DMP-1-mediated Ghr gene recombination compromises skeletal development and impairs skeletal response to intermittent PTH

Abstract

Bone minerals are acquired during growth and are key determinants of adult skeletal health. During puberty, the serum levels of growth hormone (GH) and its downstream effector IGF-1 increase and play critical roles in bone acquisition. The goal of the current study was to determine how bone cells integrate signals from the GH/IGF-1 to enhance skeletal mineralization and strength during pubertal growth. Osteocytes, the most abundant bone cells, were shown to orchestrate bone modeling during growth. We used dentin matrix protein (Dmp)-1-mediated Ghr knockout (DMP-GHRKO) mice to address the role of the GH/IGF axis in osteocytes. We found that DMP-GHRKO did not affect linear growth but compromised overall bone accrual. DMP-GHRKO mice exhibited reduced serum inorganic phosphate and parathyroid hormone (PTH) levels and decreased bone formation indices and were associated with an impaired response to intermittent PTH treatment. Using an osteocyte-like cell line along with in vivo studies, we found that PTH sensitized the response of bone to GH by increasing Janus kinase-2 and IGF-1R protein levels. We concluded that endogenously secreted PTH and GHR signaling in bone are necessary to establish radial bone growth and optimize mineral acquisition during growth.

Keywords: fibroblast growth factor-23; growth hormone receptor; microcomputed tomography; osteocyte; parathyroid hormone.

Figure 1.

DMP-GHRKO mice had normal body weight. A) Control and DMP-GHRKO mice of both sexes were weighed weekly from 3 to 16 wk of age (n > 10 mice per age, sex, and genotype). B) Serum IGF-1 levels were measured at 4, 8, and 16 wk of age in male and female control and DMP-GHRKO mice (n ≥ 6 mice per age, sex, and genotype, tested by 2-way ANOVA). C) Serum GH levels were measured at 8 and 16 wk of age in control and DMP-GHRKO female mice (male serum GH levels were not different between control and DMP-GHRKO mice; data not shown) (n ≥ 7 mice per age and genotype). NS, not significant. D) Representative micro-CT images of femurs from 16-wk-old male and female mice. Data are presented as means ± sd. *P < 0.05; Student’s t test.

Figure 2.

DMP-GHRKO mice exhibited impaired mineral metabolism. A) Serum P_i levels were measured in female control and DMP-GHRKO mice at 8 wk of age (n ≥ 10 mice per genotype). B) Serum Ca⁺² levels were measured in 8-wk-old female control and DMP-GHRKO mice (n ≥ 10 mice per genotype). C) P_i excretion in urine samples was measured in female mice between 4–5 wk of age and corrected to creatinine (n ≥ 10 mice per genotype). D) Gene expression was measured in the cortical bones of 8-wk-old control and DMP-GHRKO mice (n = 5 mice per genotype). E) Serum PTH levels were measured at 4, 8, 16, and 24 wk of age in control and DMP-GHRKO female mice. F) Serum OC levels were measured at 8 and 16 wk of age in control and DMP-GHRKO mice. G) Variation in PTH and OC levels in control and DMP-GHRKO mice at 8 and 16 wk of age. There was a linear relationship between PTH and OC in control mice, but not in DMP-GHRKO mice. H) Serum SOST levels in 8-wk-old control (floxed Ghr), DMP-GHRKO, GHR-null (GHRKO), and wild-type (C57Bl6) female mice (tested by 2-way ANOVA with Tukey’s post hoc test). Data are means ± sem. *P < 0.05; (A–F) Student’s t-test.

Figure 3.

DMP-GHRKO mice had an impaired bone response to iPTH. Female mice were injected once daily with 80 μg/kg PTH from 4 to 8 wk of age. A) Serum FGF23 levels in female control and the DMP-GHRKO mice treated with iPTH or vehicle from 4 to 8 wk. B) Serum 1,25-vitamin D levels in female control and DMP-GHRKO mice treated with iPTH or vehicle from 4 to 8 wk. C) Femurs were dissected at 8 wk of age and analyzed with micro-CT (the detailed traits are shown in Table 5). Fold change in bone traits obtained by micro-CT (in response to iPTH). The traits obtained from mice treated with PBS were used as the baseline. D) Micro-CT images of control and DMP-GHRKO bones from mice treated with PBS or iPTH. E) Gene expression in cortical bones from control and DMP-GHRKO female mice treated with PBS or iPTH from 4 to 8 wk of age. NS, not significant. Data are presented as means ± sem. *P < 0.05.

Figure 4.

PTH sensitizes IDG-SW3 osteocyte-like cells to GH. IDG-SW3 cells were seeded on collagen-coated plates and grown to 70% confluence (21 d) in differentiation medium. A–C) At 21 d in culture IDG-SW3 cells exhibited increased fluorescence (A) increased Alk-Phos staining (B), and increased Von Kossa staining (C). D) GH treatment induced STAT5b and JAK2 phosphorylation in IDG-SW3 cells in a dose-dependent manner. Cultures were serum starved overnight and stimulated with GH for 10 min. Phospho-STAT5b and phospho-JAK2 were detected by Western blot analysis. E) PTH sensitized IDG-SW3 cells to GH. Cultures were stimulated with the indicated concentrations of PTH, and 18 h later were stimulated with 50 ng/ml GH for 10 min. Phospho-STAT5b was detected by Western blot analysis. F) GH-induced STAT5 phosphorylation increased 16 h after the addition of PTH to IDG-SW3 cells. Cultures were stimulated with the indicated concentrations of PTH followed by 50 ng/ml GH for 10 min at 4, 8, and 16 h. Phospho-STAT5b was detected by Western blot analysis. G) The augmentation of GH-induced STAT5 phosphorylation by PTH was inhibited by CHX. Cultures were treated with 10⁻¹⁰ M PTH and 16 h later were stimulated with 50 ng/ml GH for 10 min in the presence or absence of CHX. Phospho (P)-STAT5b, total (T)-STAT5b, JAK2, and GHR were detected by Western blot analysis. Data are presented as means ± sem of 4 independent experiments. *P < 0.05.

Figure 5.

PTH sensitizes bone to GH in vivo. A) Four-week-old control female mice were treated with PTH (80 μg/kg/d) for 3–4 wk, left unfed overnight, injected with 0.25 mg/kg mGH, and euthanized 15 min thereafter. STAT5b phosphorylation (P-STAT5b) and total STAT5b (T-STAT5b), levels were detected by Western blot analysis. Data are presented as means ± SEM; *P < 0.05; ANOVA with Tukey’s post hoc test. B) IGF-1R and JAK2 protein levels were detected in bone protein extracts from female mice treated as described above using Western blot analysis. Four-week-old DMP-GHRKO mice were treated with iPTH (80 μg/kg/d) for 3–4 wk. NS, not significant. Data are presented as means ± sem. *P < 0.05; Student’s t test. C) DMP-GHRKO (GH resistance) in osteoblasts and osteocytes was associated with impaired trabecular and cortical bone accrual during growth. The mechanism may be related to increased skeletal FGF23 from DMP-1-mediated Ghr gene recombination (DMP-GHRKO) (a) or reductions in serum P_i levels and increased urine P_i excretion (b). Likewise, GH resistance in bone was associated with increased Sost gene expression in bone (c) and elevated serum SOST (d). Changes in FGF23 and SOST may contribute directly or indirectly to a blunted serum PTH level during pubertal growth (e). This decrease in turn can result in blunted intestinal absorption of Ca²⁺ and P_i (f) in the DMP-GHRKO mice because of the inability of 1,25 OHD to respond to PTH, possibly from changes in FGF23.