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

Sato Y, Miyauchi Y, Yoshida S, Morita M, Kobayashi T, Kanagawa H, Katsuyama E, Fujie A, Hao W, Tando T, Watanabe R, Miyamoto K, Morioka H, Matsumoto M, Toyama Y, Miyamoto T - PLoS ONE (2014)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan; Department of Musculoskeletal Reconstruction and Regeneration Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.

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.

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HIF1α protein is suppressed by ED71 but not by 1,25(OH)2D3.(A) 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) Hif1α 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 Hif1α expression relative to that of Actb ± SD (n = 5). (C) Levels of VDR transcripts in Raw264.7 cells transfected with shRNA targeting the VDR (shVDR) or control shRNA (Control) were determined by realtime PCR. Data represent mean VDR expression relative to that of Actb ± SD (n = 5). (D) 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. (E) M-CSF-dependent Ctsk Cre/Hifflox/flox 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 Ctsk and NFATc1 was then assessed by realtime PCR. Data represent mean Ctsk or NFATc1 expression relative to that of Actb ± SD (n = 5). *P<0.05; **P<0.01; ***P<0.001.
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pone-0111845-g005: HIF1α protein is suppressed by ED71 but not by 1,25(OH)2D3.(A) 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) Hif1α 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 Hif1α expression relative to that of Actb ± SD (n = 5). (C) Levels of VDR transcripts in Raw264.7 cells transfected with shRNA targeting the VDR (shVDR) or control shRNA (Control) were determined by realtime PCR. Data represent mean VDR expression relative to that of Actb ± SD (n = 5). (D) 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. (E) M-CSF-dependent Ctsk Cre/Hifflox/flox 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 Ctsk and NFATc1 was then assessed by realtime PCR. Data represent mean Ctsk or NFATc1 expression relative to that of Actb ± SD (n = 5). *P<0.05; **P<0.01; ***P<0.001.

Mentions: Next, we asked whether HIF1α is a target of ED71 in osteoclasts (Fig. 5). Interestingly, we found that in cultured osteoclasts, HIF1α protein levels were suppressed by ED71 but not by 1,25(OH)2D3 (Fig. 5A). In contrast, Hif1α mRNA expression in osteoclasts was not inhibited by either treatment (Fig. 5B), suggesting that ED71 suppresses HIF1α at the protein level as demonstrated by estrogen treatment [14]. To determine if the VDR is required for ED71-mediated HIF1α 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)2D3 (Fig. 5D). HIF1α protein suppression by ED71 seen in control cells was abrogated in both VDR knockdown lines, suggesting that HIF1α protein suppression by ED71 is VDR-dependent. We then isolated osteoclast progenitors from Ctsk Cre/Hif1αflox/flox mice, cultured them in normoxic conditions to suppress HIF1α protein, and treated cells with or without ED71 or 1,25(OH)2D3 (Fig. 5E). ED71 treatment effectively inhibited osteoclast differentiation, even in HIF1α-suppressed cells, suggesting that ED71 likely targets factors other than HIF1α protein in osteoclasts (Fig. 5E). However, ED71 was less effective than 1,25(OH)2D3 in inhibiting osteoclastogenesis in HIF1α-suppressed cells (Fig. 5E).


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

Sato Y, Miyauchi Y, Yoshida S, Morita M, Kobayashi T, Kanagawa H, Katsuyama E, Fujie A, Hao W, Tando T, Watanabe R, Miyamoto K, Morioka H, Matsumoto M, Toyama Y, Miyamoto T - PLoS ONE (2014)

HIF1α protein is suppressed by ED71 but not by 1,25(OH)2D3.(A) 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) Hif1α 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 Hif1α expression relative to that of Actb ± SD (n = 5). (C) Levels of VDR transcripts in Raw264.7 cells transfected with shRNA targeting the VDR (shVDR) or control shRNA (Control) were determined by realtime PCR. Data represent mean VDR expression relative to that of Actb ± SD (n = 5). (D) 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. (E) M-CSF-dependent Ctsk Cre/Hifflox/flox 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 Ctsk and NFATc1 was then assessed by realtime PCR. Data represent mean Ctsk or NFATc1 expression relative to that of Actb ± SD (n = 5). *P<0.05; **P<0.01; ***P<0.001.
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pone-0111845-g005: HIF1α protein is suppressed by ED71 but not by 1,25(OH)2D3.(A) 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) Hif1α 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 Hif1α expression relative to that of Actb ± SD (n = 5). (C) Levels of VDR transcripts in Raw264.7 cells transfected with shRNA targeting the VDR (shVDR) or control shRNA (Control) were determined by realtime PCR. Data represent mean VDR expression relative to that of Actb ± SD (n = 5). (D) 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. (E) M-CSF-dependent Ctsk Cre/Hifflox/flox 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 Ctsk and NFATc1 was then assessed by realtime PCR. Data represent mean Ctsk or NFATc1 expression relative to that of Actb ± SD (n = 5). *P<0.05; **P<0.01; ***P<0.001.
Mentions: Next, we asked whether HIF1α is a target of ED71 in osteoclasts (Fig. 5). Interestingly, we found that in cultured osteoclasts, HIF1α protein levels were suppressed by ED71 but not by 1,25(OH)2D3 (Fig. 5A). In contrast, Hif1α mRNA expression in osteoclasts was not inhibited by either treatment (Fig. 5B), suggesting that ED71 suppresses HIF1α at the protein level as demonstrated by estrogen treatment [14]. To determine if the VDR is required for ED71-mediated HIF1α 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)2D3 (Fig. 5D). HIF1α protein suppression by ED71 seen in control cells was abrogated in both VDR knockdown lines, suggesting that HIF1α protein suppression by ED71 is VDR-dependent. We then isolated osteoclast progenitors from Ctsk Cre/Hif1αflox/flox mice, cultured them in normoxic conditions to suppress HIF1α protein, and treated cells with or without ED71 or 1,25(OH)2D3 (Fig. 5E). ED71 treatment effectively inhibited osteoclast differentiation, even in HIF1α-suppressed cells, suggesting that ED71 likely targets factors other than HIF1α protein in osteoclasts (Fig. 5E). However, ED71 was less effective than 1,25(OH)2D3 in inhibiting osteoclastogenesis in HIF1α-suppressed cells (Fig. 5E).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopedic Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan; Department of Musculoskeletal Reconstruction and Regeneration Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo, Japan.

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.

Show MeSH
Related in: MedlinePlus