Publications by authors named "Tetsuaki Miyake"

17 Publications

  • Page 1 of 1

REEP5 depletion causes sarco-endoplasmic reticulum vacuolization and cardiac functional defects.

Nat Commun 2020 02 19;11(1):965. Epub 2020 Feb 19.

Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research, Toronto, ON, M5G1M1, Canada.

The sarco-endoplasmic reticulum (SR/ER) plays an important role in the development and progression of many heart diseases. However, many aspects of its structural organization remain largely unknown, particularly in cells with a highly differentiated SR/ER network. Here, we report a cardiac enriched, SR/ER membrane protein, REEP5 that is centrally involved in regulating SR/ER organization and cellular stress responses in cardiac myocytes. In vitro REEP5 depletion in mouse cardiac myocytes results in SR/ER membrane destabilization and luminal vacuolization along with decreased myocyte contractility and disrupted Ca cycling. Further, in vivo CRISPR/Cas9-mediated REEP5 loss-of-function zebrafish mutants show sensitized cardiac dysfunction upon short-term verapamil treatment. Additionally, in vivo adeno-associated viral (AAV9)-induced REEP5 depletion in the mouse demonstrates cardiac dysfunction. These results demonstrate the critical role of REEP5 in SR/ER organization and function as well as normal heart function and development.
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http://dx.doi.org/10.1038/s41467-019-14143-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031342PMC
February 2020

Maintenance of the Undifferentiated State in Myogenic Progenitor Cells by TGFβ Signaling is Smad Independent and Requires MEK Activation.

Int J Mol Sci 2020 02 5;21(3). Epub 2020 Feb 5.

Department of Biology, York University, 4700 Keele Street, Toronto, ON M3J 1P3, Canada.

Transforming growth factor β (TGFβ) is a pluripotent cytokine and regulates a myriad of biological processes. It has been established that TGFβ potently inhibits skeletal muscle differentiation; however, the molecular mechanism is not clearly defined. Previously, we reported that inhibition of the TGFβ canonical pathway by an inhibitory Smad, Smad7, does not reverse this effect on differentiation, suggesting that activation of receptor Smads (R-Smads) by TGFβ is not responsible for repression of myogenesis. In addition, pharmacological blockade of Smad3 activation by TGFβ did not reverse TGFβ's inhibitory effect on myogenesis. In considering other pathways, we observed that TGFβ potently activates MEK/ERK, and a pharmacological inhibitor of MEK reversed TGFβ's inhibitory effect on myogenesis, as indicated by a promoter-reporter gene, sarcomeric myosin heavy chain accumulation, and phenotypic myotube formation. Furthermore, we found that c-Jun, a known potent repressor of myogenesis, which is coincidently also a down-stream target of MEK/ERK signaling, was phosphorylated and accumulates in the nucleus in response to TGFβ activation. Taken together, these observations support a model in which TGFβ activates a MEK/ERK/c-Jun pathway to repress skeletal myogenesis, maintaining the pluripotent undifferentiated state in myogenic progenitors.
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http://dx.doi.org/10.3390/ijms21031057DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7038076PMC
February 2020

TNAP limits TGF-β-dependent cardiac and skeletal muscle fibrosis by inactivating the SMAD2/3 transcription factors.

J Cell Sci 2019 08 8;132(15). Epub 2019 Aug 8.

Division of Cell Matrix Biology & Regenerative Medicine, Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, UK

Fibrosis is associated with almost all forms of chronic cardiac and skeletal muscle diseases. The accumulation of extracellular matrix impairs the contractility of muscle cells contributing to organ failure. Transforming growth factor β (TGF-β) plays a pivotal role in fibrosis, activating pro-fibrotic gene programmes via phosphorylation of SMAD2/3 transcription factors. However, the mechanisms that control de-phosphorylation of SMAD2 and SMAD3 (SMAD2/3) have remained poorly characterized. Here, we show that tissue non-specific alkaline phosphatase (TNAP, also known as ALPL) is highly upregulated in hypertrophic hearts and in dystrophic skeletal muscles, and that the abrogation of TGF-β signalling in TNAP-positive cells reduces vascular and interstitial fibrosis. We show that TNAP colocalizes and interacts with SMAD2. The TNAP inhibitor MLS-0038949 increases SMAD2/3 phosphorylation, while TNAP overexpression reduces SMAD2/3 phosphorylation and the expression of downstream fibrotic genes. Overall our data demonstrate that TNAP negatively regulates TGF-β signalling and likely represents a mechanism to limit fibrosis.
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http://dx.doi.org/10.1242/jcs.234948DOI Listing
August 2019

Smad7:β-catenin complex regulates myogenic gene transcription.

Cell Death Dis 2019 05 16;10(6):387. Epub 2019 May 16.

Department of Biology, York University, Toronto, ON, M3J 1P3, Canada.

Recent reports indicate that Smad7 promotes skeletal muscle differentiation and growth. We previously documented a non-canonical role of nuclear Smad7 during myogenesis, independent of its role in TGF-β signaling. Here further characterization of the myogenic function of Smad7 revealed β-catenin as a Smad7 interacting protein. Biochemical analysis identified a Smad7 interaction domain (SID) between aa575 and aa683 of β-catenin. Reporter gene analysis and chromatin immunoprecipitation demonstrated that Smad7 and β-catenin are cooperatively recruited to the extensively characterized ckm promoter proximal region to facilitate its muscle restricted transcriptional activation in myogenic cells. Depletion of endogenous Smad7 and β-catenin in muscle cells reduced ckm promoter activity indicating their role during myogenesis. Deletion of the β-catenin SID substantially reduced the effect of Smad7 on the ckm promoter and exogenous expression of SID abolished β-catenin function, indicating that SID functions as a trans dominant-negative regulator of β-catenin activity. β-catenin interaction with the Mediator kinase complex through its Med12 subunit led us to identify MED13 as an additional Smad7-binding partner. Collectively, these studies document a novel function of a Smad7-MED12/13-β-catenin complex at the ckm locus, indicating a key role of this complex in the program of myogenic gene expression underlying skeletal muscle development and regeneration.
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http://dx.doi.org/10.1038/s41419-019-1615-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6522533PMC
May 2019

FMRP recruitment of β-catenin to the translation pre-initiation complex represses translation.

EMBO Rep 2018 12 25;19(12). Epub 2018 Oct 25.

Department of Biology, York University, Toronto, ON, Canada

Canonical Wnt/β-catenin signaling is an essential regulator of various cellular functions throughout development and adulthood. Aberrant Wnt/β-catenin signaling also contributes to various pathologies including cancer, necessitating an understanding of cell context-dependent mechanisms regulating this pathway. Since protein-protein interactions underpin β-catenin function and localization, we sought to identify novel β-catenin interacting partners by affinity purification coupled with tandem mass spectrometry in vascular smooth muscle cells (VSMCs), where β-catenin is involved in both physiological and pathological control of cell proliferation. Here, we report novel components of the VSMC β-catenin interactome. Bioinformatic analysis of the protein networks implies potentially novel functions for β-catenin, particularly in mRNA translation, and we confirm a direct interaction between β-catenin and the fragile X mental retardation protein (FMRP). Biochemical studies reveal a basal recruitment of β-catenin to the messenger ribonucleoprotein and translational pre-initiation complex, fulfilling a translational repressor function. Wnt stimulation antagonizes this function, in part, by sequestering β-catenin away from the pre-initiation complex. In conclusion, we present evidence that β-catenin fulfills a previously unrecognized function in translational repression.
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http://dx.doi.org/10.15252/embr.201745536DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6280795PMC
December 2018

Three-dimensional imaging reveals endo(sarco)plasmic reticulum-containing invaginations within the nucleoplasm of muscle.

Am J Physiol Cell Physiol 2018 03 22;314(3):C257-C267. Epub 2017 Nov 22.

Translational Biology and Engineering Program, Ted Rogers Centre for Heart Research , Toronto, Ontario , Canada.

The mammalian nucleus has invaginations from the cytoplasm, termed nucleoplasmic reticulum (NR). With increased resolution of cellular imaging, progress has been made in understanding the formation and function of NR. In fact, nucleoplasmic Ca homeostasis has been implicated in the regulation of gene expression, DNA repair, and cell death. However, the majority of studies focus on cross-sectional or single-plane analyses of NR invaginations, providing an incomplete assessment of its distribution and content. Here, we provided advanced imaging and three-dimensional reconstructive analyses characterizing the molecular constituents of nuclear invaginations in the nucleoplasm in HEK293 cells, murine CC muscle cells, and cardiac myocytes. We demonstrated the presence of critical Ca regulatory channels, including sarco(endo)plasmic reticulum Ca-ATPase 2a (SERCA2a), stromal interaction molecule 1 (STIM1), and Ca release-activated Ca channel protein 1 (ORAI1), in the nucleoplasm in isolated primary mouse cardiomyocytes. We have shown for the first time the presence of STIM1 and ORAI1 in the nucleoplasm, suggesting the presence of store-operated calcium entry (SOCE) mechanism in nucleoplasmic Ca regulation. These results show that nucleoplasmic invaginations contain continuous endoplasmic reticulum components, mitochondria, and intact nuclear membranes, highlighting the extremely detailed and complex nature of this organellar structure.
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http://dx.doi.org/10.1152/ajpcell.00141.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5902977PMC
March 2018

TGFβ-TAZ/SRF signalling regulates vascular smooth muscle cell differentiation.

FEBS J 2017 06 20;284(11):1644-1656. Epub 2017 Apr 20.

Department of Biology, York University, Toronto, ON, Canada.

Vascular smooth muscle cells (VSMCs) do not terminally differentiate; they modulate their phenotype between proliferative and differentiated states, which is a major factor contributing to vascular diseases. TGFβ signalling has been implicated in inducing VSMC differentiation, although the exact mechanism remains largely unknown. Our goal was to assess the network of transcription factors involved in the induction of VSMC differentiation, and to determine the role of TAZ in promoting the quiescent VSMC phenotype. TGFβ robustly induces VSMC marker genes in 10T1/2 mouse embryonic fibroblast cells and the potent transcriptional regulator TAZ has been shown to retain Smad complexes on DNA. Thus, the role of TAZ in regulation of VSMC differentiation was studied. Using primary aortic VSMCs coupled with siRNA-mediated gene silencing, our studies reveal that TAZ is required for TGFβ induction of smooth muscle genes and is also required for the differentiated VSMC phenotype; synergy between TAZ and SRF, and TAZ and Myocardin (MyoC856), in regulating smooth muscle gene activation was observed. These data provide evidence of components of a novel signalling pathway that links TGFβ signalling to induction of smooth muscle genes through a mechanism involving regulation of TAZ and SRF proteins. In addition, we report a physical interaction of TAZ and MyoC856. These observations elucidate a novel level of control of VSMC induction which may have implications for vascular diseases and congenital vascular malformations.
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http://dx.doi.org/10.1111/febs.14070DOI Listing
June 2017

Metformin increases degradation of phospholamban via autophagy in cardiomyocytes.

Proc Natl Acad Sci U S A 2015 Jun 26;112(23):7165-70. Epub 2015 May 26.

Department of Physiology, University of Toronto, Toronto, ON, Canada M5G 1L7; Toronto General Hospital, University Health Network, Toronto, ON, Canada M5G 1L7; Ted Rogers Centre for Heart Research, Toronto, ON, Canada M5G 1L7

Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca(2+) ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [(35)S]methionine pulse-chase experiments, together with lysosome (chloroquine and bafilomycin A1) and autophagic (3-methyladenine and Atg5 siRNA) antagonists. Inhibiting lysosomal and autophagic activities promoted endogenous PLN accumulation, whereas accelerating autophagy with metformin enhanced PLN degradation in CMNCs. This reduction in PLN levels was functionally correlated with an increased rate of SERCA2a activity, accounting for an inotropic effect of metformin. Metabolic labeling reaffirmed that metformin promoted wild-type and R9C PLN degradation. Immunofluorescence showed that PLN and the autophagy marker, microtubule light chain 3, became increasingly colocalized in response to chloroquine and bafilomycin treatments. Mechanistically, pentameric PLN was polyubiquitinylated at the K3 residue and this modification was required for p62-mediated selective autophagy trafficking. Consistently, attenuated autophagic flux in HECT domain and ankyrin repeat-containing E3 ubiquitin protein ligase 1-null mouse hearts was associated with increased PLN levels determined by immunoblots and immunofluorescence. Our study identifies a biological mechanism that traffics PLN to the lysosomes for degradation in mouse hearts.
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http://dx.doi.org/10.1073/pnas.1508815112DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4466728PMC
June 2015

GABARAP is a determinant of apoptosis in growth-arrested chicken embryo fibroblasts.

J Cell Physiol 2015 Jul;230(7):1475-88

Department of Biology, York University, Toronto, Ontario, M3J 1P3, Canada.

Nutrient depletion triggers a series of adaptive processes as part of the unfolded protein response or UPR. These processes reduce stress to the endoplasmic reticulum by enhancing its protein folding capacity or ability to promote the degradation of dysfunctional proteins. Failure to restore ER homeostasis causes the activation of lethal pathways. The expression of a dominant negative mutant of C/EBPβ (Δ184-C/EBPβ) alters this balance in chicken embryo fibroblasts (CEF). As a result, CEF display enhanced survival upon prolonged nutrient depletion. Starved Δ184-C/EBPβ-expressing CEF display pronounced features of autophagy characterized by the appearance of large vesicles containing amorphous material, the formation of smaller double-membrane vesicles (autophagosomes) and processing of LC3 and GABARAP. However, there were marked differences in the expression and processing of these proteins. In both normal and Δ184-C/EBPβ expressing CEF, the lipidated form of LC3 (form II) accumulated during starvation but was detectable even when cells were actively dividing in complete medium. In contrast, GABARAP expression and lipidation were strongly stimulated in response to starvation. Inhibition of LC3 expression by RNA interference led to apoptosis in normal CEF even in the absence of starvation but stable and near complete repression of GABARAP was tolerated. Moreover, the inhibition of GABARAP enhanced CEF survival and abolished the expression of the pro-apoptotic CHOP factor in conditions of starvation, suggesting a reduced level of ER stress. Therefore, GABARAP is a determinant of apoptosis in CEF subjected to prolonged nutrient depletion.
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http://dx.doi.org/10.1002/jcp.24889DOI Listing
July 2015

Structural determination of the phosphorylation domain of the ryanodine receptor.

FEBS J 2012 Oct 11;279(20):3952-64. Epub 2012 Sep 11.

Department of Physiology, University of Toronto, Toronto, Ontario, Canada.

The ryanodine receptor (RyR) is a large, homotetrameric sarcoplasmic reticulum membrane protein that is essential for Ca(2+) cycling in both skeletal and cardiac muscle. Genetic mutations in RyR1 are associated with severe conditions including malignant hyperthermia (MH) and central core disease. One phosphorylation site (Ser 2843) has been identified in a segment of RyR1 flanked by two RyR motifs, which are found exclusively in all RyR isoforms as closely associated tandem (or paired) motifs, and are named after the protein itself. These motifs also contain six known MH mutations. In this study, we designed, expressed and purified the tandem RyR motifs, and show that this domain contains a putative binding site for the Ca(2+)/calmodulin-dependent protein kinase β isoform. We present a 2.2 Å resolution crystal structure of the RyR domain revealing a two-fold, symmetric, extended four-helix bundle stabilized by a β sheet. Using mathematical modelling, we fit our crystal structure within a tetrameric electron microscopy (EM) structure of native RyR1, and propose that this domain is localized in the RyR clamp region, which is absent in its cousin protein inositol 1,4,5-trisphosphate receptor.
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http://dx.doi.org/10.1111/j.1742-4658.2012.08755.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3712973PMC
October 2012

α-Crystallin B prevents apoptosis after H2O2 exposure in mouse neonatal cardiomyocytes.

Am J Physiol Heart Circ Physiol 2012 Oct 17;303(8):H967-78. Epub 2012 Aug 17.

Department of Physiology, University of Toronto, Toronto, Ontario, Canada M5G 1L6.

α-Crystallin B (cryAB) is the most abundant small heat shock protein in cardiomyocytes (CMs) and has been shown to have potent antiapoptotic properties. Because the mechanism by which cryAB prevents apoptosis has not been fully characterized, we examined its protective effects at the cellular level by silencing cryAB in mouse neonatal CMs using lentivector-mediated transduction of short hairpin RNAs. Subcellular fractionation of whole hearts showed that cryAB is cytosolic under control conditions, and after H(2)O(2) exposure, it translocates to the mitochondria. Phosphorylated cryAB (PcryAB) is mainly associated with the mitochondria, and any residual cytosolic PcryAB translocates to the mitochondria after H(2)O(2) exposure. H(2)O(2) exposure caused increases in cryAB and PcryAB levels, and cryAB silencing resulted in increased levels of apoptosis after exposure to H(2)O(2). Coimmunoprecipitation assays revealed an apparent interaction of both cryAB and PcryAB with mitochondrial voltage-dependent anion channels (VDAC), translocase of outer mitochondrial membranes 20 kDa (TOM 20), caspase 3, and caspase 12 in mouse cardiac tissue. Our results are consistent with the conclusion that the cardioprotective effects of cryAB are mediated by its translocation from the cytosol to the mitochondria under conditions of oxidative stress and that cryAB interactions with VDAC, TOM 20, caspase 3, and caspase 12 may be part of its protective mechanism.
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http://dx.doi.org/10.1152/ajpheart.00040.2012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3706333PMC
October 2012

A method for the direct identification of differentiating muscle cells by a fluorescent mitochondrial dye.

PLoS One 2011 9;6(12):e28628. Epub 2011 Dec 9.

Department of Physiology, University of Toronto, Best Institute Medical Research, Toronto, Canada.

Identification of differentiating muscle cells generally requires fixation, antibodies directed against muscle specific proteins, and lengthy staining processes or, alternatively, transfection of muscle specific reporter genes driving GFP expression. In this study, we examined the possibility of using the robust mitochondrial network seen in maturing muscle cells as a marker of cellular differentiation. The mitochondrial fluorescent tracking dye, MitoTracker, which is a cell-permeable, low toxicity, fluorescent dye, allowed us to distinguish and track living differentiating muscle cells visually by epi-fluorescence microscopy. MitoTracker staining provides a robust and simple detection strategy for living differentiating cells in culture without the need for fixation or biochemical processing.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028628PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3235136PMC
April 2012

Nuclear function of Smad7 promotes myogenesis.

Mol Cell Biol 2010 Feb 7;30(3):722-35. Epub 2009 Dec 7.

Department of Biology, York University, St., Toronto, Ontario M3J 1P3, Canada.

In the "canonical" view of transforming growth factor beta (TGF-beta) signaling, Smad7 plays an inhibitory role. While Smad7 represses Smad3 activation by TGF-beta, it does not reverse the inhibitory effect of TGF-beta on myogenesis, suggesting a different function in myogenic cells. We previously reported a promyogenic role of Smad7 mediated by an interaction with MyoD. Based on this association, we hypothesized a possible nuclear function of Smad7 independent of its role at the level of the receptor. We therefore engineered a chimera of Smad7 with a nuclear localization signal (NLS), which serves to prevent and therefore bypass binding to the TGF-beta receptor while concomitantly constitutively localizing Smad7 to the nucleus. This Smad7-NLS did not repress Smad3 activation by TGF-beta but did retain its ability to enhance myogenic gene activation and phenotypic myogenesis, indicating that the nuclear, receptor-independent function of Smad7 is sufficient to promote myogenesis. Furthermore, Smad7 physically interacts with MyoD and antagonizes the repressive effects of active MEK on MyoD. Reporter and myogenic conversion assays indicate a pivotal regulation of MyoD transcriptional properties by the balance between Smad7 and active MEK. Thus, Smad7 has a nuclear coactivator function that is independent of TGF-beta signaling and necessary to promote myogenic differentiation.
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http://dx.doi.org/10.1128/MCB.01005-09DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2812228PMC
February 2010

Menin expression modulates mesenchymal cell commitment to the myogenic and osteogenic lineages.

Dev Biol 2009 Aug 21;332(1):116-30. Epub 2009 May 21.

Department of Biology, 327 Farquharson, LSB, York University, Toronto, M3J 1P3 Ontario, Canada.

Menin plays an established role in the differentiation of mesenchymal cells to the osteogenic lineage. Conversely, whether Menin influences the commitment of mesenschymal cells to the myogenic lineage, despite expression in the developing somite was previously unclear. We observed that Menin is down-regulated in C2C12 and C3H10T1/2 mesenchymal cells when muscle differentiation is induced. Moreover, maintenance of Menin expression by constitutive ectopic expression inhibited muscle cell differentiation. Reduction of Menin expression by siRNA technology results in precocious muscle differentiation and concomitantly attenuates BMP-2 induced osteogenesis. Reduced Menin expression antagonizes BMP-2 and TGF-beta1 mediated inhibition of myogenesis. Furthermore, Menin was found to directly interact with and potentiate the transactivation properties of Smad3 in response to TGF-beta1. Finally in concert with these observations, tissue-specific inactivation of Men1 in Pax3-expressing somite precursor cells leads to a patterning defect of rib formation and increased muscle mass in the intercostal region. These data invoke a pivotal role for Menin in the competence of mesenchymal cells to respond to TGF-beta1 and BMP-2 signals. Thus, by modulating cytokine responsiveness Menin functions to alter the balance of multipotent mesenchymal cell commitment to the osteogenic or myogenic lineages.
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http://dx.doi.org/10.1016/j.ydbio.2009.05.555DOI Listing
August 2009

Cardiotrophin-1 maintains the undifferentiated state in skeletal myoblasts.

J Biol Chem 2009 Jul 12;284(29):19679-93. Epub 2009 May 12.

Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada.

Skeletal myogenesis is potently regulated by the extracellular milieu of growth factors and cytokines. We observed that cardiotrophin-1 (CT-1), a member of the interleukin-6 (IL-6) family of cytokines, is a potent regulator of skeletal muscle differentiation. The normal up-regulation of myogenic marker genes, myosin heavy chain (MyHC), myogenic regulatory factors (MRFs), and myocyte enhancer factor 2s (MEF2s) were inhibited by CT-1 treatment. CT-1 also represses myogenin (MyoG) promoter activation. CT-1 activated two signaling pathways: signal transducer and activator of transcription 3 (STAT3), and mitogen-activated protein kinase kinase (MEK), a component of the extracellular signal-regulated MAPK (ERK) pathway. In view of the known connection between CT-1 and STAT3 activation, we surprisingly found that pharmacological blockade of STAT3 activity had no effect on the inhibition of myogenesis by CT-1 suggesting that STAT3 signaling is dispensable for myogenic repression. Conversely, MEK inhibition potently reversed the inhibition of myotube formation and attenuated the repression of MRF transcriptional activity mediated by CT-1. Taken together, these data indicate that CT-1 represses skeletal myogenesis through interference with MRF activity by activation of MEK/ERK signaling. In agreement with these in vitro observations, exogenous systemic expression of CT-1 mediated by adenoviral vector delivery increased the number of myonuclei in normal post-natal mouse skeletal muscle and also delayed skeletal muscle regeneration induced by cardiotoxin injection. The expression pattern of CT-1 in embryonic and post-natal skeletal muscle and in vivo effects of CT-1 on myogenesis implicate CT-1 in the maintenance of the undifferentiated state in muscle progenitor cells.
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http://dx.doi.org/10.1074/jbc.M109.017319DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2740593PMC
July 2009

Smad7 promotes and enhances skeletal muscle differentiation.

Mol Cell Biol 2006 Aug;26(16):6248-60

Department of Biology, 327 Farquharson, LSB, York University, 4700 Keele St., Toronto M3J 1P3 Ontario, Canada.

Transforming growth factor beta1 (TGF-beta1) and myostatin signaling, mediated by the same Smad downstream effectors, potently repress skeletal muscle cell differentiation. Smad7 inhibits these cytokine signaling pathways. The role of Smad7 during skeletal muscle cell differentiation was assessed. In these studies, we document that increased expression of Smad7 abrogates myostatin- but not TGF-beta1-mediated repression of myogenesis. Further, constitutive expression of exogenous Smad7 potently enhanced skeletal muscle differentiation and cellular hypertrophy. Conversely, targeting of endogenous Smad7 by small interfering RNA inhibited C2C12 muscle cell differentiation, indicating an essential role for Smad7 during myogenesis. Congruent with a role for Smad7 in myogenesis, we observed that the muscle regulatory factor (MyoD) binds to and transactivates the Smad7 proximal promoter region. Finally, we document that Smad7 directly interacts with MyoD and enhances MyoD transcriptional activity. Thus, Smad7 cooperates with MyoD, creating a positive loop to induce Smad7 expression and to promote MyoD driven myogenesis. Taken together, these data implicate Smad7 as a fundamental regulator of differentiation in skeletal muscle cells.
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http://dx.doi.org/10.1128/MCB.00384-06DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1592807PMC
August 2006

Opposing roles of C/EBPbeta and AP-1 in the control of fibroblast proliferation and growth arrest-specific gene expression.

J Biol Chem 2003 Oct 1;278(44):43846-54. Epub 2003 Aug 1.

Department of Biology, York University, Toronto, Ontario M3J 1P3, Canada.

Chicken embryo fibroblasts (CEF) express several growth arrest-specific (GAS) gene products in G0. In contact-inhibited cells, the expression of the most abundant of these proteins, the p20K lipocalin, is activated at the transcriptional level by C/EBPbeta. In this report, we describe the role of C/EBPbeta in CEF proliferation. We show that the expression of a dominant negative mutant of C/EBPbeta (designated Delta184-C/EBPbeta) completely inhibited p20K expression at confluence and stimulated the proliferation of CEF without inducing transformation. Mouse embryo fibroblasts nullizygous for C/EBPbeta had a proliferative advantage over cells with one or two functional copies of this gene. C/EBP inhibition enhanced the expression of the three major components of AP-1 in cycling CEF, namely c-Jun, JunD, and Fra-2, and stimulated AP-1 activity. In contrast, the over-expression of C/EBPbeta caused a dramatic reduction in the levels of AP-1 proteins. Therefore, C/EBPbeta is a negative regulator of AP-1 expression and activity in CEF. The expression of cyclin D1 and cell proliferation were stimulated by the dominant negative mutant of C/EBPbeta but not in the presence of TAM67, a dominant negative mutant of c-Jun and AP-1. CEF over-expressing c-Jun, and to a lesser extent JunD and Fra-2, did not growth arrest at high cell density and did not express p20K. Therefore, AP-1 interfered with the action of C/EBPbeta at high cell density, indicating that these factors play opposing roles in the control of GAS gene expression and CEF proliferation.
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http://dx.doi.org/10.1074/jbc.M304085200DOI Listing
October 2003