Expression of the smoothelin gene is mediated by alternative promoters

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 alpha-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.