Tetsuaki Miyake

Smad7 Promotes and Enhances Skeletal Muscle Differentiation

ABSTRACT Transforming growth factor β1 (TGF-β1) 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-β1-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.

Nuclear Function of Smad7 Promotes Myogenesis

ABSTRACT In the “canonical” view of transforming growth factor β (TGF-β) signaling, Smad7 plays an inhibitory role. While Smad7 represses Smad3 activation by TGF-β, it does not reverse the inhibitory effect of TGF-β 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-β receptor while concomitantly constitutively localizing Smad7 to the nucleus. This Smad7-NLS did not repress Smad3 activation by TGF-β 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-β signaling and necessary to promote myogenic differentiation.

Structural determination of the phosphorylation domain of the ryanodine receptor

The ryanodine receptor (RyR) is a large, homotetrameric sarcoplasmic reticulum membrane protein that is essential for Ca2+ 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 Ca2+/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.

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

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.

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

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.

Cardiotrophin-1 maintains the undifferentiated state in skeletal myoblasts

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.

A Method for the Direct Identification of Differentiating Muscle Cells by a Fluorescent Mitochondrial Dye

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.

Metformin increases degradation of phospholamban via autophagy in cardiomyocytes

Significance Phospholamban (PLN) can regulate Ca2+ uptake rates in the sarcoplasmic reticulum in cardiomyocytes. However, the mechanisms that control PLN levels are not fully understood. This study shows that PLN degradation depends on ubiquitinylation of its lysine 3 residue and p62-mediated selective autophagy. Metformin was shown to accelerate autophagy and to induce PLN degradation, resulting in increased Ca2+ uptake. These results suggest that changes in PLN degradation could account for the cardiac inotropic effects of metformin. Phospholamban (PLN) is an effective inhibitor of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA). Here, we examined PLN stability and degradation in primary cultured mouse neonatal cardiomyocytes (CMNCs) and mouse hearts using immunoblotting, molecular imaging, and [35S]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.

Signal-dependent fra-2 regulation in skeletal muscle reserve and satellite cells

Activator protein-1 (AP-1) is a ubiquitous transcription factor that paradoxically also has some tissue-specific functions. In skeletal muscle cells, we document that the AP-1 subunit, Fra-2, is expressed in the resident stem cells (Pax7-positive satellite cells) and also in the analogous undifferentiated ‘reserve’ cell population in myogenic cultures, but not in differentiated myofiber nuclei. Silencing of Fra-2 expression enhances the expression of differentiation markers such as muscle creatine kinase and myosin heavy chain, indicating a possible role of Fra-2 in undifferentiated myogenic progenitor cells. We observed that Fra-2 is a target of cytokine-mediated extracellular signal-regulated kinase-1/2 signaling in cultured muscle cells, and extensive mass spectrometry and mutational analysis identified S320 and T322 as regulators of Fra-2 protein stability. Interestingly, Fra-2 S320 phosphorylation occurs transiently in activated satellite cells and is extinguished in myogenin-positive differentiating cells. Thus, cytokine-mediated Fra-2 expression and stabilization is linked to regulation of myogenic progenitor cells having implications for the molecular regulation of adult muscle stem cells and skeletal muscle regeneration.

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

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.