PIN1 regulates calpain‐mediated degradation of cyclooxygenase‐2 in endothelial cells

Abstract

Although the peptidyl‐proline isomerase, PIN1, regulates pro‐inflammatory factors, its role in cyclooxygenase‐2 (COX‐2) expression is unknown. Here, PIN1 was depleted in murine aortic endothelial cells (MAEC) by stable expression short hairpin RNA targeting PIN1 or a control construct. E. coli endotoxin (LPS) plus interferon‐gamma (IFN) induced COX‐2 4.7 times more in PIN1 knockdown (KD) than in control MAEC. KD did not affect induction of COX‐2 mRNA. Instead, KD increased stability of COX‐2 in cycloheximide‐treated cells. Previously, it was found that PIN1 KD reduced calpain activity and increased expression of the endogenous inhibitor, calpastatin. Here, induction COX‐2 was enhanced by the calpain inhibitor, MDL 28170, and loss of COX‐2 after cycloheximide was similar in KD cells and in controls treated with MDL 28170. COX‐2 in extracts of control MAEC was more sensitive to calpain‐1 digestion in vitro than COX‐2 from knockdown cells. Immunodepletion of PIN1 from control cell extracts did not affect calpain sensitivity, and no direct interaction between COX‐2 and PIN1 was seen. These results suggest that PIN1 regulates COX‐2 degradation mainly by its indirect effect on calpain via calpastatin. Thus, PIN1 may normally restrain expression of COX‐2 and other calpain substrates in endothelial cells. Modulation of PIN1 may significantly affect calpain‐dependent vascular activity and inflammatory diseases.