The role of protein phosphatases in the expression of inducible nitric oxide synthase in the rat hepatocyte.
Previously, we demonstrated that nuclear factor-kappaB (NF-kappaB) mediates cytokine-induced hepatic inducible nitric oxide synthase (iNOS) expression. NF-kappaB 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-kappaB activation, while OA did not have a significant effect on NF-kappaB DNA binding activity. Therefore, tyrosine phosphatases are involved in the regulation of cytokine-induced activation of NF-kappaB, while serine-threonine phosphatases posttranscriptionally regulate iNOS translation. These results identify the regulatory role of specific protein phosphatases (PPases) in hepatic iNOS expression.
Taylor
B S
BS
Department of Surgery, University of Pittsburgh, Pittsburgh PA, USA. BSTA@med.pitt.edu
Liu
S
S
Villavicencio
R T
RT
Ganster
R W
RW
Geller
D A
DA
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D016207:Cytokines / Q000494:pharmacology; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D020672:I-kappa B Proteins; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation / Q000187:drug effects; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley
D016207:Cytokines; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D020672:I-kappa B Proteins; D016328:NF-kappa B; C000606206:Nfkbia protein, rat; D012333:RNA, Messenger; D000072000:NF-KappaB Inhibitor alpha; D019319:Okadaic Acid; D009569:Nitric Oxide; D004247:DNA; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; C496318:Nos2 protein, rat; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases