© 2002 by European Society of Cardiology
Copyright © 2002, European Society of Cardiology
Expression of the smoothelin gene is mediated by alternative promoters
aDepartment of Genetics and Cell Biology, Section of Molecular Genetics, University of Maastricht, P.O. Box 616, 6200 MD Maastricht, The Netherlands
bDepartment of Biochemistry, University of Amsterdam, Amsterdam, The Netherlands
cDepartment of Cardiology, University of Maastricht, Maastricht, The Netherlands
dLaboratory of Molecular Genetics, NIDCH, NIH, Bethesda, MD, USA
* Corresponding author. Tel.: +31-43-388-1355; fax: +31-43-388-4574 g.vaneys{at}gen.unimaas.nl
Objective: Two major isoforms of smoothelin have been reported, a 59-kDa smoothelin-A in visceral smooth muscle cells and a 110-kDa smoothelin-B in vascular smooth muscle cells. The present study was undertaken to investigate the expression of these smoothelin isoforms in different smooth muscle tissues and to determine how they are generated. Methods: Western blotting with a new, well-defined, smoothelin antibody was used to confirm the existence of two major smoothelin isoforms. Northern blotting, RT-PCR, primer extension and 5'RACE were applied to analyse the expression of these isoforms in human and mouse. Promoter reporter assays were carried out to establish the existence of a dual promoter system governing the expression pattern of the gene. Results: Antibody C6G confirmed the existence of two smoothelin proteins. Northern blotting showed that in vascular tissues a larger smoothelin transcript is generated than in visceral tissue. The cDNA of this larger smoothelin-B was cloned. Computer analysis of the open reading frame suggests an 
-helical structure of 130 amino acids at the amino terminus of smoothelin-B. The smoothelin gene was cloned and sequenced. It comprises about 25 kb and contains 21 exons. The translational start of smoothelin-B is located in exon 2, whereas transcription and translation of the previously described smoothelin-A starts inside exon 10. Smoothelin-A and -B were demonstrated to be generated by two physically separated promoters. Splice variants within the calponin homology domain at the 3' end of the gene were found for both isoforms. Conclusions: Two major smoothelin isoforms are generated from a single gene by a dual promoter system in a tissue specific manner. Further variation in the smoothelin proteins is achieved by alternative splicing in the calponin homology domain.
KEYWORDS Contractile apparatus; Gene expression; Smooth muscle
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