Inhibitory effect of IGF-I on type 2 nitric oxide synthase expression in Ins-1 cells and protection against activation-dependent apoptosis: involvement of phosphatidylinositol 3-kinase.
Challenge of Ins-1 cells, a rat beta-pancreatic cell line, with lipopolysaccharide (LPS) and interferon-gamma (IFN-gamma) promoted the expression of type 2 nitric oxide synthase (NOS-2) in a cooperative way. Treatment of Ins-1 cells with IGF-I significantly inhibited the expression of NOS-2, especially at subsaturating concentrations of LPS and IFN-gamma. The inhibitory effect of IGF-I on NOS-2 expression was abrogated when cells were incubated with wortmannin or LY294002, two inhibitors of phosphatidylinositol 3-kinase. Transient expression of the p110 subunit of phosphatidylinositol 3-kinase impaired the LPS and IFN-gamma-dependent NOS-2 promoter activity in cells transfected with a 1-kb fragment corresponding to the 5'-flanking region of the NOS-2 gene. However, expression of a dominant negative form of p85 abolished the inhibitory action of IGF-I on the NOS-2 promoter activity. Analysis of the decreased NOS-2 promoter activity in cells incubated with IGF-I showed a lower nuclear factor KB binding as determined by electrophoretic mobility shift assays. The synthesis of NO, produced after LPS and IFN-gamma challenge, triggered an apoptotic response in these cells. IGF-I reduced apoptosis mainly through the decreased synthesis of NO. However, in activated cells treated with N-[3-(aminomethyl)benzyl]acetamidine, a specific NOS-2 inhibitor, IGF-I completely abolished the NO-independent apoptosis. This protection from apoptosis was dependent on phosphatidylinositol 3-kinase activity. These results suggest an important anti-inflammatory and anti-apoptotic role for IGF-I in beta-pancreatic cells, with both actions depending on the activation of phosphatidylinositol 3-kinase.
Castrillo
A
A
Instituto de Bioquímica, Facultad de Farmacia, Universidad Complutense, Madrid, Spain.
Bodelón
O G
OG
Boscá
L
L
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D002460:Cell Line; D004789:Enzyme Activation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007371:Interferon-gamma / Q000494:pharmacology; D007515:Islets of Langerhans / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D008070:Lipopolysaccharides / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors*; D052247:Nitric Oxide Synthase Type II; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D051381:Rats
D004791:Enzyme Inhibitors; D008070:Lipopolysaccharides; D007334:Insulin-Like Growth Factor I; D007371:Interferon-gamma; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D019869:Phosphatidylinositol 3-Kinases