The mitogen-activated protein kinase p38 is necesssary for interleukin 1beta-induced monocyte chemoattractant protein 1 expression by human mesangial cells

Mitogen-activated protein (MAP) kinases have been suggested as potential mediators for interleukin 1beta (IL-1beta)-induced gene activation. This study investigated the role of the MAP kinases p38 and ERK2 in IL-1beta-mediated expression of the chemokine MCP-1 by human mesangial cells. Phosphorylation of p38 kinase, which is necessary for activation, increased significantly after IL-1beta treatment. p38 kinase immunoprecipitated from IL-1beta-treated cells phosphorylated target substrates to a greater extent than p38 kinase from controls. SB 203580, a selective p38 kinase inhibitor, was used to examine the role of p38 kinase in MCP-1 expression. SB 203580 decreased IL-1beta-induced MCP-1 mRNA and protein levels, but did not affect MCP-1 mRNA stability. Because NF-kappaB is necessary for MCP-1 gene expression, the effect of p38 kinase inhibition on IL-1beta induction of NF-kappaB was measured. SB 203580 (up to 25 microM) had no effect on IL-1beta-induced NF-kappaB nuclear translocation or DNA binding activity. Our previous work showed that IL-1beta also activates the MAP kinase ERK2 in human mesangial cells. PD 098059, a selective inhibitor of the ERK activating kinase MEK1, had no effect on IL-1beta-induced MCP-1 mRNA or protein levels, or on IL-1beta activation of NF-kappaB. These data indicate that p38 kinase is necessary for the induction of MCP-1 expression by IL-1beta, but is not involved at the level of cytoplasmic activation of NF-kappaB. In contrast, ERK2 does not mediate IL-1beta induced MCP-1 gene expression.     

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.     

BpV (phen) induces apoptosis of RINm5F cells by modulation of MAPKs and MKP-1

We investigated the mechanism of toxicity of peroxovanadium complex bpV (phen) in RINm5F cells. Treatment with bpV (phen) provoked cell death, predominantly by apoptosis. This compound induced strong and sustained JNK and p38 MAPK activation. However, ERK phosphorylation was not affected. The level of expression of MAPK phosphatase MKP-1 was suppressed after bpV (phen) treatment. In addition, this compound did not stimulate proteolytic processing of procaspase-3, suggesting that caspase-3 is not activated during the course of bpV (phen)-induced apoptosis. A correlative inhibition of JNK activation by immunosuppressive drug FK 506 induced ERK activation and MKP-1 expression, and suppressed RINm5F cell death. A specific p38 inhibitor SB 203580 also stimulated ERK activation and cell survival. Furthermore, simultaneous pretreatment with both FK 506 and SB 203580 almost completely abolished cell death. Thus, our results suggest that stress kinases and MKP-1 have a role in bpV (phen)-induced apoptosis of RINm5F cells.     

Amomum xanthoides extract prevents cytokine-induced cell death of RINm5F cells through the inhibition of nitric oxide formation

We previously showed that Amomum xanthoides extract prevented alloxan-induced diabetes through the suppression of NF-kappaB activation. In this study, the preventive effects of A. xanthoides extract on cytokine-induced beta-cell destruction were examined. Cytokines produced by immune cells infiltrating pancreatic islets are important mediators of beta-cell destruction in insulin-dependent diabetes mellitus. A. xanthoides extract completely protected interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma)-mediated cytotoxicity in rat insulinoma cell line (RINm5F). Incubation with A. xanthoides extract resulted in a significant reduction in IL-1beta and IFN-gamma-induced nitric oxide (NO) production, a finding that correlated well with reduced levels of the inducible form of NO synthase (iNOS) mRNA and protein. The molecular mechanism by which A. xanthoides extract inhibited iNOS gene expression appeared to involve the inhibition of NF-kappaB activation. Our results revealed the possible therapeutic value of A. xanthoides extract for the prevention of diabetes mellitus progression.     

The neuroprotective agents chlomethiazole and SB203580 inhibit IL-1beta signalling but not its biosynthesis in rat cortical glial cells

Chlomethiazole and pyridinyl imidazole compounds, exemplified by SB203580, are structurally distinct p38 mitogen-activated protein kinase inhibitors with neuroprotective properties in models of cerebral ischaemia. We have examined their effects in interleukin-1beta (IL-1beta) synthesis, release and signalling in rat cortical glial cells, given the important role of IL-1beta in cerebral ischaemia. We analysed (i) IL-1beta mRNA expression by northern blot, (ii) IL-1beta protein precursor levels within the cells by western blot, and (iii) the levels of the mature IL-1beta protein secreted into the medium by enzyme-linked immunosorbent assay (ELISA) after treatment of rat cortical glial cells with lipopolysaccharide. While the induction of IL-1beta expression by lipopolysaccharide or by IL-1beta itself was very sensitive to nuclear factor kappa B (NF-kappaB) inhibitors, chlomethiazole or SB203580 were nearly without effect, indicating a differential regulation as compared to peripheral cells, e.g. monocytes. In contrast, chlomethiazole and SB203580 potently inhibited the IL-1beta-induced expression of c-fos and inducible nitric oxide synthase, as monitored by northern blot and quantitative RT-PCR, respectively. Because IL-1beta-induced expression of c-fos and inducible nitric oxide synthase is believed to directly contribute to the pathology of cerebral ischaemic injury, the results suggest a direct mechanism for the neuroprotective effects of chlomethiazole and SB203580, and further establish the anti-inflammatory properties of chlomethiazole.     

Both p38alpha(MAPK) and JNK/SAPK pathways are important for induction of nitric-oxide synthase by interleukin-1beta in rat glomerular mesangial cells

Interleukin 1beta (IL-1beta) induces expression of the inducible nitric-oxide synthase (iNOS) with concomitant release of nitric oxide (NO) from glomerular mesangial cells. These events are preceded by activation of the c-Jun NH(2)-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38(MAPK). Our current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 SAPKbeta/JNK2 significantly reduces the iNOS protein expression and NO production induced by IL-1beta. Similarly, overexpression of the kinase-dead mutant form of p38alpha(MAPK) also inhibits IL-1beta-induced iNOS expression and NO production. In previous studies we demonstrated that IL-1beta can activate MKK4/SEK1, MKK3, and MKK6 in renal mesangial cells; therefore, we examined the role of these MAPK kinases in the modulation of iNOS induced by IL-1beta. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1beta-induced iNOS expression and NO production with inhibition of both SAPK/JNK and p38(MAPK) phosphorylation. Overexpression of the kinase-dead mutant form of MKK3 or MKK6 demonstrated that either of these two mutant kinase inhibited IL-1beta-induced p38(MAPK) (but not JNK/SAPK) phosphorylation and iNOS expression. Interestingly overexpression of wild type MKK3/6 was associated with phosphorylation of p38(MAPK); however, in the absence of IL-1beta, iNOS expression was not enhanced. This study suggests that the activation of both SAPK/JNK and p38alpha(MAPK) signaling cascades are necessary for the IL-1beta-induced expression of iNOS and production of NO in renal mesangial cells.     

HO-1 and JAK-2/STAT-1 signals are involved in preferential inhibition of iNOS over COX-2 gene expression by newly synthesized tetrahydroisoquinoline alkaloid, CKD712, in cells activated with lipopolysacchride

We found that CKD712, an S enantiomer of YS49, strongly inhibited inducible nitric oxide synthase (iNOS) and NO induction but showed a weak inhibitory effect on cyclooxygenase-2 (COX-2) and PGE(2) induction in LPS-stimulated RAW 264.7 cells. We, therefore, investigated the molecular mechanism(s) responsible for this by using CKD712 in LPS-activated RAW264.7 cells. Treatment with either SP600125, a specific JNK inhibitor or TPCK, a NF-kappaB inhibitor, but neither ERK inhibitor PD98059 nor p38 inhibitor SB203580, significantly inhibited LPS-mediated iNOS and COX-2 induction. CKD712 inhibited NF-kappaB (p65) activity and translocation but failed to prevent JNK activation. However, AG490, a specific JAK-2/STAT-1 inhibitor, efficiently prevented LPS-mediated iNOS induction but not the induction of COX-2, and CKD712 completely blocked STAT-1 phosphorylation by LPS, suggesting that the NF-kappaB and JAK-2/STAT-1 pathways but not the JNK pathway are important for CKD712 action. Interestingly, CKD712 induced heme oxygenase 1 (HO-1) gene expression in LPS-treated cells. LPS-induced NF-kappaB and STAT-1 activation was partially prevented by HO-1 overexpression. Furthermore, HO-1 siRNA partly reversed not only the LPS-induced NF-kappaB activation and STAT-1 phosphorylation but also inhibition of these actions by CKD 712. Additionally, silencing HO-1 by siRNA prevented CKD712 from inhibiting iNOS expression but not COX-2. When examined plasma NO and PGE(2) levels and iNOS and COX-2 protein levels in lung tissues of mice injected with LPS (10 mg/kg), pretreatment with CKD712 greatly prevented NO and iNOS induction in a dose-dependent manner and slightly affected PGE(2) and COX-2 production as expected. Taken together, we conclude that inhibition of JAK-2/STAT-1 pathways by CKD 712 is critical for the differential inhibition of iNOS and COX-2 by LPS in vitro and in vivo where HO-1 induction also contributes to this by partially modulating JAK-2/STAT-1 pathways.     

Cytokine Induction of Inducible Nitric Oxide Synthase in an Oligodendrocyte Cell Line

Abstract : The induction of inducible nitric oxide synthase (iNOS) by proinflammatory cytokines was studied in an oligodendrocyte progenitor cell line in relation to mitogen-activated protein kinase (MAPK) activation and cytokine-mediated cytotoxicity. When introduced individually to cultures of CG4 cells, the cytokines, i.e., tumor necrosis factor-α (TNFα), interleukin-1 (IL-1), and interferon-γ (IFNγ), had either minimal (TNFα) or no (IL-1 and IFNγ) detectable stimulatory effect on the production of nitric oxide. However, combinations of these factors, in particular, TNFα plus IFNγ, elicited a strong enhancement of nitric oxide synthesis and, as revealed by western blot and RT-PCR analysis, the expression of iNOS. TNFα and IL-1 were able to activate p38 MAPK in a time- and dose-dependent manner and together showed a combinatorial effect. In contrast, IFNγ neither activated on its own nor enhanced the activation of p38 MAPK in response to TNFα and IL-1. However, a specific inhibitor of p38 MAPK, i.e., SB203580, inhibited the induction of iNOS in cytokine combination-treated cells in a dose-dependent manner, thereby suggesting a role for the MAPK cascade in regulating the induction of iNOS gene expression in cytokine-treated cells. Blocking of nitric oxide production by an inhibitor of iNOS, i.e., nitro-L-arginine methyl ester, had a minimal protective effect against cytokine-mediated cytotoxicity that occurred before the elevation of nitric oxide levels, thereby indicating temporal and functional dissociation of nitric oxide production from cell killing.     

Amylin-induced cytotoxicity is associated with activation of caspase-3 and MAP kinases

Nanomolar concentrations of human amylin promote death of RINm5F cells in a time- and concentrationdependent manner. Morphological changes of chromatin integrity suggest that cells are predominantly undergoing apoptosis. Human amylin induces significant activation of caspase-3 and strong and sustained phosphorylation of stress-activated protein kinases, c-Jun N-terminal kinase (JNK) and p38, that precedes cell death. Extracellular signal-regulated kinase (ERK) activation was not concomitant with JNK and/or p38 activation. Activation of caspase-3 and mitogen-activated protein kinases (MAPKs) was detected by Western blot analysis. Addition of the MEK1 inhibitor PD 98059 had no effect on amylin-induced apoptosis, suggesting that ERK activation does not play a role in this apoptotic scenario. A correlative inhibition of JNK activation by the immunosuppressive drug FK506, as well as a selective inhibition of p38 MAPK activation by SB 203580, significantly suppressed procaspase-3 processing and the extent of amylin-induced cell death. Moreover, simultaneous pretreatment with both FK506 and SB 203580, or with the caspase-3 inhibitor Ac-DEVD-CHO alone, almost completely abolished procaspase-3 processing and cell death. Thus, our results suggest that amylin-induced apoptosis proceeds through sustained activation of JNK and p38 MAPK followed by caspase-3 activation.     

Angiotensin II differentially regulates interleukin-1-beta-inducible NO synthase (iNOS) and vascular cell adhesion molecule-1 (VCAM-1) expression: role of p38 MAPK

Angiotensin II is implicated in pathophysiological processes associated with vascular injury and repair, which include regulating the expression of numerous NF-kappaB-dependent genes. The present study examined the effect of angiotensin II on interleukin-1beta-induced NF-kappaB activation and the subsequent expression of inducible NO synthase (iNOS) and vascular cell adhesion molecule-1 (VCAM-1) in cultured rat vascular smooth muscle cells. Neither NF-kappaB activation nor iNOS or VCAM-1 expression was induced in cells treated with angiotensin II alone. However, when added together with interleukin-1beta, angiotensin II, through activation of the AT(1) receptor, inhibited iNOS expression and enhanced VCAM-1 expression induced by the cytokine. The inhibitory effect of angiotensin II on iNOS expression was associated with a down-regulation of the sustained activation of extracellular signal-regulated kinase (ERK) and NF-kappaB by interleukin-1beta, whereas the effect on VCAM-1 was independent of ERK activation. The effect of angiotensin II on iNOS was abolished by inhibition of p38 mitogen-activated protein kinase (MAPK) with SB203580, but not by inhibition of PI3 kinase with wortmannin or stress-activated protein kinase/c-Jun NH(2)-terminal kinase (JNK) with JNK inhibitor II. Thus, angiotensin II, by a mechanism that requires the participation of p38 MAPK, differentially regulates the expression of NF-kappaB-dependent genes in response to interleukin-1beta stimulation by controlling the duration of activation of ERK and NF-kappaB.     

Novel role of IL-6/SIL-6R signaling in the expression of inducible nitric oxide synthase (iNOS) in murine B16, metastatic melanoma clone F10.9, cells

Inducible nitric oxide synthase (iNOS) has been shown to be frequently expressed in melanomas; up-regulation of this enzyme is though to be associated with tumor progression. In this study, we investigated whether diverse cytokines such as: IL-6, TNF-alpha, IL-1beta, IFN-gamma and IL6RIL6 (a highly active fusion protein of the soluble form of the IL-6R (sIL-6R) and IL-6) enhance the iNOS gene expression in B16/F10.9 murine metastatic melanoma cells. An increase at iNOS expression and NO production was observed with the co-treatment of IL6RIL6 plus TNF-alpha. Gel shift and reporter gene analyses revealed that IL6RIL6 selectively activated AP-1; while TNF-alpha increased the activities of both NF-kappaB and AP-1. Persistent activation of AP-1 was also seen in cells treated with IL6RIL6 plus TNF-alpha. Stimulation of cells with IL6RIL6/TNF-alpha resulted in the activation of mitogen-activated protein kinases (MAPK) such as c-Jun N-terminal kinase (JNK) and p38, and the abrogation by pretreatment with JNK or p38 MAPK inhibitor. IL6RIL6 or IL6RIL6/TNFalpha-inducible AP-1 binding increase was supershifted by anti-c-Jun or c-Fos antibodies, and the activation of c-Jun and c-Fos was dependent on JNK and p38, respectively. These results suggest that IL-6/sIL-6R/gp130 complex signaling has an unexpected positive effect on iNOS gene expression through JNK/p38 MAPK mediated-AP-1 activation in melanoma cells.     

Thrombin induces nitric-oxide synthase via Galpha12/13-coupled protein kinase C-dependent I-kappaBalpha phosphorylation and JNK-mediated I-kappaBalpha degradation

An imbalance between thrombin and antithrombin III contributed to vascular hyporeactivity in sepsis, which can be attributed to excess NO production by inducible nitric-oxide synthase (iNOS). In view of the importance of the thrombin-activated coagulation pathway and excess NO as the culminating factors in vascular hyporeactivity, this study investigated the effects of thrombin on the induction of iNOS and NO production in macrophages. Thrombin induced iNOS protein in the Raw264.7 cells, which was inhibited by a thrombin inhibitor, LB30057. Thrombin increased NF-kappaB DNA binding, whose band was supershifted with anti-p65 and anti-p50 antibodies. Thrombin elicited the phosphorylation and degradation of I-kappaBalpha prior to the nuclear translocation of p65. The NF-kappaB-mediated iNOS induction was stimulated by the overexpression of activated mutants of Galpha(12/13) (Galpha(12/13)QL). Protein kinase C depletion inhibited I-kappaBalpha degradation, NF-kappaB activation, and iNOS induction by thrombin or the iNOS induction by Galpha(12/13)QL. JNK, p38 kinase, and ERK were all activated by thrombin. JNK inhibition by the stable transfection with a dominant negative mutant of JNK1 (JNK1(-)) completely suppressed the NF-kappaB-mediated iNOS induction by thrombin. Conversely, the inhibition of p38 kinase enhanced the expression of iNOS. In addition, JNK and p38 kinase oppositely controlled the NF-kappaB-mediated iNOS induction by Galpha(12/13)QL. Hence, iNOS induction by thrombin was regulated by the opposed functions of JNK and p38 kinase downstream of Galpha(12/13). In the JNK1(-) cells, thrombin did not increase either the NF-kappaB binding activity or I-kappaBalpha degradation despite I-kappaBalpha phosphorylation. These results demonstrated that thrombin induces iNOS in macrophages via Galpha(12) and Galpha(13), which leads to NF-kappaB activation involving the protein kinase C-dependent phosphorylation of I-kappaBalpha and the JNK-dependent degradation of phosphorylated I-kappaBalpha.