Endothelial cells are a primary target for injury and cardiovascular risk factors. Endothelial inflammatory activation and dysfunction has been critically implicated in diverse cardiovascular diseases. Inflammatory cytokines and bacterial products such as lipopolysaccharide (LPS) together with interferon-gamma (IFN-γ), can activate vascular endothelial cells and induce expression of pro-inflammatory proteins including such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), which may promote endothelial cell injury and play an important role in the pathogenesis of many cardiovascular diseases.
The peptidyl-proline isomerase, Protein Never in Mitosis Gene A Interacting-1 (PIN1), is a cis-trans peptidyl-prolyl isomerase that catalyzes isomerization of phospho-proteins, affects their conformation and regulates most aspects of protein function. PIN1 is known to increase levels or activity of several transcription factors that are activated by phosphorylation at serine/threonine-proline and that can induce iNOS and COX-2. PIN1 can regulate these various proteins by increasing their mRNA, by reducing their protein turnover, and by regulating their subcellular distribution as well. However, the role of PIN1 in expression of iNOS and COX-2 in endothelial cells is unexplored. Thus, the original hypothesis of my dissertation was that PIN1 is necessary for induction of inflammatory proteins in endothelial cells.
Here, the effect of PIN1 depletion and gene knockout on induction of iNOS and COX-2 by LPS and IFN-γ in murine aortic endothelial cells (MAEC) was determined. In contrast to the original hypothesis, knockdown of PIN1 with an shRNA-lentiviral system, transient transfection with small interfering RNA, and gene knockout each enhanced the induction of iNOS and COX-2 proteins by LPS/IFN. There was no effect on induction of their mRNA, suggesting a post-transcriptional effect. Knockdown of PIN1 increased the stability of iNOS and COX-2 proteins in cycloheximide-treated cells. Furthermore, the degradation of iNOS and COX-2 was also blocked by calpain inhibitors in MAEC. Consistent with these results, knockdown of PIN1 reduced basal and LPS/IFN-stimulated calpain activity. These findings launched an investigation into the regulation and roles of calpains in endothelial inflammatory activation. The depletion of PIN1 did not affect the levels of the large or small subunits of ubiquitous, heterodimeric calpains. PIN1 did not interact with these proteins either. Instead, deletion of PIN1 increased the inhibitory activity of calpastatin (CAST), the endogenous protein inhibitor of heterodimeric calpains. The PIN1 fusion protein also antagonized the ability of CAST to inhibit exogenous calpain in vitro. Co-immunoprecipitation and GST-PIN1 fusion protein pull-down results indicated that PIN1 may associate with CAST and decrease its inhibitory activity towards calpain in MAEC. My results identified CAST, as a prime target of PIN1 in endothelial cells.
Overall, the results indicate that PIN1 normally associates with calpastatin and antagonizes its inhibitory effect on heterodimeric calpain. This maintains calpain activity, consequently limiting induction of the calpain substrates, iNOS and COX-2. The work presented in my thesis will direct further investigation of the important role of PIN1 in endothelial activation and dysfunction during inflammatory conditions. These mechanistic insights into endothelial biology should indicate new strategies for therapy for based on the calpain/calpastatin system.