Vascular smooth muscle alpha-actin gene transcription during myofibroblast differentiation requires Sp1/3 protein binding proximal to the MCAT enhancer.
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.
Department of Physiology/Cell Biology, The Ohio State University College of Medicine and Public Health, Dorothy M. Davis Heart and Lung Research Institute, Columbus, Ohio 43210, USA.
John G
JG
Cogan
Sukanya V
SV
Subramanian
John A
JA
Polikandriotis
Robert J
RJ
Kelm
Arthur R
AR
Strauch
D000199:Actins / Q000096:biosynthesis* / Q000235:genetics*; D020816:Amino Acid Motifs; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D015152:Blotting, Northern; D015153:Blotting, Western; D019556:COS Cells; D002454:Cell Differentiation; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004330:Drosophila; D004730:Endothelium, Vascular / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005109:Extracellular Matrix / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000378:metabolism*; D009132:Muscles / Q000166:cytology*; D009154:Mutation; D009841:Oligonucleotides / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D008926:Plicamycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000378:metabolism; D053773:Transforming Growth Factor beta1
D000199:Actins; D004268:DNA-Binding Proteins; D009841:Oligonucleotides; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C497122:Sp3 protein, mouse; C497123:Sp3 protein, rat; C508960:TGFB1 protein, human; C508961:Tgfb1 protein, mouse; C508962:Tgfb1 protein, rat; D014157:Transcription Factors; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D051705:Sp3 Transcription Factor; D004247:DNA; C066851:mithramycin A; D008926:Plicamycin