Muscle-specific Genes Research Articles

S49 Increased skeletal muscle-specific microRNA-1 in the blood of COPD patients

<h3>Introduction</h3> Quadriceps muscle dysfunction is an important prognostic comorbidity in COPD. MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression. Skeletal muscle expresses a number of tissue-specific microRNA including miR-1, which modulates muscle phenotype. MicroRNAs can be secreted from cells and maintained in blood within exosomes. Elevated levels of circulating miR-1 have been demonstrated in a number of human and animal models of muscle disease. We hypothesised that plasma levels of miR-1 would be elevated in COPD patients and would correlate with important physiological parameters. <h3>Methods</h3> 103 COPD patients and 25 controls were studied. MiR-1 was quantified in stored plasma samples using q-RT PCR.1 MiR-16 and miR-122 were quantified as negative controls. Results were normalised to an exogenous spiked-in control. <h3>Results</h3> Characteristics as mean (SD); COPD patients: M: 67, F: 36, age=66.47 (8.4), FEV₁ % pred= 43.5 (18.6), 6-minute walk (6MW) = 394 (120). Controls: M: 14, F: 11, age=67 (8.1), FEV₁ % pred=111.2 (13.1), 6MW=613 (83). Plasma miR-1 was significantly elevated in COPD patients, p=0.002. There was no difference in miR-16 and miR-122. MiR-1 was negatively associated with FEV₁ % predicted (r =−0.3, p&lt;0.001) and with Tlco (r=−0.3, p&lt;0.001), but it was not possible to distinguish between GOLD stages using ANOVA. However, if patients were sub-divided into early GOLD stage COPD (1 and 2) or late GOLD stage COPD (3 and 4), miR-1 was significantly higher in the latter group (p=0.02). The plasma level of miR-1 was inversely correlated with daily activity measured as locomotion time (r= −0.25, p&lt;0.01) but miR-1 levels were not associated with any muscle phenotype or with muscle-specific gene expression. <h3>Conclusion</h3> Our results show that stable COPD patients have elevated plasma levels of muscle-specific miR-1. The increase in miR-1 may be due to increased muscle degradation or turnover in the COPD patients studied. Our work raises the possibility of using other muscle-specific microRNAs in the future as potential biomarkers of muscle dysfunction in patients with COPD.

A unique library of myogenic cells from facioscapulohumeral muscular dystrophy subjects and unaffected relatives: family, disease and cell function

To explore possible mechanisms of pathology in facioscapulohumeral muscular dystrophy (FSHD), we generated a novel library of myogenic cells composed of paired cultures derived from FSHD subjects and unaffected first-degree relatives. We prepared cells from biopsies of both biceps and deltoid muscles obtained from each of 10 FSHD and 9 unaffected donors. We used this new collection to determine how family background and disease affected patterns of growth and differentiation, expression of a panel of candidate, and muscle-specific genes, and responses to exogenous stressors. We found that FSHD and unaffected cells had, on average, indistinguishable patterns of differentiation, gene expression, and dose-response curves to staurosporine, paraquat, hydrogen peroxide, and glutathione depletion. Differentiated FSHD and unaffected cultures were both more sensitive to glutathione depletion than proliferating cultures, but showed similar responses to paraquat, staurosporine, and peroxide. For stress responses, the sample size was sufficient to detect a 10% change in effect at the observed variability with a power of >99%. In contrast, for each of these properties, we found significant differences among cells from different cohorts, and these differences were independent of disease status, gender, or muscle biopsied. Thus, though none of the properties we examined could be used to reliably distinguish between FSHD and unaffected cells, family of origin was an important contributor to gene-expression patterns and stressor responses in cultures of both FSHD and unaffected myogenic cells.

Loss of CYR61 in Myeloma Microenvironment Is Associated with Poor Survival

Abstract 1813Previously, we used gene expression profiling (GEP) on whole-bone biopsies (BX) and plasma cells (PCs) of newly diagnosed multiple myeloma (MM) patients and age-matched controls to identify 62 non-PC-related genes that were significantly differentially expressed in bone biopsies of MM. CYR61/CCN1 was the most significantly overexpressed gene in BX in subsets of MGUS and MM patients. We hypothesize that alteration of the microenvironment may be central to the conversion of MGUS to MM and that abnormal expression of CYR61 may precede this conversion. CYR61 has been shown to promote proliferation, migration, adhesion and angiogenesis and is expressed in many cell types. Within the bone, CYR61 is induced during osteoblast differentiation by endothelin-1, (Clines et al., 2007) and by Wnt3a (Si et al., 2006). CYR61 also inhibits the formation of multinucleate osteoclasts in vitro (Crockett et al., 2007). To investigate the role of CYR61 in MM pathogenesis CYR61 protein production in MM was analyzed in sera of 192 newly diagnosed myeloma patients by ELISA. In agreement with that observed by GEP, CYR61 is significantly elevated in a subset of MM patients (144/192) at diagnosis compared with healthy donors (p < 0.001). In Total Therapy 3 (TT3) patients low CYR61 (< 500 pg/ml) is associated with poor overall survival (p < 0.05) compared with levels greater than 500 pg/ml. CYR61 levels were also significantly elevated in MGUS, Waldenstrom’s macroglobulinemia and smoldering/indolent myeloma compared with healthy donors (p < 0.001). Patient samples were grouped into the 8 GEP-defined MM molecular subtypes (Zhan et al., 2006) and CYR61 levels were significantly different from healthy donors in all subtypes with the exception of MY that typically is associated with a better prognosis. Analysis of CYR61 levels in TT3 patients 48h post-Velcade (Vel) revealed an inverse correlation between change in CYR61 levels post-Vel and 80-gene risk in TT3a patients (p < 0.01). Likewise, the change in CYR61 levels from diagnosis to remission is also inversely correlated with risk (p < 0.01). To better understand the mechanism by which loss of CYR61 might negatively affect risk we examined the GEP from MM Bx and CD138-selected plasma cells to compare gene signatures in patients with low CYR61 (Q1) and high CYR61 (Q4). Those genes that exhibited ≥ 2-fold difference were investigated further. Patients with low CYR61 differentially expressed 179 probesets in MMBX including elevated SFRP2 (4.1-fold), a Wnt inhibitor, and COL2A1 (3.5-fold) which are associated with myeloma bone disease and osteoarthritis, respectively. SFRP2 is also elevated (4.3-fold) in MMPCs (194 probesets elevated) along with TNFSF10 (2.1-fold) indicative of the HY subtype and FGFR3 (2.1-fold) which is elevated in the MS subtype. In patients with high CYR61, many muscle-specific genes were elevated in MM BX and may indicate a predominance of osteocytes. Additionally, BGLAP, whose loss is associated with poor survival in myeloma, is elevated (2-fold) in MM BX (171 probesets) with high CYR61 and EDN1, an activator of CYR61/CCN1, is elevated (2.8-fold) in MM PCs (111 probesets) with high CYR61. The test the effect of CYR61 on cell growth, H929 cells were exposed to rCYR61 which caused a dose- and time-dependent suppression of cell growth and survival. To examine the role of CYR61 in the MM ME, H929 cells transduced with empty vector, wild type CYR61 or CYR61 cDNA with a mutated αvβ3 binding site were injected into implanted bones in SCID-hu mice. CYR61-induced effects on bone have been shown to be mediated by the αvβ3 integrin, presumably through osteoblasts. X-rays of the implanted bone and serum measurement of hIg levels were taken weekly for 5 weeks. Quantitation of change in bone mineral density (BMD) over the course of the experiment showed that CYR61 inhibits osteolysis (p<0.04), whereas CYR61 that is incapable of binding αvβ3 attenuates this effect. These data indicate that the αvβ3 integrin mediates CYR61-induced inhibition of osteolysis in MM. Additionally, quantitation of serum hIg demonstrated that CYR61 significantly reduces MM tumor growth (p<0.04) and this is not effected by mutation of the avb3 binding site. Taken together these data suggest that loss of CYR61 in myeloma may be indicative of a dysfunctional ME and thus serve as a biomarker to monitor the MM ME. Finally, therapeutics that upregulate CCN1 will be tested as a novel treatment for myeloma bone disease. Disclosures:Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

Latent Cardiogenic Potential in Endocardium and Hemogenic Endothelium Revealed in the Absence of Scl/tal1

Abstract 2362The endothelium in embryonic and extraembryonic hematopoietic tissues has the capacity to generate hematopoietic stem and progenitor cells (HS/PC). However, it is unknown how this unique endothelium is specified. Microarray analysis of endothelial cells from hematopoietic tissues of embryos deficient for the bHLH transcription factor Scl/tal1 revealed that Scl establishes a robust hematopoietic transcriptional program in the endothelium. Surprisingly, lack of Scl also induced an unexpected fate switching of the prospective hemogenic endothelium to the cardiac lineage. Scl deficient embryos displayed a dramatic upregulation of cardiac transcription factors and structural proteins within the yolk sac vasculature, resulting in the generation of spontaneously beating cardiomyocytes. Ectopic cardiac potential in Scl deficient embryos arose from endothelial-derived CD31+Pdgfrα+ cardiogenic progenitor cells (CPCs), which were present in all sites of HS/PC generation. Analysis of Runx1-deficient embryos revealed, that although Runx1 acts downstream of Scl during the emergence of definitive HS/PCs, it is not required for the suppression of the cardiac fate in the endothelium. The only wild type tissue that contained CD31+Pdgfrα+ putative CPCs was the heart, and this population was greatly expanded in Scl deficient embryos. Strikingly, endocardium in Scl−/− hearts also activated a robust cardiomyogenic transcriptional program and generated Troponin T+ cardiomyocytes both in vivo and in vitro. Although CD31+Pdgfrα+ CPCs from wild type hearts did not generate readily beating cells in culture, they produced cells expressing endothelial, smooth muscle and cardiomyocyte specific genes, implying multipotentiality of this novel CPC population. Furthermore, CD31+Pdgfrα+ CPCs were greatly reduced in Isl1−/− hearts, which fail to generate functional, multipotential CPCs. Lineage tracing using VE-cadherin Cre Rosa-YFP mouse strain demonstrated that, in addition to generating HS/PCs in hematopoietic tissues, endothelial cells are also the cell of origin for CD31+Pdgfrα+ CPCs in the heart. Together, these data suggest a broader role for embryonic endothelium as a potential source of tissue-specific stem and progenitor cells and implicate Scl/tal1 as an important regulator of endothelial fate choice. Disclosures:No relevant conflicts of interest to declare.

Transforming Growth Factor-β1-induced Transcript 1 Protein, a Novel Marker for Smooth Muscle Contractile Phenotype, Is Regulated by Serum Response Factor/Myocardin Protein

Serum response factor (SRF) plays a central role in regulating expression of smooth muscle-specific genes partly by associating with the potent tissue-specific cofactor myocardin. Previous studies have shown that transforming growth factor-β1-induced transcript 1 (TGFB1I1, also known as Hic-5) is a TGF-β-responsive gene and is involved in the cellular response to vascular injury, but the regulation of TGFB1I1 expression remains elusive. In this report, we demonstrated that TGFB1I1 is a novel marker for the smooth muscle contractile phenotype and is regulated by SRF/myocardin. We found that TGFB1I1 is specifically expressed in smooth muscle cells (SMCs) and in smooth muscle-rich tissues. Furthermore, TGFB1I1 expression is significantly down-regulated in a variety of models for smooth muscle phenotypic modulation. The TGFB1I1 promoter contains an evolutionarily conserved CArG element, and this element is indispensible for myocardin-induced transactivation of TGFB1I1 promoter. By oligonucleotide pulldown and chromatin immunoprecipitation assays, we found that SRF binds to this CArG element in vitro and in vivo. Ectopic expression of myocardin is sufficient to induce endogenous TGFB1I1 expression in multiple cell lines whereas knocking-down myocardin or SRF significantly attenuated TGFB1I1 expression in SMCs. Furthermore, our data demonstrated that SRF is essential for TGF-β-mediated induction of TGFB1I1. Finally, silencing of TGFB1I1 expression significantly promotes SMC proliferation. Collectively, this study provides the first evidence that TGFB1I1 is not only an SRF/myocardin-regulated smooth muscle marker but also critical for maintaining smooth muscle contractile phenotype by inhibiting smooth muscle proliferation.

Canonical Wnt signaling is involved in switching from cell proliferation to myogenic differentiation of mouse myoblast cells

BackgroundWnt/β-catenin signaling is involved in various aspects of skeletal muscle development and regeneration. In addition, Wnt3a and β-catenin are required for muscle-specific gene transcription in embryonic carcinoma cells and satellite-cell proliferation during adult skeletal muscle regeneration. Downstream targets of canonical Wnt signaling are cyclin D1 and c-myc. However both target genes are suppressed during differentiation of mouse myoblast cells, C2C12. Underlying molecular mechanisms of β-catenin signaling during myogenic differentiation remain unknown.ResultsUsing C2C12 cells, we examined intracellular signaling and gene transcription during myoblast proliferation and differentiation. We confirmed that several Wnt signaling components, including Wnt9a, Sfrp2 and porcupine, were consistently upregulated in differentiating C2C12 cells. Troponin T-positive myotubes were decreased by Wnt3a overexpression, but not Wnt4. TOP/FOP reporter assays revealed that co-expression with Wnt4 reduced Wnt3a-induced luciferase activity, suggesting that Wnt4 signaling counteracted Wnt3a signaling in myoblasts. FH535, a small-molecule inhibitor of β-catenin/Tcf complex formation, reduced basal β-catenin in the cytoplasm and decreased myoblast proliferation. K252a, a protein kinase inhibitor, increased both cytosolic and membrane-bound β-catenin and enhanced myoblast fusion. Treatments with K252a or Wnt4 resulted in increased cytoplasmic vesicles containing phosphorylated β-catenin (Tyr654) during myogenic differentiation.ConclusionsThese results suggest that various Wnt ligands control subcellular β-catenin localization, which regulate myoblast proliferation and myotube formation. Wnt signaling via β-catenin likely acts as a molecular switch that regulates the transition from cell proliferation to myogenic differentiation.

A Long Noncoding RNA Controls Muscle Differentiation by Functioning as a Competing Endogenous RNA

SummaryRecently, a new regulatory circuitry has been identified in which RNAs can crosstalk with each other by competing for shared microRNAs. Such competing endogenous RNAs (ceRNAs) regulate the distribution of miRNA molecules on their targets and thereby impose an additional level of post-transcriptional regulation. Here we identify a muscle-specific long noncoding RNA, linc-MD1, which governs the time of muscle differentiation by acting as a ceRNA in mouse and human myoblasts. Downregulation or overexpression of linc-MD1 correlate with retardation or anticipation of the muscle differentiation program, respectively. We show that linc-MD1 “sponges” miR-133 and miR-135 to regulate the expression of MAML1 and MEF2C, transcription factors that activate muscle-specific gene expression. Finally, we demonstrate that linc-MD1 exerts the same control over differentiation timing in human myoblasts, and that its levels are strongly reduced in Duchenne muscle cells. We conclude that the ceRNA network plays an important role in muscle differentiation.

An EDMD Mutation in C. elegans Lamin Blocks Muscle-Specific Gene Relocation and Compromises Muscle Integrity

In worms, as in other organisms, many tissue-specific promoters are sequestered at the nuclear periphery when repressed and shift inward when activated. It has remained unresolved, however, whether the association of facultative heterochromatin with the nuclear periphery, or its release, has functional relevance for cell or tissue integrity. Using ablation of the unique lamin gene in C.elegans, we show that lamin is necessary for the perinuclear positioning of heterochromatin. We then express at low levels in otherwise wild-type worms a lamin carrying a point mutation, Y59C, which in humans is linked to an autosomal-dominant form of Emery-Dreifuss muscular dystrophy. Using embryos and differentiated tissues, we track the subnuclear position of integrated heterochromatic arrays and their expression. In LMN-1 Y59C-expressing worms, we see abnormal retention at the nuclear envelope of a gene array bearing a muscle-specific promoter. This correlates with impaired activation of the array-borne myo-3 promoter and altered expression of a number of muscle-specific genes. However, an equivalent array carrying the intestine-specific pha-4 promoter is expressed normally and shifts inward when activated in gut cells of LMN-1 Y59C worms. Remarkably, adult LMN-1 Y59C animals have selectively perturbed body muscle ultrastructure and reduced muscle function. Lamin helps sequester heterochromatin at the nuclear envelope, and wild-type lamin permits promoter release following tissue-specific activation. A disease-linked point mutation in lamin impairs muscle-specific reorganization of a heterochromatic array during tissue-specific promoter activation in a dominant manner. This dominance and the correlated muscle dysfunction in LMN-1 Y59C worms phenocopies Emery-Dreifuss muscular dystrophy.

Enhancement of mDia2 activity by Rho-kinase-dependent phosphorylation of the diaphanous autoregulatory domain

It is clear that RhoA activates the DRF (diaphanous-related formin) mDia2 by disrupting the molecular interaction between the DAD (diaphanous autoregulatory domain) and the DID (diaphanous inhibitory domain). Previous studies indicate that a basic motif within the DAD contributes to mDia2 auto-inhibition, and results shown in the present study suggest these residues bind a conserved acidic region within the DID. Furthermore, we demonstrate that mDia2 is phosphorylated by ROCK (Rho-kinase) at two conserved residues (Thr(1061) and Ser(1070)) just C-terminal to the DAD basic region. Phosphomimetic mutations to these residues in the context of the full-length molecule enhanced mDia2 activity as measured by increased actin polymerization, SRF (serum response factor)-dependent smooth muscle-specific gene transcription, and nuclear localization of myocardin-related transcription factor B. Biochemical and functional data indicate that the T1061E/S1070E mutation significantly inhibited the ability of DAD to interact with DID and enhanced mDia2 activation by RhoA. Taken together, the results of the present study indicate that ROCK-dependent phosphorylation of the mDia2 DAD is an important determinant of mDia2 activity and that this signalling mechanism affects actin polymerization and smooth muscle cell-specific gene expression.

Muscle-specific gene expression is underscored by differential stressor responses and coexpression changes

Variations on the transcriptome from one skeletal muscle type to another still remain unknown. The reliable identification of stable gene coexpression networks is essential to unravel gene functions and define biological processes. The differential expression of two distinct muscles, M. flexor digitorum (FD) and M. psoas major (PM), was studied using microarrays in cattle to illustrate muscle-specific transcription patterns and to quantify changes in connectivity regarding the expected gene coexpression pattern. A total of 206 genes were differentially expressed (DE), 94 upregulated in PM and 112 in FD. The distribution of DE genes in pathways and biological functions was explored in the context of system biology. Global interactomes for genes of interest were predicted. Fast/slow twitch genes, genes coding for extracellular matrix, ribosomal and heat shock proteins, and fatty acid uptake centred the specific gene expression patterns per muscle. Genes involved in repairing mechanisms, such as ribosomal and heat shock proteins, suggested a differential ability of muscles to react to similar stressing factors, acting preferentially in slow twitch muscles. Muscle attributes do not seem to be completely explained by the muscle fibre composition. Changes in connectivity accounted for 24% of significant correlations between DE genes. Genes changing their connectivity mostly seem to contribute to the main differential attributes that characterize each specific muscle type. These results underscore the unique flexibility of skeletal muscle where a substantial set of genes are able to change their behavior depending on the circumstances.

Synergistic Activation of Cardiac Genes by Myocardin and Tbx5

Myocardial differentiation is associated with the activation and expression of an array of cardiac specific genes. However, the transcriptional networks that control cardiac gene expression are not completely understood. Myocardin is a cardiac and smooth muscle-specific expressed transcriptional coactivator of Serum Response Factor (SRF) and is able to potently activate cardiac and smooth muscle gene expression during development. We hypothesize that myocardin discriminates between cardiac and smooth muscle specific genes by associating with distinct co-factors. Here, we show that myocardin directly interacts with Tbx5, a member of the T-box family of transcription factors involved in the Holt-Oram syndrome. Tbx5 synergizes with myocardin to activate expression of the cardiac specific genes atrial natriuretic factor (ANF) and alpha myosin heavy chain (α-MHC), but not that of smooth muscle specific genes SM22 or smooth muscle myosin heavy chain (SM-MHC). We found that this synergistic activation of shared target genes is dependent on the binding sites for Tbx5, T-box factor-Binding Elements (TBEs). Myocardin and Tbx5 physically interact and their interaction domains were mapped to the basic domain and the coil domain of myocardin and Tbx5, respectively. Our analysis demonstrates that the Tbx5G80R mutation, which leads to the Holt-Oram syndrome in humans, failed to synergize with myocardin to activate cardiac gene expression. These data uncover a key role for Tbx5 and myocardin in establishing the transcriptional foundation for cardiac gene activation and suggest that the interaction of myocardin and Tbx5 maybe involved in cardiac development and diseases.

The histone methyltransferase Set7/9 promotes myoblast differentiation and myofibril assembly

The molecular events that modulate chromatin structure during skeletal muscle differentiation are still poorly understood. We report in this paper that expression of the H3-K4 histone methyltransferase Set7 is increased when myoblasts differentiate into myotubes and is required for skeletal muscle development, expression of muscle contractile proteins, and myofibril assembly. Knockdown of Set7 or expression of a dominant-negative Set7 mutant impairs skeletal muscle differentiation, accompanied by a decrease in levels of histone monomethylation (H3-K4me1). Set7 directly interacts with MyoD to enhance expression of muscle differentiation genes. Expression of myocyte enhancer factor 2 and genes encoding contractile proteins is decreased in Set7 knockdown myocytes. Furthermore, we demonstrate that Set7 also activates muscle gene expression by precluding Suv39h1-mediated H3-K9 methylation on the promoters of myogenic differentiation genes. Together, our experiments define a biological function for Set7 in muscle differentiation and provide a molecular mechanism by which Set7 modulates myogenic transcription factors during muscle differentiation.

Modeling Oculopharyngeal Muscular Dystrophy in Myotube Cultures Reveals Reduced Accumulation of Soluble Mutant PABPN1 Protein

Oculopharyngeal muscular dystrophy (OPMD) is an autosomal dominant disease caused by an alanine tract expansion mutation in poly(A) binding protein nuclear 1 (expPABPN1). To model OPMD in a myogenic and physiological context, we generated mouse myoblast cell clones stably expressing either human wild type (WT) or expPABPN1 at low levels. Transgene expression is induced on myotube differentiation and results in formation of insoluble nuclear PABPN1 aggregates that are similar to those observed in patients with OPMD. Quantitative analysis of PABPN1 in myotube cultures revealed that expPABPN1 accumulation and aggregation is greater than that of the WT protein. We found that aggregation of expPABPN1 is more affected than WT PABPN1 by inhibition of proteasome activity. Consistent with this, in myotube cultures expressing expPABPN1, deregulation of the proteasome was identified as the most significantly perturbed pathway. Differences in the accumulation of soluble WT and expPABPN1 were consistent with differences in ubiquitination and rate of protein turnover. This study demonstrates, for the first time to our knowledge, that, in myotubes, the ratio of soluble/insoluble expPABPN1 is significantly lower compared with that of the WT protein. We suggest that this difference can contribute to muscle weakness in OPMD.

M-CAT element mediates mechanical stretch-activated transcription of B-type natriuretic peptide via ERK activation

The muscle-CAT (M-CAT) promoter element is found on promoters of most muscle-specific cardiac genes, but its role in cardiac pathology is poorly understood. Here we studied whether the M-CAT element is involved in hypertrophic process activated by mechanical stretch, and identified the intracellular pathways mediating the response. When an in vitro stretch model of cultured neonatal rat cardiomyocytes and luciferase reporter construct driven by rat B-type natriuretic peptide (BNP) promoter were used, mutation of M-CAT element inhibited not only the basal reporter activity (88%), but also the stretch-activated BNP transcription (58%, p< 0.001). Stretch-induced BNP promoter activation was associated with an increase in transcriptional enhancer factor-1 (TEF-1) binding activity after 24h mechanical stretch (p< 0.05). Inhibition of mitogen-activated protein kinases ERK, JNK, or p38 attenuated stretch-induced BNP activation. Interestingly, as opposed to p38 and JNK, inhibition of ERK had no additional effect on transcriptional activity of BNP promoter harboring the M-CAT mutation, suggesting a pivotal role for ERK in regulating stretch-induced BNP transcription via M-CAT binding site. Finally, immunoprecipitation studies showed that mechanical stretch induced myocyte enhancer factor-2 (MEF-2) binding to TEF-1. These data suggest a central role for M-CAT element in regulation of mechanical stretch-induced hypertrophic response via ERK activation.

Characterization of urine-derived stem cells obtained from upper urinary tract for use in cell-based urological tissue engineering.

The goals of this study were to characterize urine-derived stem cells obtained from the upper urinary tract (uUSC), induce these cells to differentiate into urothelial and smooth muscle cells, and determine whether they could serve as a potential stem cell source for bladder tissue engineering. Urine samples were collected from five patients with normal upper urinary tracts during renal pyeloplasty. Cells were isolated from this urine and extensively expanded in vitro. The mean population doubling of uUSC was 46.5±7.7. The uUSC expressed surface markers associated with mesenchymal stem cells and pericytes. These cells could differentiate into smooth muscle-like cells that expressed smooth muscle-specific gene transcripts and proteins, including α-smooth muscle actin, desmin, and myosin, when exposed to TGF-β1 and PDGF-BB. In a collagen lattice assay, these myogenic-differentiated uUSC displayed contractile function that was similar to that seen in native smooth muscle cells. Urothelial-differentiated uUSC expressed urothelial-specific genes and proteins such as uroplakin-Ia and -III, cytokeratin (CK)-7, and CK-13. uUSC possess expansion and differentiation (urothelial and myogenic) capabilities, and can potentially be used as an alternative cell source in bladder tissue engineering for patients needing cystoplasty.

Gas1 cooperates with Cdo and promotes myogenic differentiation via activation of p38MAPK

Skeletal myogenesis is a multistep process that involves cell cycle exit, expression of muscle-specific genes and formation of multinucleated myotubes. Growth arrest specific gene 1 (Gas1) is a GPI-linked membrane protein and originally identified as a growth arrest-linked gene in fibroblasts. Promyogenic cell surface protein, Cdo functions as a component of multiprotein complexes that include other cell adhesion molecules, like Cadherins to mediate cell contact signaling. Here we report that Gas1 and Cdo are coexpressed in muscle cells and form a complex in differentiating myoblasts. Interestingly, Cdo−/− myoblasts display defects in Gas1 induction during differentiation. Overexpression or depletion of Gas1 enhances or decreases myogenic differentiation, respectively. During myoblast differentiation, Gas1 depletion causes defects in downregulation of Cdk2 and Cyclin D1 and up-regulation of miR-322, a negative regulator of Cdk2 activities. Furthermore overexpression or knockdown of Gas1 either enhances or decreases activation of p38MAPK that functions downstream of Cdo. Additionally, Gas1 overexpression in Cdo-depleted C2C12 cells restores p38MAPK activities and differentiation abilities. These data suggest that Gas1 promotes myogenic differentiation through regulation of cell cycle arrest and is critical to activate p38MAPK, most likely via association with Cdo/Cadherin multiprotein complexes.

Coordination of cell cycle, DNA repair and muscle gene expression in myoblasts exposed to genotoxic stress

Upon exposure to genotoxic stress, skeletal muscle progenitors coordinate DNA repair and the activation of the differentiation program through the DNA damage-activated differentiation checkpoint, which holds the transcription of differentiation genes while the DNA is repaired. A conceptual hurdle intrinsic to this process relates to the coordination of DNA repair and muscle-specific gene transcription within specific cell cycle boundaries (cell cycle checkpoints) activated by different types of genotoxins. Here, we show that, in proliferating myoblasts, the inhibition of muscle gene transcription occurs by either a G1- or G2-specific differentiation checkpoint. In response to genotoxins that induce G1 arrest, MyoD binds target genes but is functionally inactivated by a c-Abl-dependent phosphorylation. In contrast, DNA damage-activated G2 checkpoint relies on the inability of MyoD to bind the chromatin at the G2 phase of the cell cycle. These results indicate an intimate relationship between DNA damage-activated cell cycle checkpoints and the control of tissue-specific gene expression to allow DNA repair in myoblasts prior to the activation of the differentiation program.

Gamma Interferon Modulates Myogenesis through the Major Histocompatibility Complex Class II Transactivator, CIITA

Gamma interferon (IFN-γ) is an inflammatory cytokine that has complex effects on myogenesis. Here, we show that the IFN-γ-induced inhibition of myogenesis is mediated by the major histocompatibility complex (MHC) class II transactivator, CIITA, which binds to myogenin and inhibits its activity. In IFN-γ-treated myoblasts, the inhibition of muscle-specific genes includes the expression of myogenin itself, while in myotubes, myogenin expression is unaffected. Thus, CIITA appears to act by both repressing the expression and inhibiting the activity of myogenin at different stages of myogenesis. Stimulation by IFN-γ in skeletal muscle cells induces CIITA expression as well as MHC class II gene expression. The IFN-γ-mediated repression is reversible, with myogenesis proceeding normally upon removal of IFN-γ. Through overexpression studies, we confirm that the expression of CIITA, independent of IFN-γ, is sufficient to inhibit myogenesis. Through knockdown studies, we also demonstrate that CIITA is necessary for the IFN-γ-mediated inhibition of myogenesis. Finally, we show that CIITA, which lacks DNA binding activity, is recruited to muscle-specific promoters coincident with reductions in RNA polymerase II recruitment. Thus, this work reveals how IFN-γ modulates myogenesis and demonstrates a key role for CIITA in this process.

Transcription factor TEAD4 regulates expression of Myogenin and the unfolded protein response genes during C2C12 cell differentiation

The TEAD (1-4) transcription factors comprise the conserved TEA/ATTS DNA-binding domain recognising the MCAT element in the promoters of muscle-specific genes. Despite extensive genetic analysis, the function of TEAD factors in muscle differentiation has proved elusive due to redundancy among the family members. Expression of the TEA/ATTS DNA-binding domain that acts as a dominant negative repressor of TEAD factors in C2C12 myoblasts inhibits their differentiation, whereas selective shRNA knockdown of TEAD4 results in abnormal differentiation characterised by the formation of shortened myotubes. Chromatin immunoprecipitation coupled to array hybridisation shows that TEAD4 occupies 867 promoters including those of myogenic miRNAs. We show that TEAD factors directly induce Myogenin, CDKN1A and Caveolin 3 expression to promote myoblast differentiation. RNA-seq identifies a set of genes whose expression is strongly reduced upon TEAD4 knockdown among which are structural and regulatory proteins and those required for the unfolded protein response. In contrast, TEAD4 represses expression of the growth factor CTGF (connective tissue growth factor) to promote differentiation. Together these results show that TEAD factor activity is essential for normal C2C12 cell differentiation and suggest a role for TEAD4 in regulating expression of the unfolded protein response genes.

Ret finger protein inhibits muscle differentiation by modulating serum response factor and enhancer of polycomb1

Skeletal myogenesis is precisely regulated by multiple transcription factors. Previously, we demonstrated that enhancer of polycomb 1 (Epc1) induces skeletal muscle differentiation by potentiating serum response factor (SRF)-dependent muscle gene activation. Here, we report that an interacting partner of Epc1, ret finger protein (RFP), blocks skeletal muscle differentiation. Our findings show that RFP was highly expressed in skeletal muscles and was downregulated during myoblast differentiation. Forced expression of RFP delayed myoblast differentiation, whereas knockdown enhanced it. Epc1-induced enhancements of SRF-dependent multinucleation, transactivation of the skeletal α-actin promoter, binding of SRF to the serum response element, and muscle-specific gene induction were blocked by RFP. RFP interfered with the physical interaction between Epc1 and SRF. Muscles from rfp knockout mice (Rfp(-/-)) mice were bigger than those from wild-type mice, and the expression of SRF-dependent muscle-specific genes was upregulated. Myotube formation and myoblast differentiation were enhanced in Rfp(-/-) mice. Taken together, our findings highlight RFP as a novel regulator of muscle differentiation that acts by modulating the expression of SRF-dependent skeletal muscle-specific genes.