The Smooth Muscle -Actin Gene Promoter Is Differentially Regulated in Smooth Muscle versus Non-smooth Muscle Cells

doi:10.1074/jbc.270.13.7631

The Smooth Muscle -Actin Gene Promoter Is Differentially Regulated in Smooth Muscle versus Non-smooth Muscle Cells (*)

From the Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

^¶ To whom correspondence should be addressed:
Dept of Molecular Physiology and Biological Physics, Box 449, University of Virginia Health Sciences Center, Charlottesville, VA 22908
. Tel.: 804-924-2652; Fax: 804-982-0055.

Abstract

To identify potential regulators of smooth muscle cell (SMC) differentiation, we studied the molecular mechanisms that control the tissue-specific transcriptional expression of SM α-actin, the most abundant protein in fully differentiated SMCs. A construct containing the region from −1 to −125 of the promoter (p125CAT) had high transcriptional activity in SMCs (57-fold > promoterless) and endothelial cells (ECs) (18-fold) but not in skeletal myoblasts or myotubes. Mutation of either of two highly conserved CC(AT-rich)₆GG (CArG) motifs at −62 and −112 abolished the activity of p125CAT in SMCs but had no effect in ECs. In contrast, high transcriptional activity in skeletal myotubes, which also express SM α-actin, required at least 271 base pairs of the promoter (−1 to ≥ −271). Constructs containing 547 base pairs or more of the promoter were transcriptionally active in SMCs and skeletal myotubes but had no activity in skeletal myoblasts or ECs, cell types that do not express SM α-actin. Electrophoretic mobility shift assays provided evidence for binding of a unique serum response factor-containing complex of factors to the CArG box elements in SMCs. Results indicate that: 1) transcriptional expression of SM α-actin in SMCs requires the interaction of the CArG boxes with SMC nucleoprotein(s); 2) expression of SM α-actin in skeletal myotubes requires different cis-elements and trans-factors than in SMCs; and 3) negative-acting cis-elements are important in restricting transcription in cells that do not express SM α-actin.

Footnotes

↵^§ Supported by training Grant T32 GM-07267 from the National Institutes of Health during the performance of this work.
↵* This work was supported by the National Institutes of Health Grant RO1-HL-38854 and by the University of Virginia Cancer Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

SMC

smooth muscle cell

SM

smooth muscle

bp

base pair(s)

CArG box

CC(A/T-rich)₆GG

SRE

serum response element

SRF

serum response factor

CAT

chloramphenicol acetyltransferase

RAEC

rat aortic endothelial cells

BAEC

bovine aortic endothelial cells

PCR

polymerase chain reaction

EMSA

electrophoretic mobility shift assays.
↵² R. T. Shimizu and G. K. Owens, unpublished results.
- Received October 13, 1994.
- Revision received December 19, 1994.