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NELL-1 in the treatment of osteoporotic bone loss.

James AW, Shen J, Zhang X, Asatrian G, Goyal R, Kwak JH, Jiang L, Bengs B, Culiat CT, Turner AS, Seim Iii HB, Wu BM, Lyons K, Adams JS, Ting K, Soo C - Nat Commun (2015)

Bottom Line: Recombinant NELL-1 binds to integrin β1 and consequently induces Wnt/β-catenin signalling, associated with increased OB differentiation and inhibition of OC-directed bone resorption.When extended to a large animal model, local delivery of NELL-1 to osteoporotic sheep spine leads to significant increase in bone formation.Altogether, these findings suggest that NELL-1 deficiency plays a role in osteoporosis and demonstrate the potential utility of NELL-1 as a combination anabolic/antiosteoclastic therapeutic for bone loss.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA and Orthopaedic Hospital, University of California, Los Angeles, California 90095, USA [2] Division of Growth and Development, Section of Orthodontics, School of Dentistry, University of California, Los Angeles, California 90095, USA [3] Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.

ABSTRACT
NELL-1 is a secreted, osteoinductive protein whose expression rheostatically controls skeletal ossification. Overexpression of NELL-1 results in craniosynostosis in humans and mice, whereas lack of Nell-1 expression is associated with skeletal undermineralization. Here we show that Nell-1-haploinsufficient mice have normal skeletal development but undergo age-related osteoporosis, characterized by a reduction in osteoblast:osteoclast (OB:OC) ratio and increased bone fragility. Recombinant NELL-1 binds to integrin β1 and consequently induces Wnt/β-catenin signalling, associated with increased OB differentiation and inhibition of OC-directed bone resorption. Systemic delivery of NELL-1 to mice with gonadectomy-induced osteoporosis results in improved bone mineral density. When extended to a large animal model, local delivery of NELL-1 to osteoporotic sheep spine leads to significant increase in bone formation. Altogether, these findings suggest that NELL-1 deficiency plays a role in osteoporosis and demonstrate the potential utility of NELL-1 as a combination anabolic/antiosteoclastic therapeutic for bone loss.

No MeSH data available.


Related in: MedlinePlus

NELL-1 requires integrin β1 to activate Wnt/β-catenin signalling.(a–d) RhNELL-1 increases Wnt signalling in the M2-10B4 BMSC line. (a) Active β-catenin immunocytochemistry in M2-10B4 cells, treated with rhNELL-1, WNT3A or control (PBS). (b,c) Western blot analysis and quantification of cytoplasmic and nuclear β-catenin (N=3 wells per treatment). (d) M2-10B4 cells were transfected with TOPFLASH reporter (N=4 wells per treatment). (e,f) RhNELL-1 requires intact Wnt/β-catenin signalling for induction of OB differentiation (N=4 wells per treatment). (e) M2-10B4 cells were treated with PBS or rhNELL-1 with or without DKK-1 for 3 days. Runx2 expression measured using qRT–PCR. (f) M2-10B4 cells were transduced with Runx2-EGFP reporter lentivirus and treated with PBS or rhNELL-1 with or without XAV939. Runx2 reporter assay was performed after 3 days. (g–i) RhNELL-1 increases Wnt signalling in the RAW264.7 osteoclast cell line (N=3 wells per treatment). (g) Gene expression after 2 days with or without rhNELL-1. (h) Active β-catenin immunocytochemistry in RAW264.7 cells, treated with rhNELL-1, WNT3A or control (PBS). (i,j) Western blot and quantification with or without rhNELL-1. (k–p) RhNELL-1 requires integrin β1 to activate Wnt/β-catenin signalling (N=3 wells per treatment). (k,l) siRNA-mediated knockdown of the known NELL-1 receptor integrin β1 was performed in M2-10B4 cells, confirmed by western blot analysis and quantification. (m,n) Similar siRNA-mediated knockdown of integrin β1 was performed in RAW264.7 OC cells. (o,p) After 2 days of rhNELL-1 (300 ng ml−1) treatment, Wnt signalling gene expression was evaluated in either scramble or integrin β1 siRNA-treated M2-10B4 or RAW264.7 cells. Quantitative RT–PCR for CyclinD and Axin2 was performed. Black scale bars, 100 μm. Data points indicate the means, while error bars represent one s.e.m. In vitro experiments were performed in biological triplicate, unless otherwise described. Parametric data were analysed using an appropriate Student's t-test or a one-way ANOVA, followed by a post hoc Tukey's test. Nonparametric data were analysed with a Mann–Whitney U-test or a Kruskal–Wallis one-way analysis. *P<0.05, **P<0.01.
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f4: NELL-1 requires integrin β1 to activate Wnt/β-catenin signalling.(a–d) RhNELL-1 increases Wnt signalling in the M2-10B4 BMSC line. (a) Active β-catenin immunocytochemistry in M2-10B4 cells, treated with rhNELL-1, WNT3A or control (PBS). (b,c) Western blot analysis and quantification of cytoplasmic and nuclear β-catenin (N=3 wells per treatment). (d) M2-10B4 cells were transfected with TOPFLASH reporter (N=4 wells per treatment). (e,f) RhNELL-1 requires intact Wnt/β-catenin signalling for induction of OB differentiation (N=4 wells per treatment). (e) M2-10B4 cells were treated with PBS or rhNELL-1 with or without DKK-1 for 3 days. Runx2 expression measured using qRT–PCR. (f) M2-10B4 cells were transduced with Runx2-EGFP reporter lentivirus and treated with PBS or rhNELL-1 with or without XAV939. Runx2 reporter assay was performed after 3 days. (g–i) RhNELL-1 increases Wnt signalling in the RAW264.7 osteoclast cell line (N=3 wells per treatment). (g) Gene expression after 2 days with or without rhNELL-1. (h) Active β-catenin immunocytochemistry in RAW264.7 cells, treated with rhNELL-1, WNT3A or control (PBS). (i,j) Western blot and quantification with or without rhNELL-1. (k–p) RhNELL-1 requires integrin β1 to activate Wnt/β-catenin signalling (N=3 wells per treatment). (k,l) siRNA-mediated knockdown of the known NELL-1 receptor integrin β1 was performed in M2-10B4 cells, confirmed by western blot analysis and quantification. (m,n) Similar siRNA-mediated knockdown of integrin β1 was performed in RAW264.7 OC cells. (o,p) After 2 days of rhNELL-1 (300 ng ml−1) treatment, Wnt signalling gene expression was evaluated in either scramble or integrin β1 siRNA-treated M2-10B4 or RAW264.7 cells. Quantitative RT–PCR for CyclinD and Axin2 was performed. Black scale bars, 100 μm. Data points indicate the means, while error bars represent one s.e.m. In vitro experiments were performed in biological triplicate, unless otherwise described. Parametric data were analysed using an appropriate Student's t-test or a one-way ANOVA, followed by a post hoc Tukey's test. Nonparametric data were analysed with a Mann–Whitney U-test or a Kruskal–Wallis one-way analysis. *P<0.05, **P<0.01.

Mentions: Next, we confirmed the association between NELL-1 and Wnt/β-catenin signalling in both OB and OC cells (Fig. 4). First, the M2-10B4 mouse bone marrow stromal cell (BMSC) line was treated with rhNELL-1 or WNT3A as a positive control. By three independent methods, rhNELL-1 induced a significant increase in Wnt/β-catenin (Fig. 4a–c). First, immunocytochemistry for active β-catenin showed increased staining (Fig. 4a). Western blot analysis for cytoplasmic and nuclear fractions demonstrated increased nuclear β-catenin (Fig. 4b,c). Finally, M2-10B4 cells expressing the TOPFLASH reporter system exhibited an increase in TCF/LEF1 activity (Fig. 4d). Next, we determined whether interference with Wnt/β-catenin signalling would impede rhNELL-1-induced OB differentiation. To answer this, two antagonists of Wnt signalling were used: DKK-1 (Fig. 4e) or XAV939 (Fig. 4f). Results showed that both Wnt antagonists inhibited rhNELL-1's induction of Runx2. We next determined whether rhNELL-1 induced Wnt/β-catenin signalling in OC precursor cells, using the mouse OC line RAW264.7 (Fig. 4g–j). As assessed using immunocytochemistry, nuclear β-catenin accumulation, and gene markers of Wnt/β-catenin signalling, rhNELL-1 showed a similar induction of Wnt signalling activity in RAW264.7 cells. In summary, rhNELL-1 protein activates Wnt/β-catenin signalling in both OB and OC cell types in vitro. Moreover, NELL-1's induction of OB differentiation is dependent on intact Wnt/β-catenin signalling.


NELL-1 in the treatment of osteoporotic bone loss.

James AW, Shen J, Zhang X, Asatrian G, Goyal R, Kwak JH, Jiang L, Bengs B, Culiat CT, Turner AS, Seim Iii HB, Wu BM, Lyons K, Adams JS, Ting K, Soo C - Nat Commun (2015)

NELL-1 requires integrin β1 to activate Wnt/β-catenin signalling.(a–d) RhNELL-1 increases Wnt signalling in the M2-10B4 BMSC line. (a) Active β-catenin immunocytochemistry in M2-10B4 cells, treated with rhNELL-1, WNT3A or control (PBS). (b,c) Western blot analysis and quantification of cytoplasmic and nuclear β-catenin (N=3 wells per treatment). (d) M2-10B4 cells were transfected with TOPFLASH reporter (N=4 wells per treatment). (e,f) RhNELL-1 requires intact Wnt/β-catenin signalling for induction of OB differentiation (N=4 wells per treatment). (e) M2-10B4 cells were treated with PBS or rhNELL-1 with or without DKK-1 for 3 days. Runx2 expression measured using qRT–PCR. (f) M2-10B4 cells were transduced with Runx2-EGFP reporter lentivirus and treated with PBS or rhNELL-1 with or without XAV939. Runx2 reporter assay was performed after 3 days. (g–i) RhNELL-1 increases Wnt signalling in the RAW264.7 osteoclast cell line (N=3 wells per treatment). (g) Gene expression after 2 days with or without rhNELL-1. (h) Active β-catenin immunocytochemistry in RAW264.7 cells, treated with rhNELL-1, WNT3A or control (PBS). (i,j) Western blot and quantification with or without rhNELL-1. (k–p) RhNELL-1 requires integrin β1 to activate Wnt/β-catenin signalling (N=3 wells per treatment). (k,l) siRNA-mediated knockdown of the known NELL-1 receptor integrin β1 was performed in M2-10B4 cells, confirmed by western blot analysis and quantification. (m,n) Similar siRNA-mediated knockdown of integrin β1 was performed in RAW264.7 OC cells. (o,p) After 2 days of rhNELL-1 (300 ng ml−1) treatment, Wnt signalling gene expression was evaluated in either scramble or integrin β1 siRNA-treated M2-10B4 or RAW264.7 cells. Quantitative RT–PCR for CyclinD and Axin2 was performed. Black scale bars, 100 μm. Data points indicate the means, while error bars represent one s.e.m. In vitro experiments were performed in biological triplicate, unless otherwise described. Parametric data were analysed using an appropriate Student's t-test or a one-way ANOVA, followed by a post hoc Tukey's test. Nonparametric data were analysed with a Mann–Whitney U-test or a Kruskal–Wallis one-way analysis. *P<0.05, **P<0.01.
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4557288&req=5

f4: NELL-1 requires integrin β1 to activate Wnt/β-catenin signalling.(a–d) RhNELL-1 increases Wnt signalling in the M2-10B4 BMSC line. (a) Active β-catenin immunocytochemistry in M2-10B4 cells, treated with rhNELL-1, WNT3A or control (PBS). (b,c) Western blot analysis and quantification of cytoplasmic and nuclear β-catenin (N=3 wells per treatment). (d) M2-10B4 cells were transfected with TOPFLASH reporter (N=4 wells per treatment). (e,f) RhNELL-1 requires intact Wnt/β-catenin signalling for induction of OB differentiation (N=4 wells per treatment). (e) M2-10B4 cells were treated with PBS or rhNELL-1 with or without DKK-1 for 3 days. Runx2 expression measured using qRT–PCR. (f) M2-10B4 cells were transduced with Runx2-EGFP reporter lentivirus and treated with PBS or rhNELL-1 with or without XAV939. Runx2 reporter assay was performed after 3 days. (g–i) RhNELL-1 increases Wnt signalling in the RAW264.7 osteoclast cell line (N=3 wells per treatment). (g) Gene expression after 2 days with or without rhNELL-1. (h) Active β-catenin immunocytochemistry in RAW264.7 cells, treated with rhNELL-1, WNT3A or control (PBS). (i,j) Western blot and quantification with or without rhNELL-1. (k–p) RhNELL-1 requires integrin β1 to activate Wnt/β-catenin signalling (N=3 wells per treatment). (k,l) siRNA-mediated knockdown of the known NELL-1 receptor integrin β1 was performed in M2-10B4 cells, confirmed by western blot analysis and quantification. (m,n) Similar siRNA-mediated knockdown of integrin β1 was performed in RAW264.7 OC cells. (o,p) After 2 days of rhNELL-1 (300 ng ml−1) treatment, Wnt signalling gene expression was evaluated in either scramble or integrin β1 siRNA-treated M2-10B4 or RAW264.7 cells. Quantitative RT–PCR for CyclinD and Axin2 was performed. Black scale bars, 100 μm. Data points indicate the means, while error bars represent one s.e.m. In vitro experiments were performed in biological triplicate, unless otherwise described. Parametric data were analysed using an appropriate Student's t-test or a one-way ANOVA, followed by a post hoc Tukey's test. Nonparametric data were analysed with a Mann–Whitney U-test or a Kruskal–Wallis one-way analysis. *P<0.05, **P<0.01.
Mentions: Next, we confirmed the association between NELL-1 and Wnt/β-catenin signalling in both OB and OC cells (Fig. 4). First, the M2-10B4 mouse bone marrow stromal cell (BMSC) line was treated with rhNELL-1 or WNT3A as a positive control. By three independent methods, rhNELL-1 induced a significant increase in Wnt/β-catenin (Fig. 4a–c). First, immunocytochemistry for active β-catenin showed increased staining (Fig. 4a). Western blot analysis for cytoplasmic and nuclear fractions demonstrated increased nuclear β-catenin (Fig. 4b,c). Finally, M2-10B4 cells expressing the TOPFLASH reporter system exhibited an increase in TCF/LEF1 activity (Fig. 4d). Next, we determined whether interference with Wnt/β-catenin signalling would impede rhNELL-1-induced OB differentiation. To answer this, two antagonists of Wnt signalling were used: DKK-1 (Fig. 4e) or XAV939 (Fig. 4f). Results showed that both Wnt antagonists inhibited rhNELL-1's induction of Runx2. We next determined whether rhNELL-1 induced Wnt/β-catenin signalling in OC precursor cells, using the mouse OC line RAW264.7 (Fig. 4g–j). As assessed using immunocytochemistry, nuclear β-catenin accumulation, and gene markers of Wnt/β-catenin signalling, rhNELL-1 showed a similar induction of Wnt signalling activity in RAW264.7 cells. In summary, rhNELL-1 protein activates Wnt/β-catenin signalling in both OB and OC cell types in vitro. Moreover, NELL-1's induction of OB differentiation is dependent on intact Wnt/β-catenin signalling.

Bottom Line: Recombinant NELL-1 binds to integrin β1 and consequently induces Wnt/β-catenin signalling, associated with increased OB differentiation and inhibition of OC-directed bone resorption.When extended to a large animal model, local delivery of NELL-1 to osteoporotic sheep spine leads to significant increase in bone formation.Altogether, these findings suggest that NELL-1 deficiency plays a role in osteoporosis and demonstrate the potential utility of NELL-1 as a combination anabolic/antiosteoclastic therapeutic for bone loss.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Orthopaedic Surgery and the Orthopaedic Hospital Research Center, UCLA and Orthopaedic Hospital, University of California, Los Angeles, California 90095, USA [2] Division of Growth and Development, Section of Orthodontics, School of Dentistry, University of California, Los Angeles, California 90095, USA [3] Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California, Los Angeles, California 90095, USA.

ABSTRACT
NELL-1 is a secreted, osteoinductive protein whose expression rheostatically controls skeletal ossification. Overexpression of NELL-1 results in craniosynostosis in humans and mice, whereas lack of Nell-1 expression is associated with skeletal undermineralization. Here we show that Nell-1-haploinsufficient mice have normal skeletal development but undergo age-related osteoporosis, characterized by a reduction in osteoblast:osteoclast (OB:OC) ratio and increased bone fragility. Recombinant NELL-1 binds to integrin β1 and consequently induces Wnt/β-catenin signalling, associated with increased OB differentiation and inhibition of OC-directed bone resorption. Systemic delivery of NELL-1 to mice with gonadectomy-induced osteoporosis results in improved bone mineral density. When extended to a large animal model, local delivery of NELL-1 to osteoporotic sheep spine leads to significant increase in bone formation. Altogether, these findings suggest that NELL-1 deficiency plays a role in osteoporosis and demonstrate the potential utility of NELL-1 as a combination anabolic/antiosteoclastic therapeutic for bone loss.

No MeSH data available.


Related in: MedlinePlus