RESUMO
BACKGROUND: The Akita mutation (C96Y) in the insulin gene results in early onset diabetes in both humans and mice. Expression of mutant proinsulin (C96Y) causes endoplasmic reticulum (ER) stress in pancreatic ß-cells and consequently the cell activates the unfolded protein response (UPR). Since the proinsulin is terminally misfolded ER stress is irremediable and chronic activation of the UPR eventually activates apoptosis in some cells. Here we analyzed the IRE1-dependent activation of genes in response to misfolded proinsulin production in an inducible mutant proinsulin (C96Y) insulinoma cell line. RESULTS: The IRE1 endoribonuclease inhibitors 4µ8c and MKC-3946 prevented the splicing of the XBP1 mRNA in response to ER stress caused by mutant proinsulin production. Microarray expression analysis and qPCR validation of select genes revealed that maximal upregulation of many UPR genes in response to mutant proinsulin production required IRE1, although most were still increased above control. Interestingly, neither degradation of misfolded proinsulin via ER-associated degradation (ERAD), nor apoptosis induced by prolonged misfolded proinsulin expression were affected by inhibiting IRE1. CONCLUSIONS: Although maximal induction of most UPR genes requires IRE1, inhibition of IRE1 does not affect ERAD of misfolded proinsulin or predispose pancreatic ß-cells expressing misfolded proinsulin to chronic ER stress-induced apoptosis.
Assuntos
Apoptose/efeitos dos fármacos , Células Secretoras de Insulina/citologia , Proteínas de Membrana/antagonistas & inibidores , Proinsulina/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Proteínas de Membrana/metabolismo , Mutação Puntual , Proinsulina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteólise/efeitos dos fármacos , RatosRESUMO
The Jagn1 protein was indentified in a SILAC proteomic screen of proteins that are increased in insulinoma cells expressing a folding-deficient proinsulin. Jagn1 mRNA was detected in primary rodent islets and in insulinoma cell lines and the levels were increased in response to ER stress. The function of Jagn1 was assessed in insulinoma cells by both knock-down and overexpression approaches. Knock-down of Jagn1 caused an increase in glucose-stimulated insulin secretion resulting from an increase in proinsulin biosynthesis. In contrast, overexpression of Jagn1 in insulinoma cells resulted in reduced cellular proinsulin and insulin levels. Our results identify a novel role for Jagn1 in regulating proinsulin biosynthesis in pancreatic ß-cells. Under ER stress conditions Jagn1 is induced which might contribute to reducing proinsulin biosynthesis, in part by helping to relieve the protein folding load in the ER in an effort to restore ER homeostasis.