Role of mitogen-activated protein kinases and tyrosine kinases on IL-1-Induced NF-kappaB activation and iNOS expression in bovine articular chondrocytes.

Nitric oxide (NO), produced by the inducible isoform of the NO synthase (iNOS), plays an important role in the pathophysiology of arthritic diseases. This work aimed at elucidating the role of the mitogen-activated protein kinases (MAPK), p38MAPK and p42/44MAPK, and of protein tyrosine kinases (PTK) on interleukin-1beta (IL-1)-induced iNOS expression in bovine articular chondrocytes. The specific inhibitor of the p38MAPK, SB 203580, effectively inhibited IL-1-induced iNOS mRNA and protein synthesis, as well as NO production, while the specific inhibitor of the p42/44MAPK, PD 98059, had no effect. These responses to IL-1 were also inhibited by treatment of the cells with the tyrosine kinase inhibitors, genistein and tyrphostin B42, which also prevented IL-1-induced NF-kappaB activation. The p38MAPK inhibitor, SB 203580, had no effect on IL-1-induced NF-kappaB activation. Finally, the p42/44MAPK inhibitor, PD 98059, prevented IL-1-induced AP-1 activation in a concentration that did not inhibit iNOS expression. In conclusion, this study shows that (1) PTK are part of the signaling pathway that leads to IL-1-induced NF-kappaB activation and iNOS expression; (2) the p38MAPK cascade is required for IL-1-induced iNOS expression; (3) the p42/44MAPK and AP-1 are not involved in IL-1-induced iNOS expression; and (4) NF-kappaB and the p38MAPK lie on two distinct pathways that seem to be independently required for IL-1-induced iNOS expression. Hence, inhibition of any of these two signaling cascades is sufficient to prevent iNOS expression and the subsequent production of NO in articular chondrocytes.     

P38 MAPK, but not p42/p44 MAPK mediated inducible nitric oxide synthase expression in C6 glioma cells

Recently mitogen-activated protein kinase (MAPK) has been reported to play an important role in phosphorylation cascades governing cell growth and protein expression in numerous cell types. In order to explore the signaling mechanism by which inducible nitric oxide synthase (iNOS) is regulated in C6 glioma cells, we investigated the role of MAPK in iNOS expression by using the specific MAPK inhibitors. First the induction of nitric oxide by lipopolysaccharide (LPS), tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), alone or their combination, was studied in C6 glioma cells. Administration of LPS, TNFalpha, or IFNgamma alone had no detectable stimulatory effect on the production of nitric oxide (NO). However, combination of the three factors elicited a significant elevation of NO level in C6 cell culture medium. Subsequently pretreatment of C6 cells with a specific inhibitor of p38 MAPK, SB202190, resulted in a dose-dependent inhibition of NO production and iNOS expression, but PD98059, an inhibitor of p42/p44 MAPK activation, had no effect. These data suggest that p38 MAPK mediates iNOS expression in C6 glioma cells, but p42/p44 MAPK is not involved in this process.     

Tumor necrosis factor alpha-induced activation of downstream NF-kappaB site of the promoter mediates epithelial ICAM-1 expression and monocyte adhesion. Involvement of PKCalpha, tyrosine kinase, and IKK2, but not MAPKs, pathway

TNF-alpha induced an increase in intercellular adhesion molecule-1 (ICAM-1) expression in human A549 epithelial cells and immunofluorescence staining confirmed this result. The enhanced ICAM-1 expression was shown to increase the adhesion of U937 cells to A549 cells. Tyrosine kinase inhibitors (genistein or tyrphostin 23) or phosphatidylcholine-specific phospholipase C (PC-PLC) inhibitor (D 609) attenuated TNF-alpha-induced ICAM-1 expression. TNF-alpha produced an increase in protein kinase C (PKC) activity and this effect was inhibited by D 609. PKC inhibitors (staurosporine, Ro 31-8220, calphostin C, or Go 6976) also inhibited TNF-alpha-induced response. 12-O-Tetradecanoylphorbol-13-acetate (TPA), a PKC activator, stimulated ICAM-1 expression, this effect was inhibited by genistein or tyrphostin 23. Treatment of cells with TNF-alpha resulted in stimulation of p44/42 MAPK, p38, and JNK. However, TNF-alpha-induced ICAM-1 expression was not affected by either MEK inhibitor, PD 98059, or p38 inhibitor, SB 203580. A cell-permeable ceramide analog, C(2) ceramide, also stimulated the activation of these three MAPKs, but had no effect on ICAM-1 expression. NF-kappaB DNA-protein binding and ICAM-1 promoter activity were enhanced by TNF-alpha and these effects were inhibited by D 609, calphostin C, or tyrphostin 23, but not by PD 98059 or SB 203580. TPA also stimulated NF-kappaB DNA-protein binding and ICAM-1 promoter activity, these effects being inhibited by genistein or tyrphostin 23. TNF-alpha- or TPA-induced ICAM-1 promoter activity was inhibited by dominant negative PKCalpha or IKK2, but not IKK1 mutant. IKK activity was stimulated by both TNF-alpha and TPA, and these effects were inhibited by Ro 31-8220 or tyrphostin 23. These data suggest that, in A549 cells, TNF-alpha activates PC-PLC to induce activation of PKCalpha and protein tyrosine kinase, resulting in the stimulation of IKK2, and NF-kappaB in the ICAM-1 promoter, then initiation of ICAM-1 expression and neutrophil adhesion. However, activation of p44/42 MAPK, p38, and JNK is not involved in this event.     

Mechanism of isoproterenol-induced RGS2 up-regulation in astrocytes

Regulators of G protein signaling (RGSs) are inducibly expressed in response to various stimuli and the up-regulation of RGSs leads to significant decreases in GPCR responsiveness. Isoproterenol, an adrenergic receptor agonist, stimulated RGS2 mRNA in C6 rat astrocytoma cells. The up-regulation of RGS2 mRNA was abrogated by genistein, a protein tyrosine kinase inhibitor (PTK), and by broad-spectrum protein kinase C (PKC) inhibitors (staurosporine and GF109203X). alpha-Adrenergic antagonist (prazocin), beta-adrenergic antagonist (prazocin), and pertussis toxin only partially blocked the RGS2 up-regulation, suggesting that the RGS2 up-regulation is concomitantly mediated by Galphai, Galphas, and Galphaq. It is interesting to note that SB203580, a potent p38 mitogen-activated protein kinase (MAPK) inhibitor, completely inhibited the isoproterenol-mediated RGS2 expression. In addition, isoproterenol also markedly stimulated RGS2 mRNA in rat primary astrocytes, which were sensitive to SB203580 and staurosporine. Therefore, our data suggest that adrenergic receptor-mediated signaling (induced by isoproterenol) may be involved in the regulation of RGS2 expression in astrocytes via activating PTK, PKC, and p38 MAPK.     

Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.     

Induction of autophagy in hepatocellular carcinoma cells by SB203580 requires activation of AMPK and DAPK but not p38 MAPK

SB203580 is a well-known inhibitor of p38 mitogen-activated protein kinase (MAPK). However, it can suppress cell proliferation in a p38 MAPK independent manner. The inhibitory mechanism remains unknown. Here, we showed that SB203580 induced autophagy in human hepatocellular carcinoma (HCC) cells. SB203580 increased GFP-LC3-positive cells with GFP-LC3 dots, induced accumulation of autophagosomes, and elevated the levels of microtubule-associated protein light chain 3 and Beclin 1. It stimulated the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and p53, but inhibited the phosphorylation of death-associated protein kinase (DAPK). Inhibition of AMPK, p53, or DAPK attenuated SB203580-induced autophagy. AMPK activation appeared to predate the DAPK signal. The activation of both AMPK and DAPK prompted the phosphorylation of p53 and enhanced Beclin 1 expression. Neither the downregulation of p38 MAPK by its siRNA or chemical inhibitor nor the upregulation of p38 MAPK by p38 MAPK DNA transfection affected B203580-induced autophagy. Collectively, the findings demonstrate a novel function of SB203580 to induce autophagy via activating AMPK and DAPK but independent of p38 MAPK. The induction of autophagy can thus account for the antiproliferative effect of SB203580 in HCC cells.     

Insulin inhibits hepatocyte iNOS expression induced by cytokines by an Akt-dependent mechanism

Hepatocyte inducible nitric oxide synthese (iNOS) expression is a tightly controlled pathway that mediates hepatic inflammation and hepatocyte injury in a variety of disease states. We have shown that cyclic adenosine monophosphate (cAMP) regulates cytokine-induced hepatocyte iNOS expression through mechanisms that involve protein kinase B/Akt. We hypothesized that insulin, which activates Akt signaling in hepatocytes, as well as signaling through p38 and MAPK p42/p44, would regulate iNOS expression during inflammation. In primary rat hepatocytes, insulin inhibited cytokine-stimulated nitrite accumulation and iNOS expression in a dose-dependent manner. Inhibition of MAPK p42/p44 with PD98059 had no effect on iNOS activation, whereas SB203580 to block p38 reversed insulin's inhibitory effect. However, insulin did not increase p38 activation and inhibition of p38 signaling with a dominant negative p38 plasmid had no effect on cytokine- or insulin-mediated effects on iNOS. We found that SB203580 blocked insulin-induced Akt activation. Inhibition of Akt signaling with LY294002 or a dominant negative Akt plasmid increased cytokine-stimulated nitrite production and iNOS protein expression and blocked the inhibitory effects of insulin. NF-κB induces iNOS expression and can be regulated by Akt, but insulin had no effect on cytokine-mediated IκBα levels or NF-κB p65 translocation. Our data demonstrate that insulin inhibits cytokine-stimulated hepatocyte iNOS expression and does so through effects on Akt-mediated signaling.     

Involvement of p38 MAPK and ERK/MAPK pathways in staurosporine-induced production of macrophage inflammatory protein-2 in rat peritoneal neutrophils.

Stimulation of rat peritoneal neutrophils with staurosporine (64 nM) induced production of macrophage inflammatory protein-2 (MIP-2) and phosphorylation of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase/MAP kinase (ERK/MAPK). The staurosporine-induced MIP-2 production at 4 h was inhibited by the highly specific p38 MAPK inhibitor SB 203580 and the MAPK/ERK kinase (MEK-1) inhibitor PD 98059 in a concentration-dependent manner. By treatment with SB 203580 (1 microM) or PD 98059 (50 microM), the staurosporine-induced increase in the levels of mRNA for MIP-2 was only partially lowered, although the staurosporine-induced MIP-2 production was completely inhibited. Consistent with the inhibition by the protein synthesis inhibitor cycloheximide, SB 203580 and PD 98059 inhibited MIP-2 production at 4 h either when added simultaneously with staurosporine or 2 h after stimulation with staurosporine. In contrast, the DNA-dependent RNA polymerase inhibitor actinomycin D did not inhibit MIP-2 production at 4 h when it was added 2 h after staurosporine stimulation. Dot blot analysis demonstrated that treatment with SB 203580 or PD 98059 down-regulates the stability of MIP-2 mRNA. These results suggested that p38 MAPK and ERK/MAPK pathways are involved in translation of MIP-2 mRNA to protein and stabilization of MIP-2 mRNA.     

Penehyclidine hydrochloride attenuates LPS-induced iNOS production by inhibiting p38 MAPK activation in endothelial cells

The aim of this study was to investigate the inhibitory effect of penehyclidine hydrochloride (PHC) on lipopolysaccharide (LPS)-induced nitric oxide (NO) and inducible nitric oxide synthase (iNOS) production in human endothelial cell. Cultured endothelial cells were pretreated with PHC, followed by LPS treatment. NO activity were determined. iNOS expression and p38 mitogen-activated protein kinase (p38 MAPK) protein expression were measured by Western blot analysis. LPS treatment significantly induced p38 MAPK activation, iNOS expression, and NO production, which could be attenuated by 2 μg/ml PHC pretreatment. Furthermore, our study showed LPS-induced NO production and iNOS expression were suppressed by p38 MAPK inhibitor SB203580 pretreatment. We concluded that PHC attenuates NO production and iNOS expression by suppressing the activation of p38 MAPK pathway, thereby implicating a mechanism by which PHC may exert its protective effects against LPS-induced endothelial cell injury.     

Activation of p90RSK and cAMP response element binding protein in stimulated neutrophils: novel effects of the pyridinyl imidazole SB 203580 on activation of the extracellular signal-regulated kinase cascade.

Neutrophils stimulated with the chemoattractant FMLP or the phorbol ester PMA are known to exhibit activation of a 90-kDa renaturable protein kinase. Activation of this kinase was maximal at approximately 1-3 min after cell stimulation and the time course for activation was similar to that of the extracellular-regulated kinases (ERKs) and p38-mitogen activated protein kinase (p38MAPK). Compounds that block activation of ERK-1/2 (PD 98059) or that inhibit the activity of p38MAPK (SB 203580) blocked activation of this 90-kDa kinase. SB 203580 is a highly selective inhibitor of p38MAPK in vitro and is under intense study as a lead compound for developing novel anti-inflammatory agents. However, we demonstrate that SB 203580 at concentrations >/=10 microM can also inhibit activation of ERK-1/2 in neutrophils. An Ab to the protein kinase p90RSK2 (also referred to as MAPKAP-K1b, or p90rsk) immunoprecipitated the active 90-kDa kinase from lysates of stimulated neutrophils. No activity was observed for this enzyme in immunoprecipitates obtained from unstimulated cells, and the amounts of activity were markedly reduced if the cells were treated with PD 98059 or SB 203580 before stimulation. Neutrophils stimulated with FMLP exhibited phosphorylation of the cAMP response element binding protein (CREB), and this reaction was inhibited by SB 203580 and PD 98059. These data establish that the renaturable 90-kDa protein kinase is p90RSK2 and that CREB may be a substrate for this enzyme in these cells. Novel effects of compound SB 203580 on stimulated neutrophils are also described.     

Effect of contraction on mitogen-activated protein kinase signal transduction in skeletal muscle. Involvement Of the mitogen- and stress-activated protein kinase 1

Growing evidence suggests that activation of mitogen-activated protein kinase (MAPK) signal transduction mediates changes in muscle gene expression in response to exercise. Nevertheless, little is known about upstream or downstream regulation of MAPK in response to muscle contraction. Here we show that ex vivo muscle contraction stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2), and p38(MAPK) phosphorylation. Phosphorylation of ERK1/2 or p38(MAPK) was unaffected by protein kinase C inhibition (GF109203X), suggesting that protein kinase C is not involved in mediating contraction-induced MAPK signaling. Contraction-stimulated phosphorylation of ERK1/2 and p38(MAPK) was completely inhibited by pretreatment with PD98059 (MAPK kinase inhibitor) and SB203580 (p38(MAPK) inhibitor), respectively. Muscle contraction also activated MAPK downstream targets p90 ribosomal S6 kinase (p90(Rsk)), MAPK-activated protein kinase 2 (MAPKAP-K2), and mitogen- and stress-activated protein kinase 1 (MSK1). Use of PD98059 or SB203580 revealed that stimulation of p90(Rsk) and MAPKAP-K2 most closely reflects ERK and p38(MAPK) stimulation, respectively. Stimulation of MSK1 in contracting skeletal muscle required the activation of both ERK and p38(MAPK). These data demonstrate that muscle contraction, separate from systemic influence, activates MAPK signaling. Furthermore, we are the first to show that contractile activity stimulates MAPKAP-K2 and MSK1.     

Antioxidant effect of quercetin against acute spinal cord injury in rats and its correlation with the p38MAPK/iNOS signaling pathway

AimsThe present study aimed to investigate the correlation between quercetin (Que) and the p38 mitogen-activated protein kinase (p38MAPK)/inducible nitric oxide synthase (iNOS) signaling pathway and to explore its regulating effect on secondary oxidative stress following acute spinal cord injury (SCI), so as to elucidate the protective effects and mechanism associated with Que treatment during acute SCI.Main methodsSprague–Dawley rats were randomly divided into sham-surgery, SCI, Que, methylprednisolone (MP), and specific p38MAPK inhibitor SB203580 treatment groups. Acute SCI models were established in rats by a modified Allen's method. Real-time PCR analysis, western blot assay and immunohistochemistry for molecular changes in the p38MAPK/iNOS signaling pathway, determination of malondialdehyde (MDA) content and superoxide dismutase (SOD) activity, reflecting the levels of secondary oxidative stress, and functional or behavioral data, reflecting changes induced by Que and control treatments post-SCI were performed.Key findingsQue significantly increased Basso, Beattie and Bresnahan scores and inclined plane test scores in SCI rats similar to the positive control drug, MP. Que significantly inhibited increases in phosphorylated-p38MAPK (p-p38MAPK) and iNOS expression and reduced the rate of iNOS-positive cells in rats with SCI, similar to the effects of SB203580. In addition, both Que and SB203580 reduced MDA content and enhanced SOD activity in SCI rats, with Que effects being stronger.SignificanceThese experimental findings indicate that in SCI rats, Que has protective effects on the spinal cord by the potential mechanism of inhibiting the activation of p38MAPK/iNOS signaling pathway and thus regulating secondary oxidative stress.     

Multiple signaling cascades are differentially involved in gene induction by double stranded RNA in interferon-alpha-primed cells.

Priming with interfon (IFN)alpha enhanced the ability of the synthetic double-stranded RNA polyriboinosinic acid: polyribocytidilic acid (pI:C), but not interleukin-1 beta, to activate both p38 mitogen-activated kinase (MAPK) and extracellular signal-regulated kinase (ERK) signaling cascades. Activation by pI:C in IFN alpha-primed cells was delayed compared to activation with interleukin-1 beta, and this delay was followed by high, sustained activation of p38 MAPK and a modest elevation of ERK activation. Pharmacologic inhibition of either the ERK or the p38 MAPK pathway, using U0126 and SB203580, respectively, reduced interleukin-6 protein induction by at least 70%, and combined inhibition of both pathways fully blocked interleukin-6 protein expression and reduced interleukin-6 mRNA induction by more than 80%. In contrast, induction of double-stranded RNA-activated protein kinase (PKR) mRNA and protein by IFN alpha and/or pI:C was minimally affected by either inhibitor. Induction of interferon-regulatory factor-1 (IRF-1) by pI:C in IFN alpha primed cells was profoundly inhibited by U0126 but not by SB203580. Thus, IFN alpha priming enhances activation of p38 MAPK and ERK pathways by pI:C but not by interleukin-1 beta, thereby enhancing the expression of some, but not all, genes that are induced by pI:C.     

Lipoteichoic acid-induced nitric oxide synthase expression in RAW 264.7 macrophages is mediated by cyclooxygenase-2, prostaglandin E2, protein kinase A, p38 MAPK, and nuclear factor-kappaB pathways

We recently reported that lipoteichoic acid (LTA), a cell wall component of the gram-positive bacterium Staphylococcus aureus, stimulated inducible nitric oxide synthase (iNOS) expression, nitric oxide (NO) release, and cyclooxygenase-2 (COX-2) expression in RAW 264.7 macrophages. This study was carried out to further investigate the roles of COX-2 and prostaglandin E2 (PGE2) in LTA-induced iNOS expression and NO release in RAW 264.7 macrophages. Treatment of RAW 264.7 macrophages with LTA caused a time-dependent increase in PGE2 release. LTA-induced iNOS expression and NO release were inhibited by a non-selective COX inhibitor (indomethacin), a selective COX-2 inhibitor (NS-398), an adenylyl cyclase (AC) inhibitor (dideoxyadenosine, DDA), and a protein kinase A (PKA) inhibitor (KT-5720). Furthermore, both PGE2 and the direct PKA activator, dibutyryl-cAMP, also induced iNOS expression in a concentration-dependent manner. Stimulation of RAW 264.7 macrophages with LTA, PGE2, and dibutyryl-cAMP all caused p38 MAPK activation in a time-dependent manner. LTA-mediated p38 MAPK activation was inhibited by indomethacin, NS-398, and SB 203580, but not by PD 98059. The PGE2-mediated p38 MAPK activation was inhibited by DDA, KT-5720, and SB 203580, but not by PD 98059. LTA caused time-dependent activation of the nuclear factor-kappaB (NF-kappaB)-specific DNA-protein complex formation. The LTA-induced increase in kappaB-luciferase activity was inhibited by indomethacin, NS-398, KT-5720, and a dominant negative mutant of p38 alphaMAPK (p38 alphaMAPK DN). These results suggest that LTA-induced iNOS expression and NO release involve COX-2-generated PGE2 production, and AC, PKA, p38 MAPK, and NF-kappaB activation in RAW 264.7 macrophages.     

Pioglitazone inhibits advanced glycation end product-induced matrix metalloproteinases and apoptosis by suppressing the activation of MAPK and NF-κB

Apoptosis and degeneration coming mainly from chondrocytes are important mechanisms in the onset and progression of osteoarthritis. Specifically, advanced glycation end products (AGEs) play an important role in the pathogenesis of osteoarthritis. Pioglitazone, a peroxisome proliferator-activated receptor γ (PPARγ) agonist has a protective effect on cartilage. This study aims to evaluate the effect of pioglitazone on AGEs-induced chondrocyte apoptosis and degeneration and their underlying mechanism. The in vitro study shows that AGEs induce cleavage of caspase-3 and PARP, up-regulate MMP-13 expression, enhance chondrocyte apoptosis and down-regulate PPARγ expression in human primary chondrocytes, which is reversed by pioglitazone. Furthermore, AGEs activate phosphorylation of Erk, JNK, and p38, and pioglitazone reverses AGEs-induced phosphorylation of Erk and p38. AGEs-induced degradation of IκBα and translocation of nuclear NF-κB p65 is reversed by pioglitazone. Pretreatment of chondrocytes with SB202190 (p38 inhibitor), SP600125 (JNK inhibitor) and BAY-11-7082 (NF-κB inhibitor) inhibit AGEs-induced apoptosis and degeneration. In vivo experiments suggest that pioglitazone reverses AGEs-induced cartilage degeneration and apoptosis in a mouse model, as demonstrated by HE and Safranin O staining, immunohistochemical analyses of Type II collagen (Col II), metalloproteinases (MMPs) and caspase-3. These findings suggest that pioglitazone, a PPARγ agonist, inhibits AGEs-induced chondrocytes apoptosis and degeneration via suppressing the activation of MAPK and NF-κB.