Contribution of the Helix-Loop-Helix Factor Id2 to Regulation of Vascular Smooth Muscle Cell Proliferation

Abstract

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...

The helix–loop–helix protein ID1 localizes to centrosomes and rapidly induces abnormal centrosome numbers

“…In support of this notion, there is a Interference of ID1 with either the pRb or the p53 tumor suppressor pathway is unlikely to contribute to the observed cellular phenotype, since ID1 was able to induce centrosome abnormalities in SaOS-2 human osteosarcoma cells that contain mutated p53 and pRb (Lauricella et al, 2001). ID-proteins have been shown do inhibit the expression of the CDK-inhibitory proteins p16 INK4A , p21 Waf1/Cip1 , and p27 Kip1 (Lasorella et al, 1996;Prabhu et al, 1997;Pagliuca et al, 2000;Alani et al, 2001;Ohtani et al, 2001;Matsumura et al, 2002;Mueller et al, 2002;Tang et al, 2002). Since all IDproteins were able to inhibit expression of a p21 Waf1/Cip1 reporter construct, we consider it unlikely that ID1 induces centrosome abnormalities through inhibition of p21 Waf1/Cip1 even though repression of p21 Waf1/Cip1 using antisense constructs in human hematopoietic cells results in centrosome abnormalities (Mantel et al, 1999).…”

TWIST Family of Basic Helix-Loop-Helix Transcription Factors Mediate Human Mesenchymal Stem Cell Growth and Commitment

“…Overexpression of Id proteins in embryonic-derived fibroblasts facilitates cell cycle S-phase under serum-deprived conditions [34], where Id-1 has been reported to induce the proliferation of cochlear sensory epithelial cells via the NF-kappaB/cyclin D1 pathway [35]. Furthermore, various cancer cell lines appear to require Id-1 and Id-2 expression for their growth, survival, and metastasis via regulation of p53 and PI3K/Akt/NF-kappaB signaling pathways in a similar fashion to that described for Twist-1 and Dermo-1 [36][37][38][39][40]. More recently, Id-1 has been shown to mediate long-term repopulating hematopoietic stem cell maintenance and inhibit myeloid differentiation [41,42].…”

Over-expression of Id2 and Id3 Proteins Regulates Growth and Survival of Human Colon Carcinoma (HCT116) Cells

“…Evidence available suggests that Id proteins are localized in the cytoplasm rather than the nucleus in a variety of cells such as hemopoietic [20], neural [21], muscle [22], and renal cells [23]. These findings therefore suggest that translocation between the nucleus and the cytoplasm (nucleocytoplasmic shuttling) may be involved in the functional regulation of Id proteins.…”

“…Evidence available shows that Cyclin A-and Cyclin E-dependent CDK2 phosphorylation of Id2 and Id3 proteins at a conserved serine residue at position 5 regulates the ability of these proteins to antagonize bHLH-dependent gene expression [12]. It has also been shown that phospho-ablated Id2 Ala5 mutant where serine at position 5 is mutated to an Alanine is growth inhibitory in fibroblasts, smooth muscle cells and osteosarcoma cell lines [12,22]. Literature available also suggest that Id2-induced apoptosis is promoted through a mechanism different from what is used by other Id proteins.…”