Direct stimulation of bone mass by increased GH signalling in the osteoblasts of Socs2^−/− mice

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   Figure 1

   Socs2^−/− cortical bone phenotype. (A) Images of periosteal and endosteal cortical bone apposition rate in tibia from juvenile 6-week-old male WT and Socs2^−/− mice. Scale bar=25 μm. (B) Load vs extension curves to point of failure of tibia from juvenile 6-week-old and adult 17-week-old, male and female WT (black) and Socs2^−/− (grey hatched) mice.
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   Figure 2

   SOCS2 regulation of systemic IGF1. (A) Transcript analysis of Igf1 and Igfbp3 in liver samples of 6-week-old male and female WT and Socs2^−/− mice. Data represented relative to sex-matched WT as mean±s.e.m. (n=5). Significance from WT samples denoted by ^aP<0.05. (B) Graphs showing WT and Socs2^−/− weight gain in response to twice daily GH (3 mg/kg) from 14 to 27 days of age. Data presented as mean±s.e.m. (n≥5). Significance from age-matched WT denoted by ^aP<0.05, ^bP<0.01, ^cP<0.001. (C) Western blotting analysis of phosphorylated (P−) STAT5 in 24-day-old WT (−GH (n=3); +GH (n=3)) and Socs2^−/− (−GH (n=3); +GH (n=4)) liver samples following 15 min GH (3 mg/kg) treatment. Band intensities were quantified by densitometry and analysed for significance, as depicted in the graphs. Data presented as mean±s.e.m. (n≥3). Significance denoted by ^aP<0.05, ^cP<0.001. (D) Transcript analysis of Igf1 in liver samples from 27-day-old WT and Socs2^−/− mice following 14 days twice daily GH (3 mg/kg) treatment. Data presented relative to untreated samples as mean±s.e.m. (n=5). (E) Protein analysis by ELISA of IGF1 levels in serum extracted from 27-day-old WT and Socs2^−/− mice following 14 days twice daily GH (3 mg/kg) treatment.
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   Figure 3

   SOCS2 regulation of GH-induced STAT signalling in osteoblasts and bone. (A) Transcript analysis of Socs1, Socs2 and Socs3 in WT osteoblasts following 24 h GH (500 ng/ml) or IGF1 (50 ng/ml) challenge. Transcript data represented relative to untreated samples as means±s.e.m. Significance denoted by ^cP<0.001. (B) Western blotting analysis of SOCS1, SOCS2 and SOCS3 in WT osteoblasts following 24 and 48 h GH (500 ng/ml) or IGF1 (50 ng/ml) treatment. (C) Western blotting analysis of phosphorylated (P−) STAT1, STAT3, and STAT5 in WT and Socs2^−/− osteoblasts challenged with GH (500 ng/ml) for up to 120 min. (D) Immunofluorescence detection of pSTAT5 in WT and Socs2^−/− osteoblasts following 20 min GH (500 ng/ml) challenge. (E) Analysis of (P−), STAT5 in SOCS2 overexpressing MC3T3 osteoblast-like cells and negative control-transfected cells following challenge with GH (500 ng/ml) for up to 120 min. (F) Immunofluorescence detection of pSTAT5 in SOCS2 overexpressing MC3T3 cells following 20 min GH (500 ng/ml) challenge. Scale bars for immunofluorescence represent 50 μm. Nucleus, blue; pSTAT5, pink. (G) Analysis of (P−) STAT5 in bone samples extracted from 24-day-old WT and Socs2^−/− mice following 15 min GH (3 mg/kg) treatment. Band intensities were quantified by densitometry and analysed for significance, as depicted in the graphs. Data presented as mean±s.e.m. (n=3). Significance denoted by ^bP<0.01.
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   Figure 4

   SOCS2 regulation of GH or IGF1-induced AKT and ERK1/2 signalling. (A) Western blotting analysis of phosphorylated (P−) AKT and (P−) ERK1/2 in WT and Socs2^−/− osteoblasts challenged with GH (500 ng/ml) or IGF1 (50 ng/ml) for up to 30 min. (B) Analysis of phosphorylated (P−) STAT3 and STAT5 in WT and Socs2^−/− osteoblasts challenged with IGF1 (50 ng/ml) for up to 30 min. c, GH-treated WT osteoblast acting as a positive control. (C) Analysis of phosphorylated (P−) AKT and ERK1/2 in bone samples extracted from WT and Socs2^−/− mice following 15 min GH (3 mg/kg) treatment. Band intensities were quantified by densitometry and analysed for significance, as depicted in the graphs. Data presented as mean±s.e.m. (n=3).
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   Figure 5

   SOCS2 regulation of GH-induced IGF1 expression. (A) Transcript analysis of Igf1, Igfbp3 and Socs2 in WT and Socs2^−/− osteoblasts following 48 h GH (500 ng/ml) treatment. (B) Protein analysis of IGF1 and IGFBP3 from conditioned medium from WT and Socs2^−/− osteoblasts following 48 h GH (500 ng/ml) treatment. (C) Transcript analysis of Igf1 and Igfbp3 in bone samples extracted from 27-day-old male WT and Socs2^−/− mice following 14 days twice daily GH (3 mg/kg) treatment. (D) Transcript analysis of Igf1 and Igfbp3 in bone samples extracted from 6-week-old male and female WT and Socs2^−/− mice. All data represented as means±s.e.m. Significance from vehicle-treated samples denoted by ^aP<0.05, ^cP<0.001.

• © 2014 The authors