Contribution of serum response factor and myocardin to transcriptional regulation of smoothelins.
OBJECTIVE
Smoothelin-A and -B isoforms are highly restricted to contractile smooth muscle cells (SMCs). Serum response factor (SRF) and myocardin are essential for contractile SMC differentiation. We evaluated the contribution of SRF/myocardin to transcriptional regulation of smoothelins.


METHODS
Rat vascular SMCs were transfected with smoothelin-A and smoothelin-B promoter reporter constructs and promoter activity was analyzed. The effects of mutations in the smoothelin-A promoter CArG-boxes and co-transfections with a myocardin expression plasmid were assessed. Electrophoretic mobility shift assays and chromatin immunoprecipitations were performed to investigate SRF-binding to the smoothelin-A CArG-boxes.


RESULTS
Smoothelin promoter activity was detected in vascular SMCs. Comparative sequence analysis revealed two conserved CArG elements in the smoothelin-A promoter that bind SRF as shown by chromatin immunoprecipitation. The proximal CArG-near bound SRF stronger than CArG-far in gel shift assays. Mutagenesis studies also indicated that CArG-near is more important than CArG-far in regulating smoothelin-A promoter activity. Myocardin augmented smoothelin-A promoter activity 2.5-fold in a CArG-near-dependent manner. In contrast, myocardin had little effect on the smoothelin-B promoter.


CONCLUSIONS
Smoothelin-A expression is controlled by an intragenic promoter whose activity is, in part, dependent on two CArG boxes that bind SRF. Our data show a role for SRF/myocardin in regulating smoothelin-A whereas the higher smoothelin-B expression appears to be SRF/myocardin-independent.
Rensen
Sander S M
SS
Department of Genetics and Cell Biology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands.
Niessen
Petra M G
PM
Long
Xiaochun
X
Doevendans
Pieter A
PA
Miano
Joseph M
JM
van Eys
Guillaume J J M
GJ
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000235:genetics / Q000378:metabolism*; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D015870:Gene Expression; D005786:Gene Expression Regulation; D006801:Humans; D047468:Immunoprecipitation / Q000379:methods; D051379:Mice; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017422:Sequence Analysis, DNA; D026362:Serum Response Factor / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D000099251:Myocardin
D003598:Cytoskeletal Proteins; D009124:Muscle Proteins; D009687:Nuclear Proteins; D012333:RNA, Messenger; D026362:Serum Response Factor; D015534:Trans-Activators; D000099251:Myocardin; C491051:Smtn protein, rat