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Smooth muscle cell (SMC) proliferation plays a key role in vascular proliferative disorders. The molecular mechanisms that control cell cycle entry of SMCs in response to vascular injury are not well understood. Id2 (inhibitor of DNA binding) is a member of the helix-loophelix (HLH) family of transcription regulators that are known to promote cell cycle progression. Thus, we investigated the role of Id2 in SMC growth and cell cycle regulation. The results demonstrated that overexpression of Id2 resulted in a significant enhancement of SMC growth via increased S-phase entry. A possible mechanism of Id2-enchanced SMC growth is via regulation of p21 expression, as overexpression of Id2-inhibited transcriptional activity of a 2.3-kb p21 promoter/luciferase reporter construct as well as p21 protein levels. Id2 enhancement of SMC growth and inhibition of p21 expression were dependent on phosphorylation of Id2 by cyclin E/cdk2, as an Id2 cDNA containing a mutation in the cdk2 phosphorylation site (serine 5) failed to regulate SMC cell cycle progression or p21 promoter activity. The mechanism of cyclin E/cdk2 control of the Id2 effect may in part involve regulation of nuclear transport; unlike wild-type Id2, the Id2 mutant was not transported to the nucleus. Finally, in a rat carotid model of arterial injury, Id2 was expressed in a temporal pattern that parallels the kinetics of cellular proliferation. In summary, these results provide evidence that the Id2 protein is integrated into the cell cycle regulatory cascade that results in SMC proliferation following vascular injury and suggest that this effect is at least in part via a cdk2-dependent inhibition of p21 gene expression. Smooth muscle cell (SMC)1 proliferation is a key component of the response to vascular injury in atherosclerosis and restenosis in humans and following experimental angioplasty in animals (1-3). Several recent studies have focused interest on regulation of the cell cycle as a means of modulating this pathologic SMC growth (4 -6). Of note, recent evidence links the helix-loop-helix (HLH) factors to cell cycle control in a variety of cell types (7-9). Several of these studies have demonstrated that members of the Id class of HLH proteins are involved in cell cycle regulation. Lasorella et al. (10) identified a unique role for Id2 in cell cycle regulation. Id2 is able to bind the retinoblastoma protein (Rb) and Rb-related proteins and functions as an antagonist to the growth suppressive effects of these proteins. Through this mechanism, Id2 was able to reverse p16-and p21-induced cell cycle arrest. In contrast, Id1 and Id3 did not bind to or inhibit the function of Rb, providing evidence that specific members of the Id family have unique roles in regulating proliferation (10). Given the established paradigm for Id function, it is appealing to hypothesize that the Id proteins also affect cellular proliferation via their ability to dimerize and block transactivation of genes involved in cell cycle regulation. Indeed, recent evidence (11, 12) suggests...

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