rottlerin and Inflammation

Phospholipase A

Topics: Acetophenones; Adult; Aged; Animals; Animals, Newborn; Astrocytes; Benzopyrans; Bradykinin; Bradykinin B2 Receptor Antagonists; Brain; Brain Injuries, Traumatic; Disease Models, Animal; Enzyme Inhibitors; Epilepsy; Female; Humans; Inflammation; Male; Middle Aged; Phospholipases A2, Cytosolic; Quinolines; Rats; Rats, Sprague-Dawley; Receptor, Bradykinin B2; Up-Regulation; Young Adult

Dysregulated release of neutrophil reactive oxygen species and proteolytic enzymes contributes to both acute and chronic inflammatory diseases. Therefore, molecular regulators of these processes are potential targets for new anti-inflammatory therapies. We have shown previously that myristoylated alanine-rich C-kinase substrate (MARCKS), a well-known actin binding protein and protein kinase C (PKC) substrate, is a key regulator of neutrophil functions. In the current study, we investigate the role of PKC-mediated MARCKS phosphorylation in neutrophil migration and adhesion in vitro. We report that treatment of human neutrophils with the δ-PKC inhibitor rottlerin significantly attenuates f-Met-Leu-Phe (fMLF)-induced MARCKS phosphorylation (IC50=5.709 μM), adhesion (IC50=8.4 μM), and migration (IC50=6.7 μM), while α-, β-, and ζ-PKC inhibitors had no significant effect. We conclude that δ-PKC-mediated MARCKS phosphorylation is essential for human neutrophil migration and adhesion in vitro. These results implicate δ-PKC-mediated MARCKS phosphorylation as a key step in the inflammatory response of neutrophils.

Topics: Acetophenones; Benzopyrans; Cell Adhesion; Cell Movement; Cells, Cultured; Enzyme Inhibitors; Humans; Inflammation; Intracellular Signaling Peptides and Proteins; Membrane Proteins; Myristoylated Alanine-Rich C Kinase Substrate; N-Formylmethionine Leucyl-Phenylalanine; Neutrophil Activation; Neutrophil Infiltration; Neutrophils; Phosphorylation; Protein Isoforms; Protein Kinase C-delta

Quiescent cells are considered to be dormant. However, recent studies suggest that quiescent fibroblasts possess active metabolic profile and certain functional characteristics. We previously observed that serum-starved quiescent fibroblasts respond to proinflammatory stimuli by robust expression of cyclooxygenase-2 (COX-2), which declines after the quiescent fibroblasts are driven to proliferation. In this study, we elucidated the underlying signaling and transcriptional mechanism and identified by microarray genes with similar differential expression. By using pharmacological inhibitors coupled with gene silencing, we uncovered the key role of protein kinase C δ (PKCδ) and extracellular signal regulated protein kinase 1/2 (ERK1/2) signaling in mediating COX-2 expression in quiescent cells. Surprisingly, COX-2 expression in proliferative cells was not blocked by PKCδ or ERK1/2 inhibitors due to intrinsic inhibition of PKCδ and ERK1/2 in proliferative cells. Restrained COX-2 transcription in proliferative cells was attributable to reduced NF-κB binding. Microarray analysis identified 35 genes whose expressions were more robust in quiescent than in proliferative cells. A majority of those genes belong to proinflammatory cytokines, chemokines, adhesive molecules and metalloproteinases, which require NF-κB for transcription. Quiescent fibroblasts had a higher migratory activity than proliferative fibroblasts as determined by the transwell assay. Selective COX-2 inhibition reduced migration which was restored by prostaglandin E(2). As COX-2 and inflammatory mediators induce DNA oxidation, we measured 8-hydroxydeoxyguanosine (8-OHdG) in quiescent vs. proliferative fibroblasts. PMA-induced 8-OHdG accumulation was significantly higher in quiescent than in proliferative fibroblasts. These findings indicate that quiescent fibroblasts (and probably other quiescent cells) are at the forefront in mounting inflammatory responses through expression of an array of proinflammatory genes via the PKCδ/ERK1/2 signaling pathway.

Topics: Acetophenones; Benzopyrans; Cell Movement; Cell Proliferation; Chromones; Cyclooxygenase 2; Enzyme Inhibitors; Fibroblasts; Flavonoids; Humans; Inflammation; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 3; Morpholines; NF-kappa B; Protein Kinase C-delta; Transcription, Genetic

The aim of the study was to address the role of protein kinase C-delta (PKCdelta) on phosphorylation of signal transducer and activator of transcription 3 (STAT3) and activation of inflammatory genes in response to IL-6 in adipose cells.. Differentiated mouse 3T3-L1 adipocytes preincubated with the PKCdelta inhibitor rottlerin and mouse embryonic fibroblasts (MEFs) lacking PKCdelta were incubated with IL-6 and/or insulin. RNA was extracted and the gene expression was analysed by real-time PCR, while the proteins from total, nuclear and cytoplasmic lysates were analysed by immunoblotting.. Inhibition of PKCdelta by rottlerin significantly reduced both Ser-727 and Tyr-705 phosphorylation of STAT3. Consequently, nuclear translocation of STAT3 and the IL-6-induced gene transcription and protein release of the inflammatory molecule serum amyloid A 3 (SAA3) were reduced. Similarly, the IL-6-regulated gene transcription of Il-6 (also known as Il6) to Hp and the feedback inhibitor of IL-6, Socs3, were also attenuated by rottlerin. Furthermore, PKCdelta was found to translocate to the nucleus following IL-6 treatment and this was also reduced by rottlerin. In agreement with the effect of rottlerin, Pkcdelta (also known as Prkcd) ( -/- ) MEFs also displayed a markedly reduced ability of IL-6 to activate the transcription of Saa3, Hp, Socs3 and Il6 genes compared with wild-type MEFs. These results correlated with a reduced nuclear translocation and phosphorylation of STAT3.. These results show that PKCdelta plays a key role in the inflammatory effect of IL-6 in adipose cells and may be a suitable target for novel anti-inflammatory agents.

Topics: Acetophenones; Adipocytes; Animals; Benzopyrans; Blotting, Western; Cell Line; Cells, Cultured; Gene Expression; Inflammation; Interleukin-6; Mice; Phosphorylation; Protein Kinase C-delta; Reverse Transcriptase Polymerase Chain Reaction; Serum Amyloid A Protein; Signal Transduction; STAT3 Transcription Factor; Statistics, Nonparametric; Transcriptional Activation

Lysophosphatidic acid (LPA), a potent bioactive lipid, elicits many of its biological actions via the specific G-protein-coupled receptors LPA1, LPA2, LPA3, and LPA4. Recently, we have shown that LPA-induced transactivation of platelet-derived growth factor receptor-beta is regulated by phospholipase D2 in human bronchial epithelial cells (HBEpCs) (Wang, L., Cummings, R. J., Zhao, Y., Kazlauskas, A., Sham, J., Morris, A., Brindley, D. N., Georas, S., and Natarajan, V. (2003) J. Biol. Chem. 278, 39931-39940). Here, we report that protein kinase Cdelta (PKCdelta) mediates LPA-induced NF-kappaB transcription and interleukin-8 (IL-8) secretion in HBEpCs. Treatment of HBEpCs with LPA increased both IL-8 gene and protein expression, which was coupled to Gi and G(12/13) proteins. LPA caused a marked activation of NF-kappaB in HBEpCs as determined by IkappaB phosphorylation and of NF-kappaB nuclear translocation and a strong induction of NF-kappaB promoter-mediated luciferase activity. Furthermore, LPA-activated PKCdelta and the LPA-mediated activation of NF-kappaB and IL-8 production were attenuated by overexpression of dominant-negative PKCdelta and rottlerin. Intratracheal administration of LPA in mice resulted in elevated levels of macrophage inflammatory protein-2, a murine homolog of IL-8, and an influx of neutrophils in the bronchoalveolar lavage fluid. These results demonstrate for the first time that LPA is a potent stimulator of IL-8 production in HBEpCs, which involves PKCdelta/NF-kappaB signaling pathways.

Topics: Acetophenones; Active Transport, Cell Nucleus; Animals; Benzopyrans; Blotting, Western; Bronchoalveolar Lavage; Cell Nucleus; Cells, Cultured; Chemokine CXCL2; Chemokines; Cytokines; DNA, Complementary; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Gene Expression Regulation; Humans; Inflammation; Interleukin-8; Luciferases; Lysophospholipids; Macrophages; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; NF-kappa B; Oligonucleotide Array Sequence Analysis; Phosphorylation; Protein Isoforms; Protein Kinase C; Protein Kinase C-delta; Protein Transport; Receptor, Platelet-Derived Growth Factor beta; RNA; Signal Transduction; Time Factors; Transcriptional Activation; Transfection