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J. Biol. Chem., Vol. 280, Issue 7, 6204-6214, February 18, 2005
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Cell Cycle-mediated Regulation of Smooth Muscle 
-Actin Gene Transcription in Fibroblasts and Vascular Smooth Muscle Cells Involves Multiple Adenovirus E1A-interacting Cofactors*
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From the
Department of Medicine, University of Vermont, Burlington, Vermont 05405, Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905, and ¶Department of Physiology and Cell Biology, Dorothy M. Davis Heart and Lung Research Institute, College of Medicine and Public Health, The Ohio State University, Columbus, Ohio 43210
Expression of smooth muscle 
-actin in growth factor-induced myofibroblasts and in differentiated vascular smooth muscle cells is transcriptionally controlled by multiple positive or negative trans-acting factors interacting with distinct cis-elements in the 5'-flanking region of the gene. Because none of the transcriptional regulators reported to date is smooth muscle cell- or myofibroblast-specific per se, the dynamic interplay among many factors interacting at specific sites along the promoter appears to be a signature feature of smooth muscle -actin gene regulation in these cell types. Herein, the ability of the adenovirus E1A 12 S protein to bind and functionally inactivate specific cell regulatory factors has been exploited to identify several previously unknown coactivators of the mouse smooth muscle -actin promoter in rodent fibroblasts and vascular smooth muscle cells. In transient cotransfection assays, ectopic expression of wild type E1A suppressed promoter activity in a dose- and cis-element-dependent manner. In asynchronous cells, N-terminal E1A mutants defective in CREB-binding protein (CBP) and p300 binding capacity exhibited markedly reduced inhibitory activity toward a smooth muscle -actin promoter driven by a composite TEF-1-, SRF-, and Sp1/3-regulated enhancer. In synchronized cells, however, a more complex mutant E1A inhibitory pattern indicated that collaboration between CBP/p300 and the retinoblastoma family of pocket proteins was required to produce a fully functional enhancer. Cotransfection experiments conducted with Rb-/- fibroblasts demonstrated the necessity of pRB in augmenting smooth muscle -actin enhancer/promoter activity. Physical interaction studies with the use of purified wild type and mutant E1A proteins confirmed that CBP, p300, and pRB were targets of E1A binding in nuclear extracts of vascular smooth muscle cells and/or fibroblasts. Collectively, these results suggest that a repertoire of E1A-interacting proteins, namely CBP/p300 and pRB, serve to integrate the activities of multiple trans-acting factors to control smooth muscle -actin gene transcription in a cell type- and cell cycle-dependent manner.
Received for publication, August 18, 2004 , and in revised form, November 12, 2004.
* This study was supported by National Institutes of Health Grant HL54281 (to R. J. K.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Deceased. || To whom correspondence should be addressed: Dept. of Medicine, University of Vermont, Colchester Research Facility, 208 South Park Dr., Colchester, VT 05446. Tel.: 802-656-0329; Fax: 802-656-8969; E-mail: Robert.Kelm{at}uvm.edu.
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