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

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, HIF1α 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 HIF1α 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 RANKL-stimulated osteoclastogenesis than was 1,25(OH)2D3 in vitro. Downregulation of c-Fos protein and induction of Ifnβ 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 HIF1α 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.


Introduction
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) 2 D 3 has been demonstrated to promote osteoclastogenesis in co-cultures of osteoclast progenitor cells and osteoblastic cells [7]; in addition, 1,25(OH) 2 D 3 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) 2 D 3 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) 2 D 3 [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) 2 D 3 , respectively, in osteoclast progenitor cells [10] [11]. However, patients treated with a 1,25(OH) 2 D 3 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) 2 D 3 . Since inhibition of osteoclast activity was seen in the patients treated with ED71 but not with 1,25(OH) 2 D 3 , 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.

Quantitative PCR analysis
Total RNA was isolated from bone marrow cultures using an RNeasy mini kit (Qiagen), and cDNA synthesis was done by using oligo (dT) primers and reverse transcriptase (Wako Pure Chem-icals Industries). Quantitative PCR was performed using SYBR Premix ExTaq II reagent and a DICE Thermal cycler (Takara Bio Inc., Shiga, Japan), according to the manufacturer's instructions. b-actin (Actb) expression served as an internal control. Primers for Nfatc1, Ctsk, DC-STAMP, Ifnb and Actb were as follows.

VDR knockdown
Raw264.7 cells transduced with MISSION shRNA lentiviruses targeting the VDR or with lentiviruses harboring non-target control constructs (Sigma-Aldrich Co.) were generated according to the manufacturer's instructions.

Statistical analyses
Statistical analyses were performed using an unpaired two-tailed 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 6 SD.

1,25(OH) 2 D 3 inhibits osteoclastogenesis more potently than does ED71 in vitro
Since treatment with ED71, a vitamin D3 analogue, inhibits osteoclast activity and increases bone mineral density more effectively than does the pro-1,25(OH) 2 D 3 agent, alfacalcidol [12], we asked whether ED71 inhibited osteoclastogenesis more effectively than 1,25(OH) 2 D 3 (1,25D) in vitro (Fig. 1). To do so, we isolated osteoclast progenitor cells from wild-type mice and cultured them in the presence of M-CSF and RANKL with or without ED71 or 1,25(OH) 2 D 3 . We then evaluated osteoclastogenesis by counting multi-nuclear TRAP-positive osteoclasts and examining expression of osteoclastic genes (Fig. 1A-D). Indeed ED71 significantly inhibited osteoclast differentiation based on both TRAP and gene expression analysis, while 1,25(OH) 2 D 3 was more effective in inhibiting osteoclastogenesis than was ED71 in vitro ( Fig. 1A and B). Expression of osteoclast differentiation markers such as Cathepsin K (Ctsk), nuclear factor of activated T cells 1 (NFATc1) and dendritic cell specific transmembrane protein (DC-STAMP) was more significantly inhibited by 1,25(OH) 2 D 3 than by ED71 treatment (Fig. 1C). Induction of B lymphocyteinduced maturation protein 1 (Blimp1) followed by suppression of B cell lymphoma 6 and interferon regulatory factor 8 (Irf8) is reportedly required for osteoclastogenesis [14,17,18]. We found that treatment of osteoclast progenitors with 1,25(OH) 2 D 3 elicited more robust inhibition of Blimp1 and activation of Bcl6 and Irf8 than did treatment with ED71 (Fig. 1D), suggesting that 1,25(OH) 2 D 3 is more potent in inhibiting osteoclastogenesis induced by M-CSF and RANKL than ED71.
The VDR is required for both 1,25(OH) 2 D 3 and ED71 activity on osteoclasts  HIF1a Protein as a Target of Osteoporosis Therapy PLOS ONE | www.plosone.org both compounds act on osteoclasts via the VDR (Fig. 3). Osteoclast progenitors were isolated from wild-type and VDRdeficient mice and cultured in the presence of M-CSF and RANKL with or without 1,25(OH) 2 D 3 or ED71 (Fig. 3). Inhibitory effects of either 1,25(OH) 2 D 3 or ED71 on osteoclast differentiation were not seen in VDR-deficient osteoclasts ( Fig. 3A  and B). Similarly, inhibition of the expression of osteoclastic genes Ctsk, NFATc1 and DC-STAMP seen following 1,25(OH) 2 D 3 or ED71 treatment was absent in osteoclasts lacking the VDR (Fig. 3C).
Moreover, decreased c-Fos protein and elevated Ifnb expression seen following treatment with 1,25(OH) 2 D 3 or ED71 were abrogated in VDR-deficient osteoclasts (Fig. 4A and B), supporting the idea that both compounds act on osteoclasts via the VDR.

HIF1a is a target of ED71 but not 1,25(OH) 2 D 3 in osteoclasts
Next, we asked whether HIF1a is a target of ED71 in osteoclasts (Fig. 5). Interestingly, we found that in cultured osteoclasts, HIF1a protein levels were suppressed by ED71 but not by 1,25(OH) 2 D 3 (Fig. 5A). In contrast, Hif1a mRNA expression in osteoclasts was not inhibited by either treatment (Fig. 5B), suggesting that ED71 suppresses HIF1a at the protein level as demonstrated by estrogen treatment [14]. To determine if the VDR is required for ED71mediated HIF1a protein suppression in osteoclasts, we generated two independent VDR knockdown Raw264.7 lines using shVDR#1 and shVDR#2 as well as a control (shControl) line (Fig. 5C) and then treated cells with ED71 or 1,25(OH) 2 D 3 (Fig. 5D). HIF1a protein suppression by ED71 seen in control cells was abrogated in both VDR knockdown lines, suggesting that HIF1a protein suppression by ED71 is VDR-dependent. We then isolated osteoclast progenitors from Ctsk Cre/Hif1a flox/flox mice, cultured them in normoxic conditions to suppress HIF1a protein, and treated cells with or without ED71 or 1,25(OH) 2 D 3 (Fig. 5E). ED71 treatment effectively inhibited osteoclast differentiation, even in HIF1a-suppressed cells, suggesting that ED71 likely targets factors other than HIF1a protein in osteoclasts (Fig. 5E). However, ED71 was less effective than 1,25(OH) 2 D 3 in inhibiting osteoclastogenesis in HIF1a-suppressed cells (Fig. 5E).

Discussion
Postmenopausal osteoporosis treatment is required to prevent disruption of daily activity or adverse outcomes due to fragile fractures. Among anti-osteoporosis agents, anti-bone resorptive or bone-forming agents include bisphosphonates, selective estrogen receptor modulator (SERM), ED71 and denosumab, or teripararide, respectively. Strong inhibition of osteoclastic activity beyond physiological levels by bisphosphonates frequently causes adverse effects such as osteonecrosis of the jaw or severely suppressed bone turnover (SSBT) [19] [20]. Meanwhile, teriparatide treatment is limited to less than two years in order to prevent development of tumors, particularly osteosarcoma.
Recently, we showed that HIF1a protein accumulation in osteoclasts following estrogen-deficiency was accompanied by osteoclast activation and bone loss in mice [14]. Either osteoclast-specific HIF1a conditional knockout or wild-type mice administered a HIF1a inhibitor were protected from HIF1a Protein as a Target of Osteoporosis Therapy PLOS ONE | www.plosone.org OVX-induced osteoclast activation and bone loss. Moreover, HIF1a inhibition did not interfere with physiological osteoclast activities [14]. Thus, blocking HIF1a pharmacologically could represent an ideal treatment for postmenopausal osteoporosis, as it could target pathologically-activated osteoclasts without altering physiological osteoclastogenesis required for bone turnover. In this study, we found that both ED71, which is used as therapeutic agents for postmenopausal osteoporosis therapy, inhibits HIF1a protein expression. Indeed, patients treated with ED71 exhibit reduced osteoclastic activity and increased bone mass without adverse effects such as osteopetrosis [12], jaw osteonecrosis or SSBT, as seen in treated bisphosphonate-treated patients [19] [20].
Bone is a target tissue of vitamin D, and indeed, VDR was identified in osteoblasts [21][22][23][24]. In contrast, it is controversial whether the VDR is expressed in osteoclasts, with some authors reporting expression [25][26][27][28] and others not [21,23,29,30]. Recently, Wang et al. demonstrated that the VDR is not expressed in multi-nuclear osteoclasts using immunohistochemistry of EGTA-decalcified adult mouse bones [21]. In addition, direct effects of 1,25(OH) 2 D 3 have been demonstrated in osteoclasts and osteoclast progenitors [10] [11], and here we report that these effects are VDR-dependent (Fig. 3). Taken together, these studies suggest that extremely low levels of the VDR in osteoclasts may be sufficient to transduce vitamin D signals.
ED71 and 1,25(OH) 2 D 3 have been demonstrated to inhibit osteoclast-bone resorption activity by reducing expression of the sphigosine-1-phosphate receptor 2 (S1PR2) in circulating osteoclast precursor cells and blocking the migration of these cells to the bone surface by S1P; although differences in pharmacological action between ED71 and 1,25(OH) 2 D 3 were not demonstrated [31]. Here, we observed that, although 1,25(OH) 2 D 3 was more potent than ED71 in inhibiting osteoclastogenesis induced by M-CSF and RANKL in vitro, HIF1a inhibition in osteoclasts was specific to ED71. We also found that ED71 inhibited osteoclastogenesis even in HIF1a-suppressed cells, suggesting that ED71 likely targets factors other than HIF1a protein in osteoclasts. However, ED71 was less effective than 1,25(OH) 2 D 3 in inhibiting osteoclastogenesis in HIF1a-suppressed cells, which contrasts with observations seen in patients where the effect of ED71 on osteoclastogenesis is superior to that of 1,25(OH) 2 D 3 [12]. The cause of this difference remains to be elucidated, but the difference of potential activity to target HIF1a-protein in osteoclasts explains, at least in part, this difference. In addition, it is possible that ED71 inhibits osteoclastogenesis through effects on different cell types. Further investigations are needed to define molecular actions of vitamin D3 analogues on bone metabolism. Nonetheless, HIF1a inhibition could serve as an index to assess osteoclastogenesis in vitro when developing anti-osteoporosis agents. Moreover, our study indicates that targeting HIFa could constitute an effective treatment for osteoporosis, one that would not interfere with physiological bone turnover.