Adenosine binding to adenylyl cyclase stimulatory (A2-type) adenosine receptors (AR) induces coronary vasodilatation, inhibits aortic vascular smooth muscle cell (VSMC) proliferation, and affects arterial wall matrix production, which are important components of atherosclerosis. Aortic VSMC express both the A2aAR and A2bAR, coupled to stimulation of adenylyl cyclase, as well as the A3 adenosine receptors (A3AR), coupled to inhibition of adenylyl cyclase. Analysis of A3AR knockout mice indicated that the A3AR functions as an attenuator of effects typically mediated by the A2AR, such as vasodilation. This suggested that the relative abundance of adenosine receptors is an important determinant of adenosine-induced effects. We have recently isolated the mouse A2bAR gene and identified its active promoter region. The A2bAR gene promoter contains several putative binding sites for the proliferation-associated transcription factor, Myb, and forced expression of B-myb significantly enhances the activity of this promoter in primary VSMC. In accordance, mRNA and gene promoter activity of A2bAR, but not of A3AR or A2aAR, are upregulated in proliferating aortic VSMC compared to resting cells. On the other hand, activation of the A2bAR significantly inhibits VSMC proliferation. Our hypothesis is that during VSMC proliferation, the cells upregutate the A2bAR gene to relieve the proliferative burst. We further postulate that the regulation of expression of the A2bAR gene in VSMC plays a vital role in vascular function. Therefore, understanding the control of this gene will provide tools for modulating VSMC growth.
Four specific aims are proposed to test these contentions.
Aim 1. To define the regulation of transcription of the A2bAR gene in VSMC.
Aim 2. To explore the effect of B-Myb on A2bAR gene expression and its relation to cell cycle progression.
Aim 3. To elucidate the cell cycle mechanism by which A2bAR activation leads to inhibition of VSMC proliferation.
Aim 4. To generate an A2bAR knockout mouse model, with a knocked-in reported gene, in order to test endogenous A2bAR gene promoter activity and to examine the role of this receptor in vascular function, under control conditions or during vascular injury or atherosclerosis. These studies will focus on vascular tone, proliferation and matrix production and should shed new light on the role of A2bAR gene expression in the pathogenesis of vascular dysfunction, such as atherosclerosis.
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