Growth factors such as platelet-derived growth factor (PDGF) have been postulated to be important mediators of neointimal proliferation observed in atherosclerotic plaques and restenotic lesions following coronary interventions. Binding of growth factors to their receptors activates intrinsic receptor tyrosine kinases, resulting in tyrosine phosphorylation of receptors themselves and cellular substrate proteins. We determined that the development of intimal thickening after deendothelializing balloon catheterization of rat carotid artery was accompanied by transient 2- to 3- fold increases in the extent of tyrosine phosphorylation of platelet-derived growth factor (PDGF) alpha- and beta-receptors. The human Bcr gene was originally identified by its presence in the chimeric Bcr/Abl oncogene which is causative for leukemia. Bcr encodes a protein with serine/threonine kinase activity, CDC24/dbl homology, a GAP domain, an SH2-binding region, and a 14-3-3 binding motif. However, the normal physiological function of Bcr remains unknown. Recent studies in our laboratory have identified that Bcr is expressed in cultured vascular smooth muscle cells (VSMC) and neointimal lesions in a balloon injury model. We also determined that Bcr kinase is rapidly activated by PDGF in VSMC and Bcr overexpression enhances ERK1/2 activity in CHO cells expressing PDGF receptor. Therefore, our main hypothesis is that Bcr, activated by PDGF, may increase the VSMC migration and proliferation and thereby contribute to atherosclerosis and restenosis. Thus understanding The regulatory mechanisms by which PDGF activates Bcr should provide valuable insights into pathways which may play a critical role in cardiovascular disease. To prove this hypothesis the following aims are proposed;
Aim 1 : Characterize the stream signal mechanisms by which PDGF activates Bcr.
Aim 2 : Characterize the role of Bcr in regulating ERK1/2 in Bcr overexpression cells.
Aim 3 : Characterize the role of Bcr-Syp association in regulating PDGF beta-receptor tyrosine phosphorylation.
Aim 4 : Define the role of Bcr in regulating neointimal formation in rat carotid balloon-injury model. To determine the relative importance of the Bcr 14-3-3 binding site and SH2 binding site for Bcr kinase activation and for the effect of Bcr overexpression on PDGF signal transduction, we will transfect deletion and site-directed Bcr mutants into CHO cells expressing PDGF b-receptors. To determine potential targets of Bcr in PDGF signal transduction, we will evaluate the effects of Bcr overexpression on the activity of several PDGF signal transduction molecules, including Syp, Grb2, Ras, and Raf-1. To understand the role of Bcr in injured arteries, we will measure the magnitude and time course of Bcr mRNA and protein expression after balloon injury. We will try to establish a causative link between Bcr and neointimal formation by performing carotid injury in the bcr knock-out mouse. We propose that Bcr is a novel downstream component of PDGF receptor signaling in vascular smooth muscle, and may be a candidate gene responsible for the atherosclerosis and restenosis.
