Identification of a protective role for protein phosphatase 1cγ1 against oxidative stress-induced vascular smooth muscle cell apoptosis

The development of therapeutic strategies to inhibit reactive oxygen species (ROS)-mediated damage in blood vessels has been limited by a lack of specific targets for intervention. Targeting ROS-mediated events in the vessel wall is of interest, because ROS play important roles throughout atherogenesis. In early atherosclerosis, ROS stimulate vascular smooth muscle cell (VSMC) growth, whereas in late stages of lesion development, ROS induce VSMC apoptosis, causing atherosclerotic plaque instability. To identify putative protective genes against oxidative stress, mouse aortic VSMC were infected with a retroviral human heart cDNA expression library, and apoptosis was induced in virus-infected cells by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ) treatment. A total of 17 different, complete cDNAs were identified from the DMNQ-resistant VSMC clones by PCR amplification and sequencing. The cDNA encoding PP1c gamma 1 ( catalytic subunit of protein phosphatase 1) was present in several independent DMNQ-resistant VSMC clones. DMNQ increased mitochondrial ROS production, caspase-3/7 activity, DNA fragmentation, and decreased mitochondrial transmembrane potential in VSMC while decreasing PP1c gamma 1 activity and expression. Depletion of PP1c gamma 1 expression by short hairpin RNA significantly enhanced basal as well as DMNQ-induced VSMC apoptosis. PP1c gamma 1 overexpression abrogated DMNQ-induced JNK1 activity, p53Ser(15) phosphorylation, and Bax expression and protected VSMC against DMNQ-induced apoptosis. In addition, PP1c gamma 1 overexpression attenuated DMNQ-induced caspase-3/7 activation and DNA fragmentation. Inhibition of p53 protein expression using small interfering RNA abrogated DMNQ-induced Bax expression and significantly attenuated VSMC apoptosis. Together, these data indicate that PP1c gamma 1 overexpression promotes VSMC survival by interfering with JNK1 and p53 phosphorylation cascades involved in apoptosis.