Smooth muscle cell (VSMC) proliferation is a key component of the pathophysiologic response to vascular injury. Central to this process is the production and activation of growth factors. One such VSMC growth factor, hepatoma derived growth factor (HDGF), becomes highly expressed in the nucleus of neointimal cells after balloon injury and is co-expressed with PCNA in proliferating VSMC in human atherosclerotic plaques. However, the mechanisms by which HDGF stimulates VSMC proliferation are unknown. The overall goal of this proposal is to elucidate the nuclear mechanisms by which HDGF stimulates VSMC proliferation in vitro and in response to injury. In our pilot studies, we found that HDGF is phosphorylated by protein kinase C, a key signaling intermediate in vascular injury. We also now have evidence that HDGF is a nuclear binding protein, functioning as a transcriptional repressor. Based upon these studies, we propose to determine the mechanisms of PKC mediated post-translational regulation of HDGF mitogenic function in vitro and in vivo and as a DMA binding, transcriptional repressor. First, we will test the hypothesis that PKC phosphorylation s critical to HDGF mitogenic function in isolated VSMC and in the wire injured mouse carotid using adenoviral expression of single or combination mutations of candidate PKC serines (S) to alanine (A, loss of phosphorylation) or aspartic acid (D, phosphorylation mimic) mutations, PKCa deficient knockout mice, and phospho-specific HDGF antibodies. Second, we will test the hypotheses that HDGF is a DNA binding protein using NMR based structure/function analyses, functions as a transcriptional repressor binding to the promoters of HDGF target genes via the controversial PWWP domain, whether transcriptional repression is mediated by the HDAC pathway and whether these activities are regulated by PKC phosphorylation. The significance of the proposed studies is that by linking HDGF activation with PKC, a critical step in the function of HDGF in vascular injury is revealed and provides the basis for new therapeutic strategies in vascular disease. Relevance to public health: Cardiovascular disease is the number one cause of death in the United States despite interventions with cholesterol lowering agents and the development of endovascular stents. This is because of the continued detrimental growth of vascular cells in the damaged arteries of patients; eventually, leading to vascular insufficiency of the brain or heart. We have identified a protein (HDGF) produced by vascular cells in diseased arteries that increases the growth of vascular cells. We propose here to determine the mechanism regulating the activity of this protein to provide the basis for the future design of new drugs to treat cardiovascular disease. ? ? ? ? ?
