Zhonghong Guan

Induction of cyclooxygenase-2 by the activated MEKK1 --> SEK1/MKK4 --> p38 mitogen-activated protein kinase pathway.

The mitogen-activated protein kinase (MAPK) cascade is believed to function as an important regulator of prostaglandin biosynthesis. Previously we reported that interleukin-1β induces activation of JNK/SAPK and p38 MAPK with concomitant up-regulation of cyclooxygenase (Cox)-2 expression and prostaglandin E2 (PGE2) synthesis. Our experiments demonstrate that overexpression of ΔMEKK1 (a constitutively active truncation mutant of MEKK1 containing the C-terminal 324 amino acids) increases Cox-2 expression and PGE2 production which is completely blocked by SC68376, a pharmacologic inhibitor of p38 MAPK. ΔMEKK1 overexpression results in activation of both c-Jun N-terminal kinases/extracellular signal-regulated kinases (JNK/SAPK) and p38 MAPK. Furthermore, activation of MEKK1 increases SEK1/MKK4 but not MKK3 or MKK6 activity. These findings suggest that MEKK1 → SEK1/MKK4 may function as an upstream kinase capable of activating both p38 MAPK and JNK/SAPK with subsequent induction of Cox-2 expression and PGE2production. We also found that overexpression of the constitutively active form of SEK1 (SEK1-ED) increases both p38 MAPK and JNK/SAPK phosphorylation, and increases PGE2 production and Cox-2 expression. By comparison, overexpression of the dominant negative form of SEK1 (SEK1-AL) decreases the phosphorylation of both p38 MAPK and JNK/SAPK and reduces Cox-2 expression. Together, this data suggests a potential role for the MEKK1 → SEK1/MKK4 → p38 MAPK →→ Cox-2 cascade linking members of the MAPK pathway with prostaglandin biosynthesis.

Interleukin-1β-induced Cyclooxygenase-2 Expression Requires Activation of Both c-Jun NH2-terminal Kinase and p38 MAPK Signal Pathways in Rat Renal Mesangial Cells

The inflammatory cytokine interleukin-1β (IL-1β) induces cyclooxygenase-2 (Cox-2) expression with a concomitant release of prostaglandins from glomerular mesangial cells. We reported previously that IL-1β rapidly activates the c-Jun NH2-terminal/stress-activated protein kinases (JNK/SAPK) and p38 mitogen-activated protein kinase (MAPK) and also induces Cox-2 expression and prostaglandin E2 (PGE2) production. The current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 JNK2/SAPKβ reduces Cox-2 expression and PGE2 production stimulated by IL-1β. Similarly, overexpression of the kinase-dead form of p38 MAPK also inhibits IL-1β-induced Cox-2 expression and PGE2production. These results suggest that activation of both JNK/SAPK and p38 MAPK is required for Cox-2 expression after IL-1β activation. Furthermore, our experiments confirm that IL-1β activates MAP kinase kinase-4 (MKK4)/SEK1, MKK3, and MKK6 in renal mesangial cells. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1β- induced Cox-2 expression with inhibition of both JNK/SAPK and p38 MAPK phosphorylation. Overexpression of the kinase-dead form of MKK3 or MKK6 demonstrated that either of these two mutant kinases inhibited IL-1β-induced p38 MAPK phosphorylation and Cox-2 expression but not JNK/SAPK phosphorylation and activation. This study suggests that the activation of both JNK/SAPK and p38 MAPK signaling cascades is required for IL-1β-induced Cox-2 expression and PGE2synthesis.

p38 mitogen-activated protein kinase down-regulates nitric oxide and up-regulates prostaglandin E2 biosynthesis stimulated by interleukin-1beta.

The inflammatory cytokine interleukin 1β (IL-1β) induces both cyclooxygenase-2 (Cox-2) and the inducible nitric-oxide synthase (iNOS) with increases in the release of prostaglandins (PGs) and nitric oxide (NO) from glomerular mesangial cells. However, the intracellular signaling mechanisms by which IL-1β induces iNOS and Cox-2 expression is obscure. Our current studies demonstrate that IL-1β produces a rapid increase in p38 mitogen-activated protein kinase (MAPK) phosphorylation and activation. Serum starvation and SC68376, a drug which selectively inhibits p38 MAPK in mesangial cells, were used to investigate whether p38 MAPK contributes to the signaling mechanism of IL-1β induction of NO and PG synthesis. Serum starvation and SC68376 selectively inhibited IL-1β-induced activation of p38 MAPK. Both SC68376 and serum starvation enhanced NO biosynthesis by increasing iNOS mRNA expression, protein expression, and nitrite production. In contrast, both SC68376 and serum starvation suppressed PG release by inhibiting Cox-2 mRNA, protein expression, and PGE2 synthesis. These data demonstrate that IL-1β phosphorylates and activates p38 MAPK in mesangial cells. The activation of p38 MAPK may provide a crucial signaling mechanism, which mediates the up-regulation of PG synthesis and the down-regulation of NO biosynthesis induced by IL-1β.

Nitric oxide amplifies interleukin 1-induced cyclooxygenase-2 expression in rat mesangial cells.

Interleukin 1 and nitric oxide (NO) from infiltrating macrophages and activated mesangial cells may act in concert to sustain and promote glomerular damage. To evaluate if such synergy occurs, we evaluated the effect if IL-1 beta and NO on the formation of prostaglandin (PG)E2 and cyclooxygenase (COX) expression. The NO donors, sodium nitroprusside and S-nitroso-N-acetylpenicillamine, alone did not increase basal PGE2 formation. However, these compounds amplified IL-1 beta-induced PGE2 production. Similarly, sodium nitroprusside and S-nitroso-N-acetylpenicillamine by themselves did not induce mRNA and protein for COX-2, the inducible isoform of COX; however, they both potentiated IL-1 beta-induced mRNA and protein expression of COX-2. The stimulatory effect of NO is likely to be mediated by cGMP since (a) an inhibitor of the soluble guanylate cyclase, methylene blue, reversed the stimulatory effect of NO donors on COX-2 mRNA expression; (b) the membrane-permeable cGMP analogue, 8-Br-cGMP, mimicked the stimulatory effect of NO donors on COX-2-mRNA expression; and (c) atrial natriuretic peptide, which increases cellular cGMP by activating the membrane-bound guanylate cyclase, also amplified IL-1 beta-induced COX-2 mRNA expression. These data indicate a novel interaction between NO and COX pathways.

Both p38αMAPK and JNK/SAPK Pathways Are Important for Induction of Nitric-oxide Synthase by Interleukin-1β in Rat Glomerular Mesangial Cells

Interleukin 1β (IL-1β) 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 NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) and p38MAPK. Our current study demonstrates that overexpression of the dominant negative form of JNK1 or p54 SAPKβ/JNK2 significantly reduces the iNOS protein expression and NO production induced by IL-1β. Similarly, overexpression of the kinase-dead mutant form of p38αMAPK also inhibits IL-1β-induced iNOS expression and NO production. In previous studies we demonstrated that IL-1β 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-1β. Overexpression of the dominant negative form of MKK4/SEK1 decreases IL-1β-induced iNOS expression and NO production with inhibition of both SAPK/JNK and p38MAPKphosphorylation. Overexpression of the kinase-dead mutant form of MKK3 or MKK6 demonstrated that either of these two mutant kinase inhibited IL-1β-induced p38MAPK (but not JNK/SAPK) phosphorylation and iNOS expression. Interestingly overexpression of wild type MKK3/6 was associated with phosphorylation of p38MAPK; however, in the absence of IL-1β, iNOS expression was not enhanced. This study suggests that the activation of both SAPK/JNK and p38αMAPK signaling cascades are necessary for the IL-1β-induced expression of iNOS and production of NO in renal mesangial cells.

IGF-I and insulin amplify IL-1β-induced nitric oxide and prostaglandin biosynthesis

The inflammatory cytokine interleukin-1β (IL-1β) induces both cyclooxygenase-2 (Cox-2) and the inducible nitric oxide synthase (iNOS) with concomitant release of PGs and nitric oxide (NO) by glomerular mesangial cells. In our current studies, we determine whether insulin and IGF-I are involved in the signal transduction mechanisms resulting in IL-1β-induced NO and PGE2biosynthesis in renal mesangial cells. We demonstrate that both insulin and IGF-I increase IL-1β-induced Cox-2 and iNOS protein expression, which in turn enhance PGE2 and NO production. Our data also indicate that both insulin and IGF-I enhance IL-1β-induced p38 mitogen-activated protein kinase (MAPK) phosphorylation and SAPK activation. These findings implicate the possible role of the MAPK pathway in mediating the effects of insulin and IGF-I on the upregulation of cytokine-stimulated NO and PG biosynthesis. Together, our results indicate that IGF-I and insulin may function to modulate the renal inflammatory process.

Regulation of Cyclooxygenase-2 by the Activated p38 Mapk Signaling Pathway

Cyclooxygenase (Cox) is a ubiquitous enzyme involved in various inflammatory processes. Two isoforms of Cox have been identified, Cox-1 and Cox-2. Cox-1 is constitutively expressed in most tissues and mediates physiologic responses such as regulation of renal sodium and water reabsorption, vascular homeostasis, and cytoprotection of the stomach. By comparison Cox-2 is primarily considered an inducible immediate-early gene whose synthesis can be upregulated by mitogenic or inflammatory stimuli including: tumor promoters 1, IL-1β 2, endotoxins 3, growth factors 4, and serum5. The pathophysiological role of Cox has been the topic of much interest. Cox is the main therapeutic target for non-steroidal anti-inflammatory drugs (NSAIDs) which exhibit their antipyretic, analgesic, and anti-inflammatory effects in humans via inhibition of prostaglandin biosynthesis 6. NSAIDS have been effective in the reduction of inflammatory symptoms in carrageenan-induced rat paw inflammation models 7 and in the reduced incidence of colon cancer 8,9.