Physiological Control of Smooth Muscle-specific Gene Expression through Regulated Nuclear Translocation of Serum Response Factor

Abstract

Prolonged serum deprivation induces a structurally and functionally contractile phenotype in about 1/6 of cultured airway myocytes, which exhibit morphological elongation and accumulate abundant contractile apparatus-associated proteins. We tested the hypothesis that transcriptional activation of genes encoding these proteins accounts for their accumulation during this phenotypic transition by measuring the transcriptional activities of the murine SM22 and human smooth muscle myosin heavy chain promoters during transient transfection in subconfluent, serum fed or 7 day serum-deprived cultured canine tracheal smooth muscle cells. Contrary to our expectation, SM22 and smooth muscle myosin heavy chain promoter activities (but not viral murine sarcoma virus-long terminal repeat promoter activity) were decreased in long term serum-deprived myocytes by at least 8-fold. Because serum response factor (SRF) is a required transcriptional activator of these and other smooth muscle-specific promoters, we evaluated the expression and function of SRF in subconfluent and long term serum-deprived cells. Whole cell SRF mRNA and protein were maintained at high levels in serum-deprived myocytes, but SRF transcription-promoting activity, nuclear SRF binding to consensus CArG sequences, and nuclear SRF protein were reduced. Furthermore, immunocytochemistry revealed extranuclear redistribution of SRF in serum-deprived myocytes; nuclear localization of SRF was restored after serum refeeding. These results uncover a novel mechanism for physiological control of smooth muscle-specific gene expression through extranuclear redistribution of SRF and consequent down-regulation of its transcriptionpromoting activity.Confluent cultured, passaged canine tracheal myocytes exhibit divergent phenotypes when deprived of serum for 7 or more days. About 1/6 of these cells accumulate abundant contractile apparatus proteins, increasing whole culture contents of smooth muscle myosin heavy chain (smMHC) 1 and SM22 by 5-7-fold (1, 2). These myocytes acquire a contractile phenotype, characterized by morphological elongation, expression of functionally coupled muscarinic M 3 surface receptors, and substantial contraction (shortening) upon cholinergic stimulation. Presently, the mechanism responsible for this phenotypic differentiation is unknown.In cultured skeletal muscle, differentiation of myoblasts into myotubes depends upon up-regulation of skeletal muscle-specific gene transcription, which results in abundant contractile apparatus protein accumulation (3-6). This precedent in skeletal muscle suggested that similarly enhanced transcription of contractile apparatus genes might account for the substantial smMHC and SM22 accumulation we observed in long term serum-deprived tracheal smooth muscle cells. To test this hypothesis, we assessed transcription from the smMHC and SM22 gene promoters in both preconfluent serum fed and post confluent long term serum-deprived tracheal myocytes. Contrary to our expectation, we found markedly reduced transcription from...

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