Abstract
Testosterone (T) restores bone mass loss in postmenopausal women and osteoporotic men mainly through its bioconversion to estradiol (E2). In target tissues, T is also biotransformed to the A-ring-reduced metabolites 3α,5α-androstanediol (3α,5α-diol) and 3β,5α-androstanediol (3β,5α-diol), which are potent estrogen receptor (ER) agonists; however, their biological role in bone has not been completely elucidated. To assess if osteoblasts bioconvert T to 3α,5α-diol and to 3β,5α-diol, we studied in cultured neonatal rat osteoblasts the metabolism of [14C]-labeled T. In addition, the intrinsic estrogenic potency of diols on cell proliferation and differentiation in neonatal calvarial rat osteoblasts was also investigated. Osteoblast function was assessed by determining cell DNA, cell-associated osteocalcin, and calcium content, as well as alkaline phosphatase activity and Alp1 gene expression. The results demonstrated that diols were the major bioconversion products of T, with dihydrotestosterone being an obligatory intermediary, thus demonstrating in the rat osteoblasts the activities of 5α-steroid reductase and 3α- and 3β-hydroxysteroid dehydrogenases. The most important finding was that 3β,5α- and 3α,5α-diols induced osteoblast proliferation and differentiation, mimicking the effect of E2. The observation that osteoblast differentiation induced by diols was abolished by the presence of the antiestrogen ICI 182,780, but not by the antiandrogen 2-hydroxyflutamide, suggests that diols effects are mediated through an ER mechanism. The osteoblast capability to bioconvert T into diols with intrinsic estrogen-like potency offers new insights to understand the mechanism of action of T on bone cells and provides new avenues for hormone replacement therapy to maintain bone mass density.
This study was partially supported by grants from the School of Medicine, Universidad Nacional Autónoma de México (SDI.PTID-05-3) and by the Division of Biological and Health Sciences, UAM-I, Mexico City, Mexico. The authors thank Drs. Armando Tovar, Octavio Villanueva, and Verónica Navarro for their expert scientific and technical assistance. All authors declare that there are no actual or potential conflicts of interest, including financial, personal, or other relationships with other people or organizations that could inappropriately influence this work.
References
1. Khastgir G, Studd J, Holland N, Alaghband-Zadeh J, Fox S, Chow J. Anabolic effect of estrogen replacement on bone in postmenopausal women with osteoporosis: histomorphometric evidence in a longitudinal study. J Clin Endocrinol Metab 2001;86:289–95.10.1210/jc.86.1.289Search in Google Scholar
2. Riggs BL, Khosla S, Melton LJ III. Sex steroids and the construction and conservation of the adult skeleton. Endocr Rev 2002;23:279–302.10.1210/edrv.23.3.0465Search in Google Scholar
3. Davis SR, McCloud P, Strauss BJ, Burger H. Testosterone enhances estradiol’s effects on postmenopausal bone density and sexuality. Maturitas 2008;61:17–26.10.1016/j.maturitas.2008.09.006Search in Google Scholar
4. Vanderschueren D, Vandenput L, Boonen S, Lindberg MK, Bouillon R, Ohlsson C. Androgens and bone. Endocr Rev 2004;25:389–425.10.1210/er.2003-0003Search in Google Scholar
5. Wang C, Cunningham G, Dobs A, Iranmanesh A, Matsumoto AM, Snyder PJ, Weber T, Berman N, Hull L, Swerdloff RS. Long-term testosterone gel (AndroGel) treatment maintains beneficial effects on sexual function and mood, lean and fat mass, and bone mineral density in hypogonadal men. J Clin Endocrinol Metab 2004;89:2085–98.10.1210/jc.2003-032006Search in Google Scholar
6. Benito M, Vasilic B, Wehrli FW, Bunker B, Wald M, Gomberg B, Wright AC, Zemel B, Cucchiara A, Snyder PJ. Effect of testosterone replacement on trabecular architecture in hypogonadal men. J Bone Miner Res 2005;200:1785–91.10.1359/JBMR.050606Search in Google Scholar
7. Wiren KM. Androgens and bone growth: its location, location, location. Curr Opin Pharmacol 2005;5:626–32.10.1016/j.coph.2005.06.003Search in Google Scholar
8. Orwoll ES. Osteoporosis in men. Endocrinol Metab Clin North Am 1998;27:349–67.10.1016/S0889-8529(05)70009-8Search in Google Scholar
9. Ryan CW, Huo D, Stallings JW, Davis RI, Beer TM, McWhorter LT. Lifestyle factors and duration of androgen deprivation affect bone mineral density of patients with prostate cancer during first year of therapy. Urology 2007;70:122–6.10.1016/j.urology.2007.03.026Search in Google Scholar PubMed
10. Tanaka S, Haji M, Nishi Y, Yanase T, Takuyanagi R, Nawata H. Aromatase activity in human osteoblast-like osteosarcoma cell. Calcif Tissue Int 1993;52:107–9.10.1007/BF00308318Search in Google Scholar PubMed
11. Gennari L, Nuti R, Bilezikian JP. Aromatase activity and bone homeostasis in men. J Clin Endocrinol Metab 2004;89: 5898–907.10.1210/jc.2004-1717Search in Google Scholar
12. Smith EP, Boyd J, Frank GR, Takahashi H, Cohen RM, Specker B, Williams TC, Lubahn DB, Korach KS. Estrogen resistance caused by a mutation in the estrogen-receptor gene in a man. N Engl J Med 1994;331:1056–61.10.1056/NEJM199410203311604Search in Google Scholar
13. Morishima A, Grumbach MM, Simpson ER, Fisher C, Kennan Q. Aromatase deficiency in male and female siblings caused by a novel mutation and the physiological role of estrogens. J Clin Endocrinol Metab 1995;80:3689–98.Search in Google Scholar
14. Shimodaira K, Fujikawa H, Okura F, Shimizu Y, Saito H, Yanaihara T. Osteoblast cells (MG-63 and HOS) have aromatase and 5α-reductase activities. Biochem Mol Biol Int 1996;39: 109–16.10.1080/15216549600201111Search in Google Scholar
15. Issa S, Schnabel D, Feix M, Wolf L, Schaefer H-E, Russell DW, Schweikert H-U. Human osteoblast-like cells express predominantly steroid 5α-reductase type 1. J Clin Endocrinol Metab 2002;87:5401–7.10.1210/jc.2001-011902Search in Google Scholar
16. Vittek J, Altman K, Gordon GG, Southern AL. The metabolism of 7α-3H-testosterone by rat mandibular bone. Endocrinology 1974;94:325–9.10.1210/endo-94-2-325Search in Google Scholar
17. McCarthy TL, Hochberg RB, Labaree DC, Centrella M. 3-Ketosteroid reductase activity and expression by fetal rat osteoblast. J Biol Chem 2007;282:34003–12.10.1074/jbc.M707502200Search in Google Scholar
18. Bruchovsky N, Wilson JD. The conversion of testosterone to 5α-androstan-17β-ol-3-one by rat prostate in vivo and in vitro. J Biol Chem 1968;243:2012–21.10.1016/S0021-9258(18)93542-8Search in Google Scholar
19. Wilson JD, Gloyna RE. The intranuclear metabolism of testosterone in the accessory organs of reproduction. Recent Prog Horm Res 1970;26:309–36.Search in Google Scholar
20. Luu-The V, Bélanger A, Labrie F. Androgen biosynthetic pathways in the human prostate. Best Pract Res Clin Endocrinol Metab 2008;22:207–21.10.1016/j.beem.2008.01.008Search in Google Scholar
21. Thieulant ML, Samperez S, Jouan P. Evidence for 5alpha-androstane-3beta, 17beta-diol binding to the estrogen receptor in the cytosol from male rat pituitary. Endocrinology 1981;108:1552–60.10.1210/endo-108-4-1552Search in Google Scholar
22. Ho SM, Ofner P. Prostatic cytosol binding of 5α-androstane-3β,17β-diol and estradiol-17β. Steroids 1986;47:21–34.10.1016/0039-128X(86)90073-5Search in Google Scholar
23. Kusec V, Virdi AS, Prince R, Triffitt JT. Localization of estrogen receptor-alpha in human and rabbit skeletal tissues. J Clin Endocrinol Metab 1998;83:2421–8.Search in Google Scholar
24. Vidal O, Kindblom LG, Ohlsson C. Expression and localization of estrogen receptor-beta in murine and human bone. J Bone Miner Res 1999;14:923–9.10.1359/jbmr.1999.14.6.923Search in Google Scholar
25. Vidal O, Lindberg M, Savendahl L, Lubahn DB, Ritzen EM, Gustafsson JA, Ohlsson C. Disproportional body growth in female estrogen receptor-alpha-inactivated mice. Biochem Biophys Res Comm 1999;265:569–71.10.1006/bbrc.1999.1711Search in Google Scholar
26. Lindberg MK, Alatalo SL, Halleen JM, Mohan S, Gustafsson JÅ, Ohlsson C. Estrogen receptor specificity in the regulation of the skeleton in female mice. J Endocrinol 2001;171:229–36.10.1677/joe.0.1710229Search in Google Scholar
27. Eckstein B, Golan R, Shani J. Onset of puberty in the immature female rat induced by 5α-androstane-3β,17β-diol. Endocrinology 1973;92:941–5.10.1210/endo-92-3-941Search in Google Scholar
28. Ho SM, Levin V. Induction of progesterone receptor by androgens in the mouse uterus. Molec Cell Endocrinol 1986;46:103–8.10.1016/0303-7207(86)90088-2Search in Google Scholar
29. Pérez-Palacios G, Santillán R, García-Becerra R, Borja-Cacho E, Larrea F, Damián-Matsumura P, González L, Lemus AE. Enhanced formation of non-phenolic androgen metabolites with intrinsic oestrogen-like gene transactivation potency in human breast cancer cells: a distinctive metabolic pattern. J Endocrinol 2006;190:805–18.10.1677/joe.1.06407Search in Google Scholar
30. Enríquez J, Lemus AE, Chimal-Monroy J, Arzate H, García GA, Herrero B, Larrea F, Pérez-Palacios G. The effects of synthetic 19-norprogestins on osteoblastic cell function are mediated by their non-phenolic reduced metabolites. J Endocrinol 2007;193:493–504.10.1677/JOE-06-0038Search in Google Scholar
31. Kaplow LS. A histochemical procedure for localizing and evaluating leukocyte alkaline phosphatase activity in smears of blood and marrow. Blood 1955;10:1023–7.10.1182/blood.V10.10.1023.1023Search in Google Scholar
32. Arzate H, Alvarez-Pérez MA, Aguilar-Mendoza ME, Alvarez-Fregoso O. Human cementum tumor cells have different features from human osteoblastic cells in vitro. J Periodontal Res 1998;33:249–58.10.1111/j.1600-0765.1998.tb02197.xSearch in Google Scholar
33. Lemus AE, Santillán R, Damián-Matsumura P, García GA, Grillasca I, Pérez-Palacios G. In vitro metabolism of gestodene in target organs: formation of A-ring reduced derivatives with oestrogenic activity. Eur J Pharmacol 2001;417:249–56.10.1016/S0014-2999(01)00893-7Search in Google Scholar
34. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976;72:248–54.10.1016/0003-2697(76)90527-3Search in Google Scholar
35. Labarca C, Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 1980;102:344–52.10.1016/0003-2697(80)90165-7Search in Google Scholar
36. Stein GS, Lian JB, Owen TA. Relationship of cell growth to the regulation of tissue-specific gene expression during osteoblast differentiation. FASEB J 1990;4:3111–23.10.1096/fasebj.4.13.2210157Search in Google Scholar
37. Majeska RJ, Ryaby JT, Einhorn TA. Direct modulation of osteoblastic activity with estrogen. J Bone Joint Surg Am 1994;76:713–21.10.2106/00004623-199405000-00013Search in Google Scholar
38. Qu Q, Perälä-Heape M, Kapanen A, Dahllund J, Salo J, Väänänen HK, Härkönen P. Estrogen enhances differentiation of osteoblasts in mouse bone marrow culture. Bone 1998;22:201–9.10.1016/S8756-3282(97)00276-7Search in Google Scholar
39. Nibbering PH, Van de Gevel JS, Van Furth R. A cell-ELISA for the quantification of adherent murine macrophages and the surface expression of antigens. J Immunol Methods 1990; 131:25–32.10.1016/0022-1759(90)90228-NSearch in Google Scholar
40. Lemus AE, Enríquez J, Hernández A, Santillán R, Pérez-Palacios G. Bioconversion of norethisterone, a progesterone receptor agonist into estrogen receptor agonists in osteoblastic cells. J Endocrinol 2009;200:199–206.10.1677/JOE-08-0166Search in Google Scholar
41. Moralí G, Oropeza MV, Lemus AE, Pérez-Palacios G. Mechanisms regulating male sexual behavior in the rat: role of 3α- and 3β-androstanediols. Biol Reprod 1994;51:562–71.10.1095/biolreprod51.3.562Search in Google Scholar
42. Vilchis F, Chávez B, Pérez AE, García GA, Ángeles A, Pérez-Palacios G. Evidence that a non-aromatizable metabolite of norethisterone induces estrogen-dependent pituitary progestin receptors. J Steroid Biochem 1986;24:525–31.10.1016/0022-4731(86)90115-9Search in Google Scholar
©2013 by Walter de Gruyter Berlin Boston
