Role Of Endothelial Nitric Oxide Synthase In The Regulation Of Sterol Regulatory Element Binding Protein Activation By Oxidized Phospholipids

Oxidized-1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (Ox-PAPC), found in atherosclerotic lesions and other sites of chronic inflammation, activates endothelial cells (EC) to synthesize chemotactic factors, such as interleukin 8 (IL-8). Previously, we demonstrated that the sustained induction of IL-8 transcription by Ox-PAPC was mediated through the activation of sterol regulatory element binding protein (SREBP). We now present evidence for the role of endothelial nitric oxide synthase (eNOS) in the activation of SREBP by Ox-PAPC. Ox-PAPC treatment of EC induced a dose- and time-dependent activation of eNOS, as measured by phosphorylation of serine residue 1177 and the conversion of l-arginine to l-citrulline. Activation of eNOS by Ox-PAPC was regulated through a phosphatidylinositol-3-kinase/Akt-mediated mechanism. These studies also demonstrated that pretreatment of EC with NOS inhibitor, l-NAME, significantly inhibited Ox-PAPC-induced IL-8 synthesis. Since SREBP activation had previously been shown to regulate IL-8 transcription by Ox-PAPC, we examined the effects of l-NAME on Ox-PAPC-induced SREBP activation. Our data demonstrated that Ox-PAPC-induced SREBP activation and expression of SREBP target genes were significantly reduced by pretreatment of EC with l-NAME. Interestingly, treatment of EC with NO donor, SNAP, did not activate SREBP, suggesting that NO alone was not sufficient for SREBP activation. Rather, our findings indicated that superoxide (O2.), alone or in combination with NO, regulated SREBP activation by Ox-PAPC. We found that Ox-PAPC treatment generated O2. through an eNOS-mediated mechanism and that mercaptoethylguanidine, a peroxynitrite scavenger, reduced SREBP activation by Ox-PAPC. Taken together, these findings propose a novel role for eNOS in the activation of SREBP and SREBP-mediated inflammatory processes.