Post‐transcriptional regulation of inducible nitric oxide synthase (iNOS) by Protein Interacting with Never in Mitosis Gene A‐1 (PIN1) in murine aortic endothelial cells

Abstract

PIN1, a peptidyl‐proline isomerase, increases the level or activity of several transcription factors that can induce iNOS. Thus it was hypothesized that depletion of PIN1 would reduce induction of iNOS by lipopolysaccharide (LPS) and interferon‐gamma (IFN). Murine aortic endothelial cells (EC) were stably transfected with a vector producing a PIN1 short hairpin RNA (shRNA) or an inactive control sequence. PIN1 shRNA reduced PIN1 levels >85% compared to control. Unexpectedly, suppression of PIN 1 enhanced NO production in response to LPS/IFN, and iNOS protein was induced 4.7‐fold more. The increased iNOS was cytotoxic to EC lacking PIN1, but not to control EC, in a crystal violet staining assay. Furthermore, cytotoxicity was prevented by a nitric oxide synthase inhibitor, L‐NAME. LPS/IFN increased iNOS/β‐actin mRNA ratio as expected, but PIN1 knockdown did not affect iNOS mRNA levels measured by RT‐PCR. Instead, PIN1 depletion stabilized iNOS protein in cycloheximide‐treated EC such that the time to a 50% decrease was extended from 8 h to more than 24 h. The loss of iNOS was antagonized by the calpain inhibitor, MDL 28170, but not by the selective proteasome inhibitor, epoxomicin. These results suggest that PIN1 normally restrains the induction of iNOS by facilitating its calpain‐mediated degradation in EC. Modulation of PIN1 may significantly affect iNOS‐dependent vascular activity and inflammatory diseases.