Arterial smooth muscle cells (SMCs) typically exist in a differentiated, quiescent state that is maintained, in part, by the release of antimitogens, such as prostacyclin (PGI2), from intimal endothelial cells. Arterial injury, such as that associated with atherosclerosis and balloon angioplasty, impairs the release of these antimitogens and (together with injury-associated exposure to local mitogens and changes in the extracellular matrix) leads to the """"""""phenotypic modulation"""""""" of vascular SMCs to a proliferative state. The importance of SMC proliferation in the vascular remodeling response to injury is established, but the cell cycle regulatory mechanisms that control the quiescent and proliferative states are not. We have studied the effects of mitogens and PGI2 on the cyclin-dependent kinases (cdks; the critical enzymes regulating cell cycle progression) and find that these positive and negative regulators act largely by controlling levels of the cdk inhibitory protein, p27kip1. Moreover, this convergence is also seen in the regulation of Skp2, the E3 ubiquitin ligase that controls the degradation of p27kip1. Several studies have characterized mechanisms that regulate Skp2 post-transcriptionally, but we find that the major effect of mitogens and PGI2 is on the expression of Skp2 mRNA in both fibroblasts and SMCs. We therefore propose three specific aims to test the hypothesis that the Skp2 gene is the major focal point of mitogen and PGI2 signaling during injury-induced vascular remodeling.
In Aim 1, we will use SMCs and MEFs to characterize the mechanisms by which mitogens regulate induction of the Skp2 gene. Relevant signal transduction cascades and key promoter elements will be identified. Potential mitogen effects on Skp2 mRNA stability will be explored.
In Aim 2, we will use the same systems and parallel approaches to characterize the mechanisms by which PGI2 antagonizes mitogen-dependent induction of Skp2 mRNA.
In Aim 3, we will use femoral artery injury in knock-out and transgenic mice to determine the importance of the PGI2-IP-Skp2 pathway on SMC proliferation in vivo. Together, the results will provide new insight into the basic regulatory mechanisms controlling Skp2 gene expression and directly test the importance of Skp2 on SMC proliferation in an in vivo model with clear relevance to cardiovascular pathology. Relevance: Aberrant proliferation of smooth muscle cells is an important aspect of cardiovascular disease. This research will help to identify the genes and proteins that regulate smooth muscle cell proliferation. It will also test the importance of these molecules in an in vivo model of vascular injury. ? ? ?
