The vitamin D analogue ED71 but Not 1,25(OH)2D3 targets HIF1α protein in osteoclasts.

Yuiko Sato,Yoshiteru Miyauchi,Mayu Morita,Wu Hao,Atsuhiro Fujie,Tami Kobayashi,Toshimi Tando,Takeshi Miyamoto,Kana Miyamoto,Morio Matsumoto,Shigeyuki Yoshida,Ryuichi Watanabe,Yoshiaki Toyama,Hiroya Kanagawa,Hideo Morioka,Eri Katsuyama,Brenda Smith

doi:10.1371/journal.pone.0111845

Abstract

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.

Highlights

A cause for concern in developed countries is the increasing number of osteoporosis patients and individuals suffering fragility fractures due to osteoporosis [1]
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]
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]

Summary

Introduction

A cause for concern in developed countries is the increasing number of osteoporosis patients and individuals suffering fragility fractures due to osteoporosis [1]. 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]. Since postmenopausal osteoporosis is caused in part by estrogen-deficiency, treating of patients with estrogen is one option. 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]. Osteoclast specific HIF1a knockout or administration of a HIF1a inhibitor completely abrogated ovariectomy (OVX)induced osteoclast activation and bone loss [14]. 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

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Discussion