DIHYDROTESTOSTERONE DOWNREGULATES BONE RESORPTION ACTIVITY OF OSTEOCLASTS IN DOSE DEPENDENT MANNER: AN IN VITRO MODEL USING RAW 264.7 CELLS


Hnin Ei Thu, Zahid Hussain, Isa Naina Mohamed, Ahmad Nazrun Shuid

Abstract


Objective: Numerous studies have evidenced the bone regulatory potential of dihydrotestosterone in androgen-deficient osteoporosis. The present study was thus aimed to explore the translational mechanism of dihydrotestosterone to down-regulate the bone resorption activity of osteoclasts using RAW 264.7 cells as in vitro model.

Methods: Prior to analyze the efficacy of dihydrotestosterone (5α-DHT) to alleviate osteoclastic differentiation, their cell viability and cell proliferative ability was assessed using lactate dehydrogenase (LDH) and MTS assays. The osteoclastic differentiation capacity of dihydrotestosterone was evaluated by measuring TRAP activity and the expression of bone resorption-related proteins such as matrix metallopeptidase-9 (MMP-9), cathepsin-K, tartrate-resistant acid phosphatase (TRAP) and NFATc1. Moreover, the effects of dihydrotestosterone were also evaluated on superoxide (free radicals) generation and superoxide dismutase (SOD) activity in RANKL-induced osteoclasts.

Results: Dihydrotestosterone showed no toxicity towards RAW 264.7 cells and significantly enhanced their proliferation and growth rates in a dose-dependent fashion. It was also observed that dihydrotestosterone exhibits a remarkable inhibitory effect on differentiation, maturation and activation of osteoclasts. The marked inhibition of differentiation and activation of osteoclasts caused by 5α-DHT was due to down-regulation of the expression of MMP-9, cathepsin-K, TRAP, NFATc1, generation of superoxide and up-regulation of SOD activity in the RAW 264.7 cells.

Conclusion: Resulting data provided substantially in vitroevidence for the pronounced anti-osteoclastogenetic activity of dihydrotestosterone and its therapeutic value in treating osteoporosis and other bone-erosive disorders.

 


Keywords


Androgen-deficient osteoporosis, Dihydrotestosterone, RAW 264.7 cells, Bone resorption, Anti-osteoclastogenesis

| PDF | HTML |

References


Teitelbaum SL. Bone resorption by osteoclasts. Science 2000;289:1504–8.

Rodan GA, Martin TJ. Therapeutic approaches to bone diseases. Science 2000;289:1508–14.

Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423:337–42.

Burge R, Dawson-Hughes B, Solomon DH, Wong JB, King A, Tosteson A. Incidence and economic burden of osteoporosis-related fractures in the United States, 2005-2025. J Bone Miner Res 2007;22:465–75.

Preeti Sharma. Biochemical alterations in postmenopausal women having an osteoporic risk. Asian J Pharm Clin Res 2017;10:214–7.

Pradeep Kumar. Assessment of uric acid in post-menopausal osteoporotic women. Asian J Pharm Clin Res 2016;9:138–9.

Suda T, Takahashi N, Udagawa N, Jimi E, Gillespie MT, Martin TJ. Modulation of osteoclast differentiation and function by the new members of the tumor necrosis factor receptor and ligand families. Endocr Rev 1999;20:345–57.

Mizukami J, Takaesu G, Akatsuka H, Sakurai H, Ninomiya-Tsuji J, Matsumoto K, et al. Receptor activator of NF-kappa B ligand (RANKL) activates TAK1 mitogen-activated protein kinase kinase kinase through a signaling complex containing RANK, TAB2, and TRAF6. Mol Cell Biol 2002;22:992–1000.

Wittrant Y, Theoleyre S, Couillaud S, Dunstan C, Heymann D, Rédini F. Relevance of an in vitro osteoclastogenesis system to study receptor activator of NF-kB ligand and osteoprotegerin biological activities. Exp Cell Res 2004;293:292–301.

Komarova SV, Pereverzev A, Shum JW, Sims SM, Dixon SJ. Convergent signaling by acidosis and receptor activator of NF-kappa B ligand (RANKL) on the calcium/calcineurin/NFAT pathway in osteoclasts. Proc Natl Acad Sci USA 2005;102:2643–8.

Grigoriadis AE, Wang ZQ, Cecchini MG, Hofstetter W, Felix R, Fleisch HA, et al. c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 1994;266:443–8.

Zhao Q, Wang X, Liu Y, He A, Jia R. NFATc1: functions in osteoclasts. Int J Biochem Cell Biol 2010;42:576–9.

Choi HJ, Park YR, Nepal M, Choi BY, Cho NP, Choi SH, et al. Inhibition of osteoclastogenic differentiation by Ikarisoside A in RAW 264.7 cells via JNK and NF-kappa B signaling pathways. Eur J Pharmacol 2010;636:28–35.

Fujisaki K, Tanabe N, Suzuki N, Kawato T, Takeichi O, Tsuzukibashi O, et al. Receptor activator of NF-kappa B ligand induces the expression of carbonic anhydrase II, cathepsin K, and matrix metalloproteinase-9 in osteoclast precursor RAW264.7 cells. Life Sci 2007;80:1311–8.

Daniell HW, Dunn SR, Ferguson DW, Lomas G, Niazi Z, Stratte PT. Progressive osteoporosis during androgen deprivation therapy for prostate cancer. Br J Urol 2000;163:181–6.

Snyder PJ, Peachey H, Berlin JA, Hannoush P, Haddad G, Dlewati A, et al. Effects of testosterone replacement in hypogonadal men. J Clin Endocrinol Metab 2000;85:2670–7.

Chen Q, Kaji H, Sugimoto T, Chihara K. Testosterone inhibits osteoclast formation stimulated by parathyroid hormone through androgen receptor. FEBS Lett 2001;491:91–3.

Hofbauer LC, Khosla S, Dunstan CR, Lacey DL, Boyle WJ, Riggs BL. The roles of osteoprotegerin and osteoprotegerin ligand in the paracrine regulation of bone resorption. J Bone Miner Res 2000;15:2–12.

Vanderschueren D, Vandenput L, Boonen S, Van Herck E, Swinnen JV, Bouillon R. An aged rat model of partial androgen deficiency: prevention of both loss of bone and lean body mass by low-dose androgen replacement. Endocrinology 2000;141:1642–7.

Erben RG, Eberle J, Stahr K, Goldberg M. Androgen deficiency induces high turnover osteopenia in aged male rats: a sequential histomorphometric study. J Bone Miner Res 2000;15:1085–98.

Mohamad NV, Soelaiman IN, Chin KY. A concise review of testosterone and bone health. Clin Inter-ventions Aging 2016;11:1317–24.

Murphy S, Khaw KT, Cassidy A, Compston JE. Sex hormones and bone mineral density in elderly men. Bone Miner 1993;20:133–40.

Scopacasa F, Horowitz M, Wishart JM, Morris HA, Chatterton BE, Need AG. The relation between bone density, free androgen index, and estradiol in men 60 to 70 y old. Bone 2000;27:145–9.

Takayanagi H, Kim S, Koga T, Nishina H, Isshiki M, Yoshida H, et al. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev Cell 2002;3:889–901.

Ishida N, Hayashi K, Hoshijima M, Ogawa T, Koga S, Miyatake Y, et al. Large-scale gene expression analysis of osteoclastogenesis in vitro and elucidation of NFAT2 as a key regulator. J Biol Chem 2002;277:41147–56.

Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation. Nature 2003;423:337–42.

Favus MJ. Bisphosphonates for osteoporosis. N Engl J Med 2010;363:2027–35.

Hsu H, Lacey DL, Dunstan CR, Solovyev I, Colombero A, Timms E, et al. Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteopro¬tegerin ligand. Proc Natl Acad Sci U S A 1999;96:3540–5.

Delaissé JM, Engsig MT, Everts V, del Carmen Ovejero M, Ferreras M, Lund L, et al. Proteinases in bone resorption: obvious and less obvious roles. Clin Chim Acta 2000;291:223–34.

Tezuka K, Nemoto K, Tezuka Y, Sato T, Ikeda Y, Kobori M, et al. Identification of matrix metalloproteinase 9 in rabbit osteoclasts. J Biol Chem 1994;269:15006–9.

Andersen TL, del Carmen Ovejero M, Kirkegaard T, Lenhard T, Foged NT, Delaissé JM. A scrutiny of matrix metallopro-teinases in osteoclasts: evidence for heterogeneity and for the presence of MMPs synthesized by other cells. Bone 2004;35:1107–19.

Wilson SR, Peters C, Saftig P, Bromme D. Cathepsin K activitydependent regulation of osteoclast actin ring formation and bone resorption. J Biol Chem 2009;284:2584–92.

Zhao Q, Wang X, Liu Y, He A, Jia R. NFATc1: functions in osteoclasts. Int J Biochem Cell Biol 2010;42:576–9.

Kim K, Lee SH, Ha Kim J, Choi Y, Kim N. NFATc1 induces osteoclast fusion via up-regulation of Atp6v0d2 and the dendritic cell-specific transmembrane protein (DC-STAMP). Mol Endocrinol 2008;22:176–85.

Winslow MM, Pan M, Starbuck M, Gallo EM, Deng L, Karsenty G, et al. Calcineurin/NFAT signaling in osteoblasts regulates bone mass. Dev Cell 2006;10:771–82.

Sasaki H, Yamamoto H, Tominaga K, Masuda K, Kawai T, Teshima Kondo S, et al. NADPH oxidase derived reactive oxygen species are essential for differentiation of a mouse macrophage cell line (RAW264.7) into osteoclasts. J Med Invest 2009;56:33–41.

Yang S, Zhang Y, Ries W, Key L. Expression of Nox4 in osteoclasts. J Cell Biochem 2004;92:238–48.

Tang XL, Liu XJ, Tian Q, Zhang W. Dynamic oxidative stress and DNA damage induced by oestrogen deficiency and protective effects of puerarin and 17β oestradiol in ovariectomized rats. Basic Clin Pharmacol Toxicol 2012;111:87–91.




About this article

Title

DIHYDROTESTOSTERONE DOWNREGULATES BONE RESORPTION ACTIVITY OF OSTEOCLASTS IN DOSE DEPENDENT MANNER: AN IN VITRO MODEL USING RAW 264.7 CELLS

Keywords

Androgen-deficient osteoporosis, Dihydrotestosterone, RAW 264.7 cells, Bone resorption, Anti-osteoclastogenesis

DOI

10.22159/ijpps.2017v9i10.20651

Date

02-10-2017

Additional Links

Manuscript Submission

Journal

International Journal of Pharmacy and Pharmaceutical Sciences
Vol. 9, Issue 10, 2017 Page: 86-92

Online ISSN

0975-1491

Statistics

50 Views | 51 Downloads

Authors & Affiliations

Hnin Ei Thu
Department of Pharmacology, Faculty of Medicine, Universiti Kebangsaan Malaysia (The National University of Malaysia), Jalan Yaacob Latif 56000, Cheras, Malaysia
Malaysia

Zahid Hussain
Department of Pharmaceutics, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, Bandar Puncak Alam 42300, Selangor, Malaysia
Malaysia

Isa Naina Mohamed
Department of Pharmaceutics, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, Bandar Puncak Alam 42300, Selangor, Malaysia

Ahmad Nazrun Shuid
Department of Pharmaceutics, Faculty of Pharmacy, Universiti Teknologi MARA, Puncak Alam Campus, Bandar Puncak Alam 42300, Selangor, Malaysia


Article Tools



Refbacks

  • There are currently no refbacks.