[Show abstract][Hide abstract] ABSTRACT: Lipoma preferred partner (LPP) was recently recognized as a smooth muscle marker that plays a role in smooth muscle cell migration. In this report, we focus on the transcriptional regulation of the LPP gene. In particular, we investigate whether LPP is directly regulated by serum response factor (SRF). We show that the LPP gene contains 3 evolutionarily conserved CArG boxes and that 1 of these is part of an alternative promoter in intron 2. Quantitative RT-PCR shows that this alternative promoter directs transcription specifically to smooth muscle containing tissues in vivo. By using chromatin immunoprecipitation, we demonstrate that 2 of the CArG boxes, including the promoter-associated CArG box, bind to endogenous SRF in cultured aortic smooth muscle cells. Electrophoretic mobility-shift assays show that the conserved CArG boxes bind SRF in vitro. In reporter experiments, we show that the alternative promoter has transcriptional capacity that is dependent on SRF/myocardin and that the promoter associated CArG box is required for that activity. Finally, we show by quantitative RT-PCR that the alternative promoter is strongly downregulated in SRF-deficient embryonic stem cells and in smooth muscle tissues derived from conditional SRF knockout mice. Collectively, our data demonstrate that expression of LPP in smooth muscle is mediated by an alternative promoter that is regulated by SRF/myocardin.
Circulation Research 08/2008; 103(1):61-9. DOI:10.1161/CIRCRESAHA.108.177436 · 11.02 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: SRF (Serum Response Factor), a widely expressed transcription factor, controls expression of mitogen-responsive and muscle-specific genes, thereby regulating the contractile actin microfilament. Genetic Srf deletion studies showed SRF to be indispensable for in vivo skeletal and cardiac muscle cell development. We now investigated for the first time in vivo SRF functions in smooth muscle cells of adult mice.
We conditionally deleted a floxed Srf allele (Srf(flex1)) in adult mice by inducible activation of the CreER(T2) recombinase expressed specifically in smooth muscle cells. Tamoxifen-induced CreER(T2) activity stimulated deletion of exon 1 coding sequences of Srf(flex1), thereby abolishing full-length SRF protein expression in adult smooth muscle cells of the analyzed organs: colon, bladder, and stomach.
Smooth muscle cell-specific ablation of full-length SRF protein in adult mice showed impaired contraction of intestinal smooth muscle, resulting in defective peristalsis. Mutant mice died within 2 weeks of tamoxifen treatment, displaying clear symptoms of ileus paralyticus. Cultured primary SRF-deficient colon smooth muscle cells were viable, but displayed drastic structural alterations and elevated senescence, paralleled by degeneration of the actin microfilament and impaired expression of smooth muscle-specific genes.
SRF plays a vital role in the contractile activity and cytoskeletal architecture of adult smooth muscle cells and is therefore essential for physiologic functions of the gastrointestinal tract in vivo. Our mouse genetic model may resemble features of human chronic intestinal pseudo-obstruction.