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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

Osteoporotic phenotype of aged Nell-1-haploinsufficient mice.(a) Immunohistochemical staining for Nell-1 protein in rat spines, at 1 week, and 3 and 9 months. (b) Semiquantification of Nell-1 immunohistochemical staining, expressed as Nell-1+ bone-lining cells per B.Pm (N=20, 39 and 25 images, respectively). (c) Relative Nell-1 gene expression in mouse spines at 1 week, and 3 and 9 months, using qRT–PCR (N=3 samples per time point). (d) 3D image of liveCT and 18F radioisotope incorporation of aged (18-month old) wild-type (Nell-1+/+) and Nell-1-haploinsufficient (Nell-1+/6R) mice. (e) Quantification of BMD stratified by the lumbar vertebral level (L1–L6; N=11 and 12 mice, respectively). (f) Quantification of 18F incorporation (N=6 mice per genotype). (g) 3D coronal reconstructions of lumbar vertebrae of aged Nell-1+/+ and Nell-1+/6R mice. (h–j) Trabecular analyses of the lumbar vertebrae, including (h) trabecular bone thickness (Tb.Th), (i) number (Tb.N) and (j) spacing (Tb.Sp; N=12 and 19 vertebrae, respectively). (k–m) Serum studies among Nell-1+/+ and Nell-1+/6R aged mice including (k) serum PINP (N=19 and 17 mice), (l) serum TRAP5b (N=10 and 9 mice) and (m) serum CTX (N=12 and 22 mice). (n) Masson's trichrome staining of lumbar vertebral bodies of Nell-1+/+ and Nell-1+/6R mice, coronal section. (o–q) Histomorphometric quantifications of lumbar vertebral bodies. Measurements include (o) B.Ar (N=29 and 26 images), (p) percentage (%) B.Ar (N=29 and 26 images) and (q) B.Pm (N=30 and 29 images). (r) TRAP staining of lumbar vertebral bodies, indicating osteoclasts. Quantification of (s) Ob.N per B.Pm (N=20 images per genotype) and (t) Oc.N per B.Pm (N=26 and 39 images). Black scale bar, 25 μm; red scale bar, 10 mm; yellow scale bar, 100 μm. HU: Hounsfield Units; pCi (pico Curie). Data points indicate the means, while error bars represent one s.e.m. Experiments were performed without replicate. Parametric data were analysed using an appropriate Student's t-test or a one-way analysis of variance (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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f1: Osteoporotic phenotype of aged Nell-1-haploinsufficient mice.(a) Immunohistochemical staining for Nell-1 protein in rat spines, at 1 week, and 3 and 9 months. (b) Semiquantification of Nell-1 immunohistochemical staining, expressed as Nell-1+ bone-lining cells per B.Pm (N=20, 39 and 25 images, respectively). (c) Relative Nell-1 gene expression in mouse spines at 1 week, and 3 and 9 months, using qRT–PCR (N=3 samples per time point). (d) 3D image of liveCT and 18F radioisotope incorporation of aged (18-month old) wild-type (Nell-1+/+) and Nell-1-haploinsufficient (Nell-1+/6R) mice. (e) Quantification of BMD stratified by the lumbar vertebral level (L1–L6; N=11 and 12 mice, respectively). (f) Quantification of 18F incorporation (N=6 mice per genotype). (g) 3D coronal reconstructions of lumbar vertebrae of aged Nell-1+/+ and Nell-1+/6R mice. (h–j) Trabecular analyses of the lumbar vertebrae, including (h) trabecular bone thickness (Tb.Th), (i) number (Tb.N) and (j) spacing (Tb.Sp; N=12 and 19 vertebrae, respectively). (k–m) Serum studies among Nell-1+/+ and Nell-1+/6R aged mice including (k) serum PINP (N=19 and 17 mice), (l) serum TRAP5b (N=10 and 9 mice) and (m) serum CTX (N=12 and 22 mice). (n) Masson's trichrome staining of lumbar vertebral bodies of Nell-1+/+ and Nell-1+/6R mice, coronal section. (o–q) Histomorphometric quantifications of lumbar vertebral bodies. Measurements include (o) B.Ar (N=29 and 26 images), (p) percentage (%) B.Ar (N=29 and 26 images) and (q) B.Pm (N=30 and 29 images). (r) TRAP staining of lumbar vertebral bodies, indicating osteoclasts. Quantification of (s) Ob.N per B.Pm (N=20 images per genotype) and (t) Oc.N per B.Pm (N=26 and 39 images). Black scale bar, 25 μm; red scale bar, 10 mm; yellow scale bar, 100 μm. HU: Hounsfield Units; pCi (pico Curie). Data points indicate the means, while error bars represent one s.e.m. Experiments were performed without replicate. Parametric data were analysed using an appropriate Student's t-test or a one-way analysis of variance (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: NELL-1 expression, known to be present during skeletal development, was evaluated with skeletal aging. Nell-1 protein showed a significant reduction with age, as shown by immunohistochemistry of trabecular bone-lining OBs in the rat lumbar spine (Fig. 1a). Semiquantitative analysis demonstrated a significant decrease in Nell-1+ bone-lining cells with age (Fig. 1b). These results were further quantified using quantitative reverse transcription (qRT)–PCR, showing a progressive loss of Nell-1 in the bone with age (Fig. 1c). The consequences of Nell-1 deficiency on skeletal aging were next assessed. Deficiency was induced by the generation of a point mutation in the Nell-1 gene (Nell-16R/6R) resulting in a premature stop codon and near complete loss of transcript levels. Complete deficiency (Nell-16R/6R) is neonatal lethal with major skeletal anomalies, while the heterozygote (Nell-1+/6R) mice are without gross abnormalities at birth21. The Nell-1+/6R mouse skeleton was studied across its lifetime. The neonatal (P1 days), young adult (1 month) and adult skeleton (6 months) showed no significant skeletal phenotype, as assessed by combined live CT/fluoride radioisotope (18F) incorporation studies, and histomorphometric analyses (Supplementary Fig. 1, Supplementary Table 1). In contrast, by 18 months of life the Nell-1+/6R mouse developed significant osteoporosis (Fig. 1, Supplementary Fig. 2). This was apparent both in analysis of the lumbar spine (Fig. 1d–t) and the distal femur (Supplementary Fig. 2f–k). Analyses showed significantly reduced BMD and reduced 18F incorporation (Fig. 1d–f). Dual energy X-ray absorptiometry (DXA), high-resolution microCT and trabecular bone analyses confirmed an osteoporotic phenotype in the aged Nell-1+/6R mouse in both the lumbar spine and femur (Fig. 1g–j, Supplementary Fig. 2a–k). Further, the aged Nell-1+/6R bone showed increased bone fragility and decreased bone stiffness as shown by both computer-simulated biomechanical compression testing as well as microindentation testing (Supplementary Fig. 2l–r). Next, serum biomarkers evaluated total OB and OC activity (Fig. 1k–m). Total bone formation activity (serum Procollagen I N-terminal Propeptide (PINP)) was reduced among Nell-1+/6R mice, while total bone resorption activity was increased in Nell-1+/6R mice (serum Tartrate Resistant Acid Phosphatase 5b (TRAP5b) and C-Terminal Telopeptide (CTX)). Histological analysis confirmed a significant reduction in the vertebral body trabecular bone among Nell-1+/6R specimens (Fig. 1n–q, Supplementary Fig. 2s–y). Next, OB and OC prevalence was analysed histologically (Fig. 1r–t). OB number per bone perimeter (Ob.N per B.Pm) was significantly reduced in the Nell-1+/6R lumbar vertebrae. Next, OC numbers, evaluated by TRAP staining, showed a significant increase in osteoclast number (Oc.N) per B.Pm among Nell-1+/6R vertebrae. Thus, by radiographic, biomechanical and histologic analyses, Nell-1 haploinsufficiency results in normal skeletogenesis and development, but results in an osteoporotic defect with age. This osteoporotic defect, manifested in both sexes, is characterized by increased cortical bone fragility, altered trabecular bone morphology, decreased OB and increased OC number.


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)

Osteoporotic phenotype of aged Nell-1-haploinsufficient mice.(a) Immunohistochemical staining for Nell-1 protein in rat spines, at 1 week, and 3 and 9 months. (b) Semiquantification of Nell-1 immunohistochemical staining, expressed as Nell-1+ bone-lining cells per B.Pm (N=20, 39 and 25 images, respectively). (c) Relative Nell-1 gene expression in mouse spines at 1 week, and 3 and 9 months, using qRT–PCR (N=3 samples per time point). (d) 3D image of liveCT and 18F radioisotope incorporation of aged (18-month old) wild-type (Nell-1+/+) and Nell-1-haploinsufficient (Nell-1+/6R) mice. (e) Quantification of BMD stratified by the lumbar vertebral level (L1–L6; N=11 and 12 mice, respectively). (f) Quantification of 18F incorporation (N=6 mice per genotype). (g) 3D coronal reconstructions of lumbar vertebrae of aged Nell-1+/+ and Nell-1+/6R mice. (h–j) Trabecular analyses of the lumbar vertebrae, including (h) trabecular bone thickness (Tb.Th), (i) number (Tb.N) and (j) spacing (Tb.Sp; N=12 and 19 vertebrae, respectively). (k–m) Serum studies among Nell-1+/+ and Nell-1+/6R aged mice including (k) serum PINP (N=19 and 17 mice), (l) serum TRAP5b (N=10 and 9 mice) and (m) serum CTX (N=12 and 22 mice). (n) Masson's trichrome staining of lumbar vertebral bodies of Nell-1+/+ and Nell-1+/6R mice, coronal section. (o–q) Histomorphometric quantifications of lumbar vertebral bodies. Measurements include (o) B.Ar (N=29 and 26 images), (p) percentage (%) B.Ar (N=29 and 26 images) and (q) B.Pm (N=30 and 29 images). (r) TRAP staining of lumbar vertebral bodies, indicating osteoclasts. Quantification of (s) Ob.N per B.Pm (N=20 images per genotype) and (t) Oc.N per B.Pm (N=26 and 39 images). Black scale bar, 25 μm; red scale bar, 10 mm; yellow scale bar, 100 μm. HU: Hounsfield Units; pCi (pico Curie). Data points indicate the means, while error bars represent one s.e.m. Experiments were performed without replicate. Parametric data were analysed using an appropriate Student's t-test or a one-way analysis of variance (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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f1: Osteoporotic phenotype of aged Nell-1-haploinsufficient mice.(a) Immunohistochemical staining for Nell-1 protein in rat spines, at 1 week, and 3 and 9 months. (b) Semiquantification of Nell-1 immunohistochemical staining, expressed as Nell-1+ bone-lining cells per B.Pm (N=20, 39 and 25 images, respectively). (c) Relative Nell-1 gene expression in mouse spines at 1 week, and 3 and 9 months, using qRT–PCR (N=3 samples per time point). (d) 3D image of liveCT and 18F radioisotope incorporation of aged (18-month old) wild-type (Nell-1+/+) and Nell-1-haploinsufficient (Nell-1+/6R) mice. (e) Quantification of BMD stratified by the lumbar vertebral level (L1–L6; N=11 and 12 mice, respectively). (f) Quantification of 18F incorporation (N=6 mice per genotype). (g) 3D coronal reconstructions of lumbar vertebrae of aged Nell-1+/+ and Nell-1+/6R mice. (h–j) Trabecular analyses of the lumbar vertebrae, including (h) trabecular bone thickness (Tb.Th), (i) number (Tb.N) and (j) spacing (Tb.Sp; N=12 and 19 vertebrae, respectively). (k–m) Serum studies among Nell-1+/+ and Nell-1+/6R aged mice including (k) serum PINP (N=19 and 17 mice), (l) serum TRAP5b (N=10 and 9 mice) and (m) serum CTX (N=12 and 22 mice). (n) Masson's trichrome staining of lumbar vertebral bodies of Nell-1+/+ and Nell-1+/6R mice, coronal section. (o–q) Histomorphometric quantifications of lumbar vertebral bodies. Measurements include (o) B.Ar (N=29 and 26 images), (p) percentage (%) B.Ar (N=29 and 26 images) and (q) B.Pm (N=30 and 29 images). (r) TRAP staining of lumbar vertebral bodies, indicating osteoclasts. Quantification of (s) Ob.N per B.Pm (N=20 images per genotype) and (t) Oc.N per B.Pm (N=26 and 39 images). Black scale bar, 25 μm; red scale bar, 10 mm; yellow scale bar, 100 μm. HU: Hounsfield Units; pCi (pico Curie). Data points indicate the means, while error bars represent one s.e.m. Experiments were performed without replicate. Parametric data were analysed using an appropriate Student's t-test or a one-way analysis of variance (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: NELL-1 expression, known to be present during skeletal development, was evaluated with skeletal aging. Nell-1 protein showed a significant reduction with age, as shown by immunohistochemistry of trabecular bone-lining OBs in the rat lumbar spine (Fig. 1a). Semiquantitative analysis demonstrated a significant decrease in Nell-1+ bone-lining cells with age (Fig. 1b). These results were further quantified using quantitative reverse transcription (qRT)–PCR, showing a progressive loss of Nell-1 in the bone with age (Fig. 1c). The consequences of Nell-1 deficiency on skeletal aging were next assessed. Deficiency was induced by the generation of a point mutation in the Nell-1 gene (Nell-16R/6R) resulting in a premature stop codon and near complete loss of transcript levels. Complete deficiency (Nell-16R/6R) is neonatal lethal with major skeletal anomalies, while the heterozygote (Nell-1+/6R) mice are without gross abnormalities at birth21. The Nell-1+/6R mouse skeleton was studied across its lifetime. The neonatal (P1 days), young adult (1 month) and adult skeleton (6 months) showed no significant skeletal phenotype, as assessed by combined live CT/fluoride radioisotope (18F) incorporation studies, and histomorphometric analyses (Supplementary Fig. 1, Supplementary Table 1). In contrast, by 18 months of life the Nell-1+/6R mouse developed significant osteoporosis (Fig. 1, Supplementary Fig. 2). This was apparent both in analysis of the lumbar spine (Fig. 1d–t) and the distal femur (Supplementary Fig. 2f–k). Analyses showed significantly reduced BMD and reduced 18F incorporation (Fig. 1d–f). Dual energy X-ray absorptiometry (DXA), high-resolution microCT and trabecular bone analyses confirmed an osteoporotic phenotype in the aged Nell-1+/6R mouse in both the lumbar spine and femur (Fig. 1g–j, Supplementary Fig. 2a–k). Further, the aged Nell-1+/6R bone showed increased bone fragility and decreased bone stiffness as shown by both computer-simulated biomechanical compression testing as well as microindentation testing (Supplementary Fig. 2l–r). Next, serum biomarkers evaluated total OB and OC activity (Fig. 1k–m). Total bone formation activity (serum Procollagen I N-terminal Propeptide (PINP)) was reduced among Nell-1+/6R mice, while total bone resorption activity was increased in Nell-1+/6R mice (serum Tartrate Resistant Acid Phosphatase 5b (TRAP5b) and C-Terminal Telopeptide (CTX)). Histological analysis confirmed a significant reduction in the vertebral body trabecular bone among Nell-1+/6R specimens (Fig. 1n–q, Supplementary Fig. 2s–y). Next, OB and OC prevalence was analysed histologically (Fig. 1r–t). OB number per bone perimeter (Ob.N per B.Pm) was significantly reduced in the Nell-1+/6R lumbar vertebrae. Next, OC numbers, evaluated by TRAP staining, showed a significant increase in osteoclast number (Oc.N) per B.Pm among Nell-1+/6R vertebrae. Thus, by radiographic, biomechanical and histologic analyses, Nell-1 haploinsufficiency results in normal skeletogenesis and development, but results in an osteoporotic defect with age. This osteoporotic defect, manifested in both sexes, is characterized by increased cortical bone fragility, altered trabecular bone morphology, decreased OB and increased OC number.

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