Evidence for involvement of the proteasome complex (26S) and NFkappaB in IL-1beta-induced nitric oxide and prostaglandin production by rat islets and RINm5F cells.
Interleukin-1beta (IL-1beta) has been implicated as an effector molecule of beta-cell destruction in autoimmune diabetes. IL-1beta inhibits insulin secretion from pancreatic beta-cells by stimulating the expression of inducible nitric oxide synthase (iNOS) that generates the free radical nitric oxide. IL-1beta also induces the coexpression of the inducible isoform of cyclooxygenase (COX-2) that results in the overproduction of proinflammatory prostaglandins. The current studies were designed to characterize the involvement of protease(s) in the signaling pathway of IL-1beta-induced iNOS and COX-2 expression by rat islets and transformed rat pancreatic beta-cells. Because of the limitations of cell numbers of purified primary beta-cells obtained from rat islets, biochemical and molecular studies were performed using the rat insulinoma beta-cell line RINm5F. A serine protease inhibitor, Nalpha-P-tosyl-L-lysine chloromethyl ketone (TLCK), and a proteasome complex (26S) inhibitor, MG 132, inhibited IL-1beta-induced nitrite formation, an oxidation product of nitric oxide produced by iNOS, in a concentration-dependent manner, with complete inhibition observed at 100 micromol/l and 10 micromol/l, respectively. Both TLCK and MG 132 also inhibited iNOS gene expression at the level of mRNA and protein. In an analogous manner, TLCK (100 micromol/l) and MG 132 (10 micromol/l) inhibited IL-1beta-induced COX-2 enzyme activity (PGE2 formation) and COX-2 gene expression at the level of mRNA and protein. In human islets, the proteasome inhibitor MG 132 also inhibited the formation of the products of iNOS and COX-2 enzyme activity, nitrite, and PGE2, respectively. These findings suggest that the inhibitory action of TLCK and MG 132 on iNOS and COX-2 expression precedes transcription. The transcription factor NFkappaB is essential for activation of a number of cytokine-inducible enzymes and was evaluated as a possible site of protease action necessary for IL-1beta-induced coexpression of iNOS and COX-2. TLCK and MG 132 inhibited both IL-1beta-induced activation of NFkappaB and degradation of IkappaBalpha by islets and RINm5F cells. These results implicate protease activation as an early signaling event in IL-1beta-induced inhibition of beta-cell function. This study also suggests that IL-1beta-induced iNOS and COX-2 coexpression by pancreatic beta-cells share a common signaling pathway in utilizing the proteasome complex (26S) and the transcription factor NFkappaB, and it identifies sites of intervention to prevent the overproduction of their inflammatory products.
Kwon
G
G
Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110-8118, USA.
Corbett
J A
JA
Hauser
S
S
Hill
J R
JR
Turk
J
J
McDaniel
M L
ML
D000818:Animals; D002461:Cell Line, Transformed; D051546:Cyclooxygenase 2; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D015232:Dinoprostone / Q000096:biosynthesis*; D004790:Enzyme Induction / Q000187:drug effects; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D007375:Interleukin-1 / Q000378:metabolism*; D007515:Islets of Langerhans / Q000201:enzymology / Q000378:metabolism*; D007527:Isoenzymes / Q000096:biosynthesis; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D008565:Membrane Proteins; D016328:NF-kappa B / Q000378:metabolism*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000235:genetics; D052247:Nitric Oxide Synthase Type II; D010447:Peptide Hydrolases / Q000378:metabolism*; D010766:Phosphorylation; D011451:Prostaglandin-Endoperoxide Synthases / Q000096:biosynthesis; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger / Q000037:antagonists & inhibitors; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D015853:Cysteine Proteinase Inhibitors; D007375:Interleukin-1; D007527:Isoenzymes; D007976:Leupeptins; D008565:Membrane Proteins; D016328:NF-kappa B; D012333:RNA, Messenger; D014107:Tosyllysine Chloromethyl Ketone; D009569:Nitric Oxide; C496319:NOS2 protein, human; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D051546:Cyclooxygenase 2; C496540:PTGS2 protein, human; D011451:Prostaglandin-Endoperoxide Synthases; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D015232:Dinoprostone; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde