DNA methylation, through DNMT1, has an essential role in the development of gastrointestinal smooth muscle cells and disease.

DNA methylation is a key epigenetic modification that can regulate gene expression. Genomic DNA hypomethylation is commonly found in many gastrointestinal (GI) diseases. Dysregulated gene expression in GI smooth muscle cells (GI-SMCs) can lead to motility disorders. However, the consequences of genomic DNA hypomethylation within GI-SMCs are still elusive. Utilizing a Cre-lox murine model, we have generated SMC-restricted DNA methyltransferase 1 (Dnmt1) knockout (KO) mice and analyzed the effects of Dnmt1 deficiency. Dnmt1-KO pups are born smaller than their wild-type littermates, have shortened GI tracts, and lose peristaltic movement due to loss of the tunica muscularis in their intestine, causing massive intestinal dilation, and death around postnatal day 21. Within smooth muscle tissue, significant CpG hypomethylation occurs across the genome at promoters, introns, and exons. Additionally, there is a marked loss of differentiated SMC markers (Srf, Myh11, miR-133, miR-143/145), an increase in pro-apoptotic markers (Nr4a1, Gadd45g), loss of cellular connectivity, and an accumulation of coated vesicles within SMC. Interestingly, we observed consistent abnormal expression patterns of enzymes involved in DNA methylation between both Dnmt1-KO mice and diseased human GI tissue. These data demonstrate that DNA hypomethylation in embryonic SMC, via congenital Dnmt1 deficiency, contributes to massive dysregulation of gene expression and is lethal to GI-SMC. These results suggest that Dnmt1 has a necessary role in the embryonic, primary development process of SMC with consistent patterns being found in human GI diseased tissue.

Serum response factor regulates smooth muscle contractility via myotonic dystrophy protein kinases and L-type calcium channels.

Serum response factor (SRF) transcriptionally regulates expression of contractile genes in smooth muscle cells (SMC). Lack or decrease of SRF is directly linked to a phenotypic change of SMC, leading to hypomotility of smooth muscle in the gastrointestinal (GI) tract. However, the molecular mechanism behind SRF-induced hypomotility in GI smooth muscle is largely unknown. We describe here how SRF plays a functional role in the regulation of the SMC contractility via myotonic dystrophy protein kinase (DMPK) and L-type calcium channel CACNA1C. GI SMC expressed Dmpk and Cacna1c genes into multiple alternative transcriptional isoforms. Deficiency of SRF in SMC of Srf knockout (KO) mice led to reduction of SRF-dependent DMPK, which down-regulated the expression of CACNA1C. Reduction of CACNA1C in KO SMC not only decreased intracellular Ca2+ spikes but also disrupted their coupling between cells resulting in decreased contractility. The role of SRF in the regulation of SMC phenotype and function provides new insight into how SMC lose their contractility leading to hypomotility in pathophysiological conditions within the GI tract.

Serum Response Factor Is Essential for Prenatal Gastrointestinal Smooth Muscle Development and Maintenance of Differentiated Phenotype.

BACKGROUND/AIMS: Smooth muscle cells (SMCs) characteristically express serum response factor (SRF), which regulates their development. The role of SRF in SMC plasticity in the pathophysiological conditions of gastrointestinal (GI) tract is less characterized. METHODS: We generated SMC-specific Srf knockout mice and characterized the prenatally lethal phenotype using ultrasound biomicroscopy and histological analysis. We used small bowel partial obstruction surgeries and primary cell culture using cell-specific enhanced green fluorescent protein (EGFP) mouse lines to study phenotypic and molecular changes of SMCs by immunofluorescence, Western blotting, and quantitative polymerase chain reaction. Finally we examined SRF change in human rectal prolapse tissue by immunofluorescence. RESULTS: Congenital SMC-specific Srf knockout mice died before birth and displayed severe GI and cardiac defects. Partial obstruction resulted in an overall increase in SRF protein expression. However, individual SMCs appeared to gradually lose SRF in the hypertrophic muscle. Cells expressing low levels of SRF also expressed low levels of platelet-derived growth factor receptor alpha (PDGFRα(low)) and Ki67. SMCs grown in culture recaptured the phenotypic switch from differentiated SMCs to proliferative PDGFRα(low) cells. The immediate and dramatic reduction of Srf and Myh11 mRNA expression confirmed the phenotypic change. Human rectal prolapse tissue also demonstrated significant loss of SRF expression. CONCLUSIONS: SRF expression in SMCs is essential for prenatal development of the GI tract and heart. Following partial obstruction, SMCs down-regulate SRF to transition into proliferative PDGFRα(low) cells that may represent a phenotype responsible for their plasticity. These findings demonstrate that SRF also plays a critical role in the remodeling process following GI injury.

Smooth Muscle Cell Genome Browser: Enabling the Identification of Novel Serum Response Factor Target Genes.

Genome-scale expression data on the absolute numbers of gene isoforms offers essential clues in cellular functions and biological processes. Smooth muscle cells (SMCs) perform a unique contractile function through expression of specific genes controlled by serum response factor (SRF), a transcription factor that binds to DNA sites known as the CArG boxes. To identify SRF-regulated genes specifically expressed in SMCs, we isolated SMC populations from mouse small intestine and colon, obtained their transcriptomes, and constructed an interactive SMC genome and CArGome browser. To our knowledge, this is the first online resource that provides a comprehensive library of all genetic transcripts expressed in primary SMCs. The browser also serves as the first genome-wide map of SRF binding sites. The browser analysis revealed novel SMC-specific transcriptional variants and SRF target genes, which provided new and unique insights into the cellular and biological functions of the cells in gastrointestinal (GI) physiology. The SRF target genes in SMCs, which were discovered in silico, were confirmed by proteomic analysis of SMC-specific Srf knockout mice. Our genome browser offers a new perspective into the alternative expression of genes in the context of SRF binding sites in SMCs and provides a valuable reference for future functional studies.