Inhibition of the cardiac alpha-actin gene in embryonic cardiac myocytes by dominant-negative serum response factor.
Serum response factor (SRF), a transcription factor ubiquitously involved in the processes of cellular proliferation and differentiation, has been implicated in cardiac and skeletal muscle development because of its strong expression in embryonic muscle lineages, and its necessity for the transcription of transiently transfected muscle genes that contain SRF binding sites. This study was designed to ascertain whether SRF is required for the expression of an endogenous SRF-dependent gene during differentiation of early embryonic cardiac myocytes by introducing a dominant-negative SRF construct via retroviral delivery. Although no effect on overt cellular differentiation was detected, semi-quantitative RT-PCR revealed that expression of the SRF-dependent gene cardiac alpha-actin was inhibited, whereas expression of the non-SRF-dependent genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and cardiac troponin-C was unaffected. No effect on myocyte proliferation was detected. Curiously, immunohistochemical localization of SRF protein suggested that whereas endogenous SRF was homogeneously dispersed throughout the cytoplasm and nucleus, the dominant-negative SRF protein was concentrated in the nucleus. These results extend previous findings using transiently transfected genes to the endogenous level, indicating that SRF is required for the full expression of muscle genes that contain SRF binding sites during cardiac myocyte differentiation.
Zhu
Xiaolei
X
Department of Cell Biology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
McAllister
Donna
D
Lough
John
J
D000199:Actins / Q000235:genetics*; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002454:Cell Differentiation; D002642:Chick Embryo; D017931:DNA Primers / Q000737:chemistry; D018507:Gene Expression Regulation, Developmental; D005799:Genes, Dominant; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+) / Q000378:metabolism; D007124:Immunoenzyme Techniques; D032383:Myocytes, Cardiac / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012333:RNA, Messenger / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D026362:Serum Response Factor / Q000502:physiology*; D014158:Transcription, Genetic; D014162:Transfection; D019209:Troponin C / Q000378:metabolism
D000199:Actins; D017931:DNA Primers; D012333:RNA, Messenger; D026362:Serum Response Factor; D019209:Troponin C; D024601:Glyceraldehyde 3-Phosphate Dehydrogenase (NADP+)