Abstract
Transforming growth factor beta (TGF-beta) is implicated in the regulation of smooth muscle cell (SMC) differentiation. We previously identified a novel TGF-beta control element (TCE) in the promoters of SMC differentiation marker genes, including alpha-smooth muscle actin and SM22alpha. In this study, the importance of the TCE in regulation of SM22alpha gene expression in vivo was investigated by mutating it within the context of a mouse SM22alpha promoter-lacZ transgenic construct. Mutation of the TCE completely abolished SM22alpha promoter activity in arterial SMCs as well as in developing heart and skeletal muscle. To identify the transcription factor(s) binding to the TCE, we performed yeast one-hybrid cloning analysis and identified gut-enriched Krüppel-like factor (GKLF). However, cotransfection studies in cultured cells showed that GKLF repressed the TGF-beta-dependent increases in SM22alpha and alpha-smooth muscle actin promoter activities. Furthermore, GKLF was not highly expressed in differentiated SMCs in vivo, and TGF-beta down-regulated GKLF expression in dedifferentiated cultured SMCs. In contrast, overexpression of a related factor (BTEB2) transactivated SM22alpha promoter activity. Thus, our findings suggest a reciprocal role for related Krüppel-like transcription factors in the regulation of SMC differentiation through a TCE-dependent mechanism.
Highlights
§ To whom correspondence should be addressed: Dept. of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, P
Electrophoretic Mobility Shift Assays (EMSAs) using nuclear extracts from vehicle- and TGF--stimulated aortic smooth muscle cell (SMC) demonstrated that the SM22␣ TGF- control element (TCE) formed a TGF--dependent shift complex identical in mobility to that of the ␣-SM actin TCE (Fig. 1B, lanes 1– 4)
We have shown that the TCE is required for Ϫ445 SM22␣ promoter activity in transgenic mice and, using yeast one-hybrid cloning, identified the Kruppel-like transcription factor gut-enriched Kruppellike factor (GKLF) as a TCE-binding protein
Summary
§ To whom correspondence should be addressed: Dept. of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, P. Disruption of SMAD5 and SMAD6, intracellular mediators of signaling by TGF- superfamily ligands, has resulted in defects in vascular development [9, 10] Consistent with these in vivo studies, TGF- has been shown to stimulate expression of ␣-SM actin, SM myosin heavy chain, and SM22␣ in SMCs that have undergone partial dedifferentiation (or modulation) in culture [11]. TGF- has been shown to induce expression of these SMC differentiation marker genes in a variety of non-SM precursor cell types in culture, including multipotent embryonic 10T1/2 cells and neural crest cells [12,13,14,15] These observations indicate an important role for TGF- in promoting the differentiation of mesenchymal cells toward a contractile smooth muscle fate and in regulating changes in vascular SMC phenotype, the molecular mechanisms responsible for this effect are unknown. Unlike the endogenous SM22␣ gene that is expressed in virtually all SMC subtypes [20], this region of the promoter is active only in a subset of arterial SMCs throughout development [19, 21]
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