Identification of Novel GH-regulated Genes in C2C12 Cells

Abstract

Growth hormone (GH) is the main regulator of longitudinal growth before puberty, and treatment with human recombinant (rh) GH can increase muscle strength. Nevertheless, molecular mechanisms responsible remain mostly unknown. Many physiological effects of GH require hormone-mediated changes in gene expression. In an attempt to gain insight into the mechanism of GH action in muscle cells we evaluated the effects of rhGH on gene expression profile in a murine skeletal muscle cell line C2C12. The objective of the work was to identify changes in gene expression in the murine skeletal muscle cell line C2C12 after rGH treatment using microarray assays. C2C12 murine skeletal muscle cell cultures were differentiated during 4 days. After 16 h growing in serum-free medium, C2C12 myotubes were stimulated during 6 h with 500 ng/ml rhGH. Four independent sets of experiments were performed to identify GH-regulated genes. Total RNA was isolated and subjected to analysis. To validate changes candidate genes were analyzed by real-time quantitative polymerase chain reaction. One hundred and fifty-four differentially expressed genes were identified; 90 upregulated and 64 downregulated. Many had not been previously identified as GH-responsive. Real-time PCR in biological replicates confirmed the effect of rGH on 15 genes: Cish, Serpina3g, Socs2, Bmp4, Tnfrsf11b, Rgs2, Tgfbr3, Ugdh, Npy1r, Gbp6, Tgfbi, Tgtp, Btc, Clec3b, and Bcl6. This study shows modifications in the gene expression profile of the C2C12 cell line after rhGH exposure. In vitro and gene function analysis revealed genes involved in skeletal and muscle system as well as cardiovascular system development and function.

Key words

skeletal muscle - microarrays - growth hormone - cell cultures

*

^*  These authors contributed equally to this work.

• References

• 1 Isaksson OG, Eden S, Jansson JO. Mode of action of pituitary growth hormone on target cells. Annu Rev Physiol 1985; 47: 483-489
• 2 Ceseña TI, Cui TX, Piwien-Pilipuk G, Kaplani J, Calinescu AA, Huo JS, Iñiguez-Lluhí JA, Kwok R, Schwartz J. Multiple mechanisms of growth hormone-regulated gene transcription. Mol Genet Metab 2007; 90: 126-133
• 3 Piwien-Pilipuk G, Huo JS, Schwartz J. Growth hormone signal transduction. J Pediatr Endocrinol Metab 2002; 1: 771-786
• 4 Mathews L, Enberg B, Norstedt G. Regulation of rat growth hormone receptor gene expression. J Biol Chem 1989; 264: 9905-9910
• 5 Isaksson OG, Jansson JO, Gause IA. Growth hormone stimulates longitudinal bone growth directly. Science 1982; 216: 1237-1239
• 6 Alexopoulou O, Abs R, Maiter D. Treatment of adult growth hormone deficiency: who, why and how? A review. Acta Clin Belg 2010; 65: 1322
• 7 Isaksson OG, Lindahl A, Nilsson A, Isgaard J. Mechanism of the stimulatory effect of growth hormone on longitudinal bone growth. Endocr Rev 1987; 8: 426-438
• 8 Clemmons DR. The diagnosis and treatment of growth hormone deficiency in adults. Curr Opin Endocrinol Diabetes Obes 2010; 17: 377-383
• 9 Attanasio AF, Bates PC, Ho KK, Webb SM, Ross RJ, Strasburger CJ, Bouillon R, Crowe B, Selander K, Valle D, Lamberts SW. Hypopituitary Control and Complications Study International Advisory Board. Human growth hormone replacement in adult hypopituitary patients: long-term effects on body composition and lipid status – 3-year results from the HypoCCS Database. J Clin Endocrinol Metab 2002; 87: 1600-1606
• 10 Davidson MB. Effect of growth hormone on carbohydrate and lipid metabolism. Endocr Rev 1987; 8: 115-131
• 11 Fryburg DA, Barrett EJ. The regulation of amino acid and protein metabolism by growth hormone. In: Kostyo JL. (ed.) Hormonal Control of Growth. NY: American Physiological Soc; 1999: 515-536
• 12 Webb SM. Measurements of quality of life in patients with growth hormone deficiency. J Endocrinol Invest 2008; 31: 52-55
• 13 Huo JS, McEachin RC, Cui TX, Duggal NK, Hai T, States DJ, Schwartz J. Profiles of growth hormone (GH)-regulated genes reveal time-dependent responses and identify a mechanism for regulation of activating transcription factor 3 by GH. J Biol Chem 2006; 28: 4132-4141
• 14 Yoon JB, Berry SA, Seelig S, Towle HC. An inducible nuclear factor binds to a growth hormone-regulated gene. J Biol Chem 1990; 265: 19947-19954
• 15 Le Cam A, Pantescu V, Paquereau L, Legraverend C, Fauconnier G, Asins G. cis-Acting elements controlling transcription from rat serine protease inhibitor 2.1 gene promoter. Characterization of two growth hormone response sites and a dominant purine-rich element. J Biol Chem 1994; 269: 21532-21539
• 16 Paul C, Seiliez I, Thissen JP, Le Cam A. Regulation of expression of the rat SOCS-3 gene in hepatocytes by growth hormone, interleukin-6 and glucocorticoids mRNA analysis and promoter characterization. Eur J Biochem 2000; 267: 5849-5857
• 17 Vidal OM, Merino R, Rico-Bautista E, Fernandez-Perez L, Chia DJ, Woelfle J, Ono M, Lenhard B, Norstedt G, Rotwein P, Flores-Morales A. In vivo transcript profiling and phylogenetic analysis identifies suppressor of cytokine signaling 2 as a direct signal transducer and activator of transcription 5b target in liver. Mol Endocrinol 2007; 21: 293-311
• 18 Tollet-Egnell P, Flores-Morales A, Stavréus-Evers A, Sahlin L, Norstedt G. Growth hormone regulation of SOCS-2, SOCS-3, and CIS messenger ribonucleic acid expression in the rat. Endocrinology 1999; 140: 3693-3704
• 19 Flores-Morales A, Stahlberg N, Tollet-Egnell P, Lundeberg J, Malek RL, Quackenbush J, Lee NH, Norstedt G. Microarray analysis of the in vivo effects of hypophysectomy and growth hormone treatment on gene expression in the rat. Endocrinology 2001; 142: 3163-3176
• 20 Gil H, Lozano JJ, Alvarez-García O, Secades-Vázquez P, Rodríguez-Suárez J, García-López E, Carbajo-Pérez E, Santos F. Differential gene expression induced by growth hormone treatment in the uremic rat growth plate. Growth Horm IGF Res 2008; 18: 353-359
• 21 Chia DJ, Rotwein P. Defining the epigenetic actions of growth hormone: acute chromatin changes accompany GH-activated gene transcription. Mol Endocrinol 2010; 24: 2038-2049
• 22 Chen Y, Lin G, Huo JS, Barney D, Wang Z, Livshiz T, States DJ, Qin ZS, Schwartz J. Computational and functional analysis of growth hormone (GH)-regulated genes identifies the transcriptional repressor B-cell lymphoma 6 (Bc16) as a participant in GH-regulated transcription. Endocrinology 2009; 150: 3645-3654
• 23 Chow JC, Ling PR, Qu Z, Laviola L, Ciccarone A, Bistrian BR, Smith RJ. Growth hormone stimulates tyrosine phosphorylation of JAK2 and STAT5, but not insulin receptor substrate-1 or SHC proteins in liver and skeletal muscle of normal rats in vivo. Endocrinology 1996; 137: 2880-2886
• 24 Halevy O, Hodik V, Mett A.. The effects of growth hormone on avian skeletal muscle satellite cell proliferation and differentiation. Gen Comp Endocrinol 1996; 101: 43-52
• 25 Adamafio NA, Towns RJ, Kostyo JL. Growth hormone receptors and action in BC3H-1 myocytes. Growth Regul 1991; 1: 17-22
• 26 Webb SM, de Andrés-Aguayo I, Rojas-García R, Ortega E, Gallardo E, Mestrón A, Serrano-Munuera C, Casamitjana R, Illa I. Neuromuscular dysfunction in adult growth hormone deficiency. Clin Endocrinol (Oxf) 2003; 59: 450-458
• 27 Sadowski CL, Wheeler TT, Wang LH, Sadowski HB. GH regulation of IGF-I and suppressor of cytokine signaling gene expression in C2C12 skeletal muscle cells. Endocrinology 2001; 142: 3890-3900
• 28 Segard HB, Moulin S, Boumard S, Augier de Crémiers C, Kelly PA, Finidori J. Autocrine growth hormone production prevents apoptosis and inhibits differentiation in C2C12 myoblasts. Cell Signal 2003; 15: 615-623
• 29 Gentleman RC, Carey VJ, Bates DM, Bolstad B, Dettling M, Dudoit S, Ellis B, Gautier L, Ge Y, Gentry J, Hornik K, Hothorn T, Huber W, Iacus S, Irizarry R, Leisch F, Li C, Maechler M, Rossini AJ, Sawitzki G, Smith C, Smyth G, Tierney L, Yang JY, Zhang J. Bioconductor: open software development for computational biology and bioinformatics. Genome Biol 2004; 5: R80
• 30 Irizarry RA, Hobbs B, Collin F, Beazer-Barclay YD, Antonellis KJ, Scherf U, Speed TP. Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 2003; 4: 249-264
• 31 Cartharius K, Frech K, Grote K, Klocke B, Haltmeier M, Klingenhoff A, Frisch M, Bayerlein M, Werner T. MathInspector and beyond: promoter analysis based on transcription factor binding sites. Bioinformatics 2005; 21: 2933-2942
• 32 Sadowski CL, Wheeler TT, Wang LH, Sadowski HB. GH regulation of IGF-I and suppressor of cytokine signaling gene expression in C2C12 skeletal muscle cells. Endocrinology 2001; 142: 3890-3900
• 33 Frost RA, Nystrom GJ, Lang CH. Regulation of IGF-I mRNA and signal transducers and activators of transcription-3 and -5 (Stat-3 and -5) by GH in C2C12 myoblasts. Endocrinology 2002; 143: 492-503
• 34 Haase HR, Ivanovski S, Waters MJ, Bartold PM. Growth hormone regulates osteogenic marker mRNA expression in human periodontal fibroblasts and alveolar bone-derived cells. J Periodontal Res 2003; 38: 366-374
• 35 von Bubnoff A, Cho KW. Intracellular BMP signaling regulation in vertebrates: pathway or network?. Dev Biol 2001; 239: 1-14
• 36 Ouyang X, Fujimoto M, Nakagawa R, Serada S, Tanaka T, Nomura S, Kawase I, Kishimoto T, Naka T. SOCS-2 interferes with myotube formation and potentiates osteoblast differentiation through upregulation of JunB in C2C12 cells. J Cell Physiol 2006; 207: 428-436
• 37 Kostenuik PJ, Shalhoub V. Osteoprotegerin: a physiological and pharmacological inhibitor of bone resorption. Curr Pharm Des 2001; 7: 613-635
• 38 Mrak E, Villa I, Lanzi R, Losa M, Guidobono F, Rubinacci A. Growth hormone stimulates osteoprotegerin expression and secretion in human osteoblast-like cells. J Endocrinol 2007; 192: 639-645
• 39 Lanzi R, Losa M, Villa I, Gatti E, Sirtori M, Dal Fiume C, Rubinacci A. GH replacement therapy increases plasma osteoprotegerin levels in GH-deficient adults. Eur J Endocrinol 2003; 148: 185-191
• 40 Reinhard H, Lajer M, Gall MA, Tarnow L, Parving HH, Rasmussen LM, Rossing P. Osteoprotegerin and mortality in type 2 diabetic patients. Diabetes Care 2010; 33: 2561-2566
• 41 Corallini F, Gonelli A, D’Aurizio F, di Iasio MG, Vaccarezza M. Mesenchymal stem cells-derived vascular smooth muscle cells release abundant levels of osteoprotegerin. Eur J Histochem 2009; 53: 19-24
• 42 Morena M, Dupuy AM, Jaussent I, Vernhet H, Gahide G, Klouche K, Bargnoux AS, Delcourt C, Canaud B, Cristol JP. A cut-off value of plasma osteoprotegerin level may predict the presence of coronary artery calcifications in chronic kidney disease patients. Nephrol Dial Transplant 2009; 24: 3389-3397
• 43 Heximer SP, Knutsen RH, Sun X, Kaltenbronn KM, Rhee MH, Peng N, Oliveira-dos-Santos A, Penninger JM, Muslin AJ, Steinberg TH, Wyss JM, Mecham RP, Blumer KJ. Hypertension and prolonged vasoconstrictor signaling in RGS2-deficient mice. J Clin Invest 2003; 111: 445-452
• 44 Tang KM, Wang GR, Lu P, Karas RH, Aronovitz M, Heximer SP, Kaltenbronn KM, Blumer KJ, Siderovski DP, Zhu Y, Mendelsohn ME. Regulator of G-protein signaling-2 mediates vascular smooth muscle relaxation and blood pressure. Nat Med 2003; 9: 1506-1512
• 45 Takimoto E, Koitabashi N, Hsu S, Ketner EA, Zhang M, Nagayama T, Bedja D, Gabrielson KL, Blanton R, Siderovski DP, Mendelsohn ME, Kass DA. Regulator of G protein signaling 2 mediates cardiac compensation to pressure overload and antihypertrophic effects of PDE5 inhibition in mice. J Clin Invest 2009; 119: 408-420
• 46 Zhang W, Anger T, Su J, Hao J, Xu X, Zhu M, Gach A, Cui L, Liao R, Mende U. Selective Loss of Fine Tuning of Gq/11 Signaling by RGS2 Protein Exacerbates Cardiomyocyte Hypertrophy. J Biol Chem 2006; 281: 5811-5820
• 47 Hurst JH, Mendpara N, Hooks SB. Regulator of G-protein signalling expression and function in ovarian cancer cell lines. Cell Mol Biol Lett 2009; 14: 153-174
• 48 Smalley MJ, Iravani M, Leao M, Grigoriadis A, Kendrick H, Dexter T, Fenwick K, Regan JL, Britt K, McDonald S, Lord CJ, Mackay A, Ashworth A. Regulator of G-protein signalling 2 mRNA is differentially expressed in mammary epithelial subpopulations and over-expressed in the majority of breast cancers. Breast Cancer Res 2007; 9: R85
• 49 Wang J, Lippman SM, Lee JJ, Yang H, Khuri FR, Kim E, Lin J, Chang DW, Lotan R, Hong WK, Wu X. Genetic variations in regulator of G-protein signaling genes as susceptibility loci for second primary tumor/recurrence in head and neck squamous cell carcinoma. Carcinogenesis 2010; 31: 1755-1761
• 50 Jiang Z, Wang Z, Xu Y, Wang B, Huang W, Cai S. Analysis of RGS2 expression and prognostic significance in stage II and III colorectal cancer. Biosci Rep 2010; 30: 383-390
• 51 Orme SM, McNally RJ, Cartwright RA, Belchetz PE. Mortality and cancer incidence in acromegaly: a retrospective cohort study. United Kingdom Acromegaly Study Group. J Clin Endocrinol Metab 1998; 83: 2730-2734
• 52 Herzog H, Hort YJ, Ball HJ, Hayes G, Shine J, Selbie LA. Cloned human neuropeptide Y receptor couples to two different second messenger systems. Proc Natl Acad Sci USA 1992; 89: 5794-5798
• 53 Hodges GJ, Jackson DN, Mattar L, Johnson JM, Shoemaker JK. Neuropeptide Y and neurovascular control in skeletal muscle and skin. Am J Physiol Regul Integr Comp Physiol 2009; 297: 546-555
• 54 Baldock PA, Allison SJ, Lundberg P, Lee NJ, Slack K, Lin EJ, Enriquez RF, McDonald MM, Zhang L, During MJ, Little DG, Eisman JA, Gardiner EM, Yulyaningsih E, Lin S, Sainsbury A, Herzog H. Novel role of Y1 receptors in the coordinated regulation of bone and energy homeostasis. J Biol Chem 2007; 282: 19092-19102
• 55 Michel MC, Beck-Sickinger A, Cox H, Doods HN, Herzog H, Larhammar D, Quirion R, Schwartz T, Westfall T. XVI. International Union of Pharmacology recommendations for the nomenclature of neuropeptide Y, peptide YY, and pancreatic polypeptide receptors. Pharmacological Rev 1998; 50: 143-150
• 56 Franquinho F, Liz MA, Nunes AF, Neto E, Lamghari M, Sousa MM. Neuropeptide Y and osteoblast differentiation – the balance between the neuro-osteogenic network and local control. FEBS J 2010; 277: 3664-3674
• 57 Teixeira L, Sousa DM, Nunes AF, Sousa MM, Herzog H, Lamghari M. NPY revealed as a critical modulator of osteoblast function in vitro: new insights into the role of Y1 and Y2 receptors. J Cell Biochem 2009; 107: 908-916