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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...

Abstract-The molecular mechanisms that regulate the proliferation of smooth muscle cells (SMCs) of the vasculature in response to injury are poorly understood. Members of the inhibitor of DNA binding (Id) class of helix-loop-helix transcription factors are known to regulate the growth of a variety of cell types; however, the expression of the various Id genes in SMCs and in vascular lesions has not been examined. In the present study, the yeast 2-hybrid system was used to clone Id genes from a cultured rat aortic SMC library. By use of ubiquitous E proteins as bait, Id3 and a novel isoform of Id3 (Id3a) were cloned. Id3a is the product of alternative splicing of the Id3 gene, resulting in inclusion of a 115-bp "coding intron," which encodes a unique 29 -amino acid carboxyl terminus for the Id3a protein. Unlike Id3, Id3a mRNA was not detected in the normal rat carotid artery. However, after balloon injury, Id3a was abundantly expressed throughout the neointimal layer. In addition, mRNA of the human homologue of Id3a (Id3L) Key Words: smooth muscle cells Ⅲ helix-loop-helix factors Ⅲ atherosclerosis Ⅲ vascular injury Ⅲ alternative splicing T he phenotype of smooth muscle cells (SMCs) within atherosclerotic and restenotic lesions in humans 1,2 and lesions generated after vascular injury in experimental animals 3-5 is modulated relative to normal medial SMCs. This modulation is characterized by loss of myofilaments, formation of a large endoplasmic reticulum and the Golgi complex, migration into the neointima, and extracellular matrix secretion. 4 In addition, this phenotypic modulation is accompanied by alterations in growth factor receptor expression and growth responsiveness, resulting in enhanced proliferation. The proliferation of intimal SMCs leads to encroachment on the vessel lumen. Thus, the identification of factors that enhance and inhibit the growth of SMCs is important for understanding and potentially intervening in the SMC response that leads to vascular lesion formation. See cover and page 727