanced the intracellular production of reactive oxygen species (ROS) and increased xanthine oxidase activity in vitro. Inhibition of intracellular ROS production by N-acetylcysteine, TEMPO, Me(2)SO, 1,10-phenanthroline, or allopurinol resulted in a decreased 15dPGJ(2)-dependent HO-1 expression in the cells. Furthermore, buthionine sulfoximine, an inhibitor of reduced glutathione synthesis, or Fe(2+)/Cu(2+) ions enhanced the positive effect of 15dPGJ(2) on HO-1 expression. On the other hand, the inhibition of phosphatidylinositol 3-kinase or p38 mitogen-activated protein kinase, or the blockade of transcription factor NF-kappaB activation, hindered 15dPGJ(2)-elicited HO-1 expression. Collectively, the present data suggest that 15dPGJ(2) anti-inflammatory actions at pharmacological concentrations involve the induction of HO-1 gene expression through mechanisms independent of PPAR-gamma activation and dependent on ROS produced via the xanthine/xanthine oxidase system and/or through Fenton reactions. Both phosphatidylinositol 3-kinase and p38 mitogen-activated protein kinase signaling pathways also appear implicated in modulation of HO-1 expression by 15dPGJ(2).

Topics: Acetylcysteine; Allopurinol; Blotting, Western; Buthionine Sulfoximine; Cells, Cultured; Chromans; Cyclic N-Oxides; Cyclopentanes; Dose-Response Relationship, Drug; Gene Expression Regulation, Enzymologic; Glutathione; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; Ions; Lymphocytes; Membrane Proteins; Mitogen-Activated Protein Kinases; NF-kappa B; p38 Mitogen-Activated Protein Kinases; Phenanthrolines; Phosphatidylinositol 3-Kinases; Prostaglandin D2; Reactive Oxygen Species; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Temperature; Thiazolidinediones; Time Factors; Transcription Factors; Troglitazone; Xanthine Oxidase

Subject- Peroxisome proliferator-activated receptor (PPAR)-gamma agonists are emerging as potential protectors against inflammatory cardiovascular diseases including atherosclerosis and diabetic complications. However, their molecular mechanism of action within vasculature remains unclear. We report here that PPARgamma agonists, thiazolidinedione class drugs (TZDs), or 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) were capable of activating diacylglycerol (DAG) kinase (DGK), resulting in attenuation of DAG levels and inhibition of protein kinase C (PKC) activation. The PPARgamma agonist-induced DGK was completely blocked by a dominant-negative mutant of PPARgamma, indicating an essential receptor-dependent action. Importantly, the suppression of DAG-PKC signaling pathway was functional linkage to the anti-inflammatory properties of PPARgamma agonists in endothelial cells (EC), characterized by the inhibition of proinflammatory adhesion molecule expression and adherence of monocytes to the activated EC induced by high glucose. These findings thus demonstrate a novel molecular action of PPARgamma agonists to suppress the DAG-PKC signaling pathway via upregulation of an endogenous attenuator, DGK.

Topics: Animals; Aorta; Cattle; Cell Adhesion; Cell Adhesion Molecules; Cells, Cultured; Chromans; Diabetic Angiopathies; Diacylglycerol Kinase; Diglycerides; Endothelial Cells; Endothelium, Vascular; Enzyme Induction; Glucose; Humans; PPAR gamma; Prostaglandin D2; Protein Kinase C; Protein Kinase C beta; Protein Transport; Recombinant Fusion Proteins; Signal Transduction; Thiazolidinediones; Transfection; Troglitazone; U937 Cells; Umbilical Veins; Vasculitis

Topics: Antineoplastic Agents; Apoptosis; Chromans; Cyclin D1; Humans; Lung Neoplasms; Prostaglandin D2; Proto-Oncogene Proteins p21(ras); Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazolidinediones; Transcription Factors; Troglitazone

Background. Peroxisome proliferator activated receptors (PPARs) are ligand-activated transcription factors with multiple effects on target cell function. PPAR gamma activity is regulated by extracellular signal-regulated protein kinase (ERK), mitogen-activated protein (MAP) kinase, and PPAR gamma ligands have varying effects on activity of ERK. Different PPAR gamma ligands have been shown to have both protective and detrimental effects in the kidney. Since transcriptional activation by different PPAR agonists is ligand- and depot-specific PPAR gamma, we have examined the effects of different agonists on PPAR activity in the proximal tubule.. Opossum kidney cells were used in all experiments, transiently transfected with a PPAR response element luciferase reporter and subject to stimulation with various PPAR ligands. The role of ERK and phosphorylation in PPAR gamma activation were studied, as were the effects of PPAR agonists on ERK activation and cell proliferation.. Transcriptional activity of PPAR was not stimulated by PPAR alpha agonists, and only very modestly stimulated by a PPAR beta agonist. The PPAR gamma agonists 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), ciglitazone, and troglitazone stimulated significant transcriptional activation and phosphorylation of PPAR gamma. These effects were more marked with 15d-PGJ(2). Thiazolidinediones attenuated 15d-PGJ(2) evoked PPAR gamma activation and phosphorylation. ERK activity positively regulated PPAR activation. Only 15d-PGJ(2) stimulated ERK activity and cell proliferation, and these effects were also inhibited by thiazolidinediones.. PPAR gamma agonists exert differential effects in proximal tubule cells with thiazolidinediones behaving as partial agonists.

Topics: Animals; Cells, Cultured; Chromans; Extracellular Signal-Regulated MAP Kinases; Kidney Tubules, Proximal; Ligands; Opossums; PPAR gamma; Prostaglandin D2; Thiazolidinediones; Transcriptional Activation; Transfection; Troglitazone

Several studies have shown that PPARgamma agonists play a role in the regulation of lymphocytes function and apoptosis. However, the molecular mechanism(s) underlying the immunomodulatory effects of PPARgamma agonists are not defined yet. In this study, the effects of PPARgamma (15d PGJ2 and ciglitizone) ligands on proliferation, cytokine production and apoptosis of Jurkat and Raji cells (human T and B lymphocytes, respectively) were examined. Ciglitizone and 15d PGJ2 presented antiproliferative and cytotoxic effects on Jurkat and Raji cells as shown by [14C]-thymidine incorporation and cell viability assay. In addition, 15d PGJ2 inhibited cytokine production (IL-2 in Jurkat cells and IL-10 in Raji cells). The mechanism whereby PPARgamma agonists induced cytotoxicity is via apoptosis as shown by DNA fragmentation, nuclear condensation and phosphatidylserine externalization. The induction of apoptosis by ciglitizone and 15d PGJ2 on Jurkat and Raji cells may explain the suppression of cytokine production and the decrease in proliferation observed in both cell types. The apoptotic process was associated with a decrease in mitochondrial membrane potential and a marked down-regulation of the c-myc expression. These findings might play a key role in the apoptosis of T and B lymphocytes induced by PPARgamma agonists.

Topics: Annexin A5; Apoptosis; Benzimidazoles; Cell Membrane; Cell Proliferation; Chromatin; Cytokines; DNA Fragmentation; Fluorescent Dyes; Genes, myc; Humans; Immunohistochemistry; Jurkat Cells; Ligands; Membrane Potentials; Mitochondria; Phosphatidylserines; PPAR gamma; Prostaglandin D2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazolidinediones

Interferon-gamma (IFN-gamma) production and cytolytic activity are 2 major biologic functions of natural killer (NK) cells that are important for innate immunity. We demonstrate here that these functions are compromised in human NK cells treated with peroxisome proliferator-activated-gamma (PPAR-gamma) ligands via both PPAR-gamma-dependent and -independent pathways due to variation in PPAR-gamma expression. In PPAR-gamma-null NK cells, 15-deoxy-Delta(12,14) prostaglandin J(2) (15d-PGJ(2)), a natural PPAR-gamma ligand, reduces IFN-gamma production that can be reversed by MG132 and/or chloroquine, and it inhibits cytolytic activity of NK cells through reduction of both conjugate formation and CD69 expression. In PPARgamma-positive NK cells, PPAR-gamma activation by 15d-PGJ(2) and ciglitazone (a synthetic ligand) leads to reduction in both mRNA and protein levels of IFN-gamma. Overexpression of PPAR-gamma in PPAR-gamma-null NK cells reduces IFN-gamma gene expression. However, PPAR-gamma expression and activation has no effect on NK cell cytolytic activity. In addition, 15d-PGJ(2) but not ciglitazone reduces expression of CD69 in human NK cells, whereas CD44 expression is not affected. These results reveal novel pathways regulating NK cell biologic functions and provide a basis for the design of therapeutic agents that can regulate the function of NK cells within the innate immune response.

Topics: Humans; Hypoglycemic Agents; Interferon-gamma; Killer Cells, Natural; Ligands; PPAR gamma; Prostaglandin D2; Thiazolidinediones

There is growing evidence to show that hepatic oval cells contribute to liver regeneration, dysplastic nodule formation, and hepato-carcinogenesis. Peroxisome proliferator-activated receptors (PPARs) and their ligands play an important role in cell growth, inflammatory responses, and liver pathogenesis including fibrosis and cancer. However, little is known about the role of PPARgamma/its ligands in the growth and differentiation of hepatic oval cells. In this study, we found that OC15-5, a rat hepatic oval cell line, expressed PPARgamma at mRNA and protein levels, and a natural ligand for PPARgamma, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), and a synthetic ligand, ciglitazone, inhibited growth of OC15-5 cells by arresting at G1-S in a dose-dependent manner. Apoptosis was also induced in OC15-5 cells by 15d-PGJ2 treatment. In OC15-5 cells treated with 15d-PGJ2, the expression of CDK inhibitor, p27(Kip1), was up-regulated, while that of p21(WAF1/Cip1), p18(INK4C) CDK2, CDK4, and cyclin E was unchanged. In addition, delayed up-regulation of AFP expression was observed in OC15-5 cells after 15d-PGJ2 or ciglitazone treatment. This is the first report to show that the PPARgamma ligand was involved in the growth, cell cycle, and differentiation of hepatic oval cells, raising the possibility that the PPARgamma ligands may regulate liver regeneration and hepato-carcinogenesis.

Topics: Animals; Apoptosis; Cell Cycle; Genes, cdc; Hypoglycemic Agents; Immunologic Factors; Ligands; Liver; Prostaglandin D2; Rats; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazolidinediones; Transcription Factors; Up-Regulation

Multiple myeloma is essentially an incurable malignancy and it is therefore of great interest to develop new therapeutic approaches. We previously reported that human B cell-lymphomas express the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) and are killed by PPARgamma ligands. Herein, we investigate the therapeutic potential of PPARgamma ligands for multiple myeloma. The human multiple myeloma cell lines ANBL6 and 8226 express PPARgamma mRNA and protein. The PPARgamma ligands, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone, induced multiple myeloma cell apoptosis as determined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay, loss of mitochondrial membrane potential, and caspase activation. Importantly, the ability of PPARgamma ligands to kill both multiple myeloma cell lines was not abrogated by Interleukin-6 (IL-6), a multiple myeloma growth survival factor. Finally, the RXR ligand 9-cis retinoic acid (9-cis RA) in combination with PPARgamma ligands greatly enhanced multiple myeloma cell killing. These new findings support that PPARgamma ligands may represent a novel therapy for multiple myeloma.

Topics: Apoptosis; Blotting, Western; Caspases; Cell Line, Tumor; Enzyme Activation; Humans; Immunohistochemistry; Immunologic Factors; In Situ Nick-End Labeling; Interleukin-6; Ligands; Membrane Potentials; Mitochondria; Multiple Myeloma; PPAR gamma; Prostaglandin D2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazolidinediones; Tretinoin

Neuroblastoma is a childhood cancer, which spontaneously regresses. This has led to a search for agents that mimic this process. We show that both natural and synthetic ligands of PPARgamma (peroxisome-proliferator-activated receptor gamma) inhibit the growth of neuroblastoma cells in vitro. The degree of PPAR activation was attenuated however in the presence of the retinoblastoma protein. Addition of trichostatin A, a histone deacetylase inhibitor, abolished retinoblastoma protein repression of PPAR activity. Moreover, enhanced growth inhibition was observed when neuroblastoma cells were treated with a PPARgamma ligand and a histone deacetylase inhibitor, suggesting a combination therapy to treat neuroblastoma might prove more effective than using either agent alone.

Topics: Carboxy-Lyases; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Inhibitors; Genes, Reporter; Histones; Humans; Hydroxamic Acids; Ligands; Neuroblastoma; PPAR gamma; Prostaglandin D2; Retinoblastoma Protein; Thiazolidinediones; Time Factors; Transcription, Genetic; Transfection

During chronic inflammatory response, mono- cytes/macrophages produce 92-kDa matrix metalloproteinase-9 (MMP-9), which may contribute to their extravasation, migration and tissue remodeling. Activation of peroxisome proliferator- activated factor receptor-g (PPAR-g) has been shown to inhibit MMP-9 activity. To evaluate whether ox-LDL, a PPAR-g activator, inhibits PMA-induced MMP-9 expression and activity, and if so, whether CD36 and PPAR-g are involved in this process, we investigated the effect of ox-LDL on MMP-9 expression and activity in PMA-activated human monocytic cell line U937. PMA-induced MMP-9 expression and activity were suppressed by the treatment with ox-LDL (50 mg/ml) or PPAR-g activators such as troglitazone (5 mM), ciglitazone (5 mM), and 15d- PGJ2 (1 mM) for 24 h. This ox-LDL or PPAR-g activator-mediated inhibition of MMP-9 activity was diminished by the pre-treatment of cells with a blocking antibody to CD36, or PGF2a (0.3 mM), which is a PPAR-g inhibitor, as well as overexpression of a dominant-negative form of CD36. Taken together, these results suggest that ox-LDL suppresses PMA-induced MMP-9 expression and activity through CD36-mediated activation of PPAR-g.

Topics: Antibodies, Blocking; CD36 Antigens; Cells, Cultured; Chromans; Humans; Lipoproteins, LDL; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Monocytes; NF-kappa B; PPAR gamma; Prostaglandin D2; RNA, Messenger; Tetradecanoylphorbol Acetate; Thiazolidinediones; Transcription, Genetic; Troglitazone

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to modulate cyclooxygenase (COX)-2 expression, but the mechanisms involved are controversial and may be cell specific. We show in this study that indomethacin (Indo), flurbiprofen (Flur), and the selective COX-2 inhibitor NS-398 induced COX-2 expression and markedly enhanced IL-1beta-induced COX-2 expression in human airway smooth muscle (HASM) cells. These effects were not reversed by exogenous PGE(2), suggesting that they are prostanoid-independent. Indeed, PGE(2) also induced and enhanced IL-1beta-induced COX-2 expression. Peroxisome proliferator-activated receptor (PPAR) alpha and PPARgamma (not PPARbeta) were expressed in HASM cells. PPARgamma activators ciglitizone (Cig) and 15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), but not the PPARalpha activator WY-14643, mimicked the effect of NSAIDs on COX-2 expression. Treatment with Flur, NS-398, Cig, and 15d-PGJ(2) alone, but not Indo and WY-14643, elevated COX activity; however, neither enhanced IL-1beta-induced COX activity. Pretreatment with dexamethasone suppressed COX-2 expression, PGE(2) release, and COX activity induced by NS-398, Cig, IL-1beta, alone or in combination. Unlike IL-1beta, NS-398 and Cig did not cause NF-kappaB (p65) nuclear translocation, nor did they further enhance IL-1beta-induced NF-kappaB translocation, but they stimulated PPARgamma translocation. Indo, NS-398, Flur, and 15d-PGJ(2), but not WY-14643, induced transcriptional activity of a COX-2 reporter construct containing the peroxisome proliferator response element (PPRE) on their own and enhanced the effect of IL-1beta, but had no effect on a COX-2 reporter construct lacking the PPRE. The results suggest that COX-2 expression by NSAIDs is biologically functional, prostanoid-independent, and involves PPARgamma activation, and provide the first direct evidence that the PPRE in the promoter is required for NSAID-induced COX-2 expression.

Topics: Adjuvants, Immunologic; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Cell Survival; Cyclooxygenase 2; Dexamethasone; Dinoprostone; Flurbiprofen; Humans; Indomethacin; Isoenzymes; Lung; Membrane Proteins; Muscle, Smooth; NF-kappa B; Nitrobenzenes; Promoter Regions, Genetic; Prostaglandin Antagonists; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Prostaglandins; Protein Transport; Receptors, Cytoplasmic and Nuclear; Response Elements; Sulfonamides; Thiazoles; Thiazolidinediones; Transcription Factors; Transcriptional Activation

Peroxisome proliferator-activated receptor gamma (PPARgamma) ligands reduce lesion formation in animal models of atherosclerosis by mechanisms that have not been defined completely. We hypothesized that PPARgamma ligands stimulate endothelial-derived nitric oxide release (*NO) to protect the vascular wall.. The PPARgamma ligands, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) or ciglitazone, stimulated a PPAR response element-luciferase reporter construct in transfected porcine pulmonary artery endothelial cells (PAECs), demonstrating that PPARgamma was transcriptionally functional. Treatment with 15d-PGJ2 or ciglitazone significantly increased release of *NO from PAECs or human aortic endothelial cells and augmented calcium ionophore-induced *NO release from human umbilical vein endothelial cells measured by chemiluminescence analysis of culture media. Increases in *NO release caused by treatment with 15d-PGJ2 occurred at 24 hours, but not after 1 to 16 hours, and were abrogated by treatment with the transcriptional inhibitor alpha-amanitin. Overexpression of PPARgamma or treatment with 9-cis retinoic acid also enhanced PAEC *NO release. Neither 15d-PGJ2 nor ciglitazone altered eNOS mRNA, whereas 15d-PGJ2, but not ciglitazone, decreased eNOS protein.. Taken together, these findings demonstrate that PPARgamma ligands stimulate *NO release from endothelial cells derived from multiple vascular sites, through a transcriptional mechanism unrelated to eNOS expression.

Topics: Animals; Aorta; Cells, Cultured; Endothelium, Vascular; Enzyme Induction; Humans; Immunologic Factors; Ligands; Nitric Oxide; Nitric Oxide Synthase; Prostaglandin D2; Pulmonary Artery; Receptors, Cytoplasmic and Nuclear; Swine; Thiazoles; Thiazolidinediones; Transcription Factors; Umbilical Veins

Airway smooth muscle is actively involved in the inflammatory process in diseases such as chronic obstructive pulmonary disease and asthma by 1) contributing to airway narrowing through hyperplasia and hypertrophy and 2) the release of GM-CSF and G-CSF, which promotes the survival and activation of infiltrating leukocytes. Thus, the identification of novel anti-inflammatory pathways in airway smooth muscle will have important implications for the treatment of inflammatory airway disease. This study identifies such a pathway in the activation of peroxisome proliferator-activated receptors (PPARs). PPAR ligands are known therapeutic agents in the treatment of diabetes; however, their role in human airway disease is unknown. We demonstrate, for the first time, that human airway smooth muscle cells express PPAR alpha and -gamma subtypes. Activation of PPAR gamma by natural and synthetic ligands inhibits serum-induced cell growth more effectively than does the steroid dexamethasone, and induces apoptosis. Moreover, PPAR gamma activation, like dexamethasone, inhibits the release of GM-CSF. However, PPAR gamma ligands, but not dexamethasone, similarly inhibits G-CSF release. These results reveal a novel anti-inflammatory pathway in human airway smooth muscle, where PPAR gamma activation has additional anti-inflammatory effects to those of steroids. Hence, PPAR ligands might act as potential treatments in human respiratory diseases.

Topics: Adolescent; Adult; Anti-Inflammatory Agents; Apoptosis; Blotting, Western; Cell Division; Cells, Cultured; Dexamethasone; DNA Fragmentation; Female; Granulocyte Colony-Stimulating Factor; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Inhibitors; Humans; Interleukin-1; Ligands; Male; Middle Aged; Muscle, Smooth; Peroxisomes; Prostaglandin D2; Protein Isoforms; Pulmonary Disease, Chronic Obstructive; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazoles; Thiazolidinediones; Trachea; Transcription Factors

The peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been implicated in inhibition of the expression of proinflammatory cytokines and inducible enzymes such as cyclooxygenase-2 (COX-2). Using real-time RT-PCR the present study investigates the impact of two PPAR-gamma agonists, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) and ciglitazone, on the expression of several proinflammatory genes in lipopolysaccharide (LPS)-stimulated human blood monocytes. Stimulation of cells with LPS resulted in a profound induction of the expression of COX-2, interleukin (IL)-1, IL-6, tumor necrosis factor (TNF), and granulocyte-macrophage colony-stimulating factor (GM-CSF). Treatment of cells with 15d-PGJ(2) (10 microM) was associated with a nearly complete inhibition of the expression of all genes that remained unaltered in the presence of the PPAR-gamma antagonist bisphenol A diglycidyl ether (BADGE; 100 microM). By contrast, treatment of cells with another potent PPAR-gamma agonist, ciglitazone (50 microM), and the PPAR-alpha agonist WY-14,643 (100 microM) did not suppress LPS-induced expression of the investigated genes. Stimulation of monocytes with LPS resulted in an 88% inhibition of PPAR-gamma mRNA expression that was fully restored by 15d-PGJ(2) but only to a partial extent by ciglitazone and WY-14,643. Again, BADGE did not alter the effect of 15d-PGJ(2). Collectively, our results show that alterations of gene expression by 15d-PGJ(2) in LPS-stimulated human blood monocytes are mediated by PPAR-gamma-independent mechanisms. Moreover, it is concluded that both inhibition of proinflammatory gene expression and restoration of LPS-induced decrease of PPAR-gamma expression may contribute to the biological action of 15d-PGJ(2).

Topics: Cyclooxygenase 2; Gene Expression; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Immunologic Factors; In Vitro Techniques; Inflammation; Interleukin-1; Interleukin-6; Isoenzymes; Lipopolysaccharides; Membrane Proteins; Monocytes; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazoles; Thiazolidinediones; Transcription Factors; Tumor Necrosis Factor-alpha

Treatment of MCF-7 cells with the peroxisome proliferator-activated receptor (PPAR) gamma agonists ciglitazone or 15-deoxy-Delta 12,14-prostaglandin J2 resulted in a concentration- and time-dependent decrease of cyclin D1 and estrogen receptor (ER) alpha proteins, and this was accompanied by decreased cell proliferation and G(1)-G(0)-->S-phase progression. Down-regulation of cyclin D1 and ER alpha by PPARgamma agonists was inhibited in cells cotreated with the proteasome inhibitors MG132 and PSII, but not in cells cotreated with the protease inhibitors calpain II and calpeptin. Moreover, after treatment of MCF-7 cells with 15-deoxy-Delta 12,14-prostaglandin J2 and immunoprecipitation with cyclin D1 or ER alpha antibodies, there was enhanced formation of ubiquitinated cyclin D1 and ER alpha bands. Thus, PPARgamma-induced inhibition of breast cancer cell growth is due, in part, to proteasome-dependent degradation of cyclin D1 (and ER alpha), and this pathway may be important for other cancer cell lines.

Topics: Breast Neoplasms; Cell Division; Cyclin D1; Cysteine Endopeptidases; Down-Regulation; Estrogen Receptor alpha; G1 Phase; Humans; Multienzyme Complexes; Prostaglandin D2; Proteasome Endopeptidase Complex; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; RNA, Messenger; S Phase; Thiazoles; Thiazolidinediones; Transcription Factors; Transcription, Genetic; Tumor Cells, Cultured; Ubiquitin

To determine whether peroxisome proliferator-activated receptor (PPAR)-gamma ligands can affect the expression of interleukin-6 (IL-6) and cytokines related to the pathogenesis of endometriosis.. In vitro study to determine whether PPARs are expressed in human endometrial cells and determine the effects of various PPAR-gamma ligands on IL-6 and other cytokine expression in these cells.. Academic medical center.. Women presenting for infertility workup.. Endometrial cell cultures were treated with PPAR-gamma ligands.. Interleukin-6, IL-8, colony stimulating factor-1 (CSF-1) and macrophage chemotactic factor (MCP-1) protein secretion, messenger RNA expression of IL-6, PPAR-alpha, -beta, and -gamma.. Using a human endometrial cell line (EM42), as well as primary stromal and epithelial endometrial cells, we show the presence of PPAR-alpha, -beta, and -gamma by reverse transcription-polymerase chain reaction (RT-PCR) in these cells. PPAR-gamma ligands stimulated IL-6 secretion and induced enhancement of IL-6 mRNA levels. These ligands also stimulated the secretion of IL-8 and CSF-1.. PPAR-gamma may play a role in the pathogenesis of endometriosis related to the production of IL-6 and some other cytokines.

Topics: Benzophenones; Colony-Stimulating Factors; Cytokines; Endometrium; Epithelial Cells; Female; Humans; Interleukin-6; Ligands; Phenylacetates; Prostaglandin D2; Protein Array Analysis; Protein Isoforms; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazoles; Thiazolidinediones; Transcription Factors; Tyrosine

Peroxisome proliferator-activated receptor (PPAR) gamma is an important signaling molecule in cells of mesenchymal origin, inducing differentiation and regulating cell proliferation in several cell types such as vascular smooth muscle cells. Leiomyomas arise from smooth muscle cells of the uterine myometrium with an incidence rate as high as 70% in women of reproductive age. PPAR signaling has not been characterized in these tumors, although prostaglandins, natural PPAR ligands, are known effectors of key biological functions in the normal myometrium. Leiomyomas and tumor-derived cells isolated from a rat model for this disease were characterized by Western analysis and found to express all three PPAR isoforms, suggesting that signaling pathways mediated by these receptors were intact in this tumor type. In vitro experiments with a leiomyoma-derived cell line demonstrated that the pan-PPAR ligand cis-4,7,10,13,16,19-docosahexaenoic acid and PPARgamma-specific ligands 15-deoxy-delta(12,14)-prostaglandin J(2), troglitazone, and ciglitazone inhibited 17beta-estradiol-stimulated cell proliferation. This inhibitory effect was not observed with PPARalpha- or PPARbeta-specific ligands. Although both PPAR and estrogen receptor (ER) signaling pathways were intact in leiomyoma cells, in addition to growth inhibition, stimulation of PPARgamma signaling also inhibited ER-mediated gene expression. Human leiomyomas were also found to express all three PPAR isoforms, and primary cultures of these cells were sensitive to the inhibitory effects of PPARgamma ligands. These results suggest that in uterine leiomyomas PPARgamma activation is growth inhibitory and that this inhibition is mediated at least in part by negative cross-talk between ER and PPAR signaling pathways.

Topics: Animals; Cell Division; Chromans; Docosahexaenoic Acids; Estradiol; Female; Humans; Leiomyoma; Ligands; Prostaglandin D2; Protein Isoforms; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Estrogen; Signal Transduction; Thiazoles; Thiazolidinediones; Transcription Factors; Troglitazone; Tumor Cells, Cultured; Uterine Neoplasms

Messenger RNA for peroxisome proliferator-activated receptor gamma (PPARgamma) has been found in granulosa cells, and its expression decreases after the LH surge. We determined which developmental stage of ovarian follicle expresses mRNA for PPARgamma and evaluated the impact of PPARgamma agonists on steroidogenesis. Ovaries were collected from immature eCG/hCG-treated rats at 0 (no eCG), 24, and 48 h post-eCG and 4 and 24 h post-hCG. Ovarian tissue was serially sectioned and processed for in situ hybridization to localize mRNA corresponding to PPARgamma, aromatase, and the LH receptor, and P450 side chain cleavage (P450SCC) and to determine whether apoptotic cells were present. During follicular development, there was no correlation between the expression of mRNAs for PPARgamma and aromatase or the presence of apoptotic cells, but a general inverse correlation was observed between the expression of PPARgamma mRNA and LH receptor mRNA. At 4 h post-hCG, follicles expressing P450SCC mRNA had lost expression of PPARgamma mRNA. This inverse pattern of expression between PPARgamma and P450SCC mRNAs was also observed 24 h post-hCG, with developing luteal tissue expressing high levels of P450SCC mRNA but little or no PPARgamma mRNA. To determine the impact of PPARgamma on steroidogenesis, granulosa cells were collected from ovaries 24 h post-eCG and cultured alone, with FSH alone, or with FSH in combination with the PPARgamma agonists ciglitazone or 15-deoxy-delta 12,14-prostaglandin J2 (PGJ2). Treatment of granulosa cells with PGJ2 stimulated basal progesterone secretion, whereas ciglitazone or PGJ2 had no significant effect on FSH-stimulated steroid production. These findings suggest that 1) PPARgamma may regulate genes involved with follicular differentiation and 2) the decline in PPARgamma in response to LH is important for ovulation and/or luteinization.

Topics: Animals; Apoptosis; Cells, Cultured; Cholesterol Side-Chain Cleavage Enzyme; Cytochrome P-450 Enzyme System; Female; Follicle Stimulating Hormone; Granulosa Cells; Hypoglycemic Agents; In Situ Hybridization; Ovary; Prostaglandin D2; Radioimmunoassay; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazolidinediones; Transcription Factors

Phytohemagglutinin (PHA) elicited expression of peroxisome proliferator-activated receptor gamma (PPARgamma) in primary human T cells via the PPARgamma3 promoter, as shown by reverse transcription-polymerase chain reaction. Electrophoretic mobility shift assay demonstrated no correlation between PPARgamma expression and its activation. However, addition of specific PPARgamma agonists such as ciglitazone or 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) for 1 h following PHA pretreatment provoked PPARgamma activation verified by supershift analysis. Taking the proapoptotic properties of PPARgamma into consideration, we analyzed induction of apoptosis in activated T cells in response to PPARgamma agonists. Cells exposed to PPARgamma agonists alone revealed minor cell death compared with controls, whereas treatment with 15d-PGJ(2) or ciglitazone for 4 h subsequent to PHA stimulation significantly increased cell demise, which was attenuated by the pan-caspase inhibitor zVAD, pointing to apoptosis as the underlying mechanism. These data may be relevant for pathophysiological conditions accompanied with lymphopenia of T cells under conditions such as sepsis.

Topics: Apoptosis; Cysteine Proteinase Inhibitors; Gene Expression Regulation; Humans; Hydrazines; Jurkat Cells; Lymphocyte Activation; Lymphopenia; Nitrogen Oxides; Phytohemagglutinins; Promoter Regions, Genetic; Prostaglandin D2; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; T-Lymphocytes; Thiazoles; Thiazolidinediones; Transcription Factors

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a transcription factor in the steroid nuclear receptor superfamily. Ligand activation of PPARgamma is associated with differentiation and an inhibition of proliferation in normal and malignant cells, including adipocytes, monocytes, and tumor cells in colon, prostate, and breast cancers. The current studies were undertaken to assess both the expression and functional activity of PPARgamma in cultured normal human mammary epithelial cells (HMECs) and tissue samples. Analyses by northern hybridization, immunoblotting, and immunohistochemistry demonstrate PPARgamma gene expression in HMECs and breast tissue specimens. DNA binding and transactivation assays indicate the presence of functionally active PPARgamma in HMECs. Treatment with PPARgamma selective ligands, 15-deoxy-delta-(12,14)-prostaglandin J2 (15dPGJ2) and ciglitazone, inhibits the growth of HMECs in a dose-dependent manner. This growth inhibition is associated with alterations in cell cycle progression and the induction of apoptosis.

Topics: Apoptosis; Breast; Cell Cycle; Cell Division; Cells, Cultured; Epithelial Cells; Female; Gene Expression; Humans; Hypoglycemic Agents; Ligands; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Repressor Proteins; Thiazoles; Thiazolidinediones; Transcription Factors

Peroxisome proliferator-activated receptor gamma (PPARgamma) has been the focus of studies assessing its potential neuroprotective role. These studies have shown either neuroprotection or neurotoxicity by PPARgamma ligands. Comparison of these studies is complicated by the use of different PPARgamma ligands, mechanisms of neurotoxicity induction, and neuronal cell type. In this study, we compared the effects of the synthetic PPARgamma ligand ciglitazone with an endogenous PPARgamma ligand, 15-deoxy-delta(12,14)-prostaglandin J(2) (15-deoxy PGJ(2)), on inherent neurotoxicity and neuroprotection using a reduction in extracellular KCl in rat cultured cerebellar granule neurons (CGN). We also assessed the effects of these ligands on c-Jun protein expression, which is up-regulated on induction of low-KCl-mediated neuronal apoptosis as well as being associated with PPAR in other cell types. We showed that PPARgamma mRNA is expressed in CGN cultures and observed ciglitazone- and 15-deoxy PGJ(2)-mediated inherent neurotoxicity that was concentration and time dependent. c-Jun was only modestly increased in the presence of ciglitazone but was markedly up-regulated by 15-deoxy PGJ(2) after 12 hr. Treatment of CGN cultures with ciglitazone simultaneous with KCl withdrawal resulted in a modest, time-dependent neuroprotection. Such neuroprotection after KCl withdrawal was not observed with 15-deoxy PGJ(2). Despite the absence of neuroprotection, 15-deoxy PGJ(2) markedly inhibited the early up-regulation of c-Jun during KCl withdrawal. These studies suggest that ciglitazone and 15-deoxy PGJ(2) have markedly different effects on inherent and low-KCl-induced toxicity and c-Jun expression in CGN, indicating potential non-PPARgamma mechanisms.

Topics: Animals; Cell Survival; Cells, Cultured; Cerebellum; Dose-Response Relationship, Drug; Gene Expression Regulation; Ligands; Neurons; Prostaglandin D2; Proto-Oncogene Proteins c-jun; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Transcription Factors

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor for eicosanoids that promotes differentiation of human epithelial and mesenchymal cells in vitro and in vivo. PPARgamma was proposed as a target for drug-induced differentiation therapy of cancer. Caveolin-1 is a constituent of plasma membrane caveolae in epithelial cells that is often downregulated upon oncogenic transformation. Caveolin-1 has growth-inhibitory activities and its disruption is sufficient to induce transformation in fibroblasts. Herein we have tested the hypothesis that caveolins are transcriptional target genes for PPARgamma. In human HT-29 colon carcinoma cells, thiazolidinedione PPARgamma ligands increased the levels of caveolin-1 and caveolin-2 proteins two to fivefold in a concentration-dependent manner within 24 h. In human MCF-7 breast adenocarcinoma cells, nonthiazolidinedione PPARgamma ligands elevated caveolin-2 protein three to fourfold, while the thiazoli-dinediones were less effective. Caveolin-1 mRNA levels were found to be upregulated by PPARgamma ligands already after 3 h in both the cell lines. Ectopic expression of a dominant-negative PPARgamma construct attenuated ligand-induced upregulation of caveolins in both HT-29 and HEK-293T cells, indicating that ligand action is mediated by PPARgamma. Ligand-treated MCF-7 cells exhibited a differentiated phenotype, as evinced by analysis of cell-specific differentiation markers: protein levels of maspin were elevated and perinuclear lipid droplets accumulated. In contrast, in HT-29 cells, caveolin expression was not correlated with differentiation. Interestingly, PPARgamma partially cofractionated in lipid rafts and could be coimmunoprecipitated from cell lysates with caveolin-1, indicating that PPARgamma and caveolin-1 may coexist in a complex. Our data indicate that PPARgamma participates in the regulation of caveolin gene expression in human carcinoma cells and suggest that caveolin-1 may mediate some of the phenotypic changes induced by this nuclear receptor in cancer cells. These findings may have potentially important functional implications in the context of cancer differentiation therapy and multidrug resistance.

Topics: Adenocarcinoma; Antigens, Differentiation; Antigens, Neoplasm; Breast Neoplasms; Caveolin 1; Caveolin 2; Caveolins; Cell Differentiation; Cell Line; Chromans; Colonic Neoplasms; Dimerization; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Gene Expression Regulation, Neoplastic; Genes, Dominant; Humans; Kidney; Ligands; Macromolecular Substances; Membrane Microdomains; Neoplasm Proteins; Phenotype; Phenylacetates; Prostaglandin D2; Protein Structure, Tertiary; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; RNA, Neoplasm; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors; Transcription, Genetic; Troglitazone; Tumor Cells, Cultured

To explore the effects of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) on the growth of human lung cancer cell lines and its possible mechanism.. Human non-small cell lung cancer (NSCLC) cells of the A549 line and human small cell lung cancer (SCLC) of the LTEP-P line were cultured and were divided into 3 groups respectively: control group, 15d-PGJ(2) group (15d-PGJ(2), a PPAR-gamma activator, was added), and ciglitazone group (ciglitazone, am antidiabetic drug, was added). Twenty-four, forty-eight, and seventy-two hours later, nested RT-PCR was used to detect t the expression of PPAR-gamma mRNA, Western blotting technique was used to detect the expression of PPAR-gamma protein, MTT staining was used to observe the proliferation of cells induced by PPAR-gamma agonists, TUNEL method was used to observe the apoptosis of cells affected by the ligands of PPAR-gamma, the expressions of bax, and bcl-2 mRN and proteins were examined by in situ hybridization and immunohistochemistry, and the expression of caspase-3 was detected by immunohistochemistry.. PPAR-gamma was expressed in the two lung cancer cell lines. The cell proliferation was inhibited by 15d-PGJ(2) and ciglitazone, especially the former, in dose-dependent and time-dependent manners. The apoptosis rates were (1.9 +/- 0.5)%, (9.8 +/- 1.5)%, and (5.6 +/- 1.1)% respectively in the control, 15d-PGJ(2), and ciglitazone groups with a significant difference between ant 2 groups (all P < 0.05). The expression rate of bax were (9,2 +/- 1.5)%, (63 +/- 10)%, and (31 +/- 6)% respectively in the control, 15d-PGJ(2), and ciglitazone groups with a very significant difference between ant 2 groups (all P < 0.01). he expression rate of bcl-2 were (18 +/- 3)%, (36 +/- 9)%, and (33 +/- 7)% respectively in the control, 15d-PGJ(2), and ciglitazone groups with a very significant difference between the control group and any of the agonist-treated groups (all P < 0.01) and without significant difference between the two treated groups. The expression rates of caspase-3 were (6.5 +/- 1.0)%, (65 +/- 11)%, and (40 +/- 7)% respectively in the control, 15d-PGJ(2), and ciglitazone groups with a significant difference between any 2 group (all P < 0.01). The caspase-3 level was positively correlated with the level of apoptosis.. Activated by ligands, PPAR-gamma remarkably inhibits the growth of human lung cancer cells through inducing apoptosis. Caspase-3 and bax/bcl-2 play a role in this process, PPAR-gamma is so important in the pathogenesis and/or progression of lung cancer that it may be a novel therapeutical target against lung cancer.

Topics: Apoptosis; bcl-2-Associated X Protein; Blotting, Western; Caspase 3; Caspases; Cell Division; Cell Line, Tumor; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; In Situ Hybridization; In Situ Nick-End Labeling; Prostaglandin D2; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazolidinediones; Time Factors; Transcription Factors

The peroxisome proliferator-activated receptor gamma (PPARgamma) is a member of the nuclear receptor family of ligand-activated transcription factors. PPARgamma ligands have been shown to inhibit the growth of cells from different cancer lineages. This study was designed to evaluate the effects of PPARgamma receptor activation in human melanoma cell lines. The effects of its expression and activation on cell proliferation and apoptosis in human melanoma cell lines (early stage cancer [WM35] and metastatic tumour [A375]) were investigated. Reverse transcription-polymerase chain reaction and Western blot analysis showed that both human melanoma cell lines expressed PPARgamma mRNA and protein. Treatment of cells transfected with the luciferase gene ligated to PPAR response element constructed promoter showed that ciglitazone and prostaglandin J2 (PGJ2), selective ligands for PPARgamma, increased the luciferase activity, proving the induction of the PPARgamma reporter gene. Ciglitazone and PGJ2 inhibited melanoma cell proliferation in a dose-dependent manner. Analysis of the cellular morphology and apoptosis assayed by fluorescence microscopy after incubation of A375 cells with 10 micro M ciglitazone for 24 h indicated that this ligand not only inhibited cell proliferation but also induced apoptosis.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Cell Division; Cell Line, Tumor; DNA; Dose-Response Relationship, Drug; Humans; Hypoglycemic Agents; Ligands; Luciferases; Melanoma; Microscopy, Fluorescence; Promoter Regions, Genetic; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; Thiazolidinediones; Time Factors; Transcription Factors

Granulocyte-macrophage colony-stimulating factor (GM-CSF), one of major hematopoietic growth factors, activates mature leukocytes. GM-CSF is produced by endothelial cells stimulated with lipopolysaccharide (LPS), and the LPS-induced GM-CSF production may play an important role in the activation of neutrophils on the endothelial surface. 15-Deoxy-delta 12,14-prostaglandin J2 (15d-PGJ2) is a ligand for peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and modulates inflammatory reactions by regulating the expression of various genes. We studied the effect of 15d-PGJ2 on the LPS-induced GM-CSF expression in endothelial cells. Human umbilical vein endothelial cells (HUVEC) were cultured and the expressions of GM-CSF mRNA and protein were analyzed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. 15d-PGJ2 inhibited the LPS-induced GM-CSF expression in a concentration-dependent manner; but ciglitazone, another agonist for PPAR-gamma, had no effect. This suggests that 15d-PGJ2 inhibits GM-CSF expression through a mechanism unrelated to PPAR-gamma. 15d-PGJ2 induced, by itself, the expression of interleukin-8, a potent proinflammatory chemokine, in HUVEC. 15d-PGJ2 may regulate inflammatory reactions by controlling the balance of various cytokines.

Topics: Cells, Cultured; DNA, Complementary; Down-Regulation; Endothelial Cells; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-8; Lipopolysaccharides; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazolidinediones; Transcription Factors

Asthma is characterized by a predominant T(H)2 type immune response to airborne allergens. Controlling T(H)2 cell function has been proposed as therapy for this disease. We show here that ligands for the nuclear receptor peroxisome proliferator activated receptor (PPAR)gamma significantly reduced the immunological symptoms of allergic asthma in a murine model of this disease. A PPARgamma ligand, 15-deoxy-delta(12,14)-prostaglandin J(2), significantly inhibited production of the T(H)2 type cytokine IL-5 from T cells activated in vitro. More importantly, in a murine model of allergic asthma, mice treated orally with ciglitazone, a potent synthetic PPARgamma ligand, had significantly reduced lung inflammation and mucous production following induction of allergic asthma. T cells from these ciglitazone treated mice also produced less IFNgamma, IL-4, and IL-2 upon rechallenge in vitro with the model allergen. Our results suggest that ligands for PPARgamma may be effective treatments for asthmatic patients.

Topics: Administration, Oral; Animals; Antigen-Antibody Complex; Asthma; Cells, Cultured; Cytokines; Female; Interleukin-5; Ligands; Lymph Nodes; Male; Mice; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Reference Values; Spleen; T-Lymphocytes; Th2 Cells; Thiazolidinediones; Transcription Factors; Treatment Outcome

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) plays a critical role in adipocytes differentiation and insulin sensitivity and is also related to regulation of inflammation and cell proliferation. The aim of this study was to investigate the PPAR-gamma agonist-induced apoptosis and effects of PPAR-gamma agonist on Fas-mediated apoptosis in a human colon cancer cell line.. Cell survival and apoptosis of HT-29 cells were measured by trypan blue exclusion method and FACScan after treatment with 15d-PGJ2, ciglitazone and IgM anti-Fas antibody (CH11), respectively or simultaneously. Also, activation of caspase-3 and caspase-8 was analyzed to assess the effects of PPAR-gamma and Fas on apoptosis signaling pathways.. CH11 induced apoptosis of HT-29 cells. 15d-PGJ2 or ciglitazone alone did not induce apoptosis, but combined stimulation with CH11 synergistically induced apoptosis. Also, 15d-PGJ2 alone did not activate caspase-3, but CH11 and 15d-PGJ2 synergistically activated caspase-3. CH11 activated procaspase-8, but 15d-PGJ2 did not.. PPAR-gamma was not an enough condition to induce apoptosis of HT-29 cells. Apoptosis was induced by high dose Fas, and was enhanced with PPAR-gamma agonist. PPAR-gamma agonist seems to enhance Fas-mediated apoptosis by affecting the way between caspase-8 and caspase-3. Further research is needed to use PPAR-gamma agonists as chemopreventive and therapeutic agent for colon cancer and to find the pathways of PPAR-gamma on apoptotic cascade of colon cancer cells.

Topics: Apoptosis; Caspases; Cell Survival; Cells, Cultured; Chalcone; Chalcones; Enzyme Activation; fas Receptor; HT29 Cells; Humans; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Thiazolidinediones; Transcription Factors

We have previously reported that rat primary microglial cultures express the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma) and that several functions associated with the activation of these cells, including nitric oxide (NO) and tumor necrosis factor-alpha synthesis, are down-regulated by 15-deoxy-delta12,14-prostaglandin J2 (15d-PGJ2) and ciglitazone, two specific PPAR-gamma agonists. Here we demonstrate that microglial cells not only express a functionally active PPAR-gamma, but also synthesize large amounts of 15d-PGJ2 upon stimulation with lipopolysaccharide (LPS). In addition, we show that, although 15d-PGJ2 and ciglitazone were equally effective in reducing microglial activation when used at 1-5 microm concentrations, 15d-PGJ2, but not of ciglitazone, reduced PGE2 production at low concentration (0.1 microm) and induced a time-dependent microglial impairment and apoptosis at high concentration (10 microm). Interestingly, the inhibition of PGE2 production was achieved mainly through the inhibition of cycloxygenase-2 enzymatic activity, as the expression of this enzyme and that of the microsomal isoform of PGE synthase remained unaltered. These findings suggest that 15d-PGJ2 affects the functional state and the survival of activated microglia through mechanisms only in part dependent on PPAR-gamma and that the concentration of 15d-PGJ2 is crucial in determining the particular microglial function affected.

Topics: Animals; Apoptosis; Cell Survival; Cells, Cultured; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Isoenzymes; Lipopolysaccharides; Microglia; Nitric Oxide; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Rats; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Thiazolidinediones; Transcription Factors

Peroxisome proliferator-activated receptor-gamma (PPAR-gamma) has been implicated in the regulation of multiple inflammatory processes. However, little is known of PPAR-gamma in the regulation of interleukin (IL)-4 expression in T cells. In this study, the effects of PPAR-gamma ligands on production of IL-4, a pro-inflammatory cytokine associated with the pathophysiology of allergic diseases, were investigated. 15-Deoxy-Delta12,14 prostaglandin J2 (15d-PGJ2) and ciglitazone, two representative PPAR-gamma ligands, significantly inhibited IL-4 production in both antigen-stimulated primary CD4+ T cells and the phorbol 12-myristate 13-acetate (PMA)/ionomycin-activated EL-4 T cell line. 15d-PGJ2 and ciglitazone inhibited the activation of IL-4 gene promoter in EL-4 T cells transiently transfected with IL-4 promoter/reporter constructs, and the repressive effect mapped to a region in the IL-4 promoter containing binding sites for nuclear factor of activated T cells (NF-AT). The activation of T cells by PMA/ionomycin resulted in a marked enhancement of the binding activities to the NF-AT site that was significantly inhibited by the addition of PPAR-gamma ligands. In cotransfected EL-4 T cells, PPAR-gamma also inhibited the activation of the IL-4 promoter at multiple NF-AT sites in a ligand-dependent manner. NF-ATc1 bound PPAR-gamma both in vivo and in vitro, and the interaction interfaces involved the Rel similarity domain of NF-ATc1. In cotransfections of HeLa cells, PPAR-gamma inhibited the NF-ATc1 transactivation in a ligand-dependent manner. Coexpression of p300 or AP-1 relieved the PPAR-gamma ligand-mediated inhibition of the NF-AT transactivation. From these results, we propose that PPAR-gamma ligand-mediated suppression of IL-4 production in CD4+ T cells may involve both inhibition of the NFAT-DNA interactions and competitive recruitment of transcription integrators between NF-AT and PPAR-gamma.

Topics: Animals; Benzhydryl Compounds; Binding Sites; Carcinogens; CD4-Positive T-Lymphocytes; Cell Line; DNA-Binding Proteins; Drug Interactions; E1A-Associated p300 Protein; Epoxy Compounds; Interleukin-4; Ionomycin; Ligands; Mice; Mice, Inbred BALB C; NFATC Transcription Factors; Nuclear Proteins; Promoter Regions, Genetic; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Tetradecanoylphorbol Acetate; Thiazolidinediones; Trans-Activators; Transcription Factor AP-1; Transcription Factors; Tumor Cells, Cultured

Peroxisome proliferator activator receptor-gamma (PPARgamma) is a nuclear receptor that controls the expression of several genes involved in metabolic homeostasis. We investigated the role of PPARgamma during the inflammatory response in sepsis by the use of the PPARgamma ligands, 15-deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)) and ciglitazone. Polymicrobial sepsis was induced by cecal ligation and puncture in rats and was associated with hypotension, multiple organ failure, and 50% mortality. PPARgamma expression was markedly reduced in lung and thoracic aorta after sepsis. Immunohistochemistry showed positive staining for nitrotyrosine and poly(ADP-ribose) synthetase in thoracic aortas. Plasma levels of TNF-alpha, IL-6, and IL-10 were increased. Elevated activity of myeloperoxidase was found in lung, colon, and liver, indicating a massive infiltration of neutrophils. These events were preceded by degradation of inhibitor kappaBalpha (IkappaBalpha), activation of IkappaB kinase complex, and c-Jun NH(2)-terminal kinase and, subsequently, activation of NF-kappaB and AP-1 in the lung. In vivo treatment with ciglitazone or 15d-PGJ(2) ameliorated hypotension and survival, blunted cytokine production, and reduced neutrophil infiltration in lung, colon, and liver. These beneficial effects of the PPARgamma ligands were associated with the reduction of IkappaB kinase complex and c-Jun NH(2)-terminal kinase activation and the reduction of NF-kappaB and AP-1 DNA binding in the lung. Furthermore, treatment with ciglitazone or 15d-PGJ(2) up-regulated the expression of PPARgamma in lung and thoracic aorta and abolished nitrotyrosine formation and poly(ADP-ribose) expression in aorta. Our data suggest that PPARgamma ligands attenuate the inflammatory response in sepsis through regulation of the NF-kappaB and AP-1 pathways.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Aorta, Thoracic; Bacteremia; Blood Glucose; Blood Pressure; Down-Regulation; I-kappa B Kinase; I-kappa B Proteins; Interleukin-10; Interleukin-6; Leukocyte Count; Ligands; Lung; Male; Microbodies; Mitogen-Activated Protein Kinase 8; Mitogen-Activated Protein Kinases; Neutrophil Infiltration; NF-kappa B; NF-KappaB Inhibitor alpha; Poly(ADP-ribose) Polymerases; Prostaglandin D2; Protein Serine-Threonine Kinases; Rats; Rats, Sprague-Dawley; Receptors, Cytoplasmic and Nuclear; Sepsis; Signal Transduction; Survival Rate; Thiazolidinediones; Transcription Factor AP-1; Transcription Factors; Tumor Necrosis Factor-alpha; Tyrosine

The expression of epsilon germline transcripts (epsilon GT) induced by interleukin (IL)-4 stimulation is essential for the progression of IgE-directed class switching. In this study, we examined the effects of various ligands for their ability to bind to the peroxisome proliferator-activated receptors (PPARs) and to modify the IL-4-induced epsilon GT expression in the human B cell line DND39. We show here that the PPAR gamma ligand, 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), can suppress epsilon GT expression at 1 microM without inhibiting cell proliferation. A synthetic and PPAR gamma-specific ligand, ciglitazone, also suppressed epsilon GT expression in a dose-dependent manner at concentrations between 10 and 50 microM. Agonists for other PPAR isoforms did not affect epsilon GT expression at concentrations between 0.01 and 10 microM. We also demonstrated that 1 microM 15d-PGJ(2) was able to suppress the IL-4-induced phosphorylation of the Signal Transducer and Activator of Transcription 6 (STAT6), which is a transcription factor essential for epsilon GT expression. Therefore, the suppression of STAT6 phosphorylation by 15d-PGJ(2) is thought to participate in the inhibition of epsilon GT expression. These results suggest that PPAR gamma ligands inhibit IL-4-induced IgE class switching in B lymphocytes.

Topics: B-Lymphocytes; Cell Line; Germ Cells; Humans; Immunoglobulin E; Immunoglobulin Heavy Chains; Interleukin-4; Ligands; Phosphorylation; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; STAT6 Transcription Factor; Thiazoles; Thiazolidinediones; Trans-Activators; Transcription Factors

Peroxisome-proliferator activated receptor-gamma (PPARgamma) has been demonstrated to exert an inhibitory effect on cell growth in most cell types studied, but its role in colon cancer is still uncertain. The molecular mechanism between the activation of PPARgamma and its consequence is unknown. In the present report, we show that the expression of PPARgamma was significantly increased in tumor tissues from human colon cancer compared with non-tumor tissues and that PPARgamma ligands, 15-Deoxy-delta(12,14)prostaglandin J2 or ciglitizone, induced apoptosis in HT-29 cells, a human colon cancer cell line. The occurrence of apoptosis induced by PPARgamma ligands was sequentially accompanied by reduced levels of NF-kappaB and Bcl-2. Over-expression of Bcl-2 significantly protected the cells from apoptosis. This study suggested that a PPARgamma-Bcl-2 feedback loop may function to control the life-death continuum in colonic cells and that a deficiency in generation of PPARgamma ligands may precede the development of human colon cancer.

Topics: Apoptosis; Cell Division; Cell Survival; Colon; Colonic Neoplasms; DNA-Binding Proteins; Electrophoretic Mobility Shift Assay; HT29 Cells; Humans; Hypoglycemic Agents; Immunoenzyme Techniques; Immunologic Factors; Ligands; NF-kappa B; Promoter Regions, Genetic; Prostaglandin D2; Proto-Oncogene Proteins c-bcl-2; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Thiazoles; Thiazolidinediones; Transcription Factors; Transcription, Genetic

Metabolic disorders in HIV-infected patients, especially those receiving highly active antiretroviral therapy (HAART) regimens containing protease inhibitors, are associated with insulin resistance. These metabolic disorders include fat redistribution, diabetes, and hypertriglyceridemia. Thiazolidinediones (TZDs) are used to treat patients with diabetes secondary to insulin resistance, and TZDs are being studied in HAART-related metabolic disorders. We studied the effects of TZDs (peroxisome proliferator-activated receptor-gamma [PPARgamma] agonists) and a PPARalpha agonist on HIV replication and TNFalpha production in peripheral blood mononuclear cells (PBMCs) acutely infected with HIV-1, in a chronically infected monoblastoid cell line (U1) and in alveolar macrophages (AMs) from HIV-infected subjects and uninfected controls. Rosiglitazone, ciglitazone, troglitazone, and PgJ (PPARgamma agonists) as well as fenofibrate (PPARalpha agonist) inhibited HIV replication in both PBMCs and U1 cells. These agents also inhibited TNFalpha production, but the magnitude of TNFalpha inhibition was not directly correlated with the quantitative decreases in HIV replication. In AMs, ciglitazone, rosiglitazone, and troglitazone reduced TNFalpha production. We hypothesize that alterations in mitogen-activated protein kinase signaling pathways have contemporaneous and interrelated effects on HIV replication, cytokine production, and lipid metabolism. Modulation of these pathways using PPAR agonists may improve the metabolic alterations during HAART in conjunction with desirable decreases in HIV replication and TNFalpha production.

Topics: Acquired Immunodeficiency Syndrome; Anti-HIV Agents; Cell Line; Chromans; Fenofibrate; HIV-1; Humans; Leukocytes, Mononuclear; Macrophages, Alveolar; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors; Troglitazone; Tumor Necrosis Factor-alpha; Virus Replication

Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed in certain human cancers; ligand-induced PPARgamma activation can result in growth inhibition and differentiation in these cells. However, the precise mechanism for the antiproliferative effect of PPARgamma ligands is not entirely known.. The purpose of this study was to examine the effect of PPARgamma ligands on pancreatic cancer cell growth and invasiveness.. The effect of two PPARgamma ligands, 15 deoxy-delta12,14 prostaglandin J2 (15d-PGJ2) and ciglitazone, on the growth of four human pancreatic cancer cell lines (BxPC-3, MIA PaCa-2, Panc-1, and L3.6) was assessed. Expression of cell-cycle and apoptotic-related proteins was measured. Finally, the effect of 15d-PGJ2 on pancreatic cancer cell invasiveness and matrix metalloproteinase expression was determined.. Both 15d-PGJ2 and ciglitazone inhibited the growth of all four pancreatic cancer cell lines in a dose- and time-dependent fashion. Treatment of BxPC-3 cells with 15d-PGJ2 resulted in a time-dependent decrease in cyclin D1 expression associated with a concomitant induction of p21waf1 and p27kip1. In addition, 15d-PGJ2 treatment induced apoptosis through activation of caspase-8, -9, and -3. Moreover, pancreatic cancer cell invasiveness was significantly suppressed after treatment with a nontoxic dose of 15d-PGJ2, which was associated with a reduction of MMP-2 and MMP-9 protein levels and activity.. These results demonstrate that PPARgamma ligands have the dual advantage of inhibiting pancreatic cancer cell growth while reducing the invasiveness of the tumor cells, suggesting a potential role for these agents in the adjuvant treatment of pancreatic cancer.

Topics: 3T3 Cells; Adenocarcinoma; alpha Catenin; Animals; Antineoplastic Agents; Apoptosis; beta Catenin; Cadherins; Caspase 3; Caspase 8; Caspase 9; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Division; Collagen; Cyclin D1; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Cyclooxygenase 2; Cytoskeletal Proteins; Drug Combinations; Enzyme Induction; Gene Expression Regulation, Neoplastic; Humans; Isoenzymes; Laminin; Ligands; Matrix Metalloproteinase 2; Matrix Metalloproteinase 9; Membrane Proteins; Mice; Neoplasm Invasiveness; Neoplasm Proteins; Pancreatic Neoplasms; Prostaglandin D2; Prostaglandin-Endoperoxide Synthases; Proteoglycans; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Trans-Activators; Transcription Factors; Tumor Cells, Cultured; Tumor Suppressor Proteins

Proliferation of intrinsic glomerular cells is a common response to renal injury. Acutely, proliferation may be beneficial, but sustained glomerular hypercellularity after injury is associated with progressive renal failure. To identify endogenous factors that may be responsible for regulating glomerular cell number, the effects of J-series cyclopentenone prostaglandins (PGs) on human glomerular mesangial cell proliferation and death were examined.. Human mesangial cells were grown in the presence or absence of PGJ2 or its metabolite 15-Deoxy-Delta12,14-PGJ2 (15dPGJ2). The number of viable cells was measured by the reduction of the tetrazolium MTS to a colored formazan product. Apoptosis was assessed by caspase-3 activation and DNA fragmentation.. PGJ2 at concentrations up to 10 micromol/L caused mesangial proliferation. 15dPGJ2 also caused mesangial proliferation at low concentrations (< or =2.5 micromol/L), but induced mesangial cell death at higher concentrations (>5 micromol/L). Cell death occurred in part through apoptosis, measured as an increase in caspase-3 activity and DNA fragmentation in 15dPGJ2-treated cells. Cell death was associated with a decline in baseline phosphorylation of the survival factor Akt and increased Akt degradation, whereas 15dPGJ2-induced mesangial proliferation was blocked by inhibition of the PI 3-kinase/Akt pathway. 15dPGJ2 is a potent PPARgamma agonist. Like 15dPGJ2, treatment of mesangial cells with thiazolidinedione-type PPARgamma ligands (10 to 20 micromol/L) caused significant cell death, but at lower concentrations also caused a small degree of proliferation.. J-series prostaglandins thus may be involved in the initiation of glomerular hypercellularity through Akt-dependent proliferation, and restoration of normal glomerular architecture through PPARgamma-mediated apoptosis. Manipulation of these prostaglandins may be relevant to the treatment of progressive glomerular disease.

Topics: 1-Phosphatidylinositol 4-Kinase; Cell Death; Cell Division; Cells, Cultured; Chromans; Chromones; Enzyme Inhibitors; Glomerular Mesangium; Humans; Morpholines; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Transcription Factors; Troglitazone

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor transcription factor that regulates adipocyte differentiation and glucose homeostasis. PPARgamma agonists are potent therapeutic agents for the treatment of type 2 diabetes and obesity. PPARgamma agonists also prevent inflammation in animal models, suggesting their use for the treatment of human inflammatory diseases. Experimental allergic encephalomyelitis (EAE) is a Th1 cell-mediated inflammatory demyelinating disease model of multiple sclerosis (MS) and IL-12 plays a crucial role in the pathogenesis of EAE and MS. In this study we have examined the effect of PPARgamma agonists on the pathogenesis of EAE. In vivo treatment of SJL/J mice with PPARgamma agonists, 15-deoxydelta(12,14) prostaglandin J2 or Ciglitazone, decreased the duration and clinical severity of active immunization and adoptive transfer models of EAE. PPARgamma agonists inhibited EAE in association with a decrease in IL-12 production and differentiation of neural antigen-specific Th1 cells. In vitro treatment of activated T cells with PPARgamma agonists inhibited IL-12-induced activation of JAK-STAT signaling pathway and Th1 differentiation. These findings highlight the fact that PPARgamma agonists regulate central nervous system inflammation and demyelination by inhibiting IL-12 production, IL-12 signaling and Th1 differentiation in EAE.

Topics: Animals; Cell Differentiation; Cells, Cultured; DNA-Binding Proteins; Dose-Response Relationship, Drug; Encephalomyelitis, Autoimmune, Experimental; Immunologic Factors; Interleukin-12; Janus Kinase 2; Kinetics; Lymphocyte Activation; Macrophages; Mice; Microglia; Myelin Basic Protein; Prostaglandin D2; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Cytoplasmic and Nuclear; Signal Transduction; Spinal Cord; Th1 Cells; Thiazoles; Thiazolidinediones; Transcription Factors

15-Deoxy-Delta(12-14)-prostaglandin J(2) (dPGJ2) and thiazolidinediones are known as ligands for the peroxisome proliferator activator receptor gamma (PPAR gamma) a member of the nuclear receptor superfamily. Herein, we show that dPGJ2 activates, in cultured primary astrocytes, Erk, Jnk, p38 MAP kinase, and ASK1, a MAP kinase kinase kinase, which can be involved in the activation of Jnk and p38 MAP kinase. The activation kinetic is similar for the three MAP kinase. The activation of the MAP kinases is detectable around 0.5 h. The activation increases with dPGJ2 in a dose dependent manner (0-15 microm). A scavenger of reactive oxygenated species (ROS), N-acetylcysteine (NAC) at 20 mm, completely suppresses the activation of MAP kinases and ASK1, suggesting a role for oxidative stress in the activation mechanism. Other prostaglandin cyclopentenones than dPGJ2, A(2), and to a lesser degree, A(1) also stimulate the MAP kinases, although they do not bind to PPAR gamma. Ciglitazone (20 microm), a thiazolidinedione that mimics several effects of dPGJ2 in different cell types, also activates the three MAP kinase families and ASK1 in cultured astrocytes. However the activation is more rapid (it is detectable at 0.25 h) and more sustained (it is still strong after 4 h). NAC prevents the activation of the three MAP kinase families by ciglitazone. Another thiazolidinedione that binds to PPAR gamma, rosiglitazone, does not activate MAP kinases, indicating that the effect of ciglitazone on MAP kinases is independent of PPAR gamma. Ciglitazone and less strongly dPGJ2 activate Erk in undifferentiated cells of the adipocyte cell line 1B8. Ciglitazone also activates Jnk and p38 MAP kinase in these preadipocytes. Our findings suggest that a part of the biological effects of dPGJ2 and ciglitazone involve the activation of the three MAP kinase families probably through PPAR gamma-independent mechanisms involving ROS.

Topics: Adipocytes; Animals; Astrocytes; Cells, Cultured; Enzyme Activation; MAP Kinase Signaling System; Prostaglandin D2; Rats; Reactive Oxygen Species; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Transcription Factors

Malignant astrocytomas are among the most common brain tumours and few therapeutic options exist. It has recently been recognized that the ligand-activated nuclear receptor PPARgamma can regulate cellular proliferation and induce apoptosis in different malignant cells. We report the effect of three structurally different PPARgamma agonists inducing apoptosis in human (U87MG and A172) and rat (C6) glioma cells. The PPARgamma agonists ciglitazone, LY171 833 and prostaglandin-J2, but not the PPARalpha agonist WY14643, inhibited proliferation and induced cell death. PPARgamma agonist-induced cell death was characterized by DNA fragmentation and nuclear condensation, as well as inhibited by the synthetic receptor-antagonist bisphenol A diglycidyl ether (BADGE). In contrast, primary murine astrocytes were not affected by PPARgamma agonist treatment. The apoptotic death in the glioma cell lines treated with PPARgamma agonists was correlated with the transient up-regulation of Bax and Bad protein levels. Furthermore, inhibition of Bax expression by specific antisense oligonucleotides protected glioma cells against PPARgamma-mediated apoptosis, indicating an essential role of Bax in PPARgamma-induced apoptosis. However, PPARgamma agonists not only induced apoptosis but also caused redifferentiation as indicated by outgrowth of long processes and expression of the redifferentiation marker N-cadherin in response to PPARgamma agonists. Taken together, treatment of glioma cells with PPARgamma agonists may hold therapeutic potential for the treatment of gliomas.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; bcl-Associated Death Protein; Cadherins; Carrier Proteins; Cell Division; Cell Survival; DNA Fragmentation; Drug Evaluation, Preclinical; Glioma; Humans; Nuclear Proteins; Oligonucleotides, Antisense; Prostaglandin D2; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Pyrimidines; Rats; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Transcription Factors; Tumor Cells, Cultured

Thiazolidinediones (TZDs) are widely used for treatment of type 2 diabetes mellitus. Peroxisome proliferator-activated receptor gamma (PPAR gamma) is the molecular target of TZDs and is believed to mediate the apoptotic effects of this class of drugs in a variety of cell types, including B and T lymphocytes. The finding that TZDs induce lymphocyte death has raised concerns regarding whether TZDs might further impair immune functions in diabetics. To address this issue, we investigated the roles of PPAR gamma and TZDs in lymphocyte survival. PPAR gamma was up-regulated upon T cell activation. As previously reported, PPAR gamma agonists induced T cell death in a dose-dependent manner. However, the concentrations of TZD needed to cause T cell death were above those needed to induce PPAR gamma-dependent transcription. Surprisingly, at concentrations that induce optimal transcriptional activation, TZD activation of PPAR gamma protected cells from apoptosis following growth factor withdrawal. The survival-enhancing effects depended on both the presence and activation of PPAR gamma. Measurements of mitochondrial potential revealed that PPAR gamma activation enhanced the ability of cells to maintain their mitochondrial potential. These data indicate that activation of PPAR gamma with TZDs can promote cell survival and suggest that PPAR gamma activation may potentially augment the immune responses of diabetic patients.

Topics: Animals; Cell Death; Cell Survival; Cells, Cultured; Humans; Hypoglycemic Agents; Interleukin-3; Lymphocyte Activation; Membrane Potentials; Mice; Mice, Inbred C57BL; Mitochondria; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Recombinant Proteins; Rosiglitazone; T-Lymphocytes; Thiazoles; Thiazolidinediones; Transcription Factors; Transfection; Up-Regulation

In this study, we examined the signaling pathways for extracellular signal-related protein kinase (ERK) activation by three structurally different peroxisome proliferator activated receptor-gamma (PPARgamma) agonists. In murine C2C12 myoblasts, treatment with 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), ciglitazone, and GW1929 leads to ERK1/2 phosphorylation in a time- and concentration-dependent manner. Consistent with ERK phosphorylation, mitogen activated protein/ERK kinase (MEK) phosphorylation as well as Raf-1 kinase activity are also accordingly stimulated, while the constitutive Ser259 phosphorylation of Raf-1 is decreased. The ERK phosphorylation induced by PPARgamma agonists is not blocked by the PKC inhibitors GF109203X and Ro31-8220, the PI3K inhibitor wortmannin, the Ras inhibitor FPTI, the negative mutant of Ras, or the PPARgamma antagonist bisphenol A diglycidil ether. Expression of PPARgamma2 without DNA binding domain or with a nonphosphorylatable mutant (S112A) fails to change ERK phosphorylation by 15d-PGJ(2). On the contrary, the ERK phosphorylation by PPARgamma agonists is inhibited by the MEK inhibitor PD98059, GSH, and permeable SOD mimetic MnTBAP. Chemiluminescence study reveals that these three PPARgamma agonists are able to induce superoxide anion production, with an efficacy similar to their action on ERK phosphorylation. Consistent with this notion, we also show that superoxide anion donor 2,3-dimethoxy-1,4-naphoquinone elicits ERK phosphorylation. In this study, we for the first time demonstrate a novel mechanism, independent of Ras activation but initiated by superoxide anion production, for PPARgamma agonists to trigger the Raf-MEK-ERK1/2 signaling pathway.

Topics: Animals; Benzophenones; Cell Line; MAP Kinase Signaling System; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Peroxisomes; Phosphatidylinositol 3-Kinases; Phosphorylation; Prostaglandin D2; Protein Kinase C; Proto-Oncogene Proteins c-raf; ras Proteins; Reactive Oxygen Species; Receptors, Cytoplasmic and Nuclear; Superoxides; Thiazoles; Thiazolidinediones; Transcription Factors; Tyrosine

This study was designed to investigate the effects of various chemically distinct activators of PPAR-gamma and PPAR-alpha in a rat model of acute myocardial infarction. Using Northern blot analysis and RT-PCR in samples of rat heart, we document the expression of the mRNA for PPAR-gamma (isoform 1 but not isoform 2) as well as PPAR-beta and PPAR-alpha in freshly isolated cardiac myocytes and cardiac fibroblasts and in the left and right ventricles of the heart. Using a rat model of regional myocardial ischemia and reperfusion (in vivo), we have discovered that various chemically distinct ligands of PPAR-gamma (including the TZDs rosiglitazone, ciglitazone, and pioglitazone, as well as the cyclopentanone prostaglandins 15D-PGJ2 and PGA1) cause a substantial reduction of myocardial infarct size in the rat. We demonstrate that two distinct ligands of PPAR-alpha (including clofibrate and WY 14643) also cause a substantial reduction of myocardial infarct size in the rat. The most pronounced reduction in infarct size was observed with the endogenous PPAR-gamma ligand, 15-deoxyDelta12,14-prostagalndin J2 (15D-PGJ2). The mechanisms of the cardioprotective effects of 15D-PGJ2 may include 1) activation of PPAR-alpha, 2) activation of PPAR-gamma, 3) expression of HO-1, and 4) inhibition of the activation of NF-kappaB in the ischemic-reperfused heart. Inhibition by 15D-PGJ2 of the activation of NF-kappaB in turn results in a reduction of the 1) expression of inducible nitric oxide synthase and the nitration of proteins by peroxynitrite, 2) formation of the chemokine MCP-1, and 3) expression of the adhesion molecule ICAM-1. We speculate that ligands of PPAR-gamma and PPAR-alpha may be useful in the therapy of conditions associated with ischemia-reperfusion of the heart and other organs. Our findings also imply that TZDs and fibrates may help protect the heart against ischemia-reperfusion injury. This beneficial effect of 15D-PGJ2 was associated with a reduction in the expression of the 1) adhesion molecules ICAM-1 and P-selectin, 2) chemokine macrophage chemotactic protein 1, and 3) inducible isoform of nitric oxide synthase. 15D-PGJ2 reduced the nitration of proteins (immunohistological analysis of nitrotyrosine formation) caused by ischemia-reperfusion, likely due to the generation of peroxynitrite. Not all of the effects of 15D-PGJ2, however, are due to the activation of PPAR-gamma. For instance, exposure of rat cardiac myocytes to 15D-PGJ2, but not to rosiglitazon

Topics: Adult; Animals; Cardiotonic Agents; Cell Adhesion Molecules; Cell Line; Cells, Cultured; Chemokine CCL2; Clofibrate; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Humans; Ligands; Male; Membrane Proteins; Myocardial Infarction; Myocardial Reperfusion Injury; Pioglitazone; Prostaglandin D2; Prostaglandins A; Protein Isoforms; Pyrimidines; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors

The mitogen-activated protein (MAP) kinases mediate the response of renal glomerular mesangial cells to a variety of physiologic and pathologic stimuli. This investigation examines the effect of the cyclopentenone prostaglandin 15-deoxy-delta(12,14)-prostaglandin J2 (15d-PGJ2) on MAP kinases in human mesangial cells. We show that 15d-PGJ2 dose-dependently increases the extracellular signal-regulated kinase (ERK) activity of human mesangial cells, but has no effect on Jun-NH2-terminal kinase or p38 MAP kinase. Despite the fact that 15d-PGJ2 is a peroxisome proliferator-activated receptor (PPAR) ligand, and PPARgamma is shown to be expressed by mesangial cells, the thiazolidinedione PPARgamma agonist ciglitazone does not activate ERK. Additionally, a synthetic PPARgamma antagonist does not attenuate the activation of ERK by 15d-PGJ2. 15d-PGJ2-mediated ERK activation is however blocked by the MEK inhibitor PD 098059, appears to require phosphatidylinositol-3 kinase, but is independent of protein kinase C activation. These results demonstrate a novel effect of 15d-PGJ2 to induce ERK in human mesangial cells independently of PPARgamma.

Topics: Cells, Cultured; Cyclopentanes; Dose-Response Relationship, Drug; Enzyme Activation; Enzyme Inhibitors; Flavonoids; Glomerular Mesangium; Humans; Hypoglycemic Agents; Immunologic Factors; JNK Mitogen-Activated Protein Kinases; Ligands; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Phosphatidylinositol 3-Kinases; Prostaglandin D2; Prostaglandins; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Transcription Factors

Peroxisome proliferator-activated receptor (PPAR)-gamma is a nuclear hormone receptor that serves as a trans factor to regulate lipid metabolism. Intense interest is focused on PPAR-gamma and its ligands owing to its putative role in adipocyte differentiation. Little is known, however, about the functions of PPAR-gamma in the immune system, especially in T lymphocytes. We demonstrate that both naive and activated ovalbumin-specific T cells from DO11.10-transgenic mice express PPAR-gamma mRNA and protein. In order to determine the function of PPAR-gamma, T cells were stimulated with phorbol 12-myristate 13-acetate and ionomycin or antigen and antigen-presenting cells. Simultaneous exposure to PPAR-gamma ligands (e. g. 15-deoxy-Delta(12, 14)-prostaglandin J(2), troglitazone) showed drastic inhibition of proliferation and significant decreases in cell viability. The decrease in cell viability was due to apoptosis of the T lymphocytes, and occurred only when cells were treated with PPAR-gamma, and not PPAR-alpha agonists, revealing specificity of this response for PPAR-gamma. These observations suggest that PPAR-gamma agonists play an important role in regulating T cell-mediated immune responses by inducing apoptosis. T cell death via PPAR-gamma ligation may act as a potent anti-inflammatory signal in the immune system, and ligands could possibly be used to control disorders in which excessive inflammation occurs.

Topics: Animals; Antigen Presentation; Apoptosis; Cell Division; Cell Survival; Cells, Cultured; Chromans; Flow Cytometry; Immunohistochemistry; In Situ Nick-End Labeling; Ionomycin; Ligands; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Mice, Transgenic; Prostaglandin D2; Pyrimidines; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Specific Pathogen-Free Organisms; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thiazoles; Thiazolidinediones; Transcription Factors; Troglitazone

The peroxisome proliferator-activated receptors (PPARs) are nuclear hormone transcription factors that regulate genes associated with lipid and glucose metabolism. Recent evidence suggests that PPAR-gamma may also act as a negative immunomodulator. To investigate the potential role of PPAR-gamma in regulating airway inflammation, we characterized the expression and function of PPAR-gamma in airway epithelial cells. Airway epithelial cells constitutively express PPAR-gamma-specific messenger RNA and protein. Further, airway epithelial PPAR-gamma is inducible by interleukin (IL)-4 in NIH-A549 cells. Two PPAR-gamma agonists, the prostaglandin D2 metabolite 15-deoxy-(Delta)(12,14) prostaglandin J2 (15d-PGJ2) and a thiazolidinedione, ciglitazone, were used to study the effects of PPAR-gamma activation on airway epithelial cytokine expression. Activation of PPAR-gamma stimulated a PPAR-responsive reporter gene in a ligand-specific manner. In NIH-A549 cells, both ligands also blocked the cytokine-induced expression of the inducible form of nitric oxide synthase in a dose-dependent manner. In contrast, ciglitazone alone had a slight effect on cytokine-induced IL-8 secretion, but markedly inhibited IL-8 secretion from cells pretreated with IL-4. The demonstration of PPAR-gamma expression and function in airway epithelial cells expands the immunoregulatory role of PPARs and suggests a critical role for PPAR-gamma in antagonizing proinflammatory pathways in the airways.

Topics: Adjuvants, Immunologic; Cytokines; Down-Regulation; Humans; Inflammation Mediators; Interleukin-4; Interleukin-8; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Respiratory Mucosa; RNA, Messenger; Signal Transduction; Thiazoles; Thiazolidinediones; Transcription Factors

PPARgamma is a transcription factor of nuclear receptor superfamily, involved in the regulation of inflammation. We investigated the influence of PPARgamma-ligands, 15-deoxy-delta12,14 prostaglandin-J2 (15d-PGJ2), and ciglitazone, on the generation of interleukin-8 (IL-8) by the human microvascular endothelial cell line (HMEC- 1). Expression of PPARgamma in HMEC-1 was confirmed by RT-PCR. Both PPARgamma-ligands tested induced the activation of PPAR, but the potency of ciglitazone was higher, as evidenced by luciferase assay. Resting HMEC-1 released about 150 pg/ml of IL-8 protein. Treatment with LPS increased the IL-8 secretion up to 1 ng/ml. 15d-PGJ2 potently and dose-dependently increased both the steady-state and LPS-induced generation of IL-8 mRNA and IL-8 protein. In contrast, neither basal nor LPS-elicited expression of IL-8 was influenced by ciglitazone. We conclude, that 15d-PGJ2 is a potent inducer of IL-8 production and can be a mediator of inflammatory response, but this effect is independent of PPARgamma activation.

Topics: Cell Line; Cell Survival; Electrophoretic Mobility Shift Assay; Endothelium, Vascular; Humans; Interleukin-8; Ligands; NF-kappa B; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazoles; Thiazolidinediones; Transcription Factors; Transfection; Up-Regulation

Release of arachidonic acid from rat liver cells is stimulated after a 6-hour incubation with 9-cis retinoic acid, all trans retinoic acid, the selective peroxisome proliferator-activated receptor-gamma synthetic thiazolidinedione, ciglitazone, the cyclopentenones, 15-deoxy-Delta(12,14) PGJ2 and PGA1 and the non-steroidal anti-inflammatory drugs, celecoxib and indomethacin. The rates of the release stimulated by 15-deoxy-Delta(12,14) PGJ2 differ from those observed with celecoxib. Arachidonic acid release by9-cis retinoic acid in the presence of either ciglitazone or trans retinoic acid is synergistic. It is additive in the presence of celecoxib. Cycloheximide and actinomycin inhibit the release of arachidonic acid stimulated by 15-deoxy-Delta(12,14) PGJ2 but not by celecoxib. The findings indicate that agonists of the peroxisome proliferator-activated receptor-gamma and retinoic acid receptors stimulate the release of arachidonic acid. The mechanisms involved may differ in the cases of 15-deoxy-Delta(12,14) PGJ2 and celecoxib.

Topics: Alitretinoin; Animals; Anti-Inflammatory Agents, Non-Steroidal; Arachidonic Acid; Celecoxib; Cell Line; Cycloheximide; Dactinomycin; Dose-Response Relationship, Drug; Drug Synergism; Indomethacin; Kinetics; Liver; Prostaglandin D2; Prostaglandins A; Pyrazoles; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Sulfonamides; Thiazoles; Thiazolidinediones; Transcription Factors; Tretinoin

Apoptosis at the site of rupture has been proposed to play a role in premature rupture of the fetal membranes, a condition associated with increased risk of neonatal sepsis and preterm birth. We investigated the ability of peroxisome proliferator-activated receptor (PPAR)-gamma ligands 15-deoxy-delta12,14PGJ2 (15d-PGJ2), delta12PGJ2, ciglitizone and rosiglitazone to induce apoptosis in the amnion-like WISH cell line. 15d-PGJ2 (10 microM) induced morphological characteristics of apoptosis within 2 h, with biochemical indices (caspase activation and substrate cleavage) following shortly after; maximum cell death (approximately 60%) was observed by 16 h, with an EC50) of approximately 7 microM 15d-PGJ2. Delta12-PGJ2 also induced apoptosis but was less potent and acted at a much slower rate. While ciglitizone also induced apoptosis, rosiglitazone had no effect on cell viability. The mechanism of induction of apoptosis by 15d-PGJ2 and delta12PGJ2, which may be independent of PPAR-gamma activation, requires further elucidation.

Topics: Amino Acid Chloromethyl Ketones; Amnion; Apoptosis; Caspase Inhibitors; Caspases; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Enzyme Activation; Humans; Hypoglycemic Agents; Immunologic Factors; Ligands; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Rosiglitazone; Thiazoles; Thiazolidinediones; Time Factors; Transcription Factors

During liver injury, hepatic stellate cells (HSC) acquire a myofibroblast-like phenotype associated with reduction of lipid droplets, increased collagen synthesis, and proliferation. Peroxisome proliferator-activated receptor gamma (PPARgamma) regulates adipocyte differentiation and controls gene transcription in response to various activators including prostanoids and antidiabetic thiazolidinediones. We explored whether the presence of PPARgamma and its transcriptional activity were involved in control of HSC proliferation in vitro. PPARgamma ligands, 15-deoxy-triangle up(1214) prostaglandin J(2) (15d-PGJ(2)) and ciglitizone, significantly decrease platelet-derived growth factor (PDGF)-induced proliferation in activated human HSC and inhibit alpha smooth muscle actin (alpha-SMA) expression during HSC transdifferentiation. Treatment with 9-cis retinoic acid (9-cisRA) and LG268, ligands of the heterodimerization partner retinoic X receptor (RXR), had a negligible effect in PDGF-treated cells but caused a further reduction of proliferation when used in combination with ciglitizone. Transfection experiments with a reporter gene consisting of 3 copies of a PPAR response element (peroxisome proliferator response element [PPRE](3)-tk-luciferase) showed a progressive reduction of PPAR transcriptional activity during plastic-induced HSC transdifferentiation. Cotransfection with human PPARgamma expression vector restored the PPRE(3)-tk-luciferase reporter expression and the increased level of the receptor in activated HSC-inhibited cell proliferation in a dose-dependent manner. Incubation of human PPARgamma-cotransfected HSC with PDGF strongly inhibited luciferase activity and this effect was blocked by the inhibition of the mitogen-activated protein (MAP) kinase signal cascade. Our results indicate that depression of PPARgamma expression and activity is involved in HSC proliferation and that the PPARgamma ligand-mediated activation exerts a previously unrecognized inhibition of PDGF-induced mitogenesis in activated human HSC.

Topics: Cell Differentiation; Cell Division; Cells, Cultured; Gene Expression; Gene Expression Regulation; Humans; Liver; Platelet-Derived Growth Factor; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazoles; Thiazolidinediones; Transcription Factors; Transcription, Genetic; Transfection

The peroxisome proliferator-activated receptors (PPARs) are a family of transcription factors belonging to the nuclear receptor superfamily. Until recently, the genes regulated by PPARs were those believed to be predominantly associated with lipid metabolism. Recently, an immunomodulatory role for PPAR gamma has been described in cells critical to the innate immune system, the monocyte/macrophage. In addition, evidence for an antiinflammatory role of the PPAR gamma ligand, 15-deoxy-Delta 12,14-PGJ2 (15d-PGJ2) has been found. In the present studies, we demonstrate, for the first time, that murine helper T cell clones and freshly isolated splenocytes express PPAR gamma 1. The PPAR gamma expressed is of functional significance in that two ligands for PPAR gamma, 15d-PGJ2 and a thiazolidinedione, ciglitazone, mediate significant inhibition of proliferative responses of both the T cell clones and the freshly isolated splenocytes. This inhibition is mediated directly at the level of the T cell and not at the level of the macrophage/APC. Finally, we demonstrate that the two ligands for PPAR gamma mediate inhibition of IL-2 secretion by the T cell clones while not inhibiting IL-2-induced proliferation of such clones. The demonstration of the expression and function of PPAR gamma in T cells reveals a new level of immunoregulatory control for PPARs and significantly increases the role and importance of PPAR gamma in immunoregulation.

Topics: Adjuvants, Immunologic; Animals; Binding Sites, Antibody; CD3 Complex; Clone Cells; Female; Immune Sera; Immunosuppressive Agents; Interleukin-2; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Microbodies; Nuclear Proteins; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Spleen; T-Lymphocytes, Helper-Inducer; Thiazoles; Thiazolidinediones; Transcription Factors

The peroxisome proliferator-activated receptor (PPAR)-gamma is a nuclear lipid-activable receptor controlling the expression of genes involved in lipid metabolism and adipocyte differentiation. In order to investigate the possible role of PPAR-gamma in the differentiation of intestinal epithelial cells, we examined its expression in the human colon carcinoma cell line Caco-2, which undergoes rapid cell differentiation in the presence of butyrate. PPARs were quantified on mRNA level by RT competitive multiplex PCR, the corresponding proteins were determined by Western blot. In contrast to PPAR-alpha and PPAR-beta, PPAR-gamma mRNA and protein increased significantly in butyrate-treated Caco-2 cells in a dose- and time-dependent manner. This effect was butyrate-specific, since no change in PPAR-gamma expression could be observed after incubation with propionate or valerate. Activation of PPAR-gamma by ciglitazone further increased butyrate-induced cell differentiation dose-dependently. These data demonstrate a role for PPAR-gamma in the regulation of cell differentiation in Caco-2 cells.

Topics: Alkaline Phosphatase; Blotting, Western; Butyrates; Caco-2 Cells; Cell Differentiation; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Pentanoic Acids; Propionates; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Substrate Specificity; Thiazoles; Thiazolidinediones; Transcription Factors; Up-Regulation

We report the isolation by RT-PCR of partial cDNAs encoding the human peroxisome proliferator-activated receptor (PPAR) isoforms PPARbeta and -gamma in human primary astrocytes (HPA) as well as in the human malignant astrocytoma cell line T98G. In contrast, we failed to detect PPARalpha mRNA in either of these two cell types. Because PPARbeta is ubiquitously expressed but has, as yet, no known function, we pursued our functional studies of these cells with regard to PPARgamma. To that end, we showed that PPARgamma protein is abundantly expressed in both cell types, having a molecular weight of approximately 50 kDa. Immunocytochemistry revealed a predominantly nuclear localization of this receptor. Moreover, incubation of the two cell types with 1-12 mcM 15-deoxy PGJ(2) or 1-12 mcM ciglitazone, both of which are agonists of PPARgamma, induced loss of cellular viability as assessed by the MTT assay after a 4 hr incubation. Reduced cellular viability as a consequence of exposure to PGJ(2) or ciglitazone resulted from induction of apoptosis, as assessed by DNA fragmentation and Hoechst staining, and involves activation of the CPP32 (caspase-3) protease. These data show that modulation of the process of apoptosis is one function of PPARgamma in cells derived from the human astrocytic lineage.

Topics: Apoptosis; Astrocytes; Caspase 3; Caspases; Cell Survival; DNA Fragmentation; Gene Expression; Humans; Hypoglycemic Agents; Peroxisomes; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazoles; Thiazolidinediones; Transcription Factors

The research described herein evaluates the expression and functional significance of peroxisome proliferator activator receptor-gamma (PPAR-gamma) on B-lineage cells. Normal mouse B cells and a variety of B lymphoma cells reflective of stages of B cell differentiation (e.g., 70Z/3, CH31, WEHI-231, CH12, and J558) express PPAR-gamma mRNA and, by Western blot analysis, the 67-kDa PPAR-gamma protein. 15-Deoxy-Delta(12,14)-PGJ(2) (15d-PGJ(2)), a PPAR-gamma agonist, has a dose-dependent antiproliferative and cytotoxic effect on normal and malignant B cells as shown by [(3)H]thymidine and 3-[4,5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide assays. Only PPAR-gamma agonists (thiazolidinediones), and not PPAR-alpha agonists, mimicked the effect of 15d-PGJ(2) on B-lineage cells, indicating that the mechanism by which 15d-PGJ(2) negatively affects B-lineage cells involves in part PPAR-gamma. The mechanism by which PPAR-gamma agonists induce cytotoxicity is via apoptosis, as shown by annexin V staining and as confirmed by DNA fragmentation detected using the TUNEL assay. Interestingly, addition of PGF(2alpha), which was not known to affect lymphocytes, dramatically attenuated the deleterious effects of PPAR-gamma agonists on B lymphomas. Surprisingly, 15d-PGJ(2) induced a massive increase in nuclear mitogen-activated protein kinase activation, and pretreatment with PGF(2alpha) blunted the mitogen-activated protein kinase activation. This is the first study evaluating PPAR-gamma expression and its significance on B lymphocytes. PPAR-gamma agonists may serve as a counterbalance to the stimulating effects of other PGs, namely PGE(2), which promotes B cell differentiation. Finally, the use of PGs, such as 15d-PGJ(2), and synthetic PPAR-gamma agonists to induce apoptosis in B-lineage cells may lead to the development of novel therapies for fatal B lymphomas.

Topics: Animals; Apoptosis; B-Lymphocytes; Cell Lineage; Cells, Cultured; Chromans; Dinoprost; Hypoglycemic Agents; Lymphoma, B-Cell; Male; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Inbred DBA; Prostaglandin D2; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Thiazoles; Thiazolidinediones; Transcription Factors; Troglitazone; Tumor Cells, Cultured

Peroxisome proliferator-activated receptors-gamma (PPARgamma) are ligand-inducible transcription factors of the nuclear hormone receptor superfamily. We examined the effect of PPARgamma activation on the generation of vascular endothelial growth factor (VEGF), one of the major angiogenic agents. Rat vascular smooth muscle cells (VSMC) and murine macrophages RAW264.7 were incubated for 24 h with PPARgamma activators: prostaglandin J2 and ciglitazone. PPARgamma were expressed in VSMC and RAW cells and their activity was upregulated in the presence of PGJ2 and ciglitazone. Incubation of the cells with PPARgamma activators significantly augmented the release of VEGF protein into the media, both in resting and in IL-1beta- or LPS-stimulated cultures. The higher protein generation was connected with the increased expression of mRNA and transcriptional activation of VEGF promoter. We conclude that the activation of PPARgamma upregulates the generation of VEGF and may be involved in the regulation of angiogenesis.

Topics: Animals; Antineoplastic Agents; Cell Line; Cells, Cultured; Dose-Response Relationship, Drug; Endothelial Growth Factors; Endothelium, Vascular; Hypoglycemic Agents; Interleukin-1; Ligands; Lipopolysaccharides; Lymphokines; Macrophages; Mice; Muscle, Smooth; Promoter Regions, Genetic; Prostaglandin D2; Rats; Receptors, Cytoplasmic and Nuclear; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thiazoles; Thiazolidinediones; Transcription Factors; Transcriptional Activation; Transfection; Up-Regulation; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

Peroxisome proliferator-activated receptor gamma (PPARgamma) is a nuclear receptor that functions as a transcription factor to mediate ligand-dependent transcriptional regulation. Activation of PPARgamma by the naturally occurring ligand, 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2), or members of a new class of oral antidiabetic agents, e.g. BRL49653 and ciglitizone, has been linked to adipocyte differentiation, regulation of glucose homeostasis, inhibition of macrophage and monocyte activation, and inhibition of tumor cell proliferation. Here we report that human umbilical vein endothelial cells (HUVEC) express PPARgamma mRNA and protein. Activation of PPARgamma by the specific ligands 15d-PGJ2, BRL49653, or ciglitizone, dose dependently suppresses HUVEC differentiation into tube-like structures in three-dimensional collagen gels. In contrast, specific PPARalpha and -beta ligands do not affect tube formation although mRNA for these receptors are expressed in HUVEC. PPARgamma ligands also inhibit the proliferative response of HUVEC to exogenous growth factors. Treatment of HUVEC with 15d-PGJ2 also reduced mRNA levels of vascular endothelial cell growth factor receptors 1 (Flt-1) and 2 (Flk/KDR) and urokinase plasminogen activator and increased plasminogen activator inhibitor-1 (PAI-1) mRNA. Finally, administration of 15d-PGJ2 inhibited vascular endothelial cell growth factor-induced angiogenesis in the rat cornea. These observations demonstrate that PPARgamma ligands are potent inhibitors of angiogenesis in vitro and in vivo, and suggest that PPARgamma may be an important molecular target for the development of small-molecule inhibitors of angiogenesis.

Topics: Animals; Cell Differentiation; Cell Division; Cells, Cultured; Cornea; Eicosanoids; Endothelium, Vascular; Gene Expression Regulation; Humans; Ligands; Neovascularization, Physiologic; Prostaglandin D2; Rats; Receptors, Cytoplasmic and Nuclear; RNA, Messenger; Rosiglitazone; Thiazoles; Thiazolidinediones; Transcription Factors

The peroxisome proliferator-activated receptor-gamma (PPARgamma) is activated by 15-deoxy-delta(12,14) prostaglandin J2 (15d-PGJ2), anti-diabetic thiazolidinediones and several non-steroidal anti-inflammatory drugs (NSAIDs). In rat glial cells, lipopolysaccharide and interferon-gamma (LPS/IFN-gamma) induced expression of both inducible nitric oxide synthase (iNOS) and heme oxygenase-1 (HO-1). PPARgamma activators inhibited iNOS expression by LPS and IFN-gamma. However, PPARgamma activator alone induced HO-1 expression and further enhanced LPS/IFN-gamma-induced HO-1 expression. These results suggest that activation of PPARgamma negatively regulate iNOS expression and positively regulates HO-1 expression in glial cells.

Topics: Alitretinoin; Animals; Anti-Inflammatory Agents, Non-Steroidal; Brain; Cells, Cultured; Dinoprostone; Enzyme Induction; Gene Expression Regulation, Enzymologic; Heme Oxygenase (Decyclizing); Heme Oxygenase-1; Interferon-gamma; Lipopolysaccharides; Neuroglia; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitrites; Prostaglandin D2; Pyrimidines; Rats; Rats, Wistar; Receptors, Cytoplasmic and Nuclear; Thiazoles; Thiazolidinediones; Transcription Factors; Tretinoin

A prominent functional change during differentiation of lutein cells from follicular thecal and granulosa cells is an enhanced production and secretion of progestins. The regulation of this process is not fully understood but may be associated with the expression of transcription factors which activate genes, products of which are involved in pathways of the cholesterol and lipid metabolism. As peroxisome proliferator-activated receptors (PPARs) play a role in both pathways, we were interested in the expression of PPARgamma, a PPAR form which is involved in adipogenic differentiation. First, we were able to show the expression of PPARgamma in bovine lutein cells (day 12 of the ovarian cycle) at the mRNA and protein level by imaging, flow cytometry and blot analysis, and secondly a role of PPARgamma in the secretion of progesterone. The cells (24 h culture) responded dose dependently by increasing progesterone secretion (up to 1.5-fold of the basal level) to an endogenous ligand of PPARgamma, 15-deoxy-delta12,14 prostaglandin J2 (15-dPGJ2) and to the thiazolidinedione ciglitizone. Aurintricarboxylic acid (ATA) was found to reduce the intracellular PPARgamma level and to promote cell cycle progress, indicating that ATA can be used as a tool for experimental changes of PPARgamma proteins in intact cells and for studying the physiological consequences. The ATA-mediated decrease of PPARgamma was accompanied by reduced progesterone production and a progression of the cell cycle, suggesting a function of PPARgamma in both processes. The response to ATA was abrogated by a high dose (>490 nM) of 15-dPGJ2, suggesting that 15-dPGJ2 exerts its effect on steroidogenic activity via PPARgamma and that the 15-dPGJ2-PPARgamma system plays a role in the maintenance of a differentiated quiescent stage in lutein cells.

Topics: Analysis of Variance; Animals; Aurintricarboxylic Acid; Cattle; Cell Cycle; Dose-Response Relationship, Drug; Female; Flow Cytometry; Granulosa Cells; Hypoglycemic Agents; Luteal Cells; Luteinizing Hormone; Microscopy, Fluorescence; Progesterone; Prostaglandin D2; Protein Isoforms; Receptors, Cytoplasmic and Nuclear; Receptors, LH; RNA, Messenger; Thiazoles; Thiazolidinediones; Transcription Factors

Prostaglandins (PGs) of the J2 series form in vivo and exert effects on a variety of biological processes. While most of PGs mediate their effects through G protein-coupled receptors, the mechanism of action for the J2 series of PGs remains unclear. Here, we report the PGJ2 and its derivatives are efficacious activators of peroxisome proliferator-activated receptors alpha and gamma (PPAR alpha and PPAR gamma, respectively), orphan nuclear receptors implicated in lipid homeostasis and adipocyte differentiation. The PGJ2 metabolite 15-deoxy-delta 12,14-PGJ2 binds directly to PPAR gamma and promotes efficient differentiation of C3H10T1/2 fibroblasts to adipocytes. These data provide strong evidence that a fatty acid metabolite can function as an adipogenic agent through direct interactions with PPAR gamma and furthermore, suggest a novel mechanism of action for PGs of the J2 series.

Topics: Adipocytes; Animals; Binding, Competitive; Cell Differentiation; Cells, Cultured; Fibroblasts; Hypoglycemic Agents; Ligands; Mice; Microbodies; Prostaglandin D2; Prostaglandins; Pyrimidines; Receptors, Cytoplasmic and Nuclear; Recombinant Fusion Proteins; Rosiglitazone; Signal Transduction; Thiazoles; Thiazolidinediones; Transcription Factors; Transcriptional Activation