Aubrey R. Morrison

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.

Exaggerated prostaglandin biosynthesis and its influence on renal resistance in the isolated hydronephrotic rabbit kidney.

Basal and hormone-stimulated prostaglandin biosynthesis was compared in isolated perfused rabbit kidneys with and without ureteral obstruction. At 72 h there was enhanced responsiveness to bradykinin in the ureter-obstructed hydronephrotic kidney. The amount of prostaglandin-like substance released from the perfused kidneys by 25 ng of bradykinin was 533+/-163 ng from the ureter-obstructed, 28+/-4 ng from the contralateral, and 26+/-3 ng from the normal kidney. The enhanced response was also noted with angiotensin II and with norepinephrine. This exaggerated responsiveness by the ureter-obstructed kidney could not be explained by decreased prostaglandin (PG) destruction or by decreased renal peptide inactivation (bradykinin or angiotensin). There was no enhanced PG biosynthesis with exogenous arachidonate, suggesting there was no increase in cyclo-oxygenase activity in the ureter-obstructed kidney. Renal tubular transport of PG from medulla to cortex was apparently not essential for the enhanced PG biosynthesis to hormone stimulation since the same exaggerated responses were noted during perfusion with the ureter ligated. The cyclo-oxygenase inhibitor, indomethacin, increased basal perfusion pressure in the obstructed kidney and enhanced the magnitude and duration of the renal vasoconstriction produced by angiotensin II in the hydronephrotic kidney. These results suggest that the local exaggerated biosynthesis of PG may be occurring in the cortical resistance vessels and may be important to the alteration in blood flow and excretory function that occur in ureteral obstruction.

Antioxidants Inhibit Interleukin-1-induced Cyclooxygenase and Nitric-oxide Synthase Expression in Rat Mesangial Cells EVIDENCE FOR POST-TRANSCRIPTIONAL REGULATION

Glomerular mesangial cells produce reactive oxygen intermediates when stimulated by interleukin-1 (IL-1) or tumor necrosis factor. Recent observations suggest that reactive oxygen intermediates may play a role in IL-1 and tumor necrosis factor signaling and may up-regulate gene expression. We therefore evaluated the effects of antioxidants on IL-1β-induced cyclooxygenase-2 (Cox-2) and inducible nitric-oxide synthase (iNOS) expression in rat mesangial cells. The oxidant scavenger, pyrrolidine dithiocarbamate (PDTC), inhibited iNOS expression at the transcriptional level, since PDTC abolished iNOS mRNA accumulation. In contrast, PDTC inhibited Cox-2 expression at the post-transcriptional level, since PDTC did not affect IL-1β-induced Cox-2 mRNA levels but inhibited Cox-2 protein expression and prostaglandin E production. Another antioxidant, rotenone, which inhibits reactive oxygen intermediate production by inhibiting the mitochondrial electron transport system, did not inhibit IL-1β-induced iNOS and Cox-2 mRNA expression but inhibited iNOS and Cox-2 protein expression, suggesting a post-transcriptional target for the inhibition of iNOS and Cox-2 expression induced by IL-1β. These results suggest that not only transcriptional regulation but also post-transcriptional mechanisms are involved in redox-sensitive inhibition of cytokine induced Cox-2 and iNOS expression. These results suggest a novel approach for intervention in cytokine-mediated inflammatory processes.

Apigenin Prevents UVB-Induced Cyclooxygenase 2 Expression: Coupled mRNA Stabilization and Translational Inhibition

ABSTRACT Cyclooxygenase 2 (COX-2) is a key enzyme in the conversion of arachidonic acid to prostaglandins, and COX-2 overexpression plays an important role in carcinogenesis. Exposure to UVB strongly increased COX-2 protein expression in mouse 308 keratinocytes, and this induction was inhibited by apigenin, a nonmutagenic bioflavonoid that has been shown to prevent mouse skin carcinogenesis induced by both chemical carcinogens and UV exposure. Our previous study suggested that one pathway by which apigenin inhibits UV-induced and basal COX-2 expression is through modulation of USF transcriptional activity in the 5′ upstream region of the COX-2 gene. Here, we found that apigenin treatment also increased COX-2 mRNA stability, and the inhibitory effect of apigenin on UVB-induced luciferase reporter gene activity was dependent on the AU-rich element of the COX-2 3′-untranslated region. Furthermore, we identified two RNA-binding proteins, HuR and the T-cell-restricted intracellular antigen 1-related protein (TIAR), which were associated with endogenous COX-2 mRNA in 308 keratinocytes, and apigenin treatment increased their localization to cell cytoplasm. More importantly, reduction of HuR levels by small interfering RNA inhibited apigenin-mediated stabilization of COX-2 mRNA. Cells expressing reduced TIAR showed marked resistance to apigenins ability to inhibit UVB-induced COX-2 expression. Taken together, these results indicate that in addition to transcriptional regulation, another mechanism by which apigenin prevents COX-2 expression is through mediating TIAR suppression of translation.

Identification of RNA-binding proteins in RAW 264.7 cells that recognize a lipopolysaccharide-responsive element in the 3-untranslated region of the murine cyclooxygenase-2 mRNA

RAW 264.7 cells rapidly induce cyclooxygenase-2 (COX-2) in response to lipopolysaccharide treatment. Part of the increased COX-2 expression occurred through post-transcriptional mechanisms mediated through specific regions of the 3′-untranslated region (UTR) of the message. The proximal region of the 3′-UTR of COX-2 contains a highly conserved AU-rich element that was able to confer lipopolysaccharide regulation of a chimeric reporter-gene. Electrophoretic mobility shift assays demonstrated that the RNA-binding proteins TIAR, AUF1, HuR, and TIA-1 all form an RNA-protein complex with the first 60 nucleotides of the 3′-UTR of COX-2. Biotinylated RNA probes were used to isolate additional proteins that bind the 3′-UTR of COX-2. We identified several RNA-binding proteins including TIAR, AUF1, CBF-A, RBM3, heterogeneous nuclear ribonucleoprotein (hnRNP) A3, and hnRNP A2/B1. We identified four alternatively spliced isoforms of AUF1 which migrated at multiple isoelectric points. Likewise, we identified alternatively spliced isoforms of CBF-A, hnRNP A3, and hnRNP A2/B1. Western analysis of two-dimensional gels identified multiple isoforms of TIA-1, TIAR, and AUF1 at pI values that spanned nearly 3 pH units. Thus, through a combination of alternative splicing and post-translational modification cells are able to increase greatly the repertoire of protein species expressed at a given time or in response to extracellular stimuli.

Translation regulatory factor RBM3 is a proto-oncogene that prevents mitotic catastrophe

RNA-binding proteins play a key role in post-transcriptional regulation of mRNA stability and translation. We have identified that RBM3, a translation regulatory protein, is significantly upregulated in human tumors, including a stage-dependent increase in colorectal tumors. Forced RBM3 overexpression in NIH3T3 mouse fibroblasts and SW480 human colon epithelial cells increases cell proliferation and development of compact multicellular spheroids in soft agar suggesting the ability to induce anchorage-independent growth. In contrast, downregulating RBM3 in HCT116 colon cancer cells with specific siRNA decreases cell growth in culture, which was partially overcome when treated with prostaglandin E2, a product of cyclooxygenase (COX)-2 enzyme activity. Knockdown also resulted in the growth arrest of tumor xenografts. We have also identified that RBM3 knockdown increases caspase-mediated apoptosis coupled with nuclear cyclin B1, and phosphorylated Cdc25c, Chk1 and Chk2 kinases, implying that under conditions of RBM3 downregulation, cells undergo mitotic catastrophe. RBM3 enhances COX-2, IL-8 and VEGF mRNA stability and translation. Conversely, RBM3 knockdown results in loss in the translation of these transcripts. These data demonstrate that the RNA stabilizing and translation regulatory protein RBM3 is a novel proto-oncogene that induces transformation when overexpressed and is essential for cells to progress through mitosis.

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.