Vascular smooth muscle α-actin gene transcription during myofibroblast differentiation requires Sp1/3 protein binding proximal to the MCAT enhancer

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 β1 (TGF-β1) and accompanied by transient activation of the vascular smooth muscle α-actin (SmαA) gene. Multiple positive-regulatory elements were identified as essential mediators of basal SmαA 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-β1. However, TGF-β1 activation was not accompanied by a stoichiometric increase in protein binding to any known positive element in the SmαA 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 SmαA enhancer activity. One of the sites that bind Sp1/3 corresponds to the region of the SmαA enhancer required for TGF-β1 amplification. Additionally, the TGF-β1 receptor-regulated Smad proteins, in particular Smad3, are rate-limiting for SmαA 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 SmαA enhancer. The identification of Sp and Smad proteins as essential, independent activators of the SmαA enhancer provides new insight into the poorly understood process of myofibroblast differentiation.