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Hypoxia-inducible factor regulates osteoclast-mediated bone resorption: role of angiopoietin-like 4.

Knowles HJ, Cleton-Jansen AM, Korsching E, Athanasou NA - FASEB J. (2010)

Bottom Line: Normoxic inducers of HIF (CoCl(2), desferrioxamine, and l-mimosine) and 100 ng/ml ANGPTL4 stimulated osteoclastic resorption 2- to 3-fold in assays of lacunar dentine resorption, without affecting osteoclast viability.In the osteoblastic Saos2 cell line, ANGPTL4 caused a dose-dependent increase in proliferation (P<0.01, 100 ng/ml) and, at lower doses (1-25 ng/ml), mineralization.These results demonstrate that HIF is sufficient to enhance osteoclast-mediated bone resorption and that ANGPTL4 can compensate for HIF-1α deficiency with respect to stimulation of osteoclast activity and also augments osteoblast proliferation and differentiation.

View Article: PubMed Central - PubMed

Affiliation: Botnar Research Centre, University of Oxford, Nuffield Orthopaedic Centre, Headington, Oxford, OX3 7LD, UK. helen.knowles@ndorms.ox.ac.uk

ABSTRACT
Hypoxia and the hypoxia-inducible factor (HIF) transcription factor regulate angiogenic-osteogenic coupling and osteoclast-mediated bone resorption. To determine how HIF might coordinate osteoclast and osteoblast function, we studied angiopoietin-like 4 (ANGPTL4), the top HIF target gene in an Illumina HumanWG-6 v3.0 48k array of normoxic vs. hypoxic osteoclasts differentiated from human CD14(+) monocytes (14.3-fold induction, P<0.0004). ANGPTL4 mRNA and protein were induced by 24 h at 2% O(2) in human primary osteoclasts, monocytes, and osteoblasts. ANGPTL4 protein was observed by immunofluorescence in osteoclasts and osteoblasts in vivo. Normoxic inducers of HIF (CoCl(2), desferrioxamine, and l-mimosine) and 100 ng/ml ANGPTL4 stimulated osteoclastic resorption 2- to 3-fold in assays of lacunar dentine resorption, without affecting osteoclast viability. Isoform-specific HIF-1α small interfering RNA ablated hypoxic induction of ANGPTL4 and of resorption, which was rescued by addition of exogenous ANGPTL4 (P<0.001). In the osteoblastic Saos2 cell line, ANGPTL4 caused a dose-dependent increase in proliferation (P<0.01, 100 ng/ml) and, at lower doses (1-25 ng/ml), mineralization. These results demonstrate that HIF is sufficient to enhance osteoclast-mediated bone resorption and that ANGPTL4 can compensate for HIF-1α deficiency with respect to stimulation of osteoclast activity and also augments osteoblast proliferation and differentiation.

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ANGPTL4 is regulated by HIF-1α in osteoclasts. A) Hypoxic induction of ANGPTL4 mRNA (open bars, n=7) and protein (shaded bars, n=4). B) Induction of ANGPTL4 mRNA by 24 h exposure to CoCl2 (100 μM), desferrioxamine (DFO, 100 μM), and l-mimosine (100 μM). C) Effect of siRNA targeting HIF-1α (H1), HIF-2α (H2), or HIF-1α scrambled control (scr) on expression of HIF-1α (open bars) or HIF-2α (shaded bars) mRNA; relative expression under hypoxia, normalized to ACTB. D) Western blot showing hypoxic expression of HIF-1α and HIF-2α (5× overexposed compared with HIF-1α) in response to siRNA targeting HIF-1α, HIF-2α, or HIF-1α scrambled control. E) Effect of HIF siRNA on ANGPTL4 mRNA expression in normoxic and hypoxic osteoclasts. *P < 0.05; **P < 0.01; ***P < 0.001.
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Figure 2: ANGPTL4 is regulated by HIF-1α in osteoclasts. A) Hypoxic induction of ANGPTL4 mRNA (open bars, n=7) and protein (shaded bars, n=4). B) Induction of ANGPTL4 mRNA by 24 h exposure to CoCl2 (100 μM), desferrioxamine (DFO, 100 μM), and l-mimosine (100 μM). C) Effect of siRNA targeting HIF-1α (H1), HIF-2α (H2), or HIF-1α scrambled control (scr) on expression of HIF-1α (open bars) or HIF-2α (shaded bars) mRNA; relative expression under hypoxia, normalized to ACTB. D) Western blot showing hypoxic expression of HIF-1α and HIF-2α (5× overexposed compared with HIF-1α) in response to siRNA targeting HIF-1α, HIF-2α, or HIF-1α scrambled control. E) Effect of HIF siRNA on ANGPTL4 mRNA expression in normoxic and hypoxic osteoclasts. *P < 0.05; **P < 0.01; ***P < 0.001.

Mentions: ANGPTL4 was the most hypoxia-inducible gene on the array (14.3-fold induction, P<0.0004) and is a known HIF target gene, although its expression has not previously been described in osteoclasts. Induction of ANGPTL4 by 24 h exposure to 2% O2 was confirmed in in vitro differentiated osteoclasts at both the mRNA and protein levels (Fig. 2A). ANGPTL4 was also induced by exposure to the panel of PHD enzyme inhibitors (Fig. 2B). HIF siRNA transfection procedures were optimized to achieve up to 70% knockdown of target gene mRNA 48 h after transfection using 50 nM siRNA and RNAiMAX (Fig. 2C). No associated osteoclast toxicity was observed (data not shown). This result translated to 59–85% inhibition of HIF-1α and 31–58% inhibition of HIF-2α protein (range of inhibition from 3 independent experiments) (Fig. 2D). Interestingly, at the mRNA level HIF-1α siRNA resulted in a significant increase in expression of HIF-2α (Fig. 2C). This was not mirrored at the protein level, however, in keeping with the predominantly post-translational mechanism of HIF regulation (29). Analysis of ANGPTL4 expression in siRNA-treated osteoclasts revealed it to be induced exclusively by HIF-1α (Fig. 2E). However, HIF-2α siRNA increased hypoxic induction of ANGPTL4 further, suggesting a potential inhibitory effect of HIF-2α on ANGPTL4 expression.


Hypoxia-inducible factor regulates osteoclast-mediated bone resorption: role of angiopoietin-like 4.

Knowles HJ, Cleton-Jansen AM, Korsching E, Athanasou NA - FASEB J. (2010)

ANGPTL4 is regulated by HIF-1α in osteoclasts. A) Hypoxic induction of ANGPTL4 mRNA (open bars, n=7) and protein (shaded bars, n=4). B) Induction of ANGPTL4 mRNA by 24 h exposure to CoCl2 (100 μM), desferrioxamine (DFO, 100 μM), and l-mimosine (100 μM). C) Effect of siRNA targeting HIF-1α (H1), HIF-2α (H2), or HIF-1α scrambled control (scr) on expression of HIF-1α (open bars) or HIF-2α (shaded bars) mRNA; relative expression under hypoxia, normalized to ACTB. D) Western blot showing hypoxic expression of HIF-1α and HIF-2α (5× overexposed compared with HIF-1α) in response to siRNA targeting HIF-1α, HIF-2α, or HIF-1α scrambled control. E) Effect of HIF siRNA on ANGPTL4 mRNA expression in normoxic and hypoxic osteoclasts. *P < 0.05; **P < 0.01; ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2992372&req=5

Figure 2: ANGPTL4 is regulated by HIF-1α in osteoclasts. A) Hypoxic induction of ANGPTL4 mRNA (open bars, n=7) and protein (shaded bars, n=4). B) Induction of ANGPTL4 mRNA by 24 h exposure to CoCl2 (100 μM), desferrioxamine (DFO, 100 μM), and l-mimosine (100 μM). C) Effect of siRNA targeting HIF-1α (H1), HIF-2α (H2), or HIF-1α scrambled control (scr) on expression of HIF-1α (open bars) or HIF-2α (shaded bars) mRNA; relative expression under hypoxia, normalized to ACTB. D) Western blot showing hypoxic expression of HIF-1α and HIF-2α (5× overexposed compared with HIF-1α) in response to siRNA targeting HIF-1α, HIF-2α, or HIF-1α scrambled control. E) Effect of HIF siRNA on ANGPTL4 mRNA expression in normoxic and hypoxic osteoclasts. *P < 0.05; **P < 0.01; ***P < 0.001.
Mentions: ANGPTL4 was the most hypoxia-inducible gene on the array (14.3-fold induction, P<0.0004) and is a known HIF target gene, although its expression has not previously been described in osteoclasts. Induction of ANGPTL4 by 24 h exposure to 2% O2 was confirmed in in vitro differentiated osteoclasts at both the mRNA and protein levels (Fig. 2A). ANGPTL4 was also induced by exposure to the panel of PHD enzyme inhibitors (Fig. 2B). HIF siRNA transfection procedures were optimized to achieve up to 70% knockdown of target gene mRNA 48 h after transfection using 50 nM siRNA and RNAiMAX (Fig. 2C). No associated osteoclast toxicity was observed (data not shown). This result translated to 59–85% inhibition of HIF-1α and 31–58% inhibition of HIF-2α protein (range of inhibition from 3 independent experiments) (Fig. 2D). Interestingly, at the mRNA level HIF-1α siRNA resulted in a significant increase in expression of HIF-2α (Fig. 2C). This was not mirrored at the protein level, however, in keeping with the predominantly post-translational mechanism of HIF regulation (29). Analysis of ANGPTL4 expression in siRNA-treated osteoclasts revealed it to be induced exclusively by HIF-1α (Fig. 2E). However, HIF-2α siRNA increased hypoxic induction of ANGPTL4 further, suggesting a potential inhibitory effect of HIF-2α on ANGPTL4 expression.

Bottom Line: Normoxic inducers of HIF (CoCl(2), desferrioxamine, and l-mimosine) and 100 ng/ml ANGPTL4 stimulated osteoclastic resorption 2- to 3-fold in assays of lacunar dentine resorption, without affecting osteoclast viability.In the osteoblastic Saos2 cell line, ANGPTL4 caused a dose-dependent increase in proliferation (P<0.01, 100 ng/ml) and, at lower doses (1-25 ng/ml), mineralization.These results demonstrate that HIF is sufficient to enhance osteoclast-mediated bone resorption and that ANGPTL4 can compensate for HIF-1α deficiency with respect to stimulation of osteoclast activity and also augments osteoblast proliferation and differentiation.

View Article: PubMed Central - PubMed

Affiliation: Botnar Research Centre, University of Oxford, Nuffield Orthopaedic Centre, Headington, Oxford, OX3 7LD, UK. helen.knowles@ndorms.ox.ac.uk

ABSTRACT
Hypoxia and the hypoxia-inducible factor (HIF) transcription factor regulate angiogenic-osteogenic coupling and osteoclast-mediated bone resorption. To determine how HIF might coordinate osteoclast and osteoblast function, we studied angiopoietin-like 4 (ANGPTL4), the top HIF target gene in an Illumina HumanWG-6 v3.0 48k array of normoxic vs. hypoxic osteoclasts differentiated from human CD14(+) monocytes (14.3-fold induction, P<0.0004). ANGPTL4 mRNA and protein were induced by 24 h at 2% O(2) in human primary osteoclasts, monocytes, and osteoblasts. ANGPTL4 protein was observed by immunofluorescence in osteoclasts and osteoblasts in vivo. Normoxic inducers of HIF (CoCl(2), desferrioxamine, and l-mimosine) and 100 ng/ml ANGPTL4 stimulated osteoclastic resorption 2- to 3-fold in assays of lacunar dentine resorption, without affecting osteoclast viability. Isoform-specific HIF-1α small interfering RNA ablated hypoxic induction of ANGPTL4 and of resorption, which was rescued by addition of exogenous ANGPTL4 (P<0.001). In the osteoblastic Saos2 cell line, ANGPTL4 caused a dose-dependent increase in proliferation (P<0.01, 100 ng/ml) and, at lower doses (1-25 ng/ml), mineralization. These results demonstrate that HIF is sufficient to enhance osteoclast-mediated bone resorption and that ANGPTL4 can compensate for HIF-1α deficiency with respect to stimulation of osteoclast activity and also augments osteoblast proliferation and differentiation.

Show MeSH
Related in: MedlinePlus