IL-1beta induces serine protease inhibitor 3 (SPI-3) gene expression in rat pancreatic beta-cells. Detection by differential display of messenger RNA.
Immune-mediated beta-cell damage induces diverse intracellular signals, leading to transcription of different genes which may either contribute to beta-cell repair and/or defence or lead to cell death. The cytokine interleukin-1beta (IL-1) is a potential mediator of beta-cell dysfunction and damage in type 1 diabetes mellitus. To understand the molecular actions of this cytokine upon beta-cells, this study aimed at the cloning of genes induced in FACS-purified rat pancreatic beta-cells by a 6- or 24-h exposure to IL-1 by using differential display of mRNA with reverse transcription-polymerase chain reaction (DDRT-PCR). Among these cytokine-induced genes, a gene encoding for rat serine protease inhibitor (SPI-3) was isolated. SPI-3 may be involved in cellular defence responses against inflammatory stress. RT-PCR analysis confirmed that SPI-3 mRNA expression in rat beta-cells is increased by IL-1 at an early stage (2 h), with maximal accumulation during 6-12 h and decline after 24 h. Similar observations were made in mouse pancreatic islets and in the rat insulinoma cell line RINm5F. IFN-gamma neither increased SPI-3 gene expression nor potentiated its induction by IL-1 in rat beta-cells. The stimulatory effects of IL-1 on SPI-3 mRNA expression were decreased by co-incubation with an inhibitor of gene transcription (actinomycin D), an inhibitor of protein synthesis (cycloheximide) or an inhibitor of NF-kappaB activation (PDTC). On the other hand, a blocker of inducible nitric oxide synthase (iNOS) activity (N(G)-methyl-L-arginine) did not prevent IL-1-induced SPI-3 expression. Thus, SPI-3 mRNA expression following IL-1 exposure depends on gene transcription, protein synthesis and activation of the nuclear transcription factor NF-kappaB, but it is independent of NO formation.
Chen
M C
MC
Diabetes Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, Brussels, B-1090, Belgium.
Schuit
F
F
Pipeleers
D G
DG
Eizirik
D L
DL
D000209:Acute-Phase Proteins / Q000096:biosynthesis* / Q000235:genetics*; D000818:Animals; D002478:Cells, Cultured; D005434:Flow Cytometry; D015870:Gene Expression / Q000187:drug effects*; D007371:Interferon-gamma / Q000494:pharmacology; D007375:Interleukin-1 / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000187:drug effects* / Q000378:metabolism*; D008297:Male; D016328:NF-kappa B / Q000037:antagonists & inhibitors; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors; D052247:Nitric Oxide Synthase Type II; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015842:Serine Proteinase Inhibitors / Q000096:biosynthesis / Q000235:genetics; D015843:Serpins; D014158:Transcription, Genetic / Q000187:drug effects
D000209:Acute-Phase Proteins; D007375:Interleukin-1; D016328:NF-kappa B; D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D015842:Serine Proteinase Inhibitors; C403317:Serpina3n protein, rat; D015843:Serpins; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat