Testosterone stimulates myotube formation, but inhibits fat cell differentiation
of pluripotent stem cells.
Our laboratory is on the cutting edge of stem cell research, exploring how hormones such as testosterone can modulate the differentiation of pluripotent stem cells, isolated from muscle or fat. We showed that androgens cause the stem cells to differentiate more into muscle fibers, and inhibits their formation of fat tissue. Stem-cells are being explored as a potential form of cell-based regenerative therapy for older patients who are chronically ill with diseases such as Parkinsons, cachexia or muscle wasting, heart infarction, or Alzheimers dementia. With the recent passage of the California initiative for Stem Cell research, this field will expand and develop during the next decade. Unlike the controversial embryonic stem cells, which can differentiate into any type of tissue in the body, the adult-stem cells derived from bone marrow, muscle, or fat are pluripotent, since they can differentiate into a limited number of cell types of the adult body. Usually these adult stem cells play a role in the healing or tissue repair process. We have shown that the body's hormones and the cellular environment of these stem cells plays an important role in determining the pathways for stem cell determination, in addition to directly affecting the adult tussues. Testosterone (T) dose-dependently increases skeletal muscle mass and decreases fat mass in men (Bhasin et al, Am.J.Physiol 2001); however the mechanism of its action remains unknown. Testosterone-induced increase in muscle mass is associated with increases in muscle fiber size, and the numbers of myonuclei and satellite cells. We hypothesized that T regulates body composition by promoting the commitment of mesenchymal pluripotent cells into myogenic lineage and inhibiting their differentiation into adipogenic lineage. Mouse C3H 10T1/2 pluripotent cells were treated with T (0-300 nM) or dihydrotestosterone (DHT, 0-30 nM) for 0-14 days, and myogenic conversion was evaluated by immunochemical staining for early (MyoD) and late (myosin heavy chain II: MHC) myogenic markers, and measurements of MyoD and MHC mRNA and protein. The number of MyoD positive myogenic cells and MHC positive myotubes, and MyoD and MHC mRNA and protein levels increased dose-dependently in response to T and DHT treatment. Both T and DHT decreased the number of adipocytes and down-regulated the expression of PPAR?2 mRNA and PPAR?2 and C/EBP? proteins. Androgen receptor (AR) mRNA and protein levels were low at baseline, but increased after T or DHT treatment. The effects of T and DHT on myogenesis and adipogenesis were blocked by bicalutamide (AR antagonist). Thus, we found that T and DHT regulate lineage determination in mesenchymal pluripotent cells by promoting their commitment to the myogenic lineage and inhibiting their differentiation into the adipogenic lineage through an AR-mediated pathway (ref. 20). We have recently observed androgen inhibition of adipocyte differentiation and PPAR 2 gene expression in the pre-adipocyte cell line 3T3-L1, also by an AR-mediated pathway. The observation that differentiation of pluripotent cells is androgen-dependent provides a unifying explanation for the reciprocal effects of androgens on muscle and fat mass in men.
In addition to testing the effects of androgens T and DHT in stimulation of myogenesis, and inhibition of adipogenesis in the above pluripotent mouse cell line, we will investigate these hormonal effects upon differentiation of human and mouse primary mesenchymal stem cell cultures. It was recently shown (Zuk and Hedrick et al.) that muscle, fat, and cartilage cells can be differentiated in vitro by culture of adult stem cells that co-purify with "fibroblasts" derived from muscle or fat. As a potential human therapy, treatment of a persons own primary cultures in vitro with different hormonal growth conditions, then re-implantation of these altered cells into a patient, may allow tissue repair of cardiac or skeletal muscle.
Human myostatin: Cloning & DNA sequence of gene, promoter expression, and protein function. Myostatin is a member of the transforming growth factor (TGF- ) superfamily of growth factors and a regulator of skeletal muscle development and tissue homeostasis. Muscle hypertrophy has been observed in both cattle and mice with null mutations in the myostatin gene, thus it was hypothesized that myostatin negatively regulates skeletal muscle growth. Our laboratory has shown that myostatin is expressed predominantly in skeletal muscle tissue, and the myostatin-immunoreactive protein in serum and muscle is higher in humans or animals with muscle wasting diseases, including AIDS-HIV, hypogonadism, and sarcopenia. We showed that the gene for myostatin contains three exons, it is transcribed as a 3.1 kb mRNA, and is located in the chromosomal region 2q33.2 (published in PNAS, 1998). Myostatin is made as a 375 amino acid precursor protein, but it is processed in human skeletal muscle as a glycosylated 110 aa protein dimer of 30 kDa. We investigated how myostatin functions to inhibit muscle growth, and developed a bioassay utilizing purified myostatin protein, to detect its effects on inhibition of proliferation in cultured C2C12 muscle cells. By gene expression profiling with micro-array methods, we have determined that myostatin's downstream effects include arrest of mitotic cell cycling by induction of the cyclin inhibitor protein p21-Cip, but inhibition of myogenesis by down-regulation of the myogenic transcription factor MyoD. We are investigating whether protein synthesis in mature myotubes may also be inhibited through myostatin-signaling regulated by cortisone, and stimulated by glutamine.
In another important study, we investigated how the promoter for myostatin gene expression is regulated in muscle cells, and studied binding of transcription factors to DNA elements in the myostatin promoter. Dr. Ma in our division has cloned the human myostatin gene promoter, and determined the DNA sequence of the 4 kb region upstream of the protein coding sequence. The promoter DNA sequence contains homology to basic transcription factors such as SP1 in the proximal region, and DNA elements that bind the muscle-specific factor MEF2B, as seen by gel-retardation of nuclear proteins. Since the myostatin promoter is induced by glucocorticoids, we will analyze a hormone response element DNA fragment for interaction with the glucocorticoid and androgen receptor proteins. Elucidation of the role of myostatin in wasting diseases could potentially lead to the development of important new medicines in the future. By understanding how myostatin gene expression is regulated in muscle cells, it may be possible to ameliorate diseases such as sarcopenia by inhibition of myostatin or its expression.
Singh R., Bhasin S., Braga M., Artaza J.N., Taylor W.E., Sinha S.K., Tripathi B.R., and Jasuja R. Regulation of myogenic differentiation by androgens: Cross-talk between androgen receptor /B-catenin and Follistatin /TGF-B signaling pathways. Submit. to Proc. Natl. Acad.
Sci., Dec (2007).
Singh, R., Artaza, J. N., Taylor, W. E., Braga, M., Gonzalez-Cadavid, N. F., and Bhasin, S. 2006. Testosterone Inhibits Adipogenic Differentiation in 3T3-L1 cells: Possible Role of Androgen Receptor induced Nuclear Translocation of beta-catenin and activation of Wnt Signaling. Endocrinology 147(1):141-54. [abstract]
Salehian, B., Mahabadi, V., Bilas, J., Taylor, W.E., and Ma, K. 2006. The effect of glutamine on prevention of glucocorticoid-induced skeletal muscle atrophy is associated with myostatin suppression. Metabolism 55(9):1239-47. [abstract]
Jasuja, R., Ramaraj, P., Phong Mac, R., Singh, A.B., Storer, T.W., Artaza, J., Miller, A., Singh, R., Taylor, W.E., Lee, M.I., Davidson, T., Sinha-Hikim, I., Gonzalez-Cadavid, N.F., and Bhasin, S. 2005. Androstenedione Binds Androgen Receptor, Promotes Myogenesis in Vitro, and Increases Serum Testosterone Levels, Fat-Free Mass, and Muscle Strength in Hypogonadal Men: It Meets all the Criteria for an Anabolic Steroid. J. Clin. Endo. & Metab. 90(2):855-63. [abstract]
Wang, H., Casaburi, R., Taylor, W.E., Aboellail, H., Storer, T.W., Kopple, J.D. 2005. Skeletal muscle mRNA for IGF-IEa, IGF-II, and IGF-I receptor is decreased in sedentary chronic hemodialysis patients. Kidney Int. 68(1):352-61. [abstract]
Sinha-Hikim I, Taylor WE, Gonzalez-Cadavid NF, Zheng W, Bhasin S. Androgen receptor in the human skeletal muscle and cultured muscle satellite cells: up-regulation by androgen treatment. J Clin Endocrinol Metab. 2004 Oct: 89(10): 5245-55. [abstract]
Bhasin S, Taylor W.E., Singh R, Artaza J, Sinha-Hikim I, Jasuja R, Choi, H, Gonzalez-Cadavid N.F., The Mechanisms of Androgen Effects on Body Composition: Mesenchymal Pluripotent Cell as the Target of Androgen Action. (Review), Journal of Gerontology 58A, 1103-1110, August 2003.[abstract]
Singh, R., Artaza, J.N., Taylor, W.E., Gonzalez-Cadavid, N.F., and Bhasin, S. Androgens stimulate myogenic differentiation and inhibit adipogenesis in C3H 10T1/2 pluripotent cells through an androgen receptor-mediated pathway. Endocrinology
144, 5081-5088, (2003). [abstract]
Taylor, W.E., Bhasin S., Lalani R.., Datta A., Gonzalez-Cadavid, N.F. Alteration of gene expression profiles in skeletal muscle of rats exposed to microgravity during a spaceflight. Journal of Gravitational Physiology 9, 61-70, (2002).[abstract]
Artaza, J.N., Bhasin, S., Mallidis, C., Taylor, W.E., Ma, K., and Gonzalez-Cadavid, N.F., Endogenous expression and localization of myostatin and its relation to myosin heavy chain distribution in C2C12 skeletal muscle cells. J. of Cellular Physiology 190, 170-179 (2002). [abstract]
Kun, M., Mallidis, C., Artaza, J., Taylor, W.E., Gonzalez-Cadavid, N.F., and Bhasin, S., Characterization of 5' regulatory region of human myostatin gene: Regulation by dexamethasone in vitro. Am. J. Physiology, Endocrinol and Metab. 281, E1128-E1136 (2001). [abstract]
Taylor W.E., Bhasin S., Artaza J., Byhower F., Azam M., Willard Jr. D.H., Kull F.C., & Gonzalez-Cadavid NF. Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells. Am. J. of Physiology 280, E221-E228 (2001). [abstract]
Sinha, I., Limbo, M., Taylor, W.E., and Salehian, B. (1998). The genetic basis of male infertility. Gonadal Disorders,
Vol. 27, of Endocrinology and Metabolism Clinics of North America, pp. 783-805. [abstract]
Last updated 02/02/08