u-0126 and benzyloxycarbonylleucyl-leucyl-leucine-aldehyde

Influenza virus exploits a number of cellular signaling pathways during the course of its replication, rendering them potential targets for new therapeutic interventions. Several preclinical approaches are now focusing on cellular factors or pathways as a means of treating influenza. By targeting host factors, rather than viral mechanisms, these novel therapies may be effective against multiple virus strains and subtypes, and are less likely to elicit viral drug resistance. The most promising candidates are inhibitors of intracellular signaling cascades that are essential for virus replication. This article reviews novel approaches and compounds that target the Raf/MEK/ERK signaling pathway, NF-κB signaling, the PI3K/Akt pathway and the PKC signaling cascade. Although these new antiviral strategies are still in an early phase of preclinical development, results to date suggest they offer a new approach to the treatment of influenza, supplementing direct-acting antiviral drugs.

Topics: Animals; Antiviral Agents; Benzamides; Butadienes; Enzyme Activation; Humans; Influenza, Human; Leupeptins; MAP Kinase Signaling System; NF-kappa B; Nitriles; Orthomyxoviridae; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase Inhibitors; Virus Replication

Large conductance Ca2+-activated potassium channels (BK) are targets for research that explores therapeutic means to various diseases, owing to the roles of the channels in mediating multiple physiological processes in various cells and tissues. We investigated the pharmacological effects of curcumin, a compound isolated from the herb Curcuma longa, on BK channels. As recorded by whole-cell patch-clamp, curcumin increased BK (α) and BK (α+β1) currents in transfected HEK293 cells as well as the current density of BK in A7r5 smooth muscle cells in a dose-dependent manner. By incubating with curcumin for 24 hours, the current density of exogenous BK (α) in HEK293 cells and the endogenous BK in A7r5 cells were both enhanced notably, though the steady-state activation of the channels did not shift significantly, except for BK (α+β1). Curcumin up-regulated the BK protein expression without changing its mRNA level in A7r5 cells. The surface expression and the half-life of BK channels were also increased by curcumin in HEK293 cells. These effects of curcumin were abolished by MG-132, a proteasome inhibitor. Curcumin also increased ERK 1/2 phosphorylation, while inhibiting ERK by U0126 attenuated the curcumin-induced up-regulation of BK protein expression. We also observed that the curcumin-induced relaxation in the isolated rat aortic rings was significantly attenuated by paxilline, a BK channel specific blocker. These results show that curcumin enhances the activity of the BK channels by interacting with BK directly as well as enhancing BK protein expression through inhibiting proteasomal degradation and activating ERK signaling pathway. The findings suggest that curcumin is a potential BK channel activator and provide novel insight into its complicated pharmacological effects and the underlying mechanisms.

Topics: Animals; Butadienes; Curcumin; HEK293 Cells; Humans; Large-Conductance Calcium-Activated Potassium Channels; Leupeptins; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Nitriles; Proteasome Endopeptidase Complex; Proteolysis; Rats; Up-Regulation

High expression levels of cyclooxygenase-2 (COX-2) contribute a strong proliferative ability to human lung cancer cells, and this function is link to the epidermal growth factor receptor (EGFR) pathway, which was mediated by extracellular-signal-regulated kinase (ERK) and nuclear factor kappa B (NFκB). In this study, scutellarein, a flavonoid compound, was screened for proliferation inhibition at different concentrations (0, 5, 25 and 50 μM) at 24 h or 48 h in human lung cancer cell line A549. Results showed that A549 cell proliferation was inhibited by 50 μM scutellarein treatment in 24 h and 48 h of treatment. The expression levels of phosphorylated EGFR, phosphorylated ERK, phosphorylated NFκB and COX-2 were reduced in a dose-dependent manner after 24 h scutellarein treatments at different concentrations. Further, ERK inhibitor U0126 and NFκB inhibitor MG132 also inhibited A549 cell proliferation similar to 50 κM scutellarein treatment from 24 h to 48 h. The experimental results showed that scutellarein could inhibit proliferation of the human lung cancer cell line A549 through ERK and NFκB mediated by the EGFR pathway.

Topics: Antineoplastic Agents; Apigenin; Butadienes; Cell Line, Tumor; Cell Proliferation; Enzyme Inhibitors; ErbB Receptors; Extracellular Signal-Regulated MAP Kinases; Humans; Leupeptins; Lung Neoplasms; MAP Kinase Signaling System; NF-kappa B; Nitriles

Mechanical ventilation with high tidal volumes has been associated with pulmonary alveolar flooding. Understanding the mechanisms underlying cyclic stretch-induced increases in alveolar epithelial permeability may be important in designing preventive measures for acute lung injury. In this work, we assessed whether cyclic stretch leads to the generation of reactive oxygen species in type I-like alveolar epithelial cells, which increase monolayer permeability via activation of NF-κB and extracellular signal-regulated kinase (ERK). We cyclically stretched type I-like rat primary alveolar epithelial cells at magnitudes of 12, 25, and 37% change in surface area (ΔSA) for 10 to 120 minutes. High levels of reactive oxygen species and of superoxide and NO specifically were detected in cells stretched at 37% ΔSA for 10 to 120 minutes. Exogenous superoxide and NO stimulation increased epithelial permeability in unstretched cells, which was preventable by the NF-κB inhibitor MG132. The cyclic stretch-induced increase in permeability was decreased by the superoxide scavenger tiron and by MG132. Furthermore, tiron had a dramatic protective effect on in vivo lung permeability under mechanical ventilation conditions. Cyclic stretch increased the activation of the NF-κB signaling pathway, which was significantly decreased with the ERK inhibitor U0126. Altogether, our in vitro and in vivo data demonstrate the sensitivity of permeability to stretch- and ventilation-induced superoxide production, suggesting that using antioxidants may be helpful in the prevention and treatment of ventilator-induced lung injury.

Topics: 1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium Salt; Animals; Antioxidants; Butadienes; Cell Membrane Permeability; Epithelial Cells; Leupeptins; Male; NF-kappa B; Nitric Oxide; Nitriles; Oxidative Stress; Pulmonary Alveoli; Rats; Rats, Sprague-Dawley; Respiration, Artificial; Signal Transduction; Superoxides; Ventilator-Induced Lung Injury

To examine CD99 expression and its functional role in ICAM-1 induction in human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGECs) by activating cells with anti-CD99 monoclonal antibody, MT99/3.. Engagement of CD99 with agonistic antibodies has been shown to regulate immune responses, cell adhesion and migration, and cell death in several studies. Particularly, this engagement results in transendothelial migration of leukocytes mediated by intercellular adhesion molecule-1 (ICAM-1) induction in endothelial cells.. Total mRNA and protein were isolated from HGFs and HGECs for analyses of CD99 and ICAM-1 expression. Surface expression of CD99 and ICAM-1 was analysed by flow cytometry, and the detection of soluble ICAM-1 was assayed by immunoprecipitation and ELISA.. CD99 surface expression was constitutive on HGFs to a greater extent than that on HGECs. CD99 ligation with MT99/3 induced ICAM-1 mRNA expression in HGFs, but not in HGECs. Interestingly, CD99 ligation led to an increased level of soluble ICAM-1 detected in culture supernatant, whereas interleukin-1β (IL-1β) treatment induced expression of membrane-bound ICAM-1. Furthermore, ICAM-1 induction by CD99 engagement was demonstrated to involve the activation of the p50 subunit of nuclear factor-kappaB (NF-κB), extracellular signal-regulated kinase, and p46 c-Jun N-terminal kinase that differed from that by IL-1β treatment.. Our study has shown the involvement of CD99 ligation in the up-regulation of ICAM-1 expression and its secretion in gingival fibroblasts, which may be essential for better understanding of the pathogenesis of periodontal disease.

Topics: 12E7 Antigen; Anthracenes; Antibodies, Monoclonal; Antigens, CD; Butadienes; Cell Adhesion Molecules; Cell Movement; Cells, Cultured; Culture Media, Conditioned; Endothelial Cells; Enzyme Inhibitors; Epithelial Cells; Extracellular Signal-Regulated MAP Kinases; Fibroblasts; Flow Cytometry; Gingiva; Humans; Imidazoles; Intercellular Adhesion Molecule-1; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Jurkat Cells; Leukocytes; Leupeptins; MAP Kinase Signaling System; NF-kappa B; NF-kappa B p50 Subunit; Nitriles; p38 Mitogen-Activated Protein Kinases; Pyridines

Injury-induced cytokines act through gp130 in sympathetic neurons to suppress expression of tyrosine hydroxylase (TH) and other genes associated with noradrenergic transmission. These cytokines also trigger the local loss of TH in peri-infarct sympathetic axons after myocardial infarction, but altered gene expression cannot explain the selective loss of TH enzyme in one region of the heart. We hypothesized that inflammatory cytokines, which are highest near the infarct, stimulated local degradation of TH protein. We used cultured sympathetic neurons and neuroblastoma cells to test this hypothesis. The cytokines ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) suppressed TH content in both neurons and neuroblastoma cells. CNTF suppressed TH in a gp130-dependent manner, and decreased the half-life of TH protein by approximately 50%. CNTF stimulated the ubiquitination of TH in both neurons and neuroblastoma cells, and the proteasome inhibitors MG-132 and lactacystin prevented the CNTF-induced loss of TH protein. Inhibiting activation of extracellular signal regulated kinases 1&2 (ERK1/2) with U0126 prevented the CNTF-induced ubiquitination of TH and the associated decrease in protein half-life. Likewise, inhibiting ERK1/2 activation blunted the cytokine-stimulated loss of TH protein in sympathetic neurons, despite enhancing the loss of TH mRNA. These data suggest that gp130 cytokines stimulate proteasomal degradation of TH through an ERK1/2 dependent pathway, and may have important implications for local regulation of neurotransmission at sites of inflammation.

Topics: Animals; Animals, Newborn; Butadienes; Cells, Cultured; Ciliary Neurotrophic Factor; Cytokine Receptor gp130; Dopamine beta-Hydroxylase; Enzyme Inhibitors; Female; Ganglia, Spinal; Gene Expression Regulation; Half-Life; Humans; Immunoprecipitation; Leukemia Inhibitory Factor; Leupeptins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Mitogen-Activated Protein Kinase 3; Neurons; Nitriles; Pregnancy; Proteasome Endopeptidase Complex; Rats; Rats, Sprague-Dawley; RNA, Messenger; Statistics, Nonparametric; Tyrosine 3-Monooxygenase; Ubiquitination

The serine/threonine kinase, B-RAF, is frequently mutated in melanoma and is required for cell proliferation. Proteasomal turnover of cyclins and cyclin-dependent kinase inhibitors via E3 ubiquitin ligases regulates cell cycle progression. We previously showed that B-RAF regulates Cks1, a co-factor for the F-box protein Skp2. Recently, a second F-box protein cofactor was identified, alphaB-crystallin, that binds Fbx4 and promotes cyclin D1 degradation. Here, we demonstrate that alphaB-crystallin is down-regulated in mutant B-RAF melanoma cells compared to melanocytes in a B-RAF and MEK-dependent manner. In a subset of lines, MEK inhibition was sufficient to up-regulate alphaB-crystallin protein levels; whereas in other lines combined MEK and proteasome inhibition was required. alphaB-crystallin knockdown partially stabilized cyclin D1 in melanocytes. Expression of alphaB-crystallin in mutant B-RAF melanoma cells did not promote cyclin D1 turnover under normal conditions, but did enhance turnover following etoposide-induced DNA damage. Together, these data show that alphaB-crystallin is highly expressed in melanocytes contributing, in part, to cyclin D1 turnover. Furthermore, alphaB-crystallin is down-regulated in a B-RAF-dependent manner in melanoma cells and its re-expression regulates cyclin D1 turnover after DNA damage.

Topics: alpha-Crystallin B Chain; Bleomycin; Butadienes; Cells, Cultured; Cyclin D1; Cycloheximide; DNA Damage; Etoposide; Humans; Leupeptins; Melanocytes; Melanoma; Mutant Proteins; Mutation; Nitriles; Protein Kinase Inhibitors; Proto-Oncogene Proteins B-raf; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Structure-Activity Relationship; Tetracycline

CD40 is a costimulatory molecule linking innate and adaptive immune responses to bacterial stimuli, as well as a critical regulator of functions of other costimulatory molecules. The mechanisms regulating lipopolysaccharide (LPS)-induced CD40 expression have not been adequately characterized in human monocytic cells. In this study we used a human monocytic cell line, THP-1, to investigate the possible mechanisms of CD40 expression following LPS exposure. Exposure to LPS resulted in a dose- and time-dependent increase in CD40 expression. Further studies using immunoblotting and pharmacological inhibitors revealed that mitogen-activated protein kinases (MAPKs) and NFkappaB were activated by LPS exposure and involved in LPS-induced CD40 expression. Activation of MAPKs was not responsible for LPS-induced NFkappaB activation. TLR4 was expressed on THP-1 cells and pretreatment of cells with a Toll-like receptor 4 (TLR4) neutralizing antibody (HTA125) significantly blunted LPS-induced MAPK and NFkappaB activation and ensuing CD40 expression. Additional studies with murine macrophages expressing wild type and mutated TLR4 showed that TLR4 was implicated in LPS-induced ERK and NFkappaB activation, and CD40 expression. Moreover, blockage of MAPK and NFkappaB activation inhibited LPS-induced TLR4 expression. In summary, LPS-induced CD40 expression in monocytic cells involves MAPKs and NFkappaB.

Topics: Animals; Anthracenes; Butadienes; CD40 Antigens; Cell Line; Cell Line, Tumor; Dose-Response Relationship, Drug; Electrophoretic Mobility Shift Assay; Flow Cytometry; Humans; Imidazoles; Immunoblotting; Leupeptins; Mitogen-Activated Protein Kinases; Monocytes; NF-kappa B; Nitriles; Phosphorylation; Polysaccharides, Bacterial; Protein Kinase Inhibitors; Pyridines; Signal Transduction; Sulfones; Time Factors; Toll-Like Receptor 4

MPF and MAP kinase ERK2 are two major M-phase kinases. They interact with each other in a complex way during meiotic maturation of Xenopus laevis oocytes. Here we study their interrelationship during first mitosis in X. laevis embryo cell-free extract perturbing the polyubiquitination pathway as a tool. Recombinant ubiquitin K48R (Ub-K48R) mutant protein arrests mitotic cyclin B degradation in the extract. This results in both increased accumulation of phosphorylated form of cyclin B2 and MPF activity as well as mitotic phosphorylation of its substrates. Ub-K48R also increased the mitotic phosphorylation of ERK2. Simultaneous addition of Ub-K48R and the proteasome inhibitor MG 132 strengthened and further prolonged MPF activity, MCM4 phosphorylation and accumulation of phosphorylated forms of cyclin B2. ERK2 phosphorylation levels increased and persisted longer than upon action of Ub-K48R alone. This shows a synergistic effect of inhibition of two different steps of ubiquitin-proteasome pathway on MPF activity and mitotic phosphorylation and ubiquitination of specific M-phase proteins. On the other hand, complete inhibition of ERK2 activation using U0126 had no effect either on MPF activity or on MCM4 phosphorylation either in control or in Ub-K48R-supplemented extracts. Experimental reduction of MPF activity by addition of recombinant p21(Cip) protein resulted in significant reduction of ERK2 phosphorylation. Thus, the reciprocal feedback observed between MPF and ERK2 in meiosis is not observed during mitotic M-phase in cell-free Xenopus embryo extracts. ERK2 phosphorylation is regulated by the levels of MPF activity, however no influence of ERK2 on MPF activity could be detected. These results show a fundamental difference in the relationship between the two major M-phase kinases in meiotic and mitotic cell cycle.

Topics: Animals; Butadienes; Cell-Free System; Embryo, Nonmammalian; Feedback, Physiological; Female; Leupeptins; Maturation-Promoting Factor; Meiosis; Mitogen-Activated Protein Kinase 1; Mitosis; Mutation; Nitriles; Phosphorylation; Proteasome Inhibitors; Protein Kinase Inhibitors; Signal Transduction; Ubiquitin; Xenopus laevis; Xenopus Proteins

The purpose of this study was to investigate whether proteasome inhibition acts as a thermal sensitizing agent to induce tumor cell death in a colon cancer cell line.. HT-29 colon cancer cells were exposed to hyperthermia (43 degrees C) in the presence of proteasome inhibition for 1 h. Viable cell mass and apoptosis were measured by MTT and annexin V staining, respectively. Protein levels were determined by Western blot analysis.. A significant synergistic effect on cell viability with proteasome inhibition was noted under hyperthermic conditions compared to hyperthermia alone (p < 0.05). Increases in phosphorylated ERK and decreases in HSP27 levels were observed in the cells exposed to proteasome inhibition at 43 degrees C. Pretreatment with an inhibitor of ERK yielded an additional increase in apoptosis when used in combination with proteasome inhibition and hyperthermia. Decreased expression of HSP27 by siRNA also resulted in increased thermally induced apoptotic cell death.. Thermal sensitization through proteasome inhibition may represent a novel approach to increase the efficacy of hyperthermia as an anticancer modality.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Butadienes; Cell Proliferation; Cell Survival; Colonic Neoplasms; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Synergism; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Heat-Shock Proteins; HT29 Cells; Humans; Hyperthermia, Induced; Leupeptins; Nitriles; Protease Inhibitors; Proteasome Inhibitors; RNA, Small Interfering

A previous exposure to a non-harmful ischemic insult (preconditioning) protects the brain against subsequent harmful ischemia (ischemic tolerance). In contrast to delayed gene-mediated ischemic tolerance, little is known about the molecular mechanisms that regulate rapid ischemic tolerance, which occurs within 1 h following preconditioning. Here we have investigated the degradation of the pro-apoptotic Bcl-2 family member Bim as a mechanism of rapid ischemic tolerance. Bim protein levels were reduced 1 h following preconditioning and occurred concurrent with an increase in Bim ubiquitination. Ubiquitinated proteins are degraded by the proteasome, and inhibition of the proteasome with MG132 (a proteasome inhibitor) prevented Bim degradation and blocked rapid ischemic tolerance. Inhibition of p42/p44 mitogen-activated protein kinase activation by U0126 reduced Bim ubiquitination and Bim degradation and blocked rapid ischemic tolerance. Finally, inhibition of Bim expression using antisense oligonucleotides also reduced cell death following ischemic challenge. Our results suggest that following preconditioning ischemia, Bim is rapidly degraded by the ubiquitin-proteasome system, resulting in rapid ischemic tolerance. This suggests that the rapid degradation of cell death-promoting proteins by the ubiquitin-proteasome pathway may represent a novel therapeutic strategy to reduce cell damage following neuropathological insults, e.g. stroke.

Topics: Animals; Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Butadienes; Cell Death; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Immunoblotting; Immunohistochemistry; In Situ Nick-End Labeling; Ischemia; Ischemic Preconditioning; Leupeptins; Membrane Proteins; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Neurons; Nitriles; Oligonucleotides, Antisense; Phosphorylation; Propidium; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Rats; Rats, Sprague-Dawley; Time Factors; Ubiquitin

Treatment of transected distal axons of rat sympathetic neurons in compartmented cultures with MG132 (5 microM) and other inhibitors of proteasome activity, preserved axonal mitochondrial function, assessed by Mitotracker-Orange and MTT staining, for at least 24 h. MG132 similarly protected axons from undergoing branch elimination (pruning) in response to local NGF deprivation. Axons protected by MG132 displayed persistent phosphorylation of Erk1/2, and pharmacological inhibition of MEK activity with U0126 (50 microM) restored rapid axonal degeneration. Therefore, the proteasome does not appear to be necessary as a general effector of protein degradation during axonal degeneration. Rather, the proteasome functions in the regulation of signaling pathways that control axonal survival and degeneration. Specifically, the down-regulation of the MEK/Erk pathway by the proteasome plays roles in Wallerian degeneration of severed axons and axonal pruning in response to local NGF deprivation. Identification of the pathways that regulate axonal survival and degeneration will provide possible target sites for pharmacological treatments of neurodegenerative diseases and traumatic injury.

Topics: Animals; Animals, Newborn; Axons; Butadienes; Cell Survival; Cells, Cultured; Cysteine Proteinase Inhibitors; Down-Regulation; Enzyme Inhibitors; Leupeptins; MAP Kinase Kinase 1; MAP Kinase Signaling System; Mitogen-Activated Protein Kinase 3; Nerve Growth Factor; Nerve Regeneration; Nitriles; Phosphorylation; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Sprague-Dawley; Signal Transduction; Superior Cervical Ganglion; Sympathetic Nervous System; Wallerian Degeneration

We generated A21-13 cells expressing p14(ARF) in the presence of doxycycline in order to examine the stability of p14(ARF) protein. The effects of proteasome inhibitor MG132 on p14(ARF) protein stabilization were detectable using our experimental procedure. Introduction of mutant p53 did not affect MG132-mediated p14(ARF) protein stabilization. We found that phorbol ester TPA (12-o-tetradecanoyl-phorbol 13-acetate) stabilized p14(ARF) protein and that p53 status had no effect on TPA-mediated stabilization. TPA-mediated stabilization was abolished by staurosporine but not by lovastatin or U0126. We further investigated which isoforms of PKC were involved in TPA-mediated p14(ARF) stabilization using short-interference RNA. Knockdown of PKCalpha, but not PKCdelta, attenuated TPA-mediated p14(ARF) stabilization. These findings suggest that PKCalpha is involved in TPA-mediated stabilization of p14(ARF) protein, and this effect of TPA was not affected by the Ras/MAPK pathway or p53 status. Our results are indicative of a novel role of PKC in p14(ARF) protein stability.

Topics: Butadienes; Cell Line, Tumor; Doxycycline; Genes, p53; Humans; Leupeptins; Lovastatin; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Nitriles; Proteasome Inhibitors; Protein Kinase C; Protein Kinase C beta; Protein Kinase C-alpha; Proto-Oncogene Proteins p21(ras); RNA, Small Interfering; Staurosporine; Tetradecanoylphorbol Acetate; Tumor Suppressor Protein p14ARF

In cultured rat vascular smooth muscle cells, sustained activation of ERK is required for interleukin-1beta to persistently activate NF-kappaB. Without ERK activation, interleukin-1beta induces only acute and transient NF-kappaB activation. The present study examined whether the temporal control of NF-kappaB activation by ERK could differentially regulate the expression of NF-kappaB-dependent genes, including inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), vascular cell adhesion molecule-1 (VCAM-1), and manganese-containing superoxide dismutase (Mn-SOD). Treatment of vascular smooth muscle cells with interleukin-1beta induced the expression of iNOS, COX-2, VCAM-1, and Mn-SOD in a time-dependent manner, but with different patterns. Either PD98059 or U0126, selective inhibitors of MEK, or overexpression of a dominant negative MEK-1 inhibited interleukin-1beta- induced ERK activation and the expression of iNOS and COX-2 but had essentially no effect on the expression of VCAM-1 and Mn-SOD. The expression of these genes was inhibited when NF-kappaB activation was down-regulated by MG132, a proteasome inhibitor, or by overexpression of an I-kappaBalpha mutant that prevented both the transient and the persistent activation of NF-kappaB. Inhibition of ERK did not affect interleukin-1beta-induced I-kappaBalpha phosphorylation and degradation but attenuated I-kappaBbeta degradation. Thus, although NF-kappaB activation was essential for interleukin-1beta induction of each of the proteins studied, gene expression was differentially regulated by ERK and by the duration of NF-kappaB activation. These results reveal a novel functional role for ERK as an important temporal regulator of NF-kappaB activation and NF-kappaB-dependent gene expression.

Topics: Animals; Blotting, Western; Butadienes; Cells, Cultured; Cyclooxygenase 2; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Enzyme Inhibitors; Flavonoids; Gene Expression Regulation; Genes, Dominant; I-kappa B Proteins; Interleukin-1; Isoenzymes; Leupeptins; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Mutation; Myocytes, Smooth Muscle; NF-kappa B; NF-KappaB Inhibitor alpha; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Nitriles; Phosphorylation; Prostaglandin-Endoperoxide Synthases; Proteasome Endopeptidase Complex; Rats; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Superoxide Dismutase; Time Factors; Vascular Cell Adhesion Molecule-1

The serine/threonine kinase Raf-1 is a major regulator of the mitogen activated protein kinase (MAPK) pathway, which has been associated with the progression of prostate cancer to the more advanced and androgen-independent disease. Cdc25A phosphatase has been implicated in the regulation of Raf-1 and the MAPK pathway.. We used a novel and potent Cdc25A inhibitor, 2,3-bis-[2-hydroxyethylsulfonyl]-[1,4] naphthoquinone (NSC 95397), and its congener (2-mercaptoethanol)-3-methyl-1, 4-naphthoquinone (NSC 672121) to study the role of Cdc25A on the MAPK pathway in human prostate cancer cells.. We found Raf-1 physically interacted with Cdc25A in PC-3 and LNCap cells and inhibitors of Cdc25A induced both extracellular signal-regulated kinase (Erk) activation and Raf-1 tyrosine phosphorylation. NSC 95397 attenuated Cdc25A and Raf-1 interactions due to accelerated degradation of Cdc25A, which was mediated by proteasome degradation. The MAPK kinase (MEK) inhibitor U0126 completely inhibited Erk activation by NSC 95397 and NSC 672121.. These results indicate Cdc25A phosphatase regulates Raf-1/MEK/Erk kinase activation in human prostate cancer cells.

Topics: Blotting, Western; Butadienes; cdc25 Phosphatases; Cell Line, Tumor; Enzyme Inhibitors; Humans; Leupeptins; Male; MAP Kinase Signaling System; Mitogen-Activated Protein Kinases; Naphthoquinones; Nitriles; Phosphorylation; Prostatic Neoplasms; Proto-Oncogene Proteins c-raf

Interleukin-6 (IL-6) is a multifunctional cytokine that plays an important role in inflammatory reactions. We have addressed the possible regulation of IL-6 expression by the ubiquitin-protease system in human umbilical vein endothelial cells. Cultured endothelial cells were treated with MG-132, a protease inhibitor, and the levels of IL-6 mRNA and protein were measured by reverse transcription-PCR and ELISA. MG-132 increased the expression of IL-6 mRNA and protein;and this effect was abolished by the pretreatment of the cells with U0126, an inhibitor of MAP or ERK kinases (MEK 1/2). MG-132 treatment was also found to enhance the level of phosphorylated MEK 1/2. Treatment of the cells with actinomycin D inhibited IL-6 expression in response to MG-132, suggesting the transcriptional upregulation of IL-6 under proteasomal inhibition. We conclude that a protease inhibitor MG-132 upregulates IL-6 expression in vascular endothelial cells, at least in part, through the activation of MEK 1/2.

Topics: Butadienes; Cells, Cultured; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dactinomycin; Endothelium, Vascular; Humans; I-kappa B Proteins; Interleukin-6; Leupeptins; MAP Kinase Kinase Kinase 1; Mitogen-Activated Protein Kinase Kinases; Multienzyme Complexes; Nitriles; Proteasome Endopeptidase Complex; Protein Serine-Threonine Kinases; Umbilical Veins