The role of protein phosphatases in the expression of inducible nitric oxide synthase in the rat hepatocyte

Previously, we demonstrated that nuclear factor-kappa B (NF-kappa B) mediat es cytokine-induced hepatic inducible nitric oxide synthase (iNOS) expressi on. NF-kappa B activation is regulated by kinases and phosphatases whose fu nction is only beginning to be understood. Therefore, experiments were perf ormed to determine the role of protein phosphatases (PPase) in cytokine-ind uced iNOS expression. Hepatocytes were stimulated with cytokines in the pre sence or absence of tyrosine phosphatase inhibitors (pervanadate [PV], phen ylarsine oxide [PAO]) and a serine-threonine phosphatase inhibitor (okadaic acid [OA]), Cytokines induced hepatocyte iNOS mRNA, protein, and NO2- prod uction that was substantially decreased by the addition of the tyrosine pho sphatase inhibitors (PAO and PV), The serine-threonine phosphatase inhibito r (OA) decreased NO release and protein levels in a concentration-dependent fashion; however, iNOS mRNA levels were not significantly reduced. Nuclear run-on experiments demonstrated that protein tyrosine phosphatases (PTPase s) are required for iNOS transcription, while the serine-threonine phosphat ase inhibitor (OA) had no effect on iNOS transcription. Electromobility shi ft assays (EMSAs) revealed that the tyrosine-phosphatase inhibitors blocked cytokine-induced NF-kappa B activation, while OA did not have a significan t effect on NF-kappa B DNA binding activity. Therefore, tyrosine phosphatas es are involved in the regulation of cytokine-induced activation of NF-kapp a B, while serine-threonine phosphatases posttranscriptionally regulate iNO S translation. These results identify the regulatory role of specific prote in phosphatases (PPases) in hepatic iNOS expression.