Regulation of Inducible Nitric Oxide Synthase (iNOS) by Cyclin‐Dependent Kinase 7 (CDK7) in Endothelial Cells

Abstract

E. coli endotoxin (LPS) combined with interferon‐γ (IFN) induces inducible nitric oxide synthase (iNOS), with production of nitric oxide (NO) and reactive nitrogen species in endothelial cells. Appropriate manipulation of iNOS during onset or resolution of inflammation may be useful. Since transcription is required for induction of iNOS by LPS/IFN, we hypothesized that depletion of cyclin dependent kinase (CDK) 7, a promoter of transcription, would antagonize induction. Mouse aortic endothelial cells (MAEC) were transfected with Non‐targeted (NT) control RNA or CDK7 siRNA to deplete the kinase, followed by stimulation with LPS/IFN for 12 h. Rather than suppressing, CDK7 depletion increased induction of iNOS, and its dimerization and NO production. The effect was post‐transcriptional in that mRNA induction was unaffected, while iNOS protein turnover was reduced in the absence of CDK7. Cellular calpain activity, which is required for iNOS protein degradation in MAEC, was unaffected by CDK7 depletion, suggesting an effect on iNOS itself. Indeed, iNOS in lysates of kinase‐deficient cells was resistant to in vitro digestion with exogenous calpain. In conjunction with protease resistance, the association of iNOS with calmodulin, known to inhibit its digestion by calpain, was increased in cells lacking CDK7. These results suggest that CDK7 normally restrains iNOS induction by limiting calmodulin binding and maintaining calpain‐mediated turnover. By regulating iNOS enzyme complexes, CDK7 may serve as a novel target for manipulation of vascular NO.Support or Funding InformationSupport was received from the Popat Patil Fellowship (SS).