Protein Never in Mitosis Gene A Interacting-1 (PIN1) regulates degradation of inducible nitric oxide synthase in endothelial cells

Abstract

The peptidyl-proline isomerase Protein Never in Mitosis Gene A Interacting-1 (PIN1) increases the level or activity of several transcription factors that can induce the inducible nitric oxide (NO) synthase (iNOS). PIN1 can also regulate mRNA and protein turnover. Here, the effect of depletion of PIN1 on induction of iNOS by Escherichia coli endotoxin (LPS) and interferon-gamma (IFNgamma) in murine aortic endothelial cells (MAEC) was determined. Suppression of PIN1 by 85% with small hairpin RNA enhanced the induction of NO and iNOS protein by LPS-IFNgamma. There was no effect on induction of iNOS mRNA, suggesting a posttranscriptional effect. The enhanced levels of iNOS protein were functionally significant since LPS-IFNgamma was cytotoxic to MAEC lacking PIN1 but not MAEC harboring an inactive control construct, and because cytotoxicity was blocked by the NO synthase inhibitor N(omega)-nitro-L-arginine methyl ester. Consistent with posttranscriptional action, knockdown of PIN1 increased the stability of iNOS protein in cycloheximide-treated cells. Furthermore, loss of iNOS was blocked by the calpain inhibitor carbobenzoxy-valinyl-phenylalaninal but not by the selective proteasome inhibitor epoxomicin. Immunoprecipitation indicated that PIN1 can interact with iNOS. Pull down of iNOS with a wild-type glutathione-S-transferase-PIN1 fusion protein, but not with a mutant of the amino terminal phospho-(serine/threonine)-proline binding WW domain of PIN1, indicated that this domain mediates interaction. The results suggest that PIN1 associates with iNOS and can limit its induction by facilitating calpain-mediated degradation in MAEC.