Substitution of the degenerate smooth muscle (SM) alpha-actin CC(A/T-rich)6GG elements with c-fos serum response elements results in increased basal expression but relaxed SM cell specificity and reduced angiotensin II inducibility.
We have previously demonstrated that both CC(A/T-rich)6GG (CArG) elements A and B of the smooth muscle (SM) alpha-actin promoter are required for smooth muscle cell (SMC)-specific expression and angiotensin II (AII)-induced stimulation. Moreover, results provided evidence that AII responsiveness of SM alpha-actin was at least partially dependent on modulation of serum response factor (SRF) binding to the SM alpha-actin CArGs by the homeodomain containing protein, MHox. The goal of the present study was to investigate whether the degeneracy of the SM alpha-actin CArGs (both contain a Gua or Cyt substitution in their A/T-rich center) and their reduced SRF binding activity as compared with c-fos serum response element (SRE) is important for conferring cell type-specific expression and AII responsiveness. Transient transfection assays using SM alpha-actin reporter gene constructs in which the endogenous SM alpha-actin CArGs were replaced by c-fos SREs demonstrated the following: 1) relaxation of cell-specific expression, 2) a 50% reduction in AII responsiveness, and 3) reduced ability to be transactivated by MHox. In addition, we also showed that the position of the SM alpha-actin CArGs was important in that interchanging them abolished both basal and AII-induced activities. Taken together, these results suggest that the reduced SRF binding activities of the SM alpha-actin CArGs and CArG positional context contribute to SMC-specific expression of SM alpha-actin as well as maximal AII responsiveness.
Hautmann
M B
MB
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Madsen
C S
CS
Mack
C P
CP
Owens
G K
GK
D000199:Actins / Q000096:biosynthesis* / Q000235:genetics*; D000804:Angiotensin II / Q000494:pharmacology*; D000818:Animals; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics*; D005786:Gene Expression Regulation; D009126:Muscle Relaxation / Q000187:drug effects / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000502:physiology*; D009687:Nuclear Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D026362:Serum Response Factor; D014162:Transfection
D000199:Actins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D026362:Serum Response Factor; D000804:Angiotensin II