The Role of Protein Phosphatases in the Expression of Inducible Nitric Oxide Synthase in the Rat Hepatocyte

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Abstract
Previously, we demonstrated that nuclear factor–κB (NF–κB) mediates cytokine–induced hepatic inducible nitric oxide synthase (iNOS) expression. NF–κB activation is regulated by kinases and phosphatases whose function is only beginning to be understood. Therefore, experiments were performed to determine the role of protein phosphatases (PPase) in cytokine–induced iNOS expression. Hepatocytes were stimulated with cytokines in the presence or absence of tyrosine phosphatase inhibitors (pervanadate [PV], phenylarsine oxide [PAO]) and a serine–threonine phosphatase inhibitor (okadaic acid [OA]). Cytokines induced hepatocyte iNOS mRNA, protein, and NO2 - production that was substantially decreased by the addition of the tyrosine phosphatase inhibitors (PAO and PV). The serine–threonine phosphatase inhibitor (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 (PTPases) are required for iNOS transcription, while the serine–threonine phosphatase inhibitor (OA) had no effect on iNOS transcription. Electromobility shift assays (EMSAs) revealed that the tyrosine–phosphatase inhibitors blocked cytokine–induced NF–κB activation, while OA did not have a significant effect on NF–κB DNA binding activity. Therefore, tyrosine phosphatases are involved in the regulation of cytokine–induced activation of NF–κB, while serine–threonine phosphatases posttranscriptionally regulate iNOS translation. These results identify the regulatory role of specific protein phosphatases (PPases) in hepatic iNOS expression