The Vitamin D Analogue ED71 but Not 1,25(OH)2D3 Targets HIF1α Protein in Osteoclasts

25(OH)2D3 Targets HIF1a Protein in Osteoclasts. PLoS ONE 9(11): e111845. doi:10.1371/journal.pone.0111845 The Vitamin D Analogue ED71 but Not 1,25(OH)2D3 Targets HIF1a Protein in Osteoclasts Yuiko Sato 0 Yoshiteru Miyauchi 0 Shigeyuki Yoshida 0 Mayu Morita 0 Tami Kobayashi 0 Hiroya Kanagawa 0 Eri Katsuyama 0 Atsuhiro Fujie 0 Wu Hao 0 Toshimi Tando 0 Ryuichi Watanabe 0 Kana Miyamoto 0 Hideo Morioka 0 Morio Matsumoto 0 Yoshiaki Toyama 0 Takeshi Miyamoto 0 Brenda Smith, Oklahoma State University, United States of America 0 1 Department of Orthopedic Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan, 2 Department of Musculoskeletal Reconstruction and Regeneration Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan, 3 Department of Dentistry and Oral Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan, 4 Department of Integrated Bone Metabolism and Immunology, Keio University School of Medicine , Shinjuku-ku, Tokyo , Japan Although both an active form of the vitamin D metabolite, 1,25(OH)2D3, and the vitamin D analogue, ED71 have been used to treat osteoporosis, anti-bone resorbing activity is reportedly seen only in ED71- but not in 1,25(OH)2D3 -treated patients. In addition, how ED71 inhibits osteoclast activity in patients has not been fully characterized. Recently, HIF1a expression in osteoclasts was demonstrated to be required for development of post-menopausal osteoporosis. Here we show that ED71 but not 1,25(OH)2D3, suppress HIF1a protein expression in osteoclasts in vitro. We found that 1,25(OH)2D3 or ED71 function in osteoclasts requires the vitamin D receptor (VDR). ED71 was significantly less effective in inhibiting M-CSF and RANKLstimulated osteoclastogenesis than was 1,25(OH)2D3 in vitro. Downregulation of c-Fos protein and induction of Ifnb mRNA in osteoclasts, both of which reportedly block osteoclastogenesis induced by 1,25(OH)2D3 in vitro, were both significantly higher following treatment with 1,25(OH)2D3 than with ED71. Thus, suppression of HIF1a protein activity in osteoclasts in vitro, which is more efficiently achieved by ED71 rather than by 1,25(OH)2D3, could be a reliable read-out in either developing or screening reagents targeting osteoporosis. - . These authors contributed equally to this work. A cause for concern in developed countries is the increasing number of osteoporosis patients and individuals suffering fragility fractures due to osteoporosis [1]. Estrogen-deficiency due to menopause is a risk factor for both [2]. Vitamin D insufficiency is also reportedly observed in osteoporosis patients with fragility fractures and considered a cause of osteoporotic fractures [3]. Indeed, vitamin D is known to play a crucial role in skeletal development, and lack of the vitamin D receptor (VDR) or low vitamin D intake results in Rickets [4] [5]. Currently, active vitamin D analogues are used in several countries to treat patients with bone and mineral disorders associated with chronic renal disease or osteoporosis [6]. Interestingly, 1,25(OH)2D3 has been demonstrated to promote osteoclastogenesis in co-cultures of osteoclast progenitor cells and osteoblastic cells [7]; in addition, 1,25(OH)2D3 elevated receptor activator of nuclear factor kappa B ligand (RANKL), an essential cytokine for osteoclastogenesis, but inhibited expression of OPG, a decoy receptor of RANKL, in osteoblastic cells to promote osteoclast differentiation [8] [9]. In contrast, 1,25(OH)2D3 was shown to inhibit osteoclast differentiation in osteoblastic cell-free culture systems: osteoclast formation induced by macrophage colony stimulating factor (M-CSF) and RANKL was inhibited in the presence of 1,25(OH)2D3 [10] [11]. c-Fos protein, an essential transcription factor for osteoclast differentiation, or interferon beta (Ifnb), an inhibitor of osteoclastogenesis, was downregulated or elevated by 1,25(OH)2D3, respectively, in osteoclast progenitor cells [10] [11]. However, patients treated with a 1,25(OH)2D3 prodrug, alfacalcidol, did not show inhibition of osteoclastic activity or increased bone mass, while patients treated with the vitamin D analogue ED71 exhibited significantly reduced osteoclast activities and increased bone mass [12]. Since postmenopausal osteoporosis is caused in part by estrogen-deficiency, treating of patients with estrogen is one option. However, continuous estrogen administration is associated with adverse effects such as uterine or mammary gland tumors or cardio-vascular disease [13]. Recently, we reported that hypoxia inducible factor 1 alpha (HIF1a) is required for osteoclast activation following estrogen-deficiency and for development of postmenopausal osteoporosis in animal models [14]. We found that in pre-menopausal mice, HIF1a activity in osteoclasts is continuously suppressed by estrogen but then HIF1a accumulate in osteoclasts following estrogen deficiency due to menopause, which in turn activates osteoclastic activity and promotes bone loss. Osteoclast specific HIF1a knockout or administration of a HIF1a inhibitor completely abrogated ovariectomy (OVX)induced osteoclast activation and bone loss [14]. This study suggests that HIF1a could be a therapeutic target for postmenopausal osteoporosis. Here, we show that HIF1a is a target of ED71 in vitro. HIF1a in osteoclasts was suppressed by ED71 but not by 1,25(OH)2D3. Since inhibition of osteoclast activity was seen in the patients treated with ED71 but not with 1,25(OH)2D3, this work confirms that HIF1a could be a target to treat postmenopausal osteoporosis patients. Materials and Methods Mice C57BL/6 background wild-type mice were purchased from Sankyo Labo Service (Tokyo, Japan). VDR-deficient mice were established previously [4]. Animals were maintained under specific pathogen-free conditions in animal facilities certified by the Keio University School of Medicine animal care committee. All animal procedures were approved by the Keio University School of Medicine animal care committee. Cell culture To assess in vitro osteoclast formation, bone marrow cells isolated from Hifflox/flox or Ctsk Cre/Hifflox/flox mouse femurs and tibias were cultured for 72 hours in aMEM (Sigma-Aldrich 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) supplemented with MCSF (50 ng/ml, Kyowa Hakko Kirin Co. Tokyo, Japan). Subsequently, adherent cells were collected and cultured under indicated conditions containing M-CSF (50 ng/ml), recombinant soluble RANKL (25 ng/ml, PeproTech Ltd., Rocky Hill, NJ, USA) using 16105 cells per well in 96-well plates. Osteoclastogenesis was evaluated by TRAP staining [15] [16]. Raw264.7 cells were maintained in DMEM (Sigma-Aldrich Co.) containing 10% heat-inactivated FBS (JRH Biosciences) and GlutaMax (Invitrogen Figure 1. 1,25(OH)2D3 is a more potent inhibitor of osteoclastogenesis in vitro than is ED71. (...truncated)