199 Bioconversion of norethisterone, a progesterone receptor agonist into estrogen receptor agonists in osteoblastic cells Ana E Lemus1,2, Juana Enrı´quez2,A´ ngeles Herna´ndez1, Rene´ Santilla´n3 and Gregorio Pe´rez-Palacios3 1Department of Reproductive Biology, Universidad Auto´noma Metropolitana Iztapalapa, Mexico City P.C. 09340, Mexico 2Department of Reproductive Biology, Instituto Nacional de Ciencias Me´dicas y Nutricio´n S. Zubira´n, Mexico City P.C. 14000, Mexico 3National Institute of Perinatology and School of Medicine, Universidad Nacional Auto´noma de Me´xico, Mexico City P.C. 11000, Mexico (Correspondence should be addressed to A E Lemus; Email: [email protected]) Abstract A number of clinical studies have demonstrated that (36.4%) to 5a-reduced metabolites, including 5a-dihydro norethisterone (NET), a potent synthetic progestin, restores NET, 3a,5a-tetrahydro NET (3a,5a-NET) and 3b,5a- postmenopausal bone loss, although its mode of action on tetrahydro NET (3b,5a-NET), demonstrating the activities bone cells is not fully understood, while the effect of naturally of 5a-steroid reductase and two enzymes of the aldo-keto occurring progesterone in bone has remained controversial. reductases family. Expression of Srd5a1 in neonatal osteoblast A recent report claims that the potent effects of NET on was well demonstrated, whereas Srd5a2 expression was not osteoblastic cell proliferation and differentiation, mimicking detected. The most striking finding was that 3b,5a-NET and the action of estrogens, are mediated by non-phenolic NET 3a,5a-NET were efficient competitors of [3H]-estradiol for derivatives. To determine whether osteoblasts possess the osteoblast ER binding sites, exhibiting affinities similar to that enzymes required to bioconvert a progesterone receptor (PR) of estradiol. The results support the concept that the interplay agonist into A-ring reduced metabolites with affinity to bind of 5a-steroid reductase and aldo-keto reductases in osteo- estrogen receptor (ER), we studied the in vitro metabolism blastic cells, acting as an intracrine modulator system is of [3H]-labeled NET in cultured neonatal rat osteoblasts and capable to bioconvert a PR agonist into ER agonists, offering the interaction of its metabolic conversion products with an explanation of the molecular mechanisms NET uses to cytosolic –osteoblast ER, employing a competition analysis. enhance osteoblastic cell activities. Results indicated that NET was extensively bioconverted Journal of Endocrinology (2009) 200, 199–206 Introduction significant effects on rat osteoblast proliferation, differen- tiation, and mineralization processes, mimicking the effects of A number of clinical trials have demonstrated that adminis- estradiol (E2). The osteoblast proliferation induced by NET tration of norethisterone (NET), a synthetic 19-nor reduced derivatives was suppressed by the addition of ICI 182 progestin, to postmenopausal women prevents bone mineral 780, a potent steroidal antiestrogen, indicating that this effect loss and reduces bone resorption (Abdalla et al. 1985, is mediated by ER. The study also provides evidence that high Horowitz et al. 1993) and also prevents bone loss in young concentrations of unmodified NET exhibited estrogenic women treated with LH-releasing agonists (Riis et al. 1990). potency in osteoblastic cells, though these effects were The mechanisms of estrogen-like bone actions of NET are suppressed by finasteride, a selective steroid 5a-reductase not fully understood, particularly since this steroid molecule inhibitor (Enrı´quez et al. 2007). neither interacts with estrogen receptors (ER; Cha´vez et al. Androgen metabolizing enzymes have been demonstrated 1985) nor undergoes enzyme-mediated aromatization (Gual in bone cells; thus, several reports have documented the et al. 1962). Interestingly, a study conducted in postmeno- activities and gene expression of 5a-reductase 1 and 2 in pausal women and castrated patients with complete androgen human osteoblastic cells Shimodaira et al. (1996), Issa et al. resistance, strongly suggested that the antigonadotropic effect (2002) and Vittek et al. (1974), using testosterone as a of NET is mediated through ER (Pe´rez-Palacios et al. 1981). substrate, demonstrated the activity of 3a-hydroxysteroid Recently, evidence has been presented indicating that two dehydrogenase (3a-HSD) in rat mandibular bone. Previous reduced derivatives of NET, 3b,5a tetrahydro-NET (3b,5a- studies from our laboratory have demonstrated that synthetic NET) and 3a,5a tetrahydro-NET (3a,5a-NET), induced 19-nor progestins are extensively metabolized in target organs Journal of Endocrinology (2009) 200, 199–206 DOI: 10.1677/JOE-08-0166 0022–0795/09/0200–199 q 2009 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 09/29/2021 01:14:17PM via free access 200 A E LEMUS and others . Norethisterone metabolism in osteoblasts to A-ring reduced tetrahydro derivatives (Larrea et al. 1987, Isolation and culture of rat osteoblastic cells Lemus et al. 1992, 2001), which exert estrogen-like effects Osteoblastic calvarial cells from 1 day old rats were used (Vilchis et al. 1986, Moralı´ et al. 1990, Lemus et al. 2000, throughout the study. Calvariae were carefully dissected, Santilla´n et al. 2001). cleaned from connective tissue, periosteum and cartilaginous To assess if NET is bioconverted to non-phenolic reduced part, and sequentially digested for 60 min with 0.3% type II metabolites with intrinsic estrogenic potency in osteoblasts, we studied the metabolism of [3H]-labeled NET in rat collagenase (Robey 1995). Cells obtained in the first cultured osteoblastic cells and the interaction of NET and its treatment were discarded, while cells isolated from the 5a-reduced metabolites with the osteoblast intracellular ER. subsequent three digestions were pooled, plated, and cultured The identification of NET metabolites was established by a overnight in DMEM supplemented with 10% FBS and reverse isotope dilution technique, while their interaction 100 mM non-essential amino acids and an antibiotics– 3 antimycotic solution (100 U/ml penicillin, 100 mg/ml with ER was assessed by competition analysis using [ H]-E2 as the radioligand. In addition, the gene expression of 5a-steroid streptomycin and 250 ng/ml amphotericin-B; Gibco BRL) reductase type 1 (Srd5a1) and type 2 (Srd5a2) was studied and 50 mg/ml ascorbic acid, at 37 8C in a humidified using RT-PCR. atmosphere of 5% CO2 in air. Assessment of the phenotype of cultured rat calvarial cells was done by determining the presence of alkaline phosphatase activity and osteocalcin, according to the methods described Materials and Methods by Kaplow (1955) and Arzate et al. (1998) respectively. The results revealed the presence of these two bone-related Steroids and chemicals proteins in more than 95% of the calvarial cells, thus [6,7-3H] NET ([3H]-NET), specific activity (sp. act.) demonstrating the distinctive features of the osteoblast 55 Ci/mmol was kindly provided by Schering (Berlin, phenotype. At confluence, primary rat osteoblasts were 3 3 detached with 1 mM EDTA/0.25% trypsin solution, counted Germany), [2,4,6,7,16,17- H] estradiol ([ H]-E2), sp. act. 157 Ci/mmol was purchased from Amersham International and submitted to metabolism and ER competition studies. and their radiochemical purity was established by thin-layer chromatographic behavior. Authentic NET was kindly Norethisterone metabolism provided by Schering Mexicana, S.A. de C.V. (Mexico City, Mexico). Synthesis of 5a-dihydronorethisterone (5a- To assess the bioconversion of NET to its A-ring reduced 3 NET), 3a,5a-NET, and 3b,5a-NETwas previously reported derivatives, the metabolism of [ H]-NET in cultured (Cha´vez et al. 1985). The chemical purity of NET and its osteoblastic cells was studied. Osteoblasts were plated at a 6 reduced derivatives was assessed by their melting points, density of 2!10 /Petri dish and incubated for 24 h in culture HPLC behavior, infrared absorption, and H-nuclear mag- medium (DMEM at pH 7.4, containing 10% charcoal– netic resonance. The physical and spectroscopic constants of dextran treated (stripped) FBS, non-essential amino acids, the A-ring reduced derivatives of NET have been previously antibiotics–antimycotic solution) and 50 mg/ml ascorbic acid, described (Cha´vez et al. 1985). Other non-radioactive steroids at 37 8C in a humidified atmosphere of 5% CO2 in air. The were supplied by Sigma Chemical Co. Fetal bovine serum culture medium was removed and replaced by fresh medium 3 (FBS) was purchased from Hyclone Laboratories Inc., and containing 2 mM[ H]-NETand incubated at 37 8C, for 6, 24, phenol red-free DMEM from Life Technologies. Reverse and 48 h, under 5% CO2–air atmosphere. The optimal 3 transcriptase RT-PCR kit and TRIzol reagent were concentration of substrate ([ H]-NET) employed in these purchased from Invitrogen. TaqMan Universal PCR Master experiments, was derived from previous studies aimed to Mix and TaqMan probes and primers were obtained from characterize the kinetic constant (KM) at equilibrium of 5a- Applied Biosystems (Foster City, CA, USA). Reagents and steroid reductase in different tissues, using testosterone and solvents used were of analytical grade. gestodene, other synthetic 19-norprogestin as substrates (Lemus et al. 2001). Final incubation volume was 3 ml. Cell-free and boiled inactivated cell incubations carried out Animals under identical experimental conditions were