Double-Stranded RNA—Dependent Protein Kinase Is Not Required for Double-Stranded RNA—Induced Nitric Oxide Synthase Expression or Nuclear Factor-κB Activation by Islets

Double-Stranded RNA—Dependent Protein Kinase Is Not Required for Double-Stranded RNA—Induced Nitric Oxide Synthase Expression or Nuclear Factor-κB Activation by Islets Libby A. Blair , Monique R. Heitmeier , Anna L. Scarim , Leonard B. Maggi, Jr. and John A. Corbett From the Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri. Address correspondence and reprint requests to Dr. John A. Corbett, St. Louis University School of Medicine, Department of Biochemistry and Molecular Biology, 1402 South Grand Blvd., St. Louis, MO 63104. E-mail: corbettj{at}slu.edu . Abstract Environmental factors, such as viral infection, have been implicated in the destruction of β-cells during the development of autoimmune diabetes. Double-stranded RNA (dsRNA), produced during viral replication, is an active component of a viral infection that stimulates antiviral responses in infected cells. Previous studies have shown that treatment of rat islets with dsRNA in combination with γ-interferon (IFN-γ) results in a nitric oxide-dependent inhibition of glucose-stimulated insulin secretion. This study examines the role of nuclear factor-κB (NF-κB) and the dsRNA-dependent protein kinase (PKR) in dsRNA + IFN-γ-induced nitric oxide synthase (iNOS) expression and nitric oxide production by rat, mouse, and human islets. Treatment of rat and human islets with dsRNA in the form of polyinosinic-polycytidylic acid (poly IC) and IFN-γ resulted in iNOS expression and nitric oxide production. Inhibitors of NF-κB activation—the proteasome inhibitor MG-132 and the antioxidant pyrrolidinedithiocarbamate (PDTC)—prevented poly IC + IFN-γ-induced iNOS expression and nitric oxide production. Incubation of rat islets for 3 h or human islets for 2 h with poly IC alone or poly IC + IFN-γ resulted in NF-κB nuclear translocation and degradation of the NF-κB inhibitor protein, IκB, events that are prevented by MG-132. PKR has been shown to participate in dsRNA-induced NF-κB activation in a number of cell types, including mouse embryonic fibroblasts. However, poly IC stimulated NF-κB nuclear translocation and IκB degradation to similar levels in islets isolated from mice devoid of PKR (PKR -/- ) and wild-type mice (PKR +/+ ). Furthermore, the genetic absence of PKR did not affect dsRNA + IFN-γ-induced iNOS expression, nitric oxide production, or the inhibitory actions of these agents on glucose-stimulated insulin secretion. These results