Transcriptome profiling reveals significant changes in the gastric muscularis externa with obesity that partially overlap those that occur with idiopathic gastroparesis.

BACKGROUND: Gastric emptying is impaired in patients with gastroparesis whereas it is either unchanged or accelerated in obese individuals. The goal of the current study was to identify changes in gene expression in the stomach muscularis that may be contributing to altered gastric motility in idiopathic gastroparesis and obesity. METHODS: Quantitative real time RT-PCR and whole transcriptome sequencing were used to compare the transcriptomes of lean individuals, obese individuals and either lean or obese individuals with idiopathic gastroparesis. RESULTS: Obesity leads to an increase in mRNAs associated with muscle contractility whereas idiopathic gastroparesis leads to a decrease in mRNAs associated with PDGF BB signaling. Both obesity and idiopathic gastroparesis were also associated with similar alterations in pathways associated with inflammation. CONCLUSIONS: Our findings show that obesity and idiopathic gastroparesis result in overlapping but distinct changes in the gastric muscularis transcriptome. Increased expression of mRNAs encoding smooth muscle contractile proteins may be contributing to the increased gastric motility observed in obese subjects, whereas decreased PDGF BB signaling may be contributing to the impaired motility seen in subjects with idiopathic gastroparesis.

The TRPC6 inhibitor, larixyl acetate, is effective in protecting against traumatic brain injury-induced systemic endothelial dysfunction.

BACKGROUND: The incidence of traumatic brain injuries (TBIs) is on the rise in the USA. Concussions, or mild TBIs without skull fracture, account for about 75% of all TBIs. Mild TBIs (mTBIs) lead to memory and cognitive deficits, headaches, intraocular pressure rises, axonal degeneration, neuroinflammation, and an array of cerebrovascular dysfunctions, including increased vascular permeability and decreased cerebral blood flow. It has been recently reported that besides vascular dysfunction in the cerebral circulation, mTBI may also cause a significant impairment of endothelial function in the systemic circulation, at least within mesenteric microvessels. In this study, we investigated whether mTBI affects endothelial function in aortas and determined the contribution of transient receptor potential canonical (TRPC) channels to modulating mTBI-associated endothelial dysfunction. METHODS: We used a model of closed-head mTBI in C57BL/6, 129S, 129S-C57BL/6-F2 mice, and 129S-TRPC1 and 129S-C57BL/6-TRPC6 knockout mice to determine the effect of mTBI on endothelial function in mouse aortas employing ex vivo isometric tension measurements. Aortic tissue was also analyzed using immunofluorescence and qRT-PCR for TRPC6 expression following mTBI. RESULTS: We show that in various strains of mice, mTBI induces a pronounced and long-lasting endothelial dysfunction in the aorta. Ablation of TRPC6 protects mice from mTBI-associated aortic endothelial dysfunction, while TRPC1 ablation does not impact brain injury-induced endothelial impairment in the aorta. Consistent with a role of TRPC6 activation following mTBI, we observed improved endothelial function in wild type control mice subjected to mTBI following 7-day in vivo treatment with larixyl acetate, an inhibitor of TRPC6 channels. Conversely, in vitro treatment with the pro-inflammatory endotoxin lipopolysaccharide, which activates endothelial TRPC6 in a Toll-like receptor type 4 (TLR4)-dependent manner, worsened aortic endothelial dysfunction in wild type mice. Lipopolysaccharide treatment in vitro failed to elicit endothelial dysfunction in TRPC6 knockout mice. No change in endothelial TRPC6 expression was observed 7 days following TBI. CONCLUSIONS: These data suggest that TRPC6 activation may be critical for inducing endothelial dysfunction following closed-head mTBI and that pharmacological inhibition of the channel may be a feasible therapeutic strategy for preventing mTBI-associated systemic endothelial dysfunction.

First look: A mixed-methods study exploring women's initial experiences of their appearance after mastectomy and/or breast reconstruction✰.

OBJECTIVES: Increasing numbers of women are undergoing appearance-altering surgery for the treatment and/or prevention of breast cancer. However, women's experiences of seeing the results of their breast surgery for the first time, and the support available to them, are currently omitted from the research literature. This study aimed to explore women's initial experiences of seeing their appearance after mastectomy and/or breast reconstruction. DESIGN: An online mixed-methods survey explored participants' feelings and expectations before seeing their breast surgery for the first time, their experiences of looking at the results of their surgery, and the support they received. METHODS: Women (n = 128) who had undergone mastectomy and/or breast reconstruction following a diagnosis of invasive breast cancer, DCIS or increased risk of breast cancer participated. Data were analysed using descriptive statistics and qualitative content analysis. RESULTS: Most respondents had worried about looking at their breast/breast area for the first time, with 75% concerned about what they would see. Women found the experience moderately distressing, and younger women were particularly concerned about other people's reactions to their altered appearance. Approximately half of the women (51%) felt they received enough support, while 29% thought this aspect of care could be improved. Areas for improvement were suggested, including increased preparation, privacy and support. CONCLUSION: Women's experiences of looking at their breast/breast area and any donor site after surgery vary considerably. The results indicate important implications for provision of care and further research.

Submucosal Gland Myoepithelial Cells Are Reserve Stem Cells That Can Regenerate Mouse Tracheal Epithelium.

The mouse trachea is thought to contain two distinct stem cell compartments that contribute to airway repair-basal cells in the surface airway epithelium (SAE) and an unknown submucosal gland (SMG) cell type. Whether a lineage relationship exists between these two stem cell compartments remains unclear. Using lineage tracing of glandular myoepithelial cells (MECs), we demonstrate that MECs can give rise to seven cell types of the SAE and SMGs following severe airway injury. MECs progressively adopted a basal cell phenotype on the SAE and established lasting progenitors capable of further regeneration following reinjury. MECs activate Wnt-regulated transcription factors (Lef-1/TCF7) following injury and Lef-1 induction in cultured MECs promoted transition to a basal cell phenotype. Surprisingly, dose-dependent MEC conditional activation of Lef-1 in vivo promoted self-limited airway regeneration in the absence of injury. Thus, modulating the Lef-1 transcriptional program in MEC-derived progenitors may have regenerative medicine applications for lung diseases.

Idiopathic gastroparesis is associated with specific transcriptional changes in the gastric muscularis externa.

BACKGROUND: The molecular changes that occur in the stomach that are associated with idiopathic gastroparesis are poorly described. The aim of this study was to use quantitative analysis of mRNA expression to identify changes in mRNAs encoding proteins required for the normal motility functions of the stomach. METHODS: Full-thickness stomach biopsy samples were collected from non-diabetic control subjects who exhibited no symptoms of gastroparesis and from patients with idiopathic gastroparesis. mRNA was isolated from the muscularis externa and mRNA expression levels were determined by quantitative reverse transcriptase (RT)-PCR. KEY RESULTS: Smooth muscle tissue from idiopathic gastroparesis patients had decreased expression of mRNAs encoding several contractile proteins, such as MYH11 and MYLK1. Conversely, there was no significant change in mRNAs characteristic of interstitial cells of Cajal (ICCs) such as KIT or ANO1. There was also a significant decrease in mRNA-encoding platelet-derived growth factor receptor α (PDGFRα) and its ligand PDGFB and in Heme oxygenase 1 in idiopathic gastroparesis subjects. In contrast, there was a small increase in mRNA characteristic of neurons. Although there was not an overall change in KIT expression in gastroparesis patients, KIT expression showed a significant correlation with gastric emptying whereas changes in MYLK1, ANO1 and PDGFRα showed weak correlations to the fullness/satiety subscore of patient assessment of upper gastrointestinal disorder-symptom severity index scores. CONCLUSIONS AND INFERENCES: Our findings suggest that idiopathic gastroparesis is associated with altered smooth muscle cell contractile protein expression and loss of PDGFRα+ cells without a significant change in ICCs.

Inflammation and vascular smooth muscle cell dedifferentiation following carotid artery ligation.

Following vascular injury medial smooth muscle cells dedifferentiate and migrate through the internal elastic lamina where they form a neointima. The goal of the current study was to identify changes in gene expression that occur before the development of neointima and are associated with the early response to injury. Vascular injury was induced in C57BL/6 mice and in Myh11-creER(T2) mTmG reporter mice by complete ligation of the left carotid artery. Reporter mice were used to visualize cellular changes in the injured vessels. Total RNA was isolated from control carotid arteries or from carotid arteries 3 days following ligation of C57BL/6 mice and analyzed by Affymetrix microarray and quantitative RT-PCR. This analysis revealed decreased expression of mRNAs encoding smooth muscle-specific contractile proteins that was accompanied by a marked increase in a host of mRNAs encoding inflammatory cytokines following injury. There was also marked decrease in molecules associated with BMP, Wnt, and Hedgehog signaling and an increase in those associated with B cell, T cell, and macrophage signaling. Expression of a number of noncoding RNAs were also altered following injury with microRNAs 143/145 being dramatically downregulated and microRNAs 1949 and 142 upregulated. Several long noncoding RNAs showed altered expression that mirrored the expression of their nearest coding genes. These data demonstrate that following carotid artery ligation an inflammatory cascade is initiated that is associated with the downregulation of coding and noncoding RNAs that are normally required to maintain smooth muscle cells in a differentiated state.

Critical contribution of KV1 channels to the regulation of coronary blood flow.

Ion channels in smooth muscle control coronary vascular tone, but the identity of the potassium channels involved requires further investigation. The purpose of this study was to evaluate the functional role of KV1 channels on porcine coronary blood flow using the selective antagonist correolide. KV1 channel gene transcripts were found in porcine coronary arteries, with KCNA5 (encoding KV1.5) being most abundant (P < 0.001). Immunohistochemical staining demonstrated KV1.5 protein in the vascular smooth muscle layer of both porcine and human coronary arteries, including microvessels. Whole-cell patch-clamp experiments demonstrated significant correolide-sensitive (1-10 µM) current in coronary smooth muscle. In vivo studies included direct intracoronary infusion of vehicle or correolide into a pressure-clamped left anterior descending artery of healthy swine (n = 5 in each group) with simultaneous measurement of coronary blood flow. Intracoronary correolide (~0.3-3 µM targeted plasma concentration) had no effect on heart rate or systemic pressure, but reduced coronary blood flow in a dose-dependent manner (P < 0.05). Dobutamine (0.3-10 µg/kg/min) elicited coronary metabolic vasodilation and intracoronary correolide (3 µM) significantly reduced coronary blood flow at any given level of myocardial oxygen consumption (P < 0.001). Coronary artery occlusions (15 s) elicited reactive hyperemia and correolide (3 µM) reduced the flow volume repayment by approximately 30 % (P < 0.05). Taken together, these data support a major role for KV1 channels in modulating baseline coronary vascular tone and, perhaps, vasodilation in response to increased metabolism and transient ischemia.

Obesity alters molecular and functional cardiac responses to ischemia/reperfusion and glucagon-like peptide-1 receptor agonism.

This study tested the hypothesis that obesity alters the cardiac response to ischemia/reperfusion and/or glucagon like peptide-1 (GLP-1) receptor activation, and that these differences are associated with alterations in the obese cardiac proteome and microRNA (miRNA) transcriptome. Ossabaw swine were fed normal chow or obesogenic diet for 6 months. Cardiac function was assessed at baseline, during a 30-minutes coronary occlusion, and during 2 hours of reperfusion in anesthetized swine treated with saline or exendin-4 for 24 hours. Cardiac biopsies were obtained from normal and ischemia/reperfusion territories. Fat-fed animals were heavier, and exhibited hyperinsulinemia, hyperglycemia, and hypertriglyceridemia. Plasma troponin-I concentration (index of myocardial injury) was increased following ischemia/reperfusion and decreased by exendin-4 treatment in both groups. Ischemia/reperfusion produced reductions in systolic pressure and stroke volume in lean swine. These indices were higher in obese hearts at baseline and relatively maintained throughout ischemia/reperfusion. Exendin-4 administration increased systolic pressure in lean swine but did not affect the blood pressure in obese swine. End-diastolic volume was reduced by exendin-4 following ischemia/reperfusion in obese swine. These divergent physiologic responses were associated with obesity-related differences in proteins related to myocardial structure/function (e.g. titin) and calcium handling (e.g. SERCA2a, histidine-rich Ca(2+) binding protein). Alterations in expression of cardiac miRs in obese hearts included miR-15, miR-27, miR-130, miR-181, and let-7. Taken together, these observations validate this discovery approach and reveal novel associations that suggest previously undiscovered mechanisms contributing to the effects of obesity on the heart and contributing to the actions of GLP-1 following ischemia/reperfusion.

Forkhead box F2 regulation of platelet-derived growth factor and myocardin/serum response factor signaling is essential for intestinal development.

Alterations in the forkhead box F2 gene expression have been reported in numerous pathologies, and Foxf2(-/-) mice are perinatal lethal with multiple malformations; however, molecular mechanisms pertaining to Foxf2 signaling are severely lacking. In this study, Foxf2 requirements in murine smooth muscle cells were examined using a conditional knock-out approach. We generated novel Foxf2-floxed mice, which we bred to smMHC-Cre-eGFP mice to generate a mouse line with Foxf2 deleted specifically from smooth muscle. These mice exhibited growth retardation due to reduced intestinal length as well as inflammation and remodeling of the small intestine. Colons of Tg(smMHC-Cre-eGFP(+/-));Foxf2(-/-) mice had expansion of the myenteric nerve plexus and increased proliferation of smooth muscle cells leading to thickening of the longitudinal smooth muscle layer. Foxf2 deficiency in colonic smooth muscle was associated with increased expression of Foxf1, PDGFa, PDGFb, PDGF receptor α, and myocardin. FOXF2 bound to promoter regions of these genes indicating direct transcriptional regulation. Foxf2 repressed Foxf1 promoter activity in co-transfection experiments. We also show that knockdown of Foxf2 in colonic smooth muscle cells in vitro and in transgenic mice increased myocardin/serum response factor signaling and increased expression of contractile proteins. Foxf2 attenuated myocardin/serum response factor signaling in smooth muscle cells through direct binding to the N-terminal region of myocardin. Our results indicate that Foxf2 signaling in smooth muscle cells is essential for intestinal development and serum response factor signaling.

Regulation of 130-kDa smooth muscle myosin light chain kinase expression by an intronic CArG element.

The mylk1 gene encodes a 220-kDa nonmuscle myosin light chain kinase (MLCK), a 130-kDa smooth muscle MLCK (smMLCK), as well as the non-catalytic product telokin. Together, these proteins play critical roles in regulating smooth muscle contractility. Changes in their expression are associated with many pathological conditions; thus, it is important to understand the mechanisms regulating expression of mylk1 gene transcripts. Previously, we reported a highly conserved CArG box, which binds serum response factor, in intron 15 of mylk1. Because this CArG element is near the promoter that drives transcription of the 130-kDa smMLCK, we examined its role in regulating expression of this transcript. Results show that deletion of the intronic CArG region from a ß-galactosidase reporter gene abolished transgene expression in mice in vivo. Deletion of the CArG region from the endogenous mylk1 gene, specifically in smooth muscle cells, decreased expression of the 130-kDa smMLCK by 40% without affecting expression of the 220-kDa MLCK or telokin. This reduction in 130-kDa smMLCK expression resulted in decreased phosphorylation of myosin light chains, attenuated smooth muscle contractility, and a 24% decrease in small intestine length that was associated with a significant reduction of Ki67-positive smooth muscle cells. Overall, these data show that the CArG element in intron 15 of the mylk1 gene is necessary for maximal expression of the 130-kDa smMLCK and that the 130-kDa smMLCK isoform is specifically required to regulate smooth muscle contractility and small intestine smooth muscle cell proliferation.

The transcription factor Foxf1 binds to serum response factor and myocardin to regulate gene transcription in visceral smooth muscle cells.

Smooth muscle cells (SMCs) modulate their phenotype from a quiescent contractile state to a dedifferentiated, proliferative and migratory state during the pathogenesis of many diseases, including intestinal pseudoobstruction. Understanding how smooth muscle gene expression is regulated in these different phenotypic states is critical for unraveling the pathogenesis of these diseases. In the current study we examined the specific roles of Foxf1 in visceral SMC differentiation. Data show that Foxf1 is specifically required for expression of several contractile and regulatory proteins such as telokin, smooth muscle γ-actin, and Cav1.2b in visceral SMCs. Mechanistically, Foxf1 directly binds to and activates the telokin promoter. Foxf1 also directly binds to serum response factor (SRF) and myocardin-related transcription factors (MRTFs). Unlike Foxo4 and Foxq1, which bind to MRTFs and block their interaction with SRF, Foxf1 acts synergistically with these proteins to regulate telokin expression. Knock-out of Foxf1 specifically in SMCs results in neonatal lethality, with mice exhibiting GI tract abnormalities. Mice heterozygous for Foxf1 in SMC exhibited impaired colonic contractility and decreased expression of contractile proteins. These studies together with previous studies, suggest that different forkhead proteins can regulate gene expression in SMCs through modulating the activity of the SRF-myocardin axis to either promote or inhibit differentiation and proliferation thereby altering gastrointestinal contractility and development.

Regulation of microRNAs by Brahma-related gene 1 (Brg1) in smooth muscle cells.

MicroRNAs are involved in phenotypic switching of smooth muscle cells (SMCs). Brg1-containing SWI/SNF chromatin-remodeling complexes also play an important role in controlling the phenotype of SMCs. We thus determined whether Brg1 influences the transcription of microRNAs in SMCs. Microarray and quantitative RT-PCR analysis of smooth muscle from mice harboring smooth muscle-specific deletion of Brg1 revealed altered expression of several microRNAs, including miRs-143/145 and miR-133. Ablation of Brg1 in SMCs in vitro either by expression of dominant negative Brg1 or Brg1 knock-out attenuated miRs-143/145 expression. Knockdown of serum response factor (SRF) in SMCs significantly reduced the expression levels of miRs-143/145 and miR-133, whereas knockdown of myocardin only attenuated miRs-143/145 expression. Myocardin induced expression of miRs-143/145 and miR-133a and increased SRF binding to these genes in 10T1/2 cells. This myocardin-mediated induction was attenuated by dominant negative Brg1. In Brg1-null SW13 cells, miRs-143/145 were dramatically induced by myocardin only in the presence of Brg1, whereas miR-133 was not induced by myocardin in a Brg1-dependent manner. Chromatin immunoprecipitation assays demonstrated that in the presence of Brg1, myocardin increased SRF binding to both the miRs-143/145 and miR-133a loci. Together, these data suggest a mechanism in which Brg1-containing SWI/SNF complexes are required for myocardin to induce expression of miRs-143/145 in smooth muscle cells. In contrast, miR-133 expression appears to be regulated by Brg1-containing chromatin remodeling complexes in a partially SRF-dependent, although largely myocardin-independent manner. SWI/SNF-mediated chromatin remodeling thus regulates the phenotype of smooth muscle by affecting expression of protein-coding genes and microRNAs.

Hexosamine biosynthesis impairs insulin action via a cholesterolgenic response.

Plasma membrane cholesterol accumulation has been implicated in cellular insulin resistance. Given the role of the hexosamine biosynthesis pathway (HBP) as a sensor of nutrient excess, coupled to its involvement in the development of insulin resistance, we delineated whether excess glucose flux through this pathway provokes a cholesterolgenic response induced by hyperinsulinemia. Exposing 3T3-L1 adipocytes to physiologically relevant doses of hyperinsulinemia (250pM-5000pM) induced a dose-dependent gain in the mRNA/protein levels of 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase (HMGR). These elevations were associated with elevated plasma membrane cholesterol. Mechanistically, hyperinsulinemia increased glucose flux through the HBP and O-linked ß-N-acetylglucosamine (O-GlcNAc) modification of specificity protein 1 (Sp1), known to activate cholesterolgenic gene products such as the sterol response element-binding protein (SREBP1) and HMGR. Chromatin immunoprecipitation demonstrated that increased O-GlcNAc modification of Sp1 resulted in a higher binding affinity of Sp1 to the promoter regions of SREBP1 and HMGR. Luciferase assays confirmed that HMGR promoter activity was elevated under these conditions and that inhibition of the HBP with 6-diazo-5-oxo-l-norleucine (DON) prevented hyperinsulinemia-induced activation of the HMGR promoter. In addition, both DON and the Sp1 DNA-binding inhibitor mithramycin prevented the hyperinsulinemia-induced increases in HMGR mRNA/protein and plasma membrane cholesterol. In these mithramycin-treated cells, both cortical filamentous actin structure and insulin-stimulated glucose transport were restored. Together, these data suggest a novel mechanism whereby increased HBP activity increases Sp1 transcriptional activation of a cholesterolgenic program, thereby elevating plasma membrane cholesterol and compromising cytoskeletal structure essential for insulin action.

Altered calcium signaling in colonic smooth muscle of type 1 diabetic mice.

Seventy-six percent of diabetic patients develop gastrointestinal symptoms, such as constipation. However, the direct effects of diabetes on intestinal smooth muscle are poorly described. This study aimed to identify the role played by smooth muscle in mediating diabetes-induced colonic dysmotility. To induce type 1 diabetes, mice were injected intraperitoneally with low-dose streptozotocin once a day for 5 days. Animals developed hyperglycemia (>200 mg/dl) 1 wk after the last injection and were euthanized 7-8 wk after the last treatment. Computed tomography demonstrated decreased overall gastrointestinal motility in the diabetic mice. In vitro contractility of colonic smooth muscle rings from diabetic mice was also decreased. Fura-2 ratiometric Ca(2+) imaging showed attenuated Ca(2+) increases in response to KCl stimulation that were associated with decreased light chain phosphorylation in diabetic mice. The diabetic mice also exhibited elevated basal Ca(2+) levels, increased myosin phosphatase targeting subunit 1 expression, and significant changes in expression of Ca(2+) handling proteins, as determined by quantitative RT-PCR and Western blotting. Mice that were hyperglycemic for <1 wk also showed decreased colonic contractile responses that were associated with decreased Ca(2+) increases in response to KCl stimulation, although without an elevation in basal Ca(2+) levels or a significant change in the expression of Ca(2+) signaling molecules. These data demonstrate that type 1 diabetes is associated with decreased depolarization-induced Ca(2+) influx in colonic smooth muscle that leads to attenuated myosin light chain phosphorylation and impaired colonic contractility.

Delta-like 1-Lysine613 regulates notch signaling.

Delta ligands are important for regulating Notch signaling through transcellular stimulation of Notch receptors. The cytoplasmic tails of Delta ligands have multiple potential regulatory sites including several lysine residues that are putative targets for ubiquitination by the E3 ubiquitin ligases, Mind Bomb and Neuralized. To identify possible roles for specific lysine residues in the cytoplasmic tail of the Notch ligand Dll1 a mutational and functional analysis was performed. Examination of a panel of individual or clustered lysine mutants demonstrated that lysine 613 (K613) in the cytoplasmic tail of Dll1 is a key residue necessary for transcellular activation of Notch signaling. Multi-ubiquitination of the Dll1 mutant Dll1-K613R was altered compared to wild type Dll1, and the K613R mutation blocked the ability of Dll1 to interact with Notch1. Finally, mutation of K613 did not affect the stability of Dll1 or its ability to traffic to recycle to the plasma membrane, but did enhance the fraction associated with lipid rafts. Collectively these results suggest that the transcellular defect in Notch signaling attributed to residue K613 in cytoplasmic tail of Dll1 may result from altering its multi-ubiquitination and increasing its retention in lipid rafts.

SWI/SNF complexes containing Brahma or Brahma-related gene 1 play distinct roles in smooth muscle development.

SWI/SNF ATP-dependent chromatin-remodeling complexes containing either Brahma-related gene 1 (Brg1) or Brahma (Brm) play important roles in mammalian development. In this study we examined the roles of Brg1 and Brm in smooth muscle development, in vivo, through generation and analysis of mice harboring a smooth muscle-specific knockout of Brg1 on wild-type and Brm null backgrounds. Knockout of Brg1 from smooth muscle in Brg1(flox/flox) mice expressing Cre recombinase under the control of the smooth muscle myosin heavy-chain promoter resulted in cardiopulmonary defects, including patent ductus arteriosus, in 30 to 40% of the mice. Surviving knockout mice exhibited decreased expression of smooth muscle-specific contractile proteins in the gastrointestinal tract, impaired contractility, shortened intestines, disorganized smooth muscle cells, and an increase in apoptosis of intestinal smooth muscle cells. Although Brm knockout mice had normal intestinal structure and function, knockout of Brg1 on a Brm null background exacerbated the effects of knockout of Brg1 alone, resulting in an increase in neonatal lethality. These data show that Brg1 and Brm play critical roles in regulating development of smooth muscle and that Brg1 has specific functions within vascular and gastrointestinal smooth muscle that cannot be performed by Brm.

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 NH(2)-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.

Modulation of myocardin function by the ubiquitin E3 ligase UBR5.

Fully differentiated mature smooth muscle cells (SMCs) are characterized by the presence of a unique repertoire of smooth muscle-specific proteins. Although previous studies have shown myocardin to be a critical transcription factor for stimulating expression of smooth muscle-specific genes, the mechanisms regulating myocardin activity are still poorly understood. We used a yeast two-hybrid screen with myocardin as bait to search for factors that may regulate the transcriptional activity of the myocardin. From this screen we identified a HECT domain-containing protein UBR5 (ubiquitin protein ligase E3 component n-recognin 5) as a myocardin-binding protein. Previous studies have shown that HECT domain-containing proteins are ubiquitin E3 ligases that play an important role in protein degradation. UBR5 has, however, also been shown to regulate transcription independent of its E3 ligase activity. In the current study we demonstrated that UBR5 localized in the nuclei of SMCs and forms a complex with myocardin in vivo and in vitro. We also show that UBR5 specifically enhanced trans-activation of smooth muscle-specific promoters by the myocardin family of proteins. In addition, UBR5 significantly augmented the ability of myocardin to induce expression of endogenous SMC marker genes independent on its E3 ligase function. Conversely, depletion of endogenous UBR5 by small interfering RNA in fibroblast cells attenuated myocardin-induced smooth muscle-specific gene expression, and UBR5 knockdown in SMCs resulted in down-regulation of smooth muscle-specific genes. Furthermore, we found that UBR5 can attenuate myocardin protein degradation resulting in increased myocardin protein expression without affecting myocardin mRNA expression. The effects of UBR5 on myocardin requires only the HECT and UBR1 domains of UBR5. This study reveals an unexpected role for the ubiquitin E3 ligase UBR5 as an activator of smooth muscle differentiation through its ability to stabilize myocardin protein.

The SWI/SNF chromatin remodeling complex regulates myocardin-induced smooth muscle-specific gene expression.

OBJECTIVE: Regulatory complexes comprising myocardin and serum response factor (SRF) are critical for the transcriptional regulation of many smooth muscle-specific genes. However, little is known about the epigenetic mechanisms that regulate the activity of these complexes. In the current study, we investigated the role of SWI/SNF ATP-dependent chromatin remodeling enzymes in regulating the myogenic activity of myocardin. METHODS AND RESULTS: We found that both Brg1 and Brm are required for maintaining expression of several smooth muscle-specific genes in primary cultures of aortic smooth muscle cells. Furthermore, the ability of myocardin to induce expression of smooth muscle-specific genes is abrogated in cells expressing dominant negative Brg1. In SW13 cells, which lack endogenous Brg1 and Brm1, myocardin is unable to induce expression of smooth muscle-specific genes. Whereas, reconstitution of wild-type, or bromodomain mutant forms Brg1 or Brm1, into SW13 cells restored their responsiveness to myocardin. SWI/SNF complexes were found to be required for myocardin to increase SRF binding to the promoters of smooth muscle-specific genes. Brg1 and Brm directly bind to the N terminus of myocardin, in vitro, through their ATPase domains and Brg1 forms a complex with SRF and myocardin in vivo in smooth muscle cells. CONCLUSIONS: These data demonstrate that the ability of myocardin to induce smooth muscle-specific gene expression is dependent on its interaction with SWI/SNF ATP-dependent chromatin remodeling complexes.