A Transforming Growth Factor β (TGFβ) Control Element Drives TGFβ-induced Stimulation of Smooth Muscle α-Actin Gene Expression in Concert with Two CArG Elements*
- From the Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908
Abstract
The goal of the present study was to determine the molecular mechanism whereby transforming growth factor β (TGFβ) increases smooth muscle (SM) α-actin expression. Confluent, growth-arrested rat aortic smooth muscle cells (SMC) were transiently transfected with various SM α-actin promoter/chloramphenicol acetyltransferase deletion mutants and stimulated with TGFβ (2.5 ng/ml). Results demonstrated that the first 125 base pairs of the SM α-actin promoter were sufficient to confer TGFβ responsiveness. Three cis elements were shown to be required for TGFβ inducibility: two highly conserved CArG boxes, designated A (−62) and B (−112) and a novel TGFβ control element (TCE) (−42). Mutation of any one of these elements completely abolished TGFβ-induced reporter activity. Results of electrophoretic mobility shift assays demonstrated that nuclear extracts from TGFβ-treated SMC enhanced binding activity of serum response factor to the CArG elements and binding of an as yet unidentified factor to the TCE. Northern analysis showed that TGFβ also stimulated transcription of two other SM (SM myosin heavy chain) differentiation marker genes, SM myosin heavy chain and h1calponin, whose promoters also contained a TCE-like element. In summary, we identified a TGFβ response element in the SM α-actin promoter that may contribute to coordinate regulation of expression of multiple cell-type specific proteins during SMC differentiation.
Footnotes
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↵* This work was supported by Grants RO1 HL 38854 and PO1 HL 19242 from the National Institutes of Health (to G. K. O.) and Fellowship Grants VA-94-F-14 (to M. H.) and VA-95-F-18 (to C. M.) from the Virginia Affiliate of the American Heart Association.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Molecular Physiology and Biological Physics, Box 10011, University of Virginia Health Sciences Center, Charlottesville, VA 22906-0011. Tel.: 804-924-2652; Fax: 804-982-0055.
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↵1 The abbreviations used are: SMC, smooth muscle cell(s); SM MHC, smooth muscle myosin heavy chain; bp, base pair(s); CArG element, CC(A/T-rich)6GG; SRF, serum response factor; SRE, serum response element; CAT, chloramphenicol acetyltransferase; TGFβ, transforming growth factor β 1; TCE, TGFβ control element; kb, kilobase pair(s); MOPS, 4-morpholinepropanesulfonic acid; EMSA, electrophoretic mobility shift assay; DOTAP,N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate.
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↵2 M. M. Thompson and G. K. Owens, unpublished observations.
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↵3 M. B. Hautmann and G. K. Owens, unpublished observations.
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- Received August 6, 1996.
- Revision received December 13, 1996.
