Abstract
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.
Highlights
Ate-early response genes [1,2,3,4,5] including c-fos and has been shown to confer serum- and growth factor-induced transcriptional activation of these genes
The SM ␣-Actin CArG Elements A And B Showed Reduced SRF Binding as Compared with the c-fos SRE—There is extensive evidence showing that the internal A/T-rich center of CArG elements affects SRF binding affinities [24, 31, 32, 53]
To determine whether the pattern of SRF binding activities to the different CArG elements was similar with SMC nuclear extracts as compared with those observed with recombinant SRF, we performed gel shift assays using nuclear extracts derived from SMC grown in 10% serum
Summary
Ate-early response genes [1,2,3,4,5] including c-fos and has been shown to confer serum- and growth factor-induced transcriptional activation of these genes (reviewed in Ref. 6). In contrast to the c-fos gene which contains a single high affinity binding site for SRF in its promoter, many musclespecific genes including skeletal, cardiac, SM ␣-actin, SM MHC, and SM-22␣ contain two or more CArG elements [7, 20, 24, 28, 29]. The aim of the present study was to determine whether sequence degeneracy of the SM ␣-actin CArG elements and their reduced SRF binding activity contributes to cell-specific SM ␣-actin expression as well as the ability of the gene to be regulated in response to AII or the mesodermally restricted homeodomain containing protein MHox
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