Acta Medica Iranica 2018. 56(4):234-243.

Mechanistic Insight Into the Efficient Osteogenic Potential of Dihydrotestosterone: Exploring Sequential Expression of Bone-Related Protein Biomarkers
Hnin Ei Thu, Zahid Hussain, Isa Naina Mohamed, Ahmad Nazrun Shuid

Abstract


Numerous in vitro, in vivo and clinical studies have evidenced the outstanding potential of dihydrotestosterone (DHT) in the treatment of male osteoporosis. Despite of promising clinical efficacy of DHT in regulating the skeletal growth and homeostasis, the exact molecular and translational mechanism is yet to be explored. This study was aimed to investigate the bone-forming molecular mechanism of DHT using MC3T3-E1 cell line as in vitro model. The mechanism of bone-forming ability of DHT was assessed by evaluating the time-mannered expression of bone-related biomarkers such as bone morphogenic protein-2 (BMP-2), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx-2), osteocalcin (OCN), type I collagen, osteopontin (OPN), transforming growth factor-β1 (TGF-β1) and androgen receptor (AR). Results demonstrated a remarkable efficacy of DHT (at a dose of 0.1 ng/mL) in promoting the expression of these vital bone-forming mediators. The resulting analysis revealed that the DHT-0.1 group showed higher expression of BMP-2 (106±9 pg/mL), ALP (381±16 pg/mL), Runx-2 (664±32 pg/mL), OCN (2265±111 pg/mL), type I collagen (276±16 pg/mL), TGF-β1 (81±7 pg/mL) and AR (411±21 pg/mL) compared to the control (CN) and other DHT-treated groups. These findings provide an in vitro evidence for the bone-forming capacity of DHT and its therapeutic significance for the treatment of male osteoporosis.


Keywords


Dihydrotestosterone; Osteoporosis; Enhanced osteogenesis; Cell proliferation; Bone-related biomarkers; Time mannered expression

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Abe E, Yamamoto M, Taguchi Y, Lecka-Czernik B, O’Brien CA, Economides AN, Stahl N, Jilka RL, Manolagas SC 2000. Essential requirement of BMPs-2/4 for both osteoblast and osteoclast formation in murine bone marrow cultures from adult mice: Antagonism by noggin. J Bone Miner Res 15:663–673.

Antonio C, Alberto F. Male osteoporosis and androgenic therapy: from testosterone to SARMs. Clin Cases Miner Bone Metab. 2009; 6(3): 229-233.

Annie WCK. Androgen and bone mass in men. Asian J Androl 2003; 5: 148-154.

Balkan W, Burnstein KL, Schiller PC, Perez-Stable C, D'Ippolito G, Howard GA, Roos BA. Androgen-induced mineralization by MC3T3-E1 osteoblastic cells reveals a critical window of hormone responsiveness. Biochem Biophys Res Commun. 2005; 328(3): 783-9.

Bart L. Clarke M.D, Sundeep Khosla M.D. Androgens and Bone. Steroids. 2009; 74(3): 296–305.

Beck GR Jr1, Zerler B, Moran E. Phosphate is a specific signal for induction of osteopontin gene expression. Proc Natl Acad Sci U S A. 2000; 97(15): 8352-7.

Beloti MM, Rosa AL. Osteoblast differentiation of human bone marrow cells under continuous and discontinuous treatment with dexamethasone. Braz Dent J. 2005; 16(2): 156-61.

Bilezikian J 2002 Sex steroids, mice, and men: when androgens and estrogens get very close to each other. Journal of Bone and Mineral Research 17 563–566.

Bland R. Steroid hormone receptor expression and action in bone. 2000; Clin Sci (Lond) 98: 217–40.

Bonewald LF, Harris SE, Rosser J. Dallas MR, Dallas SL, Camacho NP, Boyan B, Boskey A. Von Kossa staining alone is not sufficient to confirm that mineralization in vitro represents bone formation. Calcif Tissue Int. 2003;72: 537-547.

Choi JY, Lee BH, Song KB, Park RW, Kim IS, Sohn KY, Jo JS, Ryoo HM. Expression patterns of bone-related proteins during osteoblastic differentiation in MC3T3-E1 cells. J Cell Biochem. 1996; 61(4): 609-18.

Cilotti A, Falchetti A. Male osteoporosis and androgenic therapy: from testosterone to SARMs. Clin Cases Miner Bone Metab. 2009; 6(3):229-233.

Clarke BL, Khosla S. New selective estrogen and androgen receptor modulators. Curr Opin Rheumatol. 2009;21(4):374-9.

Compston JE. Sex steroids and bone. Physiol Rev. 2001;81:419–47.

Derynck R, Akhurst RJ. Differentiation plasticity regulated by TGF-beta family proteins in development and disease. Nat Cell Biol. 2007;9:1000–4.

Favus M. J. Bisphosphonates for osteoporosis. N Engl J Med. 2010; 363(21):2027–2035.

Gravallese EM. Osteopontin: a bridge between bone and the immune system. J Clin Invest 2003;112:147.

Guo X, Wang XF. Signaling cross-talk between TGF-beta/BMP and other pathways. Cell Res.2009;19:71–88.

Huang CK, Lai KP, Luo J, Tsai MY, Kang HY, Chen Y, Lee SO, Chang C. Loss of androgen receptor promotes adipogenesis but suppresses osteogenesis in bone marrow stromal cells. Stem Cell Res. 2013;11(2):938-50.

Huang W, Yang S, Shao J, Li YP. Signaling and transcriptional regulation in osteoblast commitment and differentiation. Front Biosci. 2007;12:3068–92.

Huang W, Carlsen B, Rudkin G, Berry M, Ishida K, Yamaguchi DT, Miller TA. Osteopontin is a negative regulator of proliferation and differentiation in MC3T3-E1 pre-osteoblastic cells. Bone. 2004; 34(5):799-808.

Ihara H, Denhardt DT, Furuya K, et al. Parathyroid hormone-induced bone resorption does not occur in the absence of osteopontin. J Biol Chem 2001;276:13065– 71.

Ishijima M, Rittling SR, Yamashita T, et al. Enhancement of osteoclastic bone resorption and suppression of osteoblastic bone formation in response to reduced mechanical stress do not occur in the absence of osteopontin. J Exp Med 2001;193:399– 404.

Jeong JC , Lee JW , Yoon CH , Kim HM , Kim CH. Drynariae Rhizoma promotes osteoblast differentiation and mineralization in MC3T3-E1 cells through regulation of bone morphogenetic protein-2, alkaline phosphatase, type I collagen and collagenase-1. Toxicology in Vitro 18 (2004) 829–834

Kapur SP, Reddi AH. Influence of testosterone and dihydrotestosterone on bone-matrix induced endochondral bone formation. Calcif Tissue Int. 1989;44(2):108-13.

Karsenty G, Wagner EF. Reaching a genetic and molecular understanding of skeletal development. Dev Cell. 2002;2:389-406.

Kasperk CH, Wergedal JE, Farley JR, Linkhart TA, Turner RT, Baylink DJ. Androgens directly stimulate proliferation of bone cells in vitro. Endocrinology. 1989; 124(3): 1576-8.

Kim HK, Kim MG, Leem KH (2013) Osteogenic activity of collagen peptide via ERK/MAPK pathway mediated boosting of collagen synthesis and its therapeutic efficacy in osteoporotic bone by back-scattered electron imaging and microarchitecture analysis. Molecules18:15474–15489.

Komori T (2010) Regulation of bone development and extracellular matrix protein genes by RUNX2. Cell Tissue Res 339: 189–195.

Lee KE, Seymen F, Ko J, Yildirim M, Tuna EB, et al. (2013) RUNX2 mutations in cleidocranial dysplasia. Genet Mol Res 12: 4567–4574.

Leder BZ, LeBlanc KM, Schoenfeld DA, Eastell R, Finkelstein JS. Differential effects of androgens and estrogens on bone turnover in normal men. J Clin Endocrinol Metab. 2003; 88:204–10.

Lian JB, Javed A Zaidi SK, Lengner C. Montecino M, van Wijnen A, J Stein J, L. Stein GS. Regulatory controls for osteoblast growth and differentiation: role of Runx/Cbfa/AML factors. Crit Rev Eukaryot Gene Expr. 2004;14:1-41.

Lin IC , Slemp AE, Hwang C, SenaEsteves M, Nah HD, Kirschner RE. Dihydrotestosterone stimulates proliferation and differentiation of fetal calvarial osteoblasts and dural cells and induces cranial suture fusion. Plast Reconstr Surg. 2007;120(5):1137-47.

Lips P. Vitamin D physiology. Prog Biophys Mol Biol. 2006;92(1):4-8.

Liu J, Zhang B, Song S, Ma M, Si S, et al. (2014) Bovine Collagen Peptides Compounds Promote the Proliferation and Differentiation of MC3T3-E1 PreOsteoblasts. PLoS ONE 9(6): e99920.

Liu DD, Zhang JC, Yi CQ, et al. 2010. The effects of gold nanoparticles on the proliferation, differentiation, and mineralization function of MC3T3-E1 cells in vitro. Chinese Sci Bull. 2010;11(5):1013-1019.

Liu J, Si S, Qin Y, Zhang B, Song S, Guo Y. The effect of different molecular weight collagen peptides on MC3T3-E1 cells differentiation.Biomed Mater Eng. 2015;26 Suppl 1:S2041-7.

Luppen, C.A., Leclerc, N., Noh, T., Barski, A., Khokhar, A., Boskey, A.L., Smithm, E., Frenkel, B., 2003. Brief bone morphogenetic protein 2 treatment of glucocorticoid-inhibited MC3T3-E1 osteoblasts rescues commitment-associated cell cycle and mineralization without alteration of Runx2. Journal of Biological Chemistry 278,44995–45003.

Maclaughlin EJ, Sleeper RB, McNatty D, Raehl CL. Management of age-related osteoporosis and prevention of associated fractures. Ther Clin Risk Manag. 2006;2:281-295.

Nakano Y, Addison WN, Kaartinen MT. ATP induced mineralization of MC3T3-E1 osteoblast cultures. Bone. 2007;41:549-561.

Ohba S, Nakajima K, Komiyama Y, et al: A novel osteogenic helioxanthin-derivative acts in a BMP-dependent manner. Biochem Biophys Res Commun 357: 854-860, 2007.

Orimo H. The mechanism of mineralization and the role of alkaline phosphatase in health and disease. J Nippon Med Sch. 2010;77:4–12.

Owen TA, Aronow M, Shahoub V, Barone LM, Wilming L, Tassinari MS, Kennedy MB, Pockwinse S, Lian JB, Stein GS (1990): Progressive development of the rat osteoblast phenotype in vitro: Reciprocal relationships in expression of genes associated with osteoblast proliferation and differentiation during formation of the bone extracellular matrix. J Cell Physiol 143:420-430.

Picherit C, Coxam V, Oudadesse H, Martini B, Gaumet N, Davicco MJ, Lebecque P, Miller S, Irrigaray JL, Barlet JP. Dihydrotestosterone Prevents Glucocorticoid-Negative Effects on Fetal Rat Metatarsal Bone in vitro. Biol Neonate 2000;77:181–190.

Premjit Arpornmaeklong, Shelley E. Brown, Zhuo Wang, Paul H. Krebsbach. 2009. Phenotypic Characterization, Osteoblastic Differentiation, and Bone Regeneration Capacity of Human Embryonic Stem Cell–Derived Mesenchymal Stem Cells. Stem cells and development 18(7):955-968.

Raida M, Heymann AC, Gunther C, Niederwieser D. 2006. Role of bone morphogenetic protein 2 in the crosstalk between endothelial progenitor cells and mesenchymal stem cells. Int J Mol Med 18: 735–739.

Riggs BL, Khosla S, Melton III J, Sex steroids and the construction and conservation of the adult skeleton, Endocrine Rev. 23 (2002) 279–302.

Ripamonti U, Crooks J, Matsaba T, Tasker J. 2000. Induction of endochondral bone formation by recombinant human transforming growth factor‐2 in the baboon. Growth Factors 17: 269–285.

Saadiah Abdul Razak H, Shuid AN, Naina Mohamed I. Combined Effects of Eurycoma longifolia and Testosterone on Androgen-Deficient Osteoporosis in a Male Rat Model. Evid Based Complement Alternat Med. 2012; 2012:872406.

Sakamoto W, Isomura H, Fujie K, Deyama Y, Kato A, Nishihira J, Izumi H. Homocysteine attenuates the expression of osteocalcin but enhances osteopontin in MC3T3-E1preosteoblastic cells. Biochim Biophys Acta. 2005;1740(1): 12-6.

Schroder HC, Kurz L, Muller WE, Lorenz B. Polyphosphate in bone. Biochemistry. 2000; 65:296-303.

Sinnesael M, Boonen S, Claessens F, Gielen E, Vanderschueren D. Testosterone and the male skeleton: a dual mode of action. J Osteoporos. 2011;2011:240328.

Soltanoff CS, Yang S, Chen W, Li YP. Signaling networks that control the lineage commitment and differentiation of bone cells. Crit Rev Eukaryot Gene Expr. 2009;19:1–46.

Suzawa M, Takeuchi Y, Fukumoto S, Kato S, Ueno N, Miyazono K, Matsumoto T, Fujita T 1999 Extracellular matrixassociated bone morphogenetic proteins are essential for differentiation of murine osteoblastic cells in vitro. Endocrinology 140:2125–2133.

Thu HE, Mohamed IN, Hussain Z, Shuid AN. Eurycoma longifolia as a potential alternative to testosterone for the treatment of osteoporosis: Exploring time-mannered proliferative, differentiative and morphogenic modulation in osteoblasts. J Ethnopharmacol. 2016, S0378-8741(16)31654-3.

Van Pottelbergh I, Goemaere S, Zmierczak H, Kaufman J.M. Perturbed sex steroid status in men with idiopathic osteoporosis and their sons. J Clin Endocrinol Metab. 2004;89:4949–4953.

Weitao S, Mohit K. Physiological normal levels of androgen inhibit proliferation of prostate cancer cells in vitro. Asian Journal of Andrology. 2014;16:864–868.

Wilt TJ, et al. Five-alpha-reductase Inhibitors for prostate cancer prevention. Cochrane Database Syst Rev. 2008;(2):CD007091.

Wiren KM, Chapman Evans A, Zhang XW. Osteoblast differentiation influences androgen and estrogen receptor-alpha and -beta expression. J Endocrinol. 2002;175(3):683-94.

Yamaguchi A, Komori T, Suda T 2000 Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1. Endocr Rev 21:393–411.

Yarrow JF, Wronski TJ, Borst SE. Testosterone and Adult Male Bone: Actions Independent of 5α-Reductase and Aromatase. Exerc Sport Sci Rev. 2015;43(4):222-30.

Yoshida K, Shinohara H, Haneji T, Nagata T. Arachidonic acid inhibits osteoblast differentiation through cytosolic phospholipase A2-dependent pathway. Oral Dis 2007; 13(1): 32–39.

Zallone A. Direct and indirect estrogen actions on osteoblasts and osteoclasts. Ann N Y Acad Sci. 2006;1068:173-9.

Zhao J, Ohba S, Shinkai M, Chung UI and Nagamune T: Icariin induces osteogenic differentiation in vitro in a BMP- and Runx2-dependent manner. Biochem Biophys Res Commun 369: 444-448, 2008.


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