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

1,25(OH)₂D₃ is a more potent inhibitor of osteoclastogenesis <i>in vitro</i> than is ED71.

<p>(<b>A, B</b> and <b>C</b>) M-CSF-dependent osteoclast progenitor cells were isolated from wild-type mice and cultured in the presence of M-CSF (M, 50 ng/ml) + RANKL (R, 25 ng/ml) with or without indicated concentrations of ED71 or 1,25(OH)₂D₃ (1,25D) for 5 days. Cells were then stained with TRAP (<b>A</b>) and the number of multi-nuclear TRAP-positive cells was counted (<b>B</b>). Expression of <i>Ctsk</i>, <i>NFATc1</i> and <i>DC-STAMP,</i> all of which are osteoclastic genes, was analyzed by realtime PCR (<b>C</b>). Expression of <i>Blimp1</i>, <i>Bcl6</i> and <i>Irf8</i> was analyzed by realtime PCR (<b>D</b>). Data represent mean expression of each relative to <i>Actb</i> ± SD (<i>n</i> = 5). *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001; NS, not significant.</p

1,25(OH)₂D₃ is more active in promoting c-Fos protein inhibition and <i>Ifnβ</i>-induction in osteoclasts compared with ED71.

<p>(A and B) M-CSF-dependent osteoclast progenitor cells were isolated from wild-type mice and cultured in the presence of M-CSF alone (50 ng/ml) or M-CSF + RANKL (25 ng/ml) with or without 10⁻⁷ M ED71 or 1,25(OH)₂D₃ (1,25D) for 5 days. c-Fos protein was then assessed by western blot (A). <i>Ifnβ</i> expression was analyzed by realtime PCR (B). Data represent mean <i>Ifnβ</i> expression relative to that of <i>Actb</i> ± SD (<i>n</i> = 5). ***<i>P</i><0.001.</p

ED71 or 1,25(OH)₂D₃ activity requires the VDR.

<p>(<b>A, B</b> and <b>C</b>) M-CSF-dependent osteoclast progenitor cells were isolated from wild-type (WT) or VDR-deficient (VDR KO) mice and cultured in the presence of M-CSF alone (50 ng/ml) or M-CSF + RANKL (25 ng/ml) with or without indicated concentrations of ED71 or 1,25(OH)₂D₃ for 5 days. Cells were then stained with TRAP (<b>A</b>), and multi-nuclear TRAP-positive cells were counted (<b>B</b>). Expression of <i>Ctsk</i>, <i>NFATc1</i> and <i>DC-STAMP</i> was assessed by realtime PCR (<b>C</b>). Data represent mean <i>Ctsk</i>, <i>NFATc1</i> or <i>DC-STAMP</i> expression relative to that of <i>Actb</i> ± SD (<i>n</i> = 5).</p

HIF1α protein is suppressed by ED71 but not by 1,25(OH)₂D₃.

<p>(<b>A</b>) Western analysis of Raw264.7 cells cultured in hypoxic conditions with or without 10⁻⁷ M of ED71 or 1,25(OH)₂D₃ (1,25D). (<b>B</b>) <i>Hif1α</i> mRNA levels in Raw264.7 cells cultured in hypoxic conditions were analyzed by realtime PCR in the presence or absence of 10⁻⁷ M ED71 or 1,25(OH)₂D₃. Data represent mean <i>Hif1α</i> expression relative to that of <i>Actb</i> ± SD (<i>n</i> = 5). (<b>C</b>) Levels of <i>VDR</i> transcripts in Raw264.7 cells transfected with shRNA targeting the VDR (shVDR) or control shRNA (Control) were determined by realtime PCR. Data represent mean <i>VDR</i> expression relative to that of <i>Actb</i> ± SD (<i>n</i> = 5). (<b>D</b>) Western analysis of control (shControl) or VDR-suppressed (shVDR#1 or shVDR#2) Raw264.7 transformants cultured in hypoxic conditions with ED71 or 1,25(OH)₂D₃ (1,25D), both at 10⁻⁷ M. (<b>E</b>) M-CSF-dependent Ctsk Cre/<i>Hif^flox/flox</i> cells were cultured in normoxic conditions to suppress HIF1α in the presence of M-CSF (50 ng/ml) plus RANKL (25 ng/ml) with either ED71 or 1,25(OH)₂D₃ (1,25D) both at 10⁻⁷M for 4 days. Expression of <i>Ctsk</i> and <i>NFATc1</i> was then assessed by realtime PCR. Data represent mean <i>Ctsk</i> or <i>NFATc1</i> expression relative to that of <i>Actb</i> ± SD (<i>n</i> = 5). *<i>P</i><0.05; **<i>P</i><0.01; ***<i>P</i><0.001.</p