No abstract

The inflaatory cytokine interleukin 1,B(IL-113) induces both cyclooxygenase (COX) and nitric oxide synthase (NOS) with increases in the release of prostaglandin (PG) IL-1 and TNF increase nitric oxide (NO) release in macrophages (11, 12) and mesangial cells (13). The free radical NO has emerged as an important signal and effector molecule in mammalian physiology (11,12), including neurotransmission, vasodilation, and inflammation. NO is synthesized from the guanidino nitrogen of L-Arg by the catalytic reaction of NO synthase (NOS) (11,12,14,15 (21) and vascular endothelium (22)(23)(24). The cDNA for iNOS has also been cloned from a macrophage cell line (16-18).Thus inflammatory cytokines including IL-1 and TNF drive both COX and NOS pathways. These pathways share a number of similarities. A variety of cells and tissues that produce PGs simultaneously release NO in response to cytokines or other activators. Both of them are paracrine modulators of the cell functions and mediate intracellular signals via cyclic nucleotides (i.e., cAMP or cGMP). NO and some PGs dilate vascular smooth muscle and inhibit platelet aggregation (11,12). In addition, NOS and COX require heme as a cofactor (25-27) and have constitutive and cytokineinducible forms.Many effects of NO are mediated by cGMP. NO activates the soluble guanylate cyclase and increases a second messenger, cGMP, by binding to the heme moiety of the enzyme (11,12). However, it is likely that guanylate cyclase is not the only molecular target of NO. NO inhibits several enzymes in the mitochondrial electron transport system and aconitase in the citric acid cycle by interacting with iron-sulfur centers of these enzymes (12). Likewise, NO has been shown to stimulate COX activity possibly via the heme component, which binds to the active site of the COX enzyme (28-31). Thus, a close interaction between NOS and COX pathways has become evident. However, the effect of PGs on the NOS pathway has not been fully explored. Thus we determined the effects of COX inhibition and PGs on iNOS induction and expression in rat mesangial cells. MATERIALS AND METHODSMaterials. Human recombinant IL-1,B (50 half-maximal units/ng) and restriction enzymes were purchased from Boehringer Mannheim. Primers and cDNA [for the polymerase chain reaction (PCR)] for mouse iNOS and rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were from Clontech. NG-Monomethyl-L-arginine (LNMMA), aminoguanidine (AG), indomethacin (Indo), PGE2, forskolin (FSK), sulfanilamide, and naphthylethylenediamine dihydrochloride were from Sigma. A stable analogue of PGI2, carba prostacyclin, was from Cayman Chemicals (Ann Arbor, MI).

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 ␤ and NO on the formation of prostaglandin (PG) E 2 and cyclooxygenase (COX) expression. The NO donors, sodium nitroprusside and S -nitroso-N -acetylpenicillamine, alone did not increase basal PGE 2 formation. However, these compounds amplified IL-1 ␤ -induced PGE 2 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 ␤ -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 ␤ -induced COX-2 mRNA expression. These data indicate a novel interaction between NO and COX pathways. (

These studies were undertaken to examine effects of elevated glucose levels on glycolysis, sorbitol pathway activity, and the cytosolic redox state of NADH/NAD+ in isolated glomeruli. Blood-free glomeruli were isolated from kidneys of male, Sprague-Dawley rats using standard sieving techniques, then incubated for one hour at 37 degrees C, pH 7.4, pO2 approximately 500 torr, in Krebs bicarbonate/Hepes buffer containing 5 or 30 mM glucose. Elevated glucose levels increased glucose 6-phosphate, fructose 6-phosphate, total triose phosphates, lactate, the lactate/pyruvate ratio, sorbitol, and fructose, but did not affect sn-glycerol 3-phosphate, pyruvate, or myo-inositol levels. The more reduced glomerular cytosolic redox state (manifested by the tissue lactate/pyruvate ratio) induced by 30 mM glucose was completely abrogated by aldose reductase inhibitors added to the diet two to seven days prior to glomerular isolation. These observations, coupled with evidence linking glucose- and diabetes-induced glomerular dysfunction to increased sorbitol pathway metabolism, support the hypothesis that metabolic imbalances associated with a more reduced ratio of cytosolic NADH/NAD+ (resulting from increased glucose metabolism via the sorbitol pathway) play an important role in mediating glucose- and diabetes-induced glomerular dysfunction.

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 upregulate gene expression. We therefore evaluated the effects of antioxidants on IL-1␤-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-1␤-induced Cox-2 mRNA levels but inhibited Cox-2 protein expression and prostaglandin E 2 production. Another antioxidant, rotenone, which inhibits reactive oxygen intermediate production by inhibiting the mitochondrial electron transport system, did not inhibit IL-1␤-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-1␤. 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. Interleukin-1 (IL-1)1 is a cytokine which mediates a variety of processes in host defense, such as inflammation and the cellular response to injury (1). During glomerular inflammation, cytokines from infiltrating macrophages and activated mesangial cells may act to sustain and promote glomerular damage. We have previously demonstrated that IL-1␤ induces cyclooxygenase-2 (Cox-2) and the inducible nitric-oxide synthase (iNOS) with increases in proinflammatory mediators, PGE 2 (2) and NO (3), in rat mesangial cells. The molecular signaling mechanisms by which IL-1␤ induces Cox-2 and iNOS includes transcriptional activation of these genes to produce increased levels of mRNA species which are "unstable." This mRNA is then translated into protein and degraded. These intracellular events are therefore potentially subject to regulation at the transcriptional or post-transcriptional level. Furthermore, the factors which control message stability and translational efficiency are not well understood.Mesangial cells produce reactive oxygen intermediates (ROI) with stimulation by endotoxin and cytokines, including IL-1 and tumor necrosis factor (4). ROI are produced during various electron-transfer reactions. When generated in excess, ROI can damage cells by peroxidizing lipids and disrupting proteins and nucleic acids. However, ROI may exert signaling functions and regulate gene expression at moderate concentrations (5-10). During glomerular inflammation, ROI from activated mesangial cells may act as signaling molecules. We have therefore evaluated the mechanisms ...

We investigated whether JNK is activated by interleukin-1 beta (IL-1 beta) in mesangial cells. We performed in-gel kinase assays with His-c-jun-(1-79), which contains the amino-terminal activation domain of c-jun and a mutant His-c-jun in which Ser-63 and Ser-73 of His-c-jun were mutated to Ala as the substrates. JNK1 (p45) and JNK2 (p54) isoforms phosphorylated His-c-jun in mesangial cells. IL-1 beta produced a time- and concentration-dependent increase in JNK activity. IL-1 beta did not phosphorylated the mutant, His-c-jun. The IL-1 beta-activated JNK activity was independent of serum and suppressed by neither tyrosine kinase inhibitors nor protein kinase C inhibitors. JNK was also stimulated by anisomycin and okadaic acid but not by phorbol 12-myristate 13-acetate. The protein synthesis inhibitors and okadaic acid potentiated the IL-1 beta-induced JNK activity. Together, these studies indicate that the novel JNK group of protein kinases may play an important role in the signal transduction pathway initiated by proinflammatory cytokines, such as IL-1 beta in mesangial cells.

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.

Cyclooxygenase (COX) catalyzes the formation of prostaglandins from arachidonic acid. Nitric oxide synthase catalyzes the production of nitric oxide, a short-lived messenger molecule involved in many diverse cellular processes. Both of these enzymes have inducible forms [COX-2 and inducible nitric oxide synthase (iNOS), respectively] that respond to environmental stresses, chemicals, and extracellular ligands such as interleukin-1, epidermal growth factor, and platelet-derived growth factor. The precise cascade of intracellular events that leads to the expression of either COX-2 or iNOS is not known. Protein kinase C (PKC) is a family of 11 serine-threonine kinases conserved throughout eukaryotic species that transduce a wide variety of signals critical for cellular functions. Using a retroviral vector to overexpress the zeta-isoform of PKC in rat mesangial cells, we demonstrate markedly increased COX-2, prostaglandin E2 (PGE2), iNOS, and altered cellular morphology compared with mesangial cells expressing a control retroviral vector and untransfected mesangial cells. NIH/3T3 cells overexpressing PKC-zeta showed no change in morphology, PGE2 production, COX-2 expression, or iNOS expression at basal conditions. This suggests a role for PKC-zeta in the expression of these enzymes in mesangial cells.