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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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Osteoblasts and ANGPTL4. A, B) Effect of 24 h exposure to 2% O2 (shaded bars) on ANGPTL4 expression assessed by real-time PCR (A) and ELISA (B) vs. the normoxic (open bars) control. Mean fold hypoxic induction is indicated over the bars for each cell type (n/a, not calculated, normoxic levels undetectable). *P < 0.05; **P < 0.01; ***P < 0.001. C) Western blot showing hypoxic induction of ANGPTL4 in the same cell panel and detecting full-length ANGPTL4 of the predicted 41- to 45-kDa molecular mass [FL(41–45)], full-length ANGPTL4 of 50–55 kDa at which it normally runs in reducing conditions [FL(55)], and the FLD domain at 20–35 kDa (FLD). OC, in vitro differentiated osteoclasts; MON, CD14+ monocytes; THP1, monocytic cell line; HOb, primary human osteoblasts; GCTB stromal cells, mononuclear stromal component of GCTB; MG-63 and Saos2, osteoblastic cell lines.
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Figure 7: Osteoblasts and ANGPTL4. A, B) Effect of 24 h exposure to 2% O2 (shaded bars) on ANGPTL4 expression assessed by real-time PCR (A) and ELISA (B) vs. the normoxic (open bars) control. Mean fold hypoxic induction is indicated over the bars for each cell type (n/a, not calculated, normoxic levels undetectable). *P < 0.05; **P < 0.01; ***P < 0.001. C) Western blot showing hypoxic induction of ANGPTL4 in the same cell panel and detecting full-length ANGPTL4 of the predicted 41- to 45-kDa molecular mass [FL(41–45)], full-length ANGPTL4 of 50–55 kDa at which it normally runs in reducing conditions [FL(55)], and the FLD domain at 20–35 kDa (FLD). OC, in vitro differentiated osteoclasts; MON, CD14+ monocytes; THP1, monocytic cell line; HOb, primary human osteoblasts; GCTB stromal cells, mononuclear stromal component of GCTB; MG-63 and Saos2, osteoblastic cell lines.

Mentions: The relative level of ANGPTL4 expression in osteoclasts was compared with that in monocytes and osteoblasts in vitro, using both primary human cells and cell lines. At both the mRNA (Fig. 7A) and protein (Fig. 7B) levels, osteoblastic cells expressed considerably more ANGPTL4 than monocytes or osteoclasts, although the magnitude of hypoxic induction remained greatest in osteoclasts. ANGPTL4 can be proteolytically processed, in a cell- and species-specific manner, from the full-length protein into an N-terminal coiled-coil domain (CCD) and a C-terminal fibrinogen-like domain (FLD) fragment (16). The ELISA used is based on a polyclonal antibody raised against the full-length recombinant immunogen and is therefore unable to distinguish these forms. Western blotting with an antibody raised against the FLD enabled confirmation of hypoxic induction of full-length ANGPTL4 in osteoclasts, monocytes, and, most strongly, osteoblastic cells (Fig. 7C). The FLD fragment was also detected in osteoblastic cells, indicating that osteoblasts are capable of proteolytic processing of ANGPTL4, although expression of this cleaved form was not apparently hypoxia-regulated. An FLD fragment was not detected in either monocytes or osteoclasts, although this may be due to low levels of expression of ANGPTL4 in these cells.


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)

Osteoblasts and ANGPTL4. A, B) Effect of 24 h exposure to 2% O2 (shaded bars) on ANGPTL4 expression assessed by real-time PCR (A) and ELISA (B) vs. the normoxic (open bars) control. Mean fold hypoxic induction is indicated over the bars for each cell type (n/a, not calculated, normoxic levels undetectable). *P < 0.05; **P < 0.01; ***P < 0.001. C) Western blot showing hypoxic induction of ANGPTL4 in the same cell panel and detecting full-length ANGPTL4 of the predicted 41- to 45-kDa molecular mass [FL(41–45)], full-length ANGPTL4 of 50–55 kDa at which it normally runs in reducing conditions [FL(55)], and the FLD domain at 20–35 kDa (FLD). OC, in vitro differentiated osteoclasts; MON, CD14+ monocytes; THP1, monocytic cell line; HOb, primary human osteoblasts; GCTB stromal cells, mononuclear stromal component of GCTB; MG-63 and Saos2, osteoblastic cell lines.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Osteoblasts and ANGPTL4. A, B) Effect of 24 h exposure to 2% O2 (shaded bars) on ANGPTL4 expression assessed by real-time PCR (A) and ELISA (B) vs. the normoxic (open bars) control. Mean fold hypoxic induction is indicated over the bars for each cell type (n/a, not calculated, normoxic levels undetectable). *P < 0.05; **P < 0.01; ***P < 0.001. C) Western blot showing hypoxic induction of ANGPTL4 in the same cell panel and detecting full-length ANGPTL4 of the predicted 41- to 45-kDa molecular mass [FL(41–45)], full-length ANGPTL4 of 50–55 kDa at which it normally runs in reducing conditions [FL(55)], and the FLD domain at 20–35 kDa (FLD). OC, in vitro differentiated osteoclasts; MON, CD14+ monocytes; THP1, monocytic cell line; HOb, primary human osteoblasts; GCTB stromal cells, mononuclear stromal component of GCTB; MG-63 and Saos2, osteoblastic cell lines.
Mentions: The relative level of ANGPTL4 expression in osteoclasts was compared with that in monocytes and osteoblasts in vitro, using both primary human cells and cell lines. At both the mRNA (Fig. 7A) and protein (Fig. 7B) levels, osteoblastic cells expressed considerably more ANGPTL4 than monocytes or osteoclasts, although the magnitude of hypoxic induction remained greatest in osteoclasts. ANGPTL4 can be proteolytically processed, in a cell- and species-specific manner, from the full-length protein into an N-terminal coiled-coil domain (CCD) and a C-terminal fibrinogen-like domain (FLD) fragment (16). The ELISA used is based on a polyclonal antibody raised against the full-length recombinant immunogen and is therefore unable to distinguish these forms. Western blotting with an antibody raised against the FLD enabled confirmation of hypoxic induction of full-length ANGPTL4 in osteoclasts, monocytes, and, most strongly, osteoblastic cells (Fig. 7C). The FLD fragment was also detected in osteoblastic cells, indicating that osteoblasts are capable of proteolytic processing of ANGPTL4, although expression of this cleaved form was not apparently hypoxia-regulated. An FLD fragment was not detected in either monocytes or osteoclasts, although this may be due to low levels of expression of ANGPTL4 in these cells.

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