A transforming growth factor beta (TGFbeta) control element drives TGFbeta-induced stimulation of smooth muscle alpha-actin gene expression in concert with two CArG elements.
The goal of the present study was to determine the molecular mechanism whereby transforming growth factor beta (TGFbeta) increases smooth muscle (SM) alpha-actin expression. Confluent, growth-arrested rat aortic smooth muscle cells (SMC) were transiently transfected with various SM alpha-actin promoter/chloramphenicol acetyltransferase deletion mutants and stimulated with TGFbeta (2.5 ng/ml). Results demonstrated that the first 125 base pairs of the SM alpha-actin promoter were sufficient to confer TGFbeta responsiveness. Three cis elements were shown to be required for TGFbeta inducibility: two highly conserved CArG boxes, designated A (-62) and B (-112) and a novel TGFbeta control element (TCE) (-42). Mutation of any one of these elements completely abolished TGFbeta-induced reporter activity. Results of electrophoretic mobility shift assays demonstrated that nuclear extracts from TGFbeta-treated SMC enhanced binding activity of serum response factor to the CArG elements and binding of an as yet unidentified factor to the TCE. Northern analysis showed that TGFbeta also stimulated transcription of two other SM (SM myosin heavy chain) differentiation marker genes, SM myosin heavy chain and h1 calponin, whose promoters also contained a TCE-like element. In summary, we identified a TGFbeta response element in the SM alpha-actin promoter that may contribute to coordinate regulation of expression of multiple cell-type specific proteins during SMC differentiation.
Hautmann
M B
MB
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, USA.
Madsen
C S
CS
Owens
G K
GK
D000199:Actins / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D001011:Aorta; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D016384:Consensus Sequence; D017124:Conserved Sequence; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D051379:Mice; D008840:Microfilament Proteins; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D018995:Myosin Heavy Chains / Q000096:biosynthesis; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012045:Regulatory Sequences, Nucleic Acid; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection; D016212:Transforming Growth Factor beta / Q000494:pharmacology*; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D015500:Chloramphenicol O-Acetyltransferase; D008840:Microfilament Proteins; D018995:Myosin Heavy Chains; D011993:Recombinant Fusion Proteins; D016212:Transforming Growth Factor beta; D000096985:Calponins