Abstract
The conversion of stromal fibroblasts into contractile myofibroblasts is an essential feature of the wound-healing response that is mediated by transforming growth factor beta1 (TGF-beta1) and accompanied by transient activation of the vascular smooth muscle alpha-actin (SmalphaA) gene. Multiple positive-regulatory elements were identified as essential mediators of basal SmalphaA enhancer activity in mouse AKR-2B stromal fibroblasts. Three of these elements bind transcriptional activating proteins of known identity in fibroblasts. A fourth site, shown previously to be susceptible to single-strand modifying agents in myofibroblasts, was additionally required for enhancer response to TGF-beta1. However, TGF-beta1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the SmalphaA enhancer. By using oligonucleotide affinity isolation, DNA-binding site competition, gel mobility shift assays, and protein overexpression in SL2 and COS7 cells, we demonstrate that the transcription factors Sp1 and Sp3 can stimulate SmalphaA enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the SmalphaA enhancer required for TGF-beta1 amplification. Additionally, the TGF-beta1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for SmalphaA enhancer activation. Whereas Smad proteins collaborate with Sp1 in activating several stromal cell-associated promoters, they appear to operate independently from the Sp1/3 proteins in activating the SmalphaA enhancer. The identification of Sp and Smad proteins as essential, independent activators of the SmalphaA enhancer provides new insight into the poorly understood process of myofibroblast differentiation.
Highlights
Myofibroblasts have phenotypic properties intermediate between fibroblasts and smooth muscle cells and are thought to provide mechanical force necessary for wound contraction and closure [1,2,3,4]
At the low cell density required for efficient transfection, VSMP8 transcription was increased ϳ4-fold by transforming growth factor 1 (TGF-1), indicating that elements contained within the 5Јflanking/first intronic region mediated growth factor responsiveness (Fig. 1, b and c)
A subfragment of the 5Ј-flanking region, located between Ϫ191 and ϩ46 relative to the start of transcription (Fig. 1b), contains a potent core enhancer element previously shown to contain a MCAT motif required for highlevel constitutive expression and serum inducibility in stromal fibroblasts [21, 26], aortic smooth muscle cells [25, 29], and undifferentiated striated muscle myoblasts [21]
Summary
Myofibroblasts have phenotypic properties intermediate between fibroblasts and smooth muscle cells and are thought to provide mechanical force necessary for wound contraction and closure [1,2,3,4]. TGF-1 induces expression of Sm␣A in cultured rat aortic smooth muscle cells, bovine aortic endothelial cells, and rat fibroblasts, where specific transcriptional control elements contained within the rat Sm␣A gene promoter were shown to mediate TGF-1-dependent activation [16, 17]. The sequence context and position of DNA-regulatory elements identified in studies on the rat Sm␣A promoter and aortic smooth muscle cells differed from those in the mouse Sm␣A enhancer that undergoes TGF-1-dependent chromatin conformational changes in AKR-2B stromal fibroblasts [15]. Previous studies from our laboratories have indicated that the mouse Sm␣A gene is regulated by an array of both positive and negative cis-acting elements that behaved differently in fibroblasts compared with muscle cells (20 –27) These studies resulted in the identification of a minimal enhancer element that was constitutively active in fibroblasts, immature myoblasts, and cultured aortic smooth muscle cells, but not in quiescent, differentiated skeletal muscle myocytes. A rate-limiting role in the activation of the mouse Sm␣A enhancer is indicated for the TGF-1 receptor-regulated Smad proteins previously shown to govern transcription of TGF-1dependent genes associated with wound healing and extracellular matrix remodeling in fibroblasts
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