Dihydrotestosterone, a robust promoter of osteoblastic proliferation and differentiation: understanding of time-mannered and dose-dependent control of bone forming cells

Document Type : Original Article

Authors

1 Department of Pharmacology, Faculty of Medicine, University Kebangsaan Malaysia (The National University of Malaysia), Jalan Yaacob Latif 56000, Cheras, Malaysia

2 Department of Pharmaceutics, Faculty of Pharmacy, University Teknologi MARA, Puncak Alam Campus, Bandar Puncak Alam 42300, Selangor, Malaysia

Abstract

Objective(s): The present study was aimed to evaluate the time-mannered and dose-dependent effects of 5α-dihydrotestosterone (5α-DHT) on the proliferation and differentiation of bone forming cells using MC3T3-E1 cells.
Materials and Methods: Cell proliferation was analyzed using MTS and phase contrast microscopic assays. Osteogenic differentiation was assessed through a series of in vitro experiments including crystal violet staining, alkaline phosphatase (ALP) activity, and Van Gieson (VG) staining. Taken together, the efficiency of bone mineralization was examined by using alizarin red s (ARS) staining, Von Kossa staining, scanning electron microscopy (SEM) and energy dispersive x-ray (EDX) analysis.
Results: The resulting data revealed that 5α-DHT exhibits promising potential particularly at a dose of 0.1 ng/ml, in promoting the growth of MC3T3-E1 cells compared to the control group (CN). Moreover, a significantly higher ALP activity was evident in the experimental group treated with 5α-DHT compared to the CN group at various time intervals. MC3T3-E1 cells treated with 5α-DHT also expressed a remarkably higher collagen deposition and mineralization (calcium and phosphate contents) compared to the CN group at various time intervals.
Conclusion: Conclusively, we suggest that 5α-DHT exhibits outstanding potential of promoting proliferation and differentiation in osteoblasts which could be the in vitro basis for the efficacy of 5α-DHT in the treatment of androgen-deficient male osteoporosis.

Keywords

References

1.Goto T, Hagiwara K, Shirai N, Yoshida K, Hagiwara H. Apigenin inhibits osteoblastogenesis and osteoclasto-genesis and prevents bone loss in ovariectomized mice. Cytotechnology 2015; 67:357–365.
2.Dennison E, Mohamed MA, Cooper C. Epidemiology of osteoporosis. Rheum Dis Clin North Am 2006; 32:617–629.
3.Beloti MM, Rosa AL. Osteoblast differentiation of human bone marrow cells under continuous and discontinuous treatment with dexamethasone. Braz Dent J 2005; 16:156–161.
4.Lian JB, Javed A, Zaidi SK, Lengner C, Montecino M, van Wijnen AJ, et al. Regulatory controls for osteoblast growth and differentiation: role of Runx/Cbfa/AML factors. Crit Rev Eukaryot Gene Expr 2004; 14:1–41.
5.Yan XZ, Yang W, Yang F, Kersten-Niessen M, Jansen JA, Both SK. Effects of continuous passaging on mineralization of MC3T3-E1 cells with improved osteogenic culture protocol. Tissue Eng Part C Methods 2014; 20:198–204.
6.Yazid MD, Ariffin SH, Senafi S, Razak MA, Wahab RM. Determination of the differentiation capacities of murines' primary mononucleated cells and MC3T3-E1 cells. Cancer Cell Int 2010; 10:42.
7.Wang J, de Boer J, de Groot K. Proliferation and differentiation of MC3T3-E1 cells on calcium phosphate/chitosan coatings. J Dent Res 2008; 87:650–654.
8.Seo HJ, Cho YE, Kim T, Shin HI, Kwun IS. Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. Nutr Res Pract 2010; 4:356–361.
9.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; 88:204–210.
10.Lips P. Vitamin D physiology. Prog Biophys Mol Biol 2006; 92:4–8.
11.Clarke BL, Khosla S. New selective estrogen and androgen receptor modulators. Curr Opin Rheumatol 2009; 21:374–379.
12.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.
13.Huang CK, Lai KP, Luo J, Tsai MY, Kang HY, Chen Y, et al. Loss of androgen receptor promotes adipogenesis but suppresses osteogenesis in bone marrow stromal cells. Stem Cell Res 2013; 11:938–950.
14.Cilotti A, Falchetti A. Male osteoporosis and androgenic therapy: from testosterone to SARMs. Clin Cases Miner Bone Metab 2009; 6:229–233.
15.Lin IC, Slemp AE, Hwang C, Sena-Esteves 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; 120:1137–1147.
16.Clarke BL, Khosla S. Androgens and bone. Steroids 2009; 74:296–305.
17.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.
18.Yarrow JF, Wronski TJ, Borst SE. Testosterone and adult male bone: actions independent of 5α-reductase and aromatase. Exerc Sport Sci Rev 2015; 43:222–230.
19.Ho MH, Liao MH, Lin YL, Lai CH, Lin PI, Chen RM. Improving effects of chitosan nanofiber scaffolds on osteoblast proliferation and maturation. Int J Nanomedicine 2014; 9:4293–4304.
20.Yamakawa K, Iwasaki H, Masuda I, Ohjimi Y, Honda I, Saeki K, et al. The utility of alizarin red s staining in calcium pyrophosphate dehydrate crystal deposition disease. J Rheumatol 2003; 30:1032–1035.
21.Liu J, Lee J, Salazar Hernandez MA, Mazitschek R, Ozcan U. Treatment of obesity with celastrol. Cell 2015; 161:999–1011.
22.Hofbauer LC, Khosla S. Androgen effects on bone metabolism, recent progress and controversies. Eur J Endocrinol 1999; 140:271–286.
23.Coxam V, Bowman BM, Mecham M, Roth CM, Miller MA, Miller SC. Effects of dihydrotestosterone alone and combined with estrogen on bone mineral density, bone growth, and formation rates in ovariectomized rats. Bone 1996; 19:107–114.
24.Balkan W, Burnstein KL, Schiller PC, Perez-Stable C, D'Ippolito G, Howard GA, et al. Androgen-induced mineralization by MC3T3-E1 osteoblastic cells reveals a critical window of hormone responsiveness. Biochem Biophys Res Commun 2005; 328:783–789.
25.Vanderschueren D, Vandenput L, Boonen S. Lindberg MK, Bouillon R, Ohlsson C. Androgens and bone. Endocr Rev 2004; 25:389–425.
26.Lee YS, Choi EM. Costunolide stimulates the function of osteoblastic MC3T3-E1 cells. Int Immunopharmacol 2011; 11:712–718.
27.Kang HY, Cho CL, Huang KL, Wang JC, Hu YC, Lin HK, et al. Nongenomic androgen activation of phosphatidylinositol 3-kinase/Akt signaling pathway in MC3T3-E1 osteoblasts. J Bone Miner Res 2004; 19:1181–1190.
28.Krum SA. Direct transcriptional targets of sex steroid hormones in bone. J Cell Biochem 2011; 112:401–408.
29.Orimo H. The mechanism of mineralization and the role of alkaline phosphatase in health and disease. J Nippon Med Sch 2010; 77:4–12.
30.Nakano Y, Addison WN, Kaartinen MT. ATP induced mineralization of MC3T3-E1 osteoblast cultures. Bone 2007; 41:549–561.
31.Bonewald LF, Harris SE, Rosser J, Dallas MR, Dallas SL, Camacho NP, et al. Von Kossa staining alone is not sufficient to confirm that mineralization in vitro represents bone formation. Calcif Tissue Int 2003; 72:537–547.
32.Tsutsumi K, Saito N, Kawazoe Y, Ooi HK, Shiba T. Morphogenetic study on the maturation of osteoblastic cells as induced by inorganic polyphosphate. PLoS One 2014; 9:e86834.
33.Ali MM, Yoshizawa T, Ishibashi O, Matsuda A, Shimomura J, Mera H, et al. PIASx beta is a key regulator of osterix transcriptional activity and matrix mineralization in osteoblasts. J Cell Sci 2007; 120:2565–2573.
34.McKee MD, Addison WN, Kaartinen MT. Heirarchies of extracellular matrix and mineral organization in bone of the craniofacial complex and skeleton. Cells Tissues Organs 2005; 181:176–188.
35.Chen P, Satterwhite JH, Licata AA, Lewiecki EM, Sipos AA, Misurski DM, et al. Early changes in biochemical 
markers of bone formation predict BMD response to teriparatide in postmenopausal women with osteoporosis. J Bone Miner Res 2005; 20:962–970.
36.Bu SY, Hunt TS, Smith BJ. Dried plum polyphenols attenuate the detrimental effects of TNF-alpha on osteoblast function coincident with up-regulation of Runx2, Osterix and IGF-I. J Nutr Biochem 2009; 20:35–44.
37.Salasznyk RM, Williams WA, Boskey A, Batorsky A, Plopper GE. Adhesion to vitronectin and collagen I promotes osteogenic differentiation of human mesenchymal stem cells. J Biomed Biotechnol 2004; 2004:24–34.
38.Wang W, Olson D, Liang G, Franceschi RT, Li C, Wang B, et al. Collagen XXIV (Col24α1) promotes osteoblastic differentiation and mineralization through TGF-β/Smads signaling pathway. Int J Biol Sci 2012; 8:1310–1322.
39.Siddiqui S, Arshad M. Osteogenic potential of punica granatum through matrix mineralization, cell cycle progression and runx2 gene expression in primary rat osteoblasts. Daru 2014; 20:72.
40.Hoemann CD, El-Gabalawy H, McKee MD. In vitro osteogenesis assays: influence of the primary cell source on alkaline phosphatase activity and mineralization. Pathol Biol 2009; 57:318–323.
41.Zhi J, Sommerfeldt DW, Rubin CT, Hadjiargyrou M. Differential expression of neuroleukin in osseous tissues and its involvement in mineralization during osteoblast differentiation. J Bone Miner Res 2001; 16:1994–2004.
Iranian Journal of Basic Medical Sciences
Volume 20, Issue 8
August 2017
Pages 894-904

Files

Share

How to cite

Statistics