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Hif1α is required for osteoclast activation and bone loss in male osteoporosis
Biochemical and Biophysical Research Communications 470 (2016) 391e396
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Hif1a is required for osteoclast activation and bone loss in male osteoporosis Toshimi Tando a, 1, Yuiko Sato a, b, 1, Kana Miyamoto a, 1, Mayu Morita c, 1, Tami Kobayashi a, Atsushi Funayama a, Arihiko Kanaji a, Wu Hao a, Ryuichi Watanabe a, Takatsugu Oike a, Masaya Nakamura a, Morio Matsumoto a, Yoshiaki Toyama a, Takeshi Miyamoto a, * a b c
Department of Orthopedic Surgery, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan Department of Musculoskeletal Reconstruction and Regeneration Surgery, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 December 2015 Accepted 6 January 2016 Available online 11 January 2016
The number of osteoporosis patients is increasing not only in women but in men. Male osteoporosis occurs due to aging or androgen depletion therapies, leading to fractures. However, molecular mechanisms underlying male osteoporosis remain unidentified. Here, we show that hypoxia inducible factor 1 alpha (Hif1a) is required for development of testosterone deficiency-induced male osteoporosis. We found that in mice Hif1a protein accumulates in osteoclasts following orchidectomy (ORX) in vivo. In vitro, Hif1a protein accumulated in osteoclasts cultured in hypoxic conditions, but Hif1a protein rather than mRNA levels were suppressed by testosterone treatment, even in hypoxia. Administration of a Hif1a inhibitor to ORX mice abrogated testosterone deficiency-induced osteoclast activation and bone loss but did not alter osteoclast activities or bone phenotypes in sham-operated, testosterone-sufficient animals. We conclude that Hif1a protein accumulation due to testosterone-deficiency promotes development of male osteoporosis. Thus Hif1a protein could be targeted to inhibit pathologically-activated osteoclasts under testosterone-deficient conditions to treat male osteoporosis patients. © 2016 Elsevier Inc. All rights reserved.
Keywords: Testosterone Osteoporosis Hif1a Male Osteoclasts Bone
1. Introduction The size of the aging population in developed countries is growing, increasing the number of male and female osteoporosis patients [1,2]. Osteoporosis frequently causes bone fragility fractures [3], a condition reportedly associated with increased mortality risk for both men and women. Although osteoporosis is more common in women than in men, particularly in postmenopausal elderly women [1,4,5], the risk of mortality following fragility fracture is reportedly greater in men with the condition [6,7], suggesting that preventing osteoporosis-associated fractures is crucial for the health of elderly men. Osteoporosis in men is promoted by factors such as aging or hypogonadism [8,9]. Testosterone deficiency resulting from androgen depletion therapy in prostate cancer also reportedly promotes osteoporosis development [10].
* Corresponding author. E-mail address: [email protected] (T. Miyamoto). 1 T. Tando, Y. Sato, K. Miyamoto and M. Morita contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2016.01.033 0006-291X/© 2016 Elsevier Inc. All rights reserved.
However, pathogenesis of male osteoporosis development remains to be defined [8,9]. Recently, we proposed that hypoxia inducible factor 1 alpha (Hif1a), a hypoxia-responsive transcription factor, was required for development of estrogen-deficient postmenopausal osteoporosis [11]. Bone surfaces where bone-resorbing osteoclasts reside are reportedly hypoxic [12]; however, we found that Hif1a expressed in those cells was suppressed by estrogen in estrogen-sufficient, premenopausal conditions [11]. We also showed that estrogendeficient conditions in female mice caused by bilateral ovariectomy (OVX) promoted Hif1a protein accumulation in osteoclasts, and that administration of a Hif1a inhibitor blocked osteoporosis development normally seen in OVX mice [11]. However, the role of Hif1a in male osteoporosis development remained unexamined. Here, we show that Hif1a protein accumulates in osteoclasts in testosterone-deficient conditions established by ORX in male mice in vivo. Our in vitro studies showed that Hif1a protein accumulation in hypoxic conditions in osteoclasts was suppressed by testosterone treatment, although Hif1a mRNA levels were unchanged. ORX mice treated with a Hif1a inhibitor showed significantly abrogated
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osteoclast activation and bone loss, but comparable treatment did not alter bone status in sham-operated, testosterone-sufficient mice. Thus, Hif1a protein could be targeted to block pathological osteoclast activation as a treatment for male osteoporosis, particularly in conditions of testosterone depletion. 2. Materials and methods 2.1. Mice Wild type C57BL/6 mice were purchased from CLEA Japan (Meguro, Tokyo, Japan) and maintained under specific pathogenfree conditions in animal facilities certified by Keio University School of Medicine animal care committee. All experimental procedures involving mice were approved by the Keio University School of Medicine animal care committee. Twelve-week-old male mice were subjected to ORX or sham surgery, followed in some cases by 2ME2 (EMD Millipore Corporation) treatment. 2ME2 was prepared at 15 mg/mL in a 0.5% methylcellulose solution (Wako Pure Chemicals Industries) and administered daily at 75 mg/kg via intragastric infusion for 4 weeks. Controls were treated with vehicle only. At the end of drug administration, ORX or sham-operated mice treated with 2ME2 or vehicle were necropsied, and hindlimb bones were collected, fixed with 70% ethanol, and subjected to dualenergy X-ray absorptiometry (DEXA) analysis to measure bone mineral density (BMD) and subjected to bone histomorphometric analysis. 2.2. Immunohistochemical analysis Femurs were dissected from ORX or sham-operated mice, fixed in 10% buffered formalin, decalcified in 8% Na2EDTA, paraffinembedded, sectioned, and subjected to immunohistochemistry. Antigen was retrieved by treatment of tissue sections on slides with 0.4 mg/mL Proteinase K for 8 min at room temperature. Sections were incubated in 5% BSAePBS and then incubated with antibodies against HIF1a (#NB100-479, Novus Biologicals, Littleton, CO, USA) and Cathepsin K (Ctsk, #F-95, Daiichi Finechemical Co. Toyama, Japan). Nuclei were stained by TOTO-3 (#T3604, Invitrogen Corp., Carlsbad, CA, USA). Areas of oxygen tension in tissue were detected using pimonidazole (Hypoxyprobe-1 Kit, Hypoxyprobe Inc., Burlington, MA, USA) according to the manufacturer's instructions. Stained specimens were observed under a confocal laser scanning microscope (FV1000, OLYMPUS Corp., Tokyo, Japan). 2.3. Cell culture Raw264.7 cells were maintained in DMEM (SigmaeAldrich Co., St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (FBS, JRH Biosciences Lenexa, KS, USA) and GlutaMax (Invitrogen Corp., Carlsbad, CA, USA). Cells were cultured in normoxic or hypoxic conditions. In normoxic conditions, cells were gently shaken in the incubator. Hypoxic culture was performed at either 1% or 5% O2 and 5% CO2 using an INVIVO2 hypoxia workstation (Ruskin Technology Ltd., Bridgend, UK) in the presence of RANKL (PeproTech Ltd., Rocky Hill, NJ, USA) with or without 106 M testosterone (Tes; Wako Pure Chemicals Industries, Osaka, Japan), and cells were lysed in the hypoxic chamber. 2.4. Quantitative PCR analysis Total RNA was isolated from cultured cells using an RNeasy mini kit (Qiagen, Venlo, Limburg, The Netherlands), and cDNA synthesis was performed using oligo (dT) primers and reverse transcriptase
(Takara Bio Inc., Shiga, Japan). Quantitative PCR was done using SYBR Premix ExTaq II reagent and a DICE Thermal cycler (Takara Bio Inc., Shiga, Japan), according to the manufacturer's instructions. bactin (Actb) expression served as an internal control. Primers used for realtime PCR were: Hif1a-forward: 50 - AAAGCTTCTGTTATGAGGCTCA-30 Hif1a-reverse: 50 - CAGTCCATCTGTGCCTTCATCT-30 Actb-forward: 50 -TGAGAGGGAAATCGTGCGTGAC-30 Actb-reverse: 50 -AAGAAGGAAGGCTGGAAAAGAG-30
2.5. Immunoblotting Whole cell lysates were prepared using RIPA buffer (1% Triton X100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 10 mM TriseHCl, pH7.5) supplemented with a protease inhibitor cocktail (SigmaeAldrich Co.) and MG-132 (EMD Millipore Corporation). The insoluble fraction was removed by centrifugation and then lysate protein content was measured using the Bradford reagent (Bio-Rad Laboratories Inc., Tokyo, Japan). Equivalent amounts of protein were separated by SDS-PAGE, transferred to a PVDF membrane (EMD Millipore Corporation) and immunoblotted with anti-HIF1a (#NB100-479, Novus Biologicals) or anti-Vinculin (#V9131, SigmaeAldrich Co.) antibodies. 2.6. Statistical analyses Statistical analyses were performed using the unpaired twotailed Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.005; NS, not significant, throughout the paper). All data are expressed as the mean ± SD. 3. Results 3.1. Testosterone depletion stabilizes Hif1a protein in osteoclasts in vivo Recently, we showed that Hif1a protein was stabilized in osteoclasts in estrogen-deficient OVX female mice in vivo [11]. Thus, we initially examined expression of Hif1a protein in osteoclasts from testosterone-depleted ORX male mice. We could not detect Hif1a protein in osteoclasts of sham-operated control mice but did observe Hif1a protein accumulation in osteoclasts from ORX mice, based on staining with the osteoclast marker cathepsin K (Ctsk) (Fig. 1A). Notably, the hypoxic state of bone tissue was unchanged by ORX, as determined by staining with the hypoxia probe pimonidazole (Pimo) (Fig. 1B). These results suggest that Hif1a protein in osteoclasts is regulated by testosterone without a change in oxygen tension in vivo. 3.2. Hif1a protein but not mRNA is suppressed in osteoclasts by testosterone treatment in vitro We next analyzed direct effects of testosterone on Hif1a expression in cultured pre-osteoclast Raw264.7 cells in vitro (Fig. 2). Hif1a protein, as detected by western blot, increased following culture of untreated cells in hypoxic conditions, but Hif1a protein levels were suppressed by testosterone in hypoxic conditions at either 5% or 1% O2 (Fig. 2A and C). This decrease was not due to decreased Hif1a mRNA expression (Fig. 2B and D). These results suggest that Hif1a protein levels, not mRNA expression, are negatively regulated by testosterone in osteoclasts even under hypoxic conditions.
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Fig. 1. Hif1a protein accumulates in osteoclasts in ORX mice. (A) Bone sections from ORX- or sham-operated control mice were incubated with rabbit anti-Hif1a and mouse antibodies to the osteoclast marker Ctsk, followed by staining with Alexa488-conjugated goat anti-rabbit and Alexa546-conjugated goat anti-mouse antibodies plus the nuclear marker TOTO3 and observed under a fluorescence microscope. Arrows represent Ctsk/Hif1a double-positive cells. (B) Hypoxic areas in bone sections from ORX- or sham-operated mice were stained with anti-pimonidazole (Pimo). Nuclei are stained with TOTO3. Representative data of two independent experiments are shown.
Fig. 2. Hif1a is suppressed by testosterone at protein but not mRNA levels. (A and B) Cell lysates or total RNA were isolated from Raw264.7 cells cultured with or without 106 M testosterone in normoxic or hypoxic (either 5% or 1% O2) conditions, and western blot analysis for Hif1a and vinculin protein (A and C) or realtime PCR for Hif1a mRNA expression (B and D) was performed. In (A and C) vinculin serves as a loading control. Data in (B and D) represents mean Hif1a expression relative to Actb ± SD (NS: Not Significant; n ¼ 5). Representative data of at least three independent experiments are shown.
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3.3. Administration of a Hif1a-inhibitor protects bone from ORXinduced bone loss To determine the consequences of Hif1a protein accumulation in osteoclasts under testosterone-deficient conditions, ORX mice were administered the Hif1a inhibitor 2-methoxyestradiol (2ME2) and assessed for potential bone phenotypes (Fig. 3). Interestingly, the reduced bone mass normally detected by dual-energy X-ray absorptiometry (DEXA) in ORX mice was completely abrogated by 2ME2 treatment (Fig. 3A). Histological examination demonstrated that reduced trabecular bone mass and accelerated osteoclast formation following ORX demonstrated by toluidine blue staining and tartrate resistance acid phosphatase (TRAP), respectively, was blocked by 2ME2 administration (Fig. 3B and C). Based on DEXA analysis 2ME2 administration to sham-operated mice did not increase bone mass, suggesting that the effect of blocking Hif1a is specific to testosterone-deficient conditions (Fig. 3). Bone morphometric analysis also revealed that bone phenotypes induced by testosterone-deficiency in ORX mice were abrogated by 2ME2 treatment (Fig. 4). Reduced bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and increased trabecular separation (Tb.Sp), all of which
represent reduced bone mass, normally seen in ORX relative to sham-operated mice were abrogated in ORX mice administered 2ME2 (Fig. 4). Elevated proportions of eroded relative to bone surface seen in ORX mice was also abrogated by 2ME2 treatment (Fig. 4), suggesting that osteoclast activity accelerated by testosterone deficiency in ORX mice was blocked by administration of the Hif1a inhibitor. By contrast, DEXA analysis showed that 2ME2 administration to sham-operated control mice did not increase bone mass or inhibit osteoclast activities (Fig. 3). We conclude that Hif1a inhibition is specific in antagonizing pathological osteoclast activation and subsequent loss of bone mass in testosteronedeficient conditions. 4. Discussion To date, various drugs have been developed and launched to treat osteoporosis [13]. However, molecular mechanisms underlying osteoporosis in men are not well characterized [8,9]. Recently, using female mouse models, we reported that Hif1a is required for development of postmenopausal osteoporosis, and that administration of a Hif1a-inhibitor completely abrogated estrogen deficiency-induced osteoclast activation and bone loss in OVX mice
Fig. 3. Treatment of mice with a Hif1a inhibitor antagonizes ORX-induced bone loss. (A, B and C) ORX- or sham-operated mice were treated with the Hif1a inhibitor 2ME2 or with vehicle, and femur bone mineral density (BMD) was analyzed by DEXA (A), and the tibia was analyzed by toluidine blue (B) and TRAP (C) staining. Representative data of at least three independent experiments are shown. Bar ¼ 100 mm.
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Fig. 4. ORX-induced bone loss is abrogated by Hif1a inhibitor treatment. ORX- or sham-operated mice were treated with the Hif1a inhibitor 2ME2 or vehicle and then evaluated using bone morphometric analysis. Bone volume per tissue volume (BV/TV, %), trabecular thickness (Tb.Th, mm), trabecular number (Tb.N, mm), trabecular separation (Tb.Sp, mm), and eroded surface per bone surface (ES/BS, %) are shown. Data represents mean of indicated parameter ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, NS: Not Significant; n.
[11]. In the current study, we showed that Hif1a accumulates in osteoclasts in testosterone-deficient conditions in ORX mice, and that administration of a Hif1a-inhibitor abrogated osteoclast activation and bone loss in this model. Thus, Hif1a could serve as a common molecular target to treat either estrogen or testosterone deficiency-induced osteoporosis. Recent therapies to treat prostate and breast cancer have improved outcomes and increased lifespan for patients with these cancers: however, hormone-depleting therapies for such tumors frequently cause development of osteoporosis in patients [10,14e16]. Bone fragility fractures are occasionally seen in these patients [15e17], strongly suggesting that controlling bone homeostasis is crucial for the well-being of cancer patients treated with hormone-altering therapies. Our study may shed light on novel types of bone health management in male and female patients with both idiopathic aging- and hormone depletion-induced osteoporosis. Several studies have addressed the physiological role of estrogen in men [4,5,18e21]. Indeed, estrogen is synthesized via testosterone, and testosterone deficiency may result in estrogen loss. Estrogen has been described as a major regulator of bone metabolism not only in women but in men [4,5]. Furthermore, estradiol has been demonstrated to act as a more potent osteoclast inhibitor than testosterone [5,18,19], and, interestingly, bone loss in older men is reportedly associated with low estradiol levels [19e21]. Testosterone treatment has also been demonstrated effective in increasing bone mineral density (BMD) in men with low serum testosterone levels [22e24]. In the current study, we showed that Hif1a in osteoclasts is a target of testosterone. Nonetheless, treatment with either estrogen or testosterone is potentially associated with adverse outcomes, such as cardiovascular events [25,26]. Thus, identification of a target factor driving estrogen- or androgen-deficient bone loss is crucial to treat bone pathologies emerging from sex hormone deficiency. Our previous studies and this report support the idea that Hif1a is a potential therapeutic target for patients with either estrogen- or androgen-deficient osteoporosis [11]. Furthermore, vitamin D3 deficiency is reportedly a common risk factor for both male and female osteoporosis [27], and we recently found that Hif1a is also a target of a vitamin D3 analogue, ED71 [28]. In postmenopausal osteoporosis patients, osteoclast activity is known to be relatively higher than osteoblast activity. Recent advances in the understanding of osteoclast differentiation indicate that receptor activator of nuclear factor kappa B ligand (RANKL) is required for osteoclastogenesis [29,30]. Thus, bone resorptioninhibiting reagents such as bisphosphonates, osteoclast inhibitors, and denosumab, a RANKL neutralizing antibody, have been used to prevent bone fragility fractures [13,31]. However, strong inhibition of osteoclast activity beyond physiological levels reportedly promotes a condition known as severely suppressive bone turnover
(SSBT), leading to complications such as osteonecrosis of the jaws or atypical femoral fractures [32e35]. Indeed, administration of either the bisphosphonate alendronate or a RANKL-neutralizing antibody to normal mice significantly inhibits osteoclast activity and elevates bone mass [36]. However, here we found that administration of a Hif1a inhibitor to sham-operated normal mice promoted neither osteoclast inhibition nor elevated bone mass. This outcome is likely due to the fact that Hif1a protein is already suppressed by testosterone in normal conditions, and that treatment with a Hif1a inhibitor cannot therefore promote a further additive effect in inhibiting physiological osteoclast activity. Thus, Hif1a could serve as a therapeutic target specific for osteoclasts pathologically activated in osteoporosis under sex hormonedeficient conditions. Acknowledgments We thank Prof. M. Suematsu and Dr. Y.A. Minamishima for technical support in performing hypoxic culture. Y. Sato and T. Miyamoto were supported by a Grant-in-aid for Scientific Research, Japan, and Chugai Scientific foundation. The authors declare that they have no conflicts of interest with the contents of this article. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.01.033. References [1] N. Yoshimura, S. Muraki, H. Oka, A. Mabuchi, Y. En-Yo, M. Yoshida, A. Saika, H. Yoshida, T. Suzuki, S. Yamamoto, H. Ishibashi, H. Kawaguchi, K. Nakamura, T. Akune, Prevalence of knee osteoarthritis, lumbar spondylosis, and osteoporosis in Japanese men and women: the research on osteoarthritis/osteoporosis against disability study, J. Bone Min. Metab. 27 (2009) 620e628. [2] N. Yoshimura, S. Muraki, H. Oka, H. Kawaguchi, K. Nakamura, T. Akune, Cohort profile: research on osteoarthritis/osteoporosis against disability study, Int. J. Epidemiol. 39 (2010) 988e995. [3] E.S. Siris, Y.T. Chen, T.A. Abbott, E. Barrett-Connor, P.D. Miller, L.E. Wehren, M.L. Berger, Bone mineral density thresholds for pharmacological intervention to prevent fractures, Arch. Intern Med. 164 (2004) 1108e1112. [4] S. Khosla, L.J. Melton 3rd, B.L. Riggs, The unitary model for estrogen deficiency and the pathogenesis of osteoporosis: is a revision needed? J. Bone Min. Res. 26 (2011) 441e451. [5] S. Khosla, M.J. Oursler, D.G. Monroe, Estrogen and the skeleton, Trends Endocrinol. Metab. 23 (2012) 576e581. [6] D. Bliuc, N.D. Nguyen, V.E. Milch, T.V. Nguyen, J.A. Eisman, J.R. Center, Mortality risk associated with low-trauma osteoporotic fracture and subsequent fracture in men and women, JAMA. 301 (2009) 513e521. n-Emeric, D. Vanderschueren, K. Milisen, [7] P. Haentjens, J. Magaziner, C.S. Colo B. Velkeniers, S. Boonen, Meta-analysis: excess mortality after hip fracture among older women and men, Ann. Intern Med. 152 (2010) 380e390. [8] I.S. Stathopoulos, E.G. Ballas, K. Lampropoulou-Adamidou, G. Trovas, Osteoporosis in men, Hormones (Athens) 13 (2014) 441e457. [9] J.S. Walsh, R. Eastell, Osteoporosis in men, Nat. Rev. Endocrinol. 9 (2013) 637e645. [10] A.C. Lassemillante, S.A. Doi, J.D. Hooper, J.B. Prins, O.R. Wright, Prevalence of
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Hif1a is required for osteoclast activation and bone loss in male osteoporosis Toshimi Tando a, 1, Yuiko Sato a, b, 1, Kana Miyamoto a, 1, Mayu Morita c, 1, Tami Kobayashi a, Atsushi Funayama a, Arihiko Kanaji a, Wu Hao a, Ryuichi Watanabe a, Takatsugu Oike a, Masaya Nakamura a, Morio Matsumoto a, Yoshiaki Toyama a, Takeshi Miyamoto a, * a b c
Department of Orthopedic Surgery, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan Department of Musculoskeletal Reconstruction and Regeneration Surgery, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan Department of Dentistry and Oral Surgery, Keio University School of Medicine, 35 Shinano-machi, Shinjuku-ku, Tokyo 160-8582, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 31 December 2015 Accepted 6 January 2016 Available online 11 January 2016
The number of osteoporosis patients is increasing not only in women but in men. Male osteoporosis occurs due to aging or androgen depletion therapies, leading to fractures. However, molecular mechanisms underlying male osteoporosis remain unidentified. Here, we show that hypoxia inducible factor 1 alpha (Hif1a) is required for development of testosterone deficiency-induced male osteoporosis. We found that in mice Hif1a protein accumulates in osteoclasts following orchidectomy (ORX) in vivo. In vitro, Hif1a protein accumulated in osteoclasts cultured in hypoxic conditions, but Hif1a protein rather than mRNA levels were suppressed by testosterone treatment, even in hypoxia. Administration of a Hif1a inhibitor to ORX mice abrogated testosterone deficiency-induced osteoclast activation and bone loss but did not alter osteoclast activities or bone phenotypes in sham-operated, testosterone-sufficient animals. We conclude that Hif1a protein accumulation due to testosterone-deficiency promotes development of male osteoporosis. Thus Hif1a protein could be targeted to inhibit pathologically-activated osteoclasts under testosterone-deficient conditions to treat male osteoporosis patients. © 2016 Elsevier Inc. All rights reserved.
Keywords: Testosterone Osteoporosis Hif1a Male Osteoclasts Bone
1. Introduction The size of the aging population in developed countries is growing, increasing the number of male and female osteoporosis patients [1,2]. Osteoporosis frequently causes bone fragility fractures [3], a condition reportedly associated with increased mortality risk for both men and women. Although osteoporosis is more common in women than in men, particularly in postmenopausal elderly women [1,4,5], the risk of mortality following fragility fracture is reportedly greater in men with the condition [6,7], suggesting that preventing osteoporosis-associated fractures is crucial for the health of elderly men. Osteoporosis in men is promoted by factors such as aging or hypogonadism [8,9]. Testosterone deficiency resulting from androgen depletion therapy in prostate cancer also reportedly promotes osteoporosis development [10].
* Corresponding author. E-mail address: [email protected] (T. Miyamoto). 1 T. Tando, Y. Sato, K. Miyamoto and M. Morita contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2016.01.033 0006-291X/© 2016 Elsevier Inc. All rights reserved.
However, pathogenesis of male osteoporosis development remains to be defined [8,9]. Recently, we proposed that hypoxia inducible factor 1 alpha (Hif1a), a hypoxia-responsive transcription factor, was required for development of estrogen-deficient postmenopausal osteoporosis [11]. Bone surfaces where bone-resorbing osteoclasts reside are reportedly hypoxic [12]; however, we found that Hif1a expressed in those cells was suppressed by estrogen in estrogen-sufficient, premenopausal conditions [11]. We also showed that estrogendeficient conditions in female mice caused by bilateral ovariectomy (OVX) promoted Hif1a protein accumulation in osteoclasts, and that administration of a Hif1a inhibitor blocked osteoporosis development normally seen in OVX mice [11]. However, the role of Hif1a in male osteoporosis development remained unexamined. Here, we show that Hif1a protein accumulates in osteoclasts in testosterone-deficient conditions established by ORX in male mice in vivo. Our in vitro studies showed that Hif1a protein accumulation in hypoxic conditions in osteoclasts was suppressed by testosterone treatment, although Hif1a mRNA levels were unchanged. ORX mice treated with a Hif1a inhibitor showed significantly abrogated
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osteoclast activation and bone loss, but comparable treatment did not alter bone status in sham-operated, testosterone-sufficient mice. Thus, Hif1a protein could be targeted to block pathological osteoclast activation as a treatment for male osteoporosis, particularly in conditions of testosterone depletion. 2. Materials and methods 2.1. Mice Wild type C57BL/6 mice were purchased from CLEA Japan (Meguro, Tokyo, Japan) and maintained under specific pathogenfree conditions in animal facilities certified by Keio University School of Medicine animal care committee. All experimental procedures involving mice were approved by the Keio University School of Medicine animal care committee. Twelve-week-old male mice were subjected to ORX or sham surgery, followed in some cases by 2ME2 (EMD Millipore Corporation) treatment. 2ME2 was prepared at 15 mg/mL in a 0.5% methylcellulose solution (Wako Pure Chemicals Industries) and administered daily at 75 mg/kg via intragastric infusion for 4 weeks. Controls were treated with vehicle only. At the end of drug administration, ORX or sham-operated mice treated with 2ME2 or vehicle were necropsied, and hindlimb bones were collected, fixed with 70% ethanol, and subjected to dualenergy X-ray absorptiometry (DEXA) analysis to measure bone mineral density (BMD) and subjected to bone histomorphometric analysis. 2.2. Immunohistochemical analysis Femurs were dissected from ORX or sham-operated mice, fixed in 10% buffered formalin, decalcified in 8% Na2EDTA, paraffinembedded, sectioned, and subjected to immunohistochemistry. Antigen was retrieved by treatment of tissue sections on slides with 0.4 mg/mL Proteinase K for 8 min at room temperature. Sections were incubated in 5% BSAePBS and then incubated with antibodies against HIF1a (#NB100-479, Novus Biologicals, Littleton, CO, USA) and Cathepsin K (Ctsk, #F-95, Daiichi Finechemical Co. Toyama, Japan). Nuclei were stained by TOTO-3 (#T3604, Invitrogen Corp., Carlsbad, CA, USA). Areas of oxygen tension in tissue were detected using pimonidazole (Hypoxyprobe-1 Kit, Hypoxyprobe Inc., Burlington, MA, USA) according to the manufacturer's instructions. Stained specimens were observed under a confocal laser scanning microscope (FV1000, OLYMPUS Corp., Tokyo, Japan). 2.3. Cell culture Raw264.7 cells were maintained in DMEM (SigmaeAldrich Co., St. Louis, MO, USA) containing 10% heat-inactivated fetal bovine serum (FBS, JRH Biosciences Lenexa, KS, USA) and GlutaMax (Invitrogen Corp., Carlsbad, CA, USA). Cells were cultured in normoxic or hypoxic conditions. In normoxic conditions, cells were gently shaken in the incubator. Hypoxic culture was performed at either 1% or 5% O2 and 5% CO2 using an INVIVO2 hypoxia workstation (Ruskin Technology Ltd., Bridgend, UK) in the presence of RANKL (PeproTech Ltd., Rocky Hill, NJ, USA) with or without 106 M testosterone (Tes; Wako Pure Chemicals Industries, Osaka, Japan), and cells were lysed in the hypoxic chamber. 2.4. Quantitative PCR analysis Total RNA was isolated from cultured cells using an RNeasy mini kit (Qiagen, Venlo, Limburg, The Netherlands), and cDNA synthesis was performed using oligo (dT) primers and reverse transcriptase
(Takara Bio Inc., Shiga, Japan). Quantitative PCR was done using SYBR Premix ExTaq II reagent and a DICE Thermal cycler (Takara Bio Inc., Shiga, Japan), according to the manufacturer's instructions. bactin (Actb) expression served as an internal control. Primers used for realtime PCR were: Hif1a-forward: 50 - AAAGCTTCTGTTATGAGGCTCA-30 Hif1a-reverse: 50 - CAGTCCATCTGTGCCTTCATCT-30 Actb-forward: 50 -TGAGAGGGAAATCGTGCGTGAC-30 Actb-reverse: 50 -AAGAAGGAAGGCTGGAAAAGAG-30
2.5. Immunoblotting Whole cell lysates were prepared using RIPA buffer (1% Triton X100, 1% sodium deoxycholate, 0.1% SDS, 150 mM NaCl, 5 mM EDTA, 1 mM dithiothreitol, 10 mM TriseHCl, pH7.5) supplemented with a protease inhibitor cocktail (SigmaeAldrich Co.) and MG-132 (EMD Millipore Corporation). The insoluble fraction was removed by centrifugation and then lysate protein content was measured using the Bradford reagent (Bio-Rad Laboratories Inc., Tokyo, Japan). Equivalent amounts of protein were separated by SDS-PAGE, transferred to a PVDF membrane (EMD Millipore Corporation) and immunoblotted with anti-HIF1a (#NB100-479, Novus Biologicals) or anti-Vinculin (#V9131, SigmaeAldrich Co.) antibodies. 2.6. Statistical analyses Statistical analyses were performed using the unpaired twotailed Student's t-test (*P < 0.05; **P < 0.01; ***P < 0.005; NS, not significant, throughout the paper). All data are expressed as the mean ± SD. 3. Results 3.1. Testosterone depletion stabilizes Hif1a protein in osteoclasts in vivo Recently, we showed that Hif1a protein was stabilized in osteoclasts in estrogen-deficient OVX female mice in vivo [11]. Thus, we initially examined expression of Hif1a protein in osteoclasts from testosterone-depleted ORX male mice. We could not detect Hif1a protein in osteoclasts of sham-operated control mice but did observe Hif1a protein accumulation in osteoclasts from ORX mice, based on staining with the osteoclast marker cathepsin K (Ctsk) (Fig. 1A). Notably, the hypoxic state of bone tissue was unchanged by ORX, as determined by staining with the hypoxia probe pimonidazole (Pimo) (Fig. 1B). These results suggest that Hif1a protein in osteoclasts is regulated by testosterone without a change in oxygen tension in vivo. 3.2. Hif1a protein but not mRNA is suppressed in osteoclasts by testosterone treatment in vitro We next analyzed direct effects of testosterone on Hif1a expression in cultured pre-osteoclast Raw264.7 cells in vitro (Fig. 2). Hif1a protein, as detected by western blot, increased following culture of untreated cells in hypoxic conditions, but Hif1a protein levels were suppressed by testosterone in hypoxic conditions at either 5% or 1% O2 (Fig. 2A and C). This decrease was not due to decreased Hif1a mRNA expression (Fig. 2B and D). These results suggest that Hif1a protein levels, not mRNA expression, are negatively regulated by testosterone in osteoclasts even under hypoxic conditions.
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Fig. 1. Hif1a protein accumulates in osteoclasts in ORX mice. (A) Bone sections from ORX- or sham-operated control mice were incubated with rabbit anti-Hif1a and mouse antibodies to the osteoclast marker Ctsk, followed by staining with Alexa488-conjugated goat anti-rabbit and Alexa546-conjugated goat anti-mouse antibodies plus the nuclear marker TOTO3 and observed under a fluorescence microscope. Arrows represent Ctsk/Hif1a double-positive cells. (B) Hypoxic areas in bone sections from ORX- or sham-operated mice were stained with anti-pimonidazole (Pimo). Nuclei are stained with TOTO3. Representative data of two independent experiments are shown.
Fig. 2. Hif1a is suppressed by testosterone at protein but not mRNA levels. (A and B) Cell lysates or total RNA were isolated from Raw264.7 cells cultured with or without 106 M testosterone in normoxic or hypoxic (either 5% or 1% O2) conditions, and western blot analysis for Hif1a and vinculin protein (A and C) or realtime PCR for Hif1a mRNA expression (B and D) was performed. In (A and C) vinculin serves as a loading control. Data in (B and D) represents mean Hif1a expression relative to Actb ± SD (NS: Not Significant; n ¼ 5). Representative data of at least three independent experiments are shown.
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3.3. Administration of a Hif1a-inhibitor protects bone from ORXinduced bone loss To determine the consequences of Hif1a protein accumulation in osteoclasts under testosterone-deficient conditions, ORX mice were administered the Hif1a inhibitor 2-methoxyestradiol (2ME2) and assessed for potential bone phenotypes (Fig. 3). Interestingly, the reduced bone mass normally detected by dual-energy X-ray absorptiometry (DEXA) in ORX mice was completely abrogated by 2ME2 treatment (Fig. 3A). Histological examination demonstrated that reduced trabecular bone mass and accelerated osteoclast formation following ORX demonstrated by toluidine blue staining and tartrate resistance acid phosphatase (TRAP), respectively, was blocked by 2ME2 administration (Fig. 3B and C). Based on DEXA analysis 2ME2 administration to sham-operated mice did not increase bone mass, suggesting that the effect of blocking Hif1a is specific to testosterone-deficient conditions (Fig. 3). Bone morphometric analysis also revealed that bone phenotypes induced by testosterone-deficiency in ORX mice were abrogated by 2ME2 treatment (Fig. 4). Reduced bone volume per tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and increased trabecular separation (Tb.Sp), all of which
represent reduced bone mass, normally seen in ORX relative to sham-operated mice were abrogated in ORX mice administered 2ME2 (Fig. 4). Elevated proportions of eroded relative to bone surface seen in ORX mice was also abrogated by 2ME2 treatment (Fig. 4), suggesting that osteoclast activity accelerated by testosterone deficiency in ORX mice was blocked by administration of the Hif1a inhibitor. By contrast, DEXA analysis showed that 2ME2 administration to sham-operated control mice did not increase bone mass or inhibit osteoclast activities (Fig. 3). We conclude that Hif1a inhibition is specific in antagonizing pathological osteoclast activation and subsequent loss of bone mass in testosteronedeficient conditions. 4. Discussion To date, various drugs have been developed and launched to treat osteoporosis [13]. However, molecular mechanisms underlying osteoporosis in men are not well characterized [8,9]. Recently, using female mouse models, we reported that Hif1a is required for development of postmenopausal osteoporosis, and that administration of a Hif1a-inhibitor completely abrogated estrogen deficiency-induced osteoclast activation and bone loss in OVX mice
Fig. 3. Treatment of mice with a Hif1a inhibitor antagonizes ORX-induced bone loss. (A, B and C) ORX- or sham-operated mice were treated with the Hif1a inhibitor 2ME2 or with vehicle, and femur bone mineral density (BMD) was analyzed by DEXA (A), and the tibia was analyzed by toluidine blue (B) and TRAP (C) staining. Representative data of at least three independent experiments are shown. Bar ¼ 100 mm.
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Fig. 4. ORX-induced bone loss is abrogated by Hif1a inhibitor treatment. ORX- or sham-operated mice were treated with the Hif1a inhibitor 2ME2 or vehicle and then evaluated using bone morphometric analysis. Bone volume per tissue volume (BV/TV, %), trabecular thickness (Tb.Th, mm), trabecular number (Tb.N, mm), trabecular separation (Tb.Sp, mm), and eroded surface per bone surface (ES/BS, %) are shown. Data represents mean of indicated parameter ± SD (*P < 0.05, **P < 0.01, ***P < 0.001, NS: Not Significant; n.
[11]. In the current study, we showed that Hif1a accumulates in osteoclasts in testosterone-deficient conditions in ORX mice, and that administration of a Hif1a-inhibitor abrogated osteoclast activation and bone loss in this model. Thus, Hif1a could serve as a common molecular target to treat either estrogen or testosterone deficiency-induced osteoporosis. Recent therapies to treat prostate and breast cancer have improved outcomes and increased lifespan for patients with these cancers: however, hormone-depleting therapies for such tumors frequently cause development of osteoporosis in patients [10,14e16]. Bone fragility fractures are occasionally seen in these patients [15e17], strongly suggesting that controlling bone homeostasis is crucial for the well-being of cancer patients treated with hormone-altering therapies. Our study may shed light on novel types of bone health management in male and female patients with both idiopathic aging- and hormone depletion-induced osteoporosis. Several studies have addressed the physiological role of estrogen in men [4,5,18e21]. Indeed, estrogen is synthesized via testosterone, and testosterone deficiency may result in estrogen loss. Estrogen has been described as a major regulator of bone metabolism not only in women but in men [4,5]. Furthermore, estradiol has been demonstrated to act as a more potent osteoclast inhibitor than testosterone [5,18,19], and, interestingly, bone loss in older men is reportedly associated with low estradiol levels [19e21]. Testosterone treatment has also been demonstrated effective in increasing bone mineral density (BMD) in men with low serum testosterone levels [22e24]. In the current study, we showed that Hif1a in osteoclasts is a target of testosterone. Nonetheless, treatment with either estrogen or testosterone is potentially associated with adverse outcomes, such as cardiovascular events [25,26]. Thus, identification of a target factor driving estrogen- or androgen-deficient bone loss is crucial to treat bone pathologies emerging from sex hormone deficiency. Our previous studies and this report support the idea that Hif1a is a potential therapeutic target for patients with either estrogen- or androgen-deficient osteoporosis [11]. Furthermore, vitamin D3 deficiency is reportedly a common risk factor for both male and female osteoporosis [27], and we recently found that Hif1a is also a target of a vitamin D3 analogue, ED71 [28]. In postmenopausal osteoporosis patients, osteoclast activity is known to be relatively higher than osteoblast activity. Recent advances in the understanding of osteoclast differentiation indicate that receptor activator of nuclear factor kappa B ligand (RANKL) is required for osteoclastogenesis [29,30]. Thus, bone resorptioninhibiting reagents such as bisphosphonates, osteoclast inhibitors, and denosumab, a RANKL neutralizing antibody, have been used to prevent bone fragility fractures [13,31]. However, strong inhibition of osteoclast activity beyond physiological levels reportedly promotes a condition known as severely suppressive bone turnover
(SSBT), leading to complications such as osteonecrosis of the jaws or atypical femoral fractures [32e35]. Indeed, administration of either the bisphosphonate alendronate or a RANKL-neutralizing antibody to normal mice significantly inhibits osteoclast activity and elevates bone mass [36]. However, here we found that administration of a Hif1a inhibitor to sham-operated normal mice promoted neither osteoclast inhibition nor elevated bone mass. This outcome is likely due to the fact that Hif1a protein is already suppressed by testosterone in normal conditions, and that treatment with a Hif1a inhibitor cannot therefore promote a further additive effect in inhibiting physiological osteoclast activity. Thus, Hif1a could serve as a therapeutic target specific for osteoclasts pathologically activated in osteoporosis under sex hormonedeficient conditions. Acknowledgments We thank Prof. M. Suematsu and Dr. Y.A. Minamishima for technical support in performing hypoxic culture. Y. Sato and T. Miyamoto were supported by a Grant-in-aid for Scientific Research, Japan, and Chugai Scientific foundation. The authors declare that they have no conflicts of interest with the contents of this article. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2016.01.033. References [1] N. Yoshimura, S. Muraki, H. Oka, A. Mabuchi, Y. En-Yo, M. Yoshida, A. Saika, H. Yoshida, T. Suzuki, S. Yamamoto, H. Ishibashi, H. Kawaguchi, K. Nakamura, T. Akune, Prevalence of knee osteoarthritis, lumbar spondylosis, and osteoporosis in Japanese men and women: the research on osteoarthritis/osteoporosis against disability study, J. Bone Min. Metab. 27 (2009) 620e628. [2] N. Yoshimura, S. Muraki, H. Oka, H. Kawaguchi, K. Nakamura, T. Akune, Cohort profile: research on osteoarthritis/osteoporosis against disability study, Int. J. Epidemiol. 39 (2010) 988e995. [3] E.S. Siris, Y.T. Chen, T.A. Abbott, E. Barrett-Connor, P.D. Miller, L.E. Wehren, M.L. Berger, Bone mineral density thresholds for pharmacological intervention to prevent fractures, Arch. Intern Med. 164 (2004) 1108e1112. [4] S. Khosla, L.J. Melton 3rd, B.L. Riggs, The unitary model for estrogen deficiency and the pathogenesis of osteoporosis: is a revision needed? J. Bone Min. Res. 26 (2011) 441e451. [5] S. Khosla, M.J. Oursler, D.G. Monroe, Estrogen and the skeleton, Trends Endocrinol. Metab. 23 (2012) 576e581. [6] D. Bliuc, N.D. Nguyen, V.E. Milch, T.V. Nguyen, J.A. Eisman, J.R. Center, Mortality risk associated with low-trauma osteoporotic fracture and subsequent fracture in men and women, JAMA. 301 (2009) 513e521. n-Emeric, D. Vanderschueren, K. Milisen, [7] P. Haentjens, J. Magaziner, C.S. Colo B. Velkeniers, S. Boonen, Meta-analysis: excess mortality after hip fracture among older women and men, Ann. Intern Med. 152 (2010) 380e390. [8] I.S. Stathopoulos, E.G. Ballas, K. Lampropoulou-Adamidou, G. Trovas, Osteoporosis in men, Hormones (Athens) 13 (2014) 441e457. [9] J.S. Walsh, R. Eastell, Osteoporosis in men, Nat. Rev. Endocrinol. 9 (2013) 637e645. [10] A.C. Lassemillante, S.A. Doi, J.D. Hooper, J.B. Prins, O.R. Wright, Prevalence of
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