Effect of serum and mechanical stretch on skeletal alpha-actin gene regulation in cultured primary muscle cells.
The purpose of this study was to determine whether mechanical stretch or serum availability alters pretranslational regulation of skeletal alpha-actin (SkA) in cultured striated muscle cells. Chicken primary skeletal myoblasts and cardiac myocytes were plated on collagenized Silastic membranes adherent to nylon supports and stretched 8-20% of initial length 96 h postplating. Serum dependence of SkA gene regulation was determined by maintaining differentiated muscle cells in growth/differentiation (G/D; skeletal myotubes, 10% horse serum-2% chick embryo extract; cardiac myocytes, 10% horse serum) or growth-limiting (G-L; 0.5% horse serum) medium. Skeletal myotubes had higher SkA mRNA and SkA promoter activity in G/D than in G-L medium. Cardiac myocyte SkA mRNA was higher in G-L than in G/D medium. Serum response factor (SRF) protein binding to serum response element 1 (SRE1) of SkA promoter increased in skeletal cultures in G/D compared with G-L medium. Western blot analysis demonstrated that increased SRF-SRE1 binding was due, in part, to increased SRF protein. Stretching skeletal myotubes in G-L medium reduced SkA mRNA and repressed SkA promoter activity. The first 100 bp of SkA promoter were sufficient for stretch-induced repression of SkA promoter activity, and an intact transcriptional enhancer factor 1 (TEF-1) binding site was necessary for this response. Serum and stretch appear to repress SkA promoter activity in skeletal myotubes through different DNA binding elements, the SRE1 and TEF-1 sites, respectively. Stretching increased SkA mRNA in cardiac myocytes in G-L medium but did not alter SkA mRNA level in cardiac cells in G/D medium. These results demonstrate that stretch and serum interact differently to alter SkA expression in cultured cardiac and skeletal muscle cells.
Carson
J A
JA
Department of Integrative Biology, Pharmacology, and Physiology, University of Texas Health Science Center, Houston, Texas 77030, USA.
Booth
F W
FW
D000199:Actins / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001790:Blood Physiological Phenomena; D001842:Bone and Bones / Q000378:metabolism*; D002478:Cells, Cultured; D002642:Chick Embryo; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology*; D006321:Heart / Q000196:embryology / Q000502:physiology*; D009124:Muscle Proteins / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology / Q000196:embryology / Q000502:physiology*; D009206:Myocardium / Q000166:cytology; D009687:Nuclear Proteins / Q000235:genetics; D009994:Osmolar Concentration; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D026362:Serum Response Factor; D013314:Stress, Mechanical; D000091262:TEA Domain Transcription Factors; D014157:Transcription Factors / Q000378:metabolism
D000199:Actins; D004268:DNA-Binding Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D026362:Serum Response Factor; D000091262:TEA Domain Transcription Factors; C057182:TEAD1 protein, human; D014157:Transcription Factors