Differentiation and contractility of colon smooth muscle under normal and diabetic conditions

Indiana University-Purdue University Indianapolis (IUPUI)Intestinal smooth muscle development involves complex transcriptional regulation leading to cell differentiation of the circular, longitudinal and muscularis mucosae layers. Differentiated intestinal smooth muscle cells express high levels of smooth muscle-specific contractile and regulatory proteins, including telokin. Telokin is regulatory protein that is highly expressed in visceral smooth muscle. Analysis of cis-elements required for transcriptional regulation of the telokin promoter by using hypoxanthine-guanine phosphoribosyltransferase (Hprt)-targeted reporter transgenes revealed that a 10 base pair large CC(AT)₆GG ciselement, called CArG box is required for promoter activity in all tissues. We also determined that an additional 100 base pair region is necessary for transgene activity in intestinal smooth muscle cells. To examine how transcriptional regulation of intestinal smooth muscle may be altered under pathological conditions we examined the effects of diabetes on colonic smooth muscle. Approximately 76% of diabetic patients develop gastrointestinal (GI) symptoms such as constipation due to intestinal dysmotility. Mice were treated with low-dose streptozotocin to induce a type 1 diabetes-like hyperglycemia. CT scans revealed decreased overall GI tract motility after 7 weeks of hyperglycemia. Acute (1 week) and chronic (7 weeks) diabetic mice also had decreased potassium chloride (KCl)-induced colon smooth muscle contractility. We hypothesized that decreased smooth muscle contractility at least in part, was due to alteration of contractile protein gene expression. However, diabetic mice showed no changes in mRNA or protein levels of smooth muscle contractile proteins. We determined that the decreased colonic contractility was associated with an attenuated intracellular calcium increase, as measured by ratio-metric imaging of Fura-2 fluorescence in isolated colonic smooth muscle strips. This attenuated calcium increase resulted in decreased myosin light chain phosphorylation, thus explaining the decreased contractility of the colon. Chronic diabetes was also associated with increased basal calcium levels. Western blotting and quantitative real time polymerase chain reaction (qRT-PCR) analysis revealed significant changes in calcium handling proteins in chronic diabetes that were not seen in the acute state.These changes most likely reflect compensatory mechanisms activated by the initial impaired calcium response. Overall my results suggest that type 1 diabetes in mice leads to decreased colon motility in part due to altered calcium handling without altering contractile protein expression

Regulation of serum response factor activity and smooth muscle cell apoptosis by chromodomain helicase DNA-binding protein 8

Serum response factor (SRF) is a widely expressed protein that plays a key role in the regulation of smooth muscle differentiation, proliferation, migration, and apoptosis. It is generally accepted that one mechanism by which SRF regulates these diverse functions is through pathway-specific cofactor interactions. A novel SRF cofactor, chromodomain helicase DNA binding protein 8 (CHD8), was isolated from a yeast two-hybrid screen using SRF as bait. CHD8 is highly expressed in adult smooth muscle tissues. Coimmunoprecipitation assays from A10 smooth muscle cells demonstrated binding of endogenous SRF and CHD8. Data from GST-pulldown assays indicate that the NH2-terminus of CHD8 can interact directly with the MADS domain of SRF. Adenoviral-mediated knockdown of CHD8 in smooth muscle cells resulted in attenuated expression of SRF-dependent, smooth muscle-specific genes. Knockdown of CHD8, SRF, or CTCF, a previously described binding partner of CHD8, in A10 VSMCs also resulted in a marked induction of apoptosis. Mechanistically, apoptosis induced by CHD8 knockdown was accompanied by attenuated expression of the anti-apoptotic proteins, Birc5, and CARD10, whereas SRF knockdown attenuated expression of CARD10 and Mcl-1, but not Birc5, and CTCF knockdown attenuated expression of Birc5. These data suggest that CHD8 plays a dual role in smooth muscle cells modulating SRF activity toward differentiation genes and promoting cell survival through interactions with both SRF and CTCF to regulate expression of Birc5 and CARD10