Abstract 158: A Novel Approach for Identifying Genes that Protect Against Oxidative Stress: Protection Against Reactive Oxygen Species-Mediated Apoptosis by Protein Phosphatase 1
Reactive oxygen species (ROS) play an important role in atherogenesis: in early atherogenesis ROS stimulate vascular smooth muscle cells (VSMC) growth; however increased ROS production at later stages of atherosclerotic lesion development is associated with VSMC apoptosis, which likely contributes to atherosclerotic plaque instability. To identify putative protective genes against oxidative stress, we infected mouse aortic VSMC with a retroviral human heart cDNA expression library and then induced apoptosis in virus-infected cells by 2,3-Dimethoxy-1,4-naphthoquinone (DMNQ) treatment. The cell-permeable non-alkylating, non-thiol quinone DMNQ generates ROS by redox cycling and induces VSMC cell death reproducibly in a dose dependent manner. Seventy eight individual VSMC clones survived DMNQ treatment. A total of 14 different, complete cDNAs were identified from the DMNQ resistant VSMC clones by PCR amplification and sequencing. One of the most common cDNAs encoded the catalytic subunit of protein phosphatase 1 gamma 1 (PP1Cgamma1). To confirm that PP1C protected VSMC against DMNQ-induced apoptosis, VSMC were infected with retrovirus containing a full length PP1Cgamma1 cDNA and then treated with DMNQ. PP1Cgamma1 overexpression significantly reduced DMNQ-induced apoptosis. To investigate the mechanisms of PP1Cgamma1 cell protection we analyzed expression and phosphorylation of several genes involved in apoptosis signaling pathways (Bcl-2, Bax, p53, p38, JNK1). PP1 Cgamma1 overexpression decreased DMNQ-induced phosphorylation of JNK1 and p53 (Ser15) in VSMC. Further, PP1Cgamma1 directly binds to JNK1 as observed by immunoprecipitation and recombinant PP1Cgamma1 dephosphorylates JNK1. These findings suggest that PP1Cgamma1 might play an important role in the survival of VSMC in advanced atherosclerotic lesions via suppression of JNK1 and p53 pathways.