© 2001 American Heart Association, Inc.
Editorials |
A Role for an Alternatively Spliced Id3 Isoform in Vascular Lesions?
From the Department of Molecular Medicine, Institute of Biotechnology, University of Texas Health Science Center at San Antonio.
Correspondence to Dr Barbara Christy, Institute of Biotechnology, UT Health Science Center at San Antonio, 15355 Lambda Dr, San Antonio, TX 78245. E-mail christy{at}uthscsa.edu
Key Words: helix-loop-helix • vascular smooth muscle • alternative splicing • atherosclerosis
Alterations in proliferation, migration, and function of vascular smooth muscle cells (SMCs) are important in the formation of atherosclerotic and restenotic lesions.1 Although of tremendous clinical importance, the precise molecular mechanisms controlling the proliferation and differentiation of SMCs are not completely understood. Regulation of growth and differentiation in skeletal and cardiac muscle involves a class of transcription factors called helix-loop-helix (HLH) proteins, but the situation in smooth muscle is less clear. The study by Matsumura et al2 in this issue of Arteriosclerosis, Thrombosis, and Vascular Biology investigates a particular HLH protein in vascular SMCs in culture and in vivo after vascular injury.
HLH transcriptional regulatory proteins are implicated in control of cell growth and terminal differentiation in a number of cell types.3 The HLH motif mediates dimer formation among HLH proteins. Most HLH proteins contain a region of basic amino acids adjacent to the HLH domain that constitutes the DNA-binding domain; heterodimers or homodimers of basic HLH (bHLH) proteins generally bind to a conserved DNA binding site (E-box). A subfamily of HLH proteins (Inhibitor of DNA binding [Id] proteins) contain an HLH dimerization domain but lack the basic region and thus are not able to bind DNA.4 Id proteins interact with selected DNA-binding bHLH proteins and negatively regulate their transcriptional activity by preventing formation of functional DNA-binding dimers. Although other potential Id protein functions and interactions have been described,4 this dominant negative activity is thought to be a major mechanism of Id protein action. Four members of the Id subfamily of HLH proteins have been identified in mammals (Id1 to 4). A major target for negative regulation by Id proteins is E proteins, widely expressed bHLH proteins that form heterodimers with tissue-restricted bHLH proteins and are important in regulation of cellular differentiation.3 4 Ectopic Id expression inhibits differentiation of several cell types in culture.4 In general, Id proteins are thought to be positive regulators of cell growth that suppress growth-inhibitory effects of bHLH proteins involved in differentiation.4 At least 1 Id protein (Id2) can affect cell growth by interaction with non-HLH proteins of the retinoblastoma (pRb) tumor suppressor family.5 Increased Id2 suppresses growth inhibition by pRb, whereas a lack of Id2 in Rb-null mice can (partially) counteract the embryonic lethal phenotype.5 Although Id1 and Id3 cannot interact with pRb under similar conditions, they may exert their growth effects by influencing another molecule in the same pathway. Id1 in particular can inhibit transcription of the p21 cell cycle inhibitor mediated by E proteins6 and thus may be stimulating cell proliferation by this mechanism.
The present study implicates one of the Id-family proteins, Id3, in proliferation of vascular SMCs and their response to injury. Id3 was initially isolated as an immediate-early gene after mitogenic stimulation of quiescent fibroblasts; expression is induced dramatically by growth factors, including some that are important in vascular SMCs.7 A yeast 2-hybrid screen was performed by use of a SMC cDNA library with 2 different E proteins as bait to identify smooth muscle HLH proteins. All of the clones isolated in this screen corresponded to Id3. Interestingly, 2 forms of Id3 were isolated: the previously described Id3 and a splice variant that includes a "coding intron." The protein product of this isoform (Id3a) is identical in the N-terminal and HLH regions to the previously described Id3 protein but contains a unique C-terminus with no homology to the C-terminus of fully spliced Id3. The unique isoform of the rat Id3 protein (Id3a) identified in this work is analogous to an alternatively spliced version of human Id3 (Id3L) described previously.8 Both rat Id3a and human Id3L incorporate coding introns generating a unique C-terminus, but the amino acids encoded in the C-termini are divergent. The human Id3L isoform has an altered function relative to "normal" Id3 in vitro; its ability to interact with and inhibit DNA binding by the bHLH protein E47 was greatly impaired.8 A similar alternatively spliced Id1 retained an analogous coding intron9 ; this alternative Id1 has increased propensity to homodimerize relative to normal Id1. Although the dimerization affinities and ability to negatively regulate bHLH protein activation were not investigated for the rat Id3a isoform described here, the authors do show a functional difference between the 2 isoforms when ectopically expressed in cultured SMCs. Infection of cells with an adenovirus vector expressing Id3a that is unable to be spliced into normal Id3 resulted in a significant increase in cells undergoing apoptotic cell death relative to an adenovirus expressing normal Id3. Id proteins have previously been implicated in apoptotic cell death,4 although it is not yet clear whether these findings pertain mainly to cells in which Ids are expressed ectopically or whether they play a role in apoptosis in vivo.
The most exciting finding is that the 2 Id3 isoforms are differentially expressed in SMCs under certain conditions. Both forms are detected in cultured SMCs, endothelial cells, and fibroblasts, but Id3a is expressed at a much lower level. In vivo, no Id3a mRNA is detected in uninjured carotid arteries or 24 hours after balloon endothelial denudation of the artery. In contrast, at later time points (6 to 28 days), Id3a is detected in the growing neointima and the periluminal medial layers. This pattern of expression is much different from that seen for fully spliced Id3, which is expressed diffusely throughout the media of the uninjured carotid and at 24 hours after balloon injury but is decreased in this region at later times. Expression of the human alternatively spliced Id3 (Id3L) was also investigated in human carotid artery atherosclerotic lesions from surgical specimens. Significantly, Id3L mRNA was detected in atherosclerotic plaques from these specimens, localized to regions of plaque containing SMCs.
Id3 and Id1 have been implicated in angiogenesis and tumor vascularization.10 Although complete inactivation of both results in embryonic lethality, there are vascular defects in animals with reduced copy numbers of the 2 genes. Id proteins have also been implicated in other pathological states, including tumors, in which they are thought to contribute to altered growth control. This work is the first indication that Id proteins may play a role in atherosclerosis and restenosis. Because the alternatively spliced Id3a is relatively specific for the area of developing lesions, it may be possible to develop inhibitors specific for Id3a/Id3L that do not alter function of normal Id3. This work describes an intriguing area for study, although many questions remain to be answered. Are there functional differences between the 2 Id3 isoforms? Can differences in activity be accounted for by the absence of the normal Id3 C-terminus (shown previously to contribute to stability and activity11 ), or is the alternative C-terminus important? Do the 2 isoforms interact differently with either HLH or non-HLH proteins, and what are their transcriptional effects? Do they affect cell growth and differentiation differently? Does Id3a play a role in normal SMCs, or is it a marker for a pathological state?
Acknowledgments
Dr Christy was supported by a grant-in-aid from the American Heart Association, Texas Affiliate, and by a grant from the Children’s Cancer Research Center, San Antonio, Tex.
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