Genome-wide analysis of glucocorticoid receptor-binding sites in myotubes identifies gene networks modulating insulin signaling

Glucocorticoids elicit a variety of biological responses in skeletal muscle, including inhibiting protein synthesis and insulin-stimulated glucose uptake and promoting proteolysis. Thus, excess or chronic glucocorticoid exposure leads to muscle atrophy and insulin resistance. Glucocorticoids propagate their signal mainly through glucocorticoid receptors (GR), which, upon binding to ligands, translocate to the nucleus and bind to genomic glucocorticoid response elements to regulate the transcription of nearby genes. Using a combination of chromatin immunoprecipitation sequencing and microarray analysis, we identified 173 genes in mouse C2C12 myotubes. The mouse genome contains GR-binding regions in or near these genes, and gene expression is regulated by glucocorticoids. Eight of these genes encode proteins known to regulate distinct signaling events in insulin/insulin-like growth factor 1 pathways. We found that overexpression of p85α, one of these eight genes, caused a decrease in C2C12 myotube diameters, mimicking the effect of glucocorticoids. Moreover, reducing p85α expression by RNA interference in C2C12 myotubes significantly compromised the ability of glucocorticoids to inhibit Akt and p70 S6 kinase activity and reduced glucocorticoid induction of insulin receptor substrate 1 phosphorylation at serine 307. This phosphorylation is associated with insulin resistance. Furthermore, decreasing p85α expression abolished glucocorticoid inhibition of protein synthesis and compromised glucocorticoid-induced reduction of cell diameters in C2C12 myotubes. Finally, a glucocorticoid response element was identified in the p85α GR-binding regions. In summary, our studies identified GR-regulated transcriptional networks in myotubes and showed that p85α plays a critical role in glucocorticoid-induced insulin resistance and muscle atrophy in C2C12 myotubes.

[1]  S. Bodine,et al.  Muscle sparing in muscle RING finger 1 null mice: response to synthetic glucocorticoids , 2011, The Journal of physiology.

[2]  M. Tong,et al.  Over-expression of NYGGF4 (PID1) inhibits glucose transport in skeletal myotubes by blocking the IRS1/PI3K/AKT insulin pathway. , 2011, Molecular genetics and metabolism.

[3]  Keiichi Fukuda,et al.  Crosstalk between glucocorticoid receptor and nutritional sensor mTOR in skeletal muscle. , 2011, Cell metabolism.

[4]  B. Walker,et al.  Glucocorticoid-Mediated Inhibition of Angiogenic Changes in Human Endothelial Cells Is Not Caused by Reductions in Cell Proliferation or Migration , 2010, PloS one.

[5]  Terence P. Speed,et al.  Genome-Wide Analysis of Glucocorticoid Receptor Binding Regions in Adipocytes Reveal Gene Network Involved in Triglyceride Homeostasis , 2010, PloS one.

[6]  S. Price,et al.  FOXO3a mediates signaling crosstalk that coordinates ubiquitin and atrogin‐1/MAFbx expression during glucocorticoid‐induced skeletal muscle atrophy , 2010, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[7]  W. Mitch,et al.  Endogenous glucocorticoids and impaired insulin signaling are both required to stimulate muscle wasting under pathophysiological conditions in mice. , 2009, The Journal of clinical investigation.

[8]  D. Sabatini,et al.  DEPTOR Is an mTOR Inhibitor Frequently Overexpressed in Multiple Myeloma Cells and Required for Their Survival , 2009, Cell.

[9]  W. Bauman,et al.  Dependence of dexamethasone-induced Akt/FOXO1 signaling, upregulation of MAFbx, and protein catabolism upon the glucocorticoid receptor. , 2009, Biochemical and biophysical research communications.

[10]  S. Bodine,et al.  The glucocorticoid receptor and FOXO1 synergistically activate the skeletal muscle atrophy-associated MuRF1 gene. , 2008, American journal of physiology. Endocrinology and metabolism.

[11]  P. Hasselgren,et al.  Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes , 2008, Journal of cellular biochemistry.

[12]  M. Karin,et al.  p53 Target Genes Sestrin1 and Sestrin2 Connect Genotoxic Stress and mTOR Signaling , 2008, Cell.

[13]  Pan Du,et al.  lumi: a pipeline for processing Illumina microarray , 2008, Bioinform..

[14]  M. Birnbaum,et al.  The role of FoxO in the regulation of metabolism , 2008, Oncogene.

[15]  J. Thissen,et al.  Mechanisms of glucocorticoid-induced myopathy. , 2008, The Journal of endocrinology.

[16]  L. Gathercole,et al.  Glucocorticoid modulation of insulin signaling in human subcutaneous adipose tissue. , 2007, The Journal of clinical endocrinology and metabolism.

[17]  Hao Li,et al.  Cell- and gene-specific regulation of primary target genes by the androgen receptor. , 2007, Genes & development.

[18]  Panayiotis V. Benos,et al.  STAMP: a web tool for exploring DNA-binding motif similarities , 2007, Nucleic Acids Res..

[19]  M. Scadeng,et al.  Bone marrow–specific Cap gene deletion protects against high-fat diet–induced insulin resistance , 2007, Nature Medicine.

[20]  L. Ellisen,et al.  Dexamethasone Represses Signaling through the Mammalian Target of Rapamycin in Muscle Cells by Enhancing Expression of REDD1* , 2006, Journal of Biological Chemistry.

[21]  B. Draznin Molecular Mechanisms of Insulin Resistance: Serine Phosphorylation of Insulin Receptor Substrate-1 and Increased Expression of p85α , 2006, Diabetes.

[22]  Jun S. Song,et al.  CEAS: cis-regulatory element annotation system. , 2006, Nucleic acids research.

[23]  L. Cantley,et al.  Loss of class IA PI3K signaling in muscle leads to impaired muscle growth, insulin response, and hyperlipidemia. , 2006, Cell metabolism.

[24]  F. Buttgereit,et al.  Non-genomic glucocorticoid effects to provide the basis for new drug developments , 2006, Molecular and Cellular Endocrinology.

[25]  C. Kahn,et al.  Increased P85α Is a Potent Negative Regulator of Skeletal Muscle Insulin Signaling and Induces in Vivo Insulin Resistance Associated with Growth Hormone Excess* , 2005, Journal of Biological Chemistry.

[26]  J. Ofrecio,et al.  Increased p85/55/50 expression and decreased phosphotidylinositol 3-kinase activity in insulin-resistant human skeletal muscle. , 2005, Diabetes.

[27]  T. Fujita,et al.  Dexamethasone inhibits insulin‐induced chondrogenesis of ATDC5 cells by preventing PI3K‐Akt signaling and DNA binding of Runx2 , 2004, Journal of cellular biochemistry.

[28]  T. Hiragun,et al.  Dexamethasone Suppresses Antigen-Induced Activation of Phosphatidylinositol 3-Kinase and Downstream Responses in Mast Cells , 2004, The Journal of Immunology.

[29]  G. Yancopoulos,et al.  The IGF-1/PI3K/Akt pathway prevents expression of muscle atrophy-induced ubiquitin ligases by inhibiting FOXO transcription factors. , 2004, Molecular cell.

[30]  Marco Sandri,et al.  Foxo Transcription Factors Induce the Atrophy-Related Ubiquitin Ligase Atrogin-1 and Cause Skeletal Muscle Atrophy , 2004, Cell.

[31]  M. White,et al.  Insulin receptor substrate proteins and diabetes , 2004, Archives of pharmacal research.

[32]  J. Friedman,et al.  Human placental growth hormone increases expression of the p85 regulatory unit of phosphatidylinositol 3-kinase and triggers severe insulin resistance in skeletal muscle. , 2004, Endocrinology.

[33]  Gordon K Smyth,et al.  Statistical Applications in Genetics and Molecular Biology Linear Models and Empirical Bayes Methods for Assessing Differential Expression in Microarray Experiments , 2011 .

[34]  J. Klein,et al.  Positive and Negative Roles of p85α and p85β Regulatory Subunits of Phosphoinositide 3-Kinase in Insulin Signaling* , 2003, Journal of Biological Chemistry.

[35]  K. Yamamoto,et al.  Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA , 2003, Nature.

[36]  G. Haegeman,et al.  The interplay between the glucocorticoid receptor and nuclear factor-kappaB or activator protein-1: molecular mechanisms for gene repression. , 2003, Endocrine reviews.

[37]  F. Liu,et al.  Grb10 Inhibits Insulin-stimulated Insulin Receptor Substrate (IRS)-Phosphatidylinositol 3-Kinase/Akt Signaling Pathway by Disrupting the Association of IRS-1/IRS-2 with the Insulin Receptor* , 2003, The Journal of Biological Chemistry.

[38]  D J Glass,et al.  Identification of Ubiquitin Ligases Required for Skeletal Muscle Atrophy , 2001, Science.

[39]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[40]  D. Garrel,et al.  Activation of the ubiquitin pathway in rat skeletal muscle by catabolic doses of glucocorticoids. , 1997, The American journal of physiology.

[41]  G. Dimitriadis,et al.  Effects of glucocorticoid excess on the sensitivity of glucose transport and metabolism to insulin in rat skeletal muscle. , 1997, The Biochemical journal.

[42]  J. Wang,et al.  Hepatic nuclear factor 3 is an accessory factor required for the stimulation of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids. , 1996, Molecular endocrinology.

[43]  J. Vaughan,et al.  Development of Cushing's syndrome in corticotropin-releasing factor transgenic mice. , 1992, Endocrinology.

[44]  S. Egginton,et al.  Angiogenesis in skeletal and cardiac muscle. , 1992, Physiological reviews.

[45]  Young-Sun Lin,et al.  GAL4 derivatives function alone and synergistically with mammalian activators in vitro , 1988, Cell.

[46]  中尾 玲子 Ubiquitin ligase Cbl-b is a negative regulator for insulin-like growth factor 1 signaling during muscle atrophy caused by unloading , 2010 .

[47]  D. Laber,et al.  11beta-hydroxysteroid dehydrogenase type 1 regulates glucocorticoid-induced insulin resistance in skeletal muscle. , 2009, Diabetes.

[48]  Michael Karin,et al.  p53 Target Genes Sestrin1 and Sestrin2 Connect Genotoxic Stress and mTOR Signaling , 2009, Cell.

[49]  Jae-Hyoo Kim,et al.  Apoptosis of skeletal muscle on steroid-induced myopathy in rats. , 2005, The Journal of nutrition.

[50]  Y. Le Marchand-Brustel,et al.  Positive and negative regulation of insulin signaling through IRS-1 phosphorylation. , 2005, Biochimie.

[51]  J. Klein,et al.  Positive and negative roles of p85 alpha and p85 beta regulatory subunits of phosphoinositide 3-kinase in insulin signaling. , 2003, The Journal of biological chemistry.

[52]  Kohjiro Ueki,et al.  Reduced expression of the murine p85alpha subunit of phosphoinositide 3-kinase improves insulin signaling and ameliorates diabetes. , 2002, The Journal of clinical investigation.

[53]  C. Wilson,et al.  Dexamethasone inhibits insulin-stimulated recruitment of GLUT4 to the cell surface in rat skeletal muscle. , 1998, Metabolism: clinical and experimental.

[54]  K. Yamamoto,et al.  Regulatory crosstalk at composite response elements. , 1991, Trends in biochemical sciences.

[55]  G. Bray,et al.  Effects of dexamethasone on glucose transport by skeletal muscles of obese (ob/ob) mice. , 1989, International journal of obesity.

[56]  R. Eston,et al.  Body composition analysis: a defense of anthropometry in overweight female dieters and controls. , 1989, International journal of obesity.