Research ArticleDISEASES AND DISORDERS

Regulation of body length and bone mass by Gpr126/Adgrg6

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Science Advances  20 Mar 2020:
Vol. 6, no. 12, eaaz0368
DOI: 10.1126/sciadv.aaz0368
  • Fig. 1 Body length was decreased and embryonic bone formation was delayed in Osx-Cre;Gpr126fl/fl mice.

    (A) Images of body size at embryonic (E) day 14.5 (E14.5), E16.5, and E18.5 and postnatal (P) day 30 (P30) of Osx-Cre;Gpr126fl/fl mice and control (Ctrl) littermates. Scale bars, 5 mm. (B) Whole skeletal preparation of Osx-Cre;Gpr126fl/fl mice and Ctrl littermates at E14.5, E16.5, and E18.5. Black arrows indicate the delayed Alizarin red staining (of bone) in the skull, ribs, and phalanges in Osx-Cre;Gpr126fl/fl embryos. Scale bars, 5 mm. (C) Body part skeletal preparation of Osx-Cre;Gpr126fl/fl mice and Ctrl littermates at E18.5. Black arrows indicate the delayed Alizarin red staining (of bone) in the skull (top), sternum (middle), and phalanges (bottom) in Osx-Cre;Gpr126fl/fl embryos. Scale bars, 2 mm. (D) In situ hybridization analysis for expression of osteoblast differentiation markers collagen type I, alpha 1 (Col1a1) (top) and osteocalcin (Ocn) (bottom) in Osx-Cre;Gpr126fl/fl and Ctrl littermate femurs at E14.5, E15.5, and E16.5. Red arrows indicate that the signal intensity was decreased in Osx-Cre;Gpr126fl/fl embryos. Scale bars, 200 μm. (E) Relative positive area of Col1a1 and Ocn in Osx-Cre;Gpr126fl/fl and control littermate femurs at E14.5, E15.5, and E16.5 from in situ hybridization assay. **P < 0.01. n = 2 per group per time point. (F) Von Kossa staining analysis for bone mineralization in E14.5 (left), E16.5 (middle), and E18.5 (right) embryonic femurs of Osx-Cre;Gpr126fl/fl and Ctrl littermates. Red arrows indicate that there was no signal at E14.5 in Osx-Cre;Gpr126fl/fl embryos. Scale bars, 100 μm (at E14.5, left), 50 μm (at E14.5, right), and 1 mm (at E16.5 and E18.5). Photo credit: Peng Sun, East China Normal University.

  • Fig. 2 Postnatal bone mass and bone strength were decreased in Osx-Cre;Gpr126fl/fl mice.

    (A) Representative images of dorsal (top) and lateral (bottom) x-rays of 4-month-old mice (n = 3). Yellow lines indicate that the vertebral column was shorter in Osx-Cre;Gpr126fl/fl mice; yellow arrows indicate the decreased bone density in Osx-Cre;Gpr126fl/fl mice. Scale bars, 2 cm. (B) Representative μCT images of femurs from 1-month-old mice show the proximal femur (top; scale bars, 500 μm) and trabecular bone of the femur metaphysis (bottom; scale bars, 200 μm). (C) Quantitative μCT analysis of trabecular bone parameters of femurs from 1-month-old mice. BMD, bone mineral density; BV/TV, bone-volume/tissue-volume ratio; Tb.N, trabecular number; Tb.Sp, trabecular separation; Tb.Th, trabecular thickness. *P < 0.05, **P < 0.01, ***P < 0.001. n = 5. (D) Maximal loading (Max load) of humeral diaphysis from 1-month-old mice by three-point bending assay. n = 5. (E) Representative image of von Kossa staining of lumbar sections of 6-week-old mice (top) and trabecular bone parameters (bottom). Scale bars, 500 μm. n = 7. (F) Bone formation rate was decreased in Osx-Cre;Gpr126fl/fl mice. Representative images of calcein double labeling of the spinal trabecular bone of 6-week-old mice (top). Bone formation parameters from spinal sections of 6-week-old mice (bottom). BFR/BS, bone formation rate per bone surface; MAR, mineral apposition rate. Scale bars, 10 μm. *P < 0.05, **P < 0.01. n = 7. (G) Osteoid formation was suppressed in Osx-Cre;Gpr126fl/fl mice. Representative images of Goldner’s staining of 6-week-old mouse spinal trabecular bone. Spinal bone histomorphometric parameters. Ob.S/BS, osteoblast surface per bone surface; OS/BS, osteoid per bone surface; O.Th, osteoid thickness. Scale bars, 50 μm. *P < 0.05. n = 7.

  • Fig. 3 Gpr126 regulates osteoblast proliferationdifferentiation, and mineralization.

    (A and B) Bone marrow stromal cell (BMSC) proliferation and differentiation were inhibited in Gpr126-deficient osteoblasts as determined by colony-forming unit (CFU) assay. BMSCs from 1-month-old Osx-Cre;Gpr126fl/fl mice and Ctrl littermates were cultured for 14 days and then subjected to crystal violet staining (CFU-F, left) or Alizarin red staining (CFU-Ob, right). Representative images are shown (A). The number (n) of colonies per well for CFU-F and CFU-Ob was counted (B). Scale bars, 10 mm. *P < 0.05. n = 3. (C) BMSC differentiation and mineralization were suppressed in Gpr126 deletion osteoblasts. BMSCs were isolated from 1-month-old Osx-Cre;Gpr126fl/fl mice and Ctrl littermates and subjected to ALP staining (7 day), von Kossa staining (14th day), and Alizarin red staining (21st day) assays. Scale bars, 5 mm. (D) Gpr126 KO inhibited ALP enzyme activity (n = 5) and Ocn relative mRNA expression (n = 2) in osteoblasts. BMSCs were isolated from 1-month-old Osx-Cre;Gpr126fl/fl mice and Ctrl littermates and differentiated into osteoblasts. The cells were harvested at days 7 and 14 of differentiation for ALP enzyme activity assay and Ocn mRNA quantitation by real-time PCR, respectively. *P < 0.05, ***P < 0.001. n = 5.

  • Fig. 4 Gpr126 regulates osteoblast differentiation and function through the cAMP-CREB signaling pathway.

    (A) Intracellular cAMP level was decreased in Gpr126 deletion osteoblasts. BMSCs were isolated from 1-month-old Osx-Cre;Gpr126fl/fl mice and Ctrl littermates and differentiated into osteoblasts. The cells were harvested and subjected to the cAMP enzyme-linked immunosorbent assay (ELISA) assay at day 7 of differentiation. **P < 0.01. n = 3. (B) Western blot analysis of the p-CREB, CREB, and OCN levels in BMSCs from indicated mice after 14 days of osteoblast differentiation. (C) Intracellular cAMP level was restored in Gpr126 deletion osteoblasts when treated with FSK. BMSCs were isolated from 1-month-old Osx-Cre;Gpr126fl/fl mice and Ctrl littermates and differentiated into osteoblasts. The cells were treated with 10 μM FSK or vehicle control for 14 days, harvested, and subjected to the cAMP ELISA assay at day 14 of differentiation. **P < 0.01, ***P < 0.001. n = 3. (D) ALP enzyme activity (N = 5), ALP mRNA expression (n = 2), and OCN mRNA expression (n = 2) were restored in Gpr126 deletion osteoblasts treated with 1 μM FSK. The cells were harvested and subjected to ALP enzyme activity assay (at day 14) or real-time PCR assay (ALP at day 7 and OCN at day14). **P < 0.01, ***P < 0.001. (E) Osteoblast differentiation and mineralization were rescued in Gpr126 deletion osteoblasts when treated with FSK. BMSCs were isolated from 1-month-old Osx-Cre;Gpr126fl/fl mice and control (Ctrl) littermates and differentiated into osteoblasts. ALP staining, von Kossa staining, and Alizarin red staining of BMSCs were performed after 7, 14, and 21 days of differentiation, respectively, while treated with or without 10 μM FSK. Scale bars, 5 mm. (F) ALP enzyme activity (N = 5), Alp mRNA expression (n = 2), and Ocn mRNA expression (n = 2) were restored in Gpr126 deletion osteoblasts treated with PTH(1–34). The cells were harvested and subjected to ALP enzyme activity assay (at day 14) or real-time PCR assay (Alp at day 7 and Ocn at day14). **P < 0.01, ***P < 0.001. (G) Osteoblast differentiation and mineralization were rescued in Gpr126 deletion osteoblasts when treated with PTH(1–34). BMSCs were isolated from 1-month-old Osx-Cre;Gpr126fl/fl mice and Ctrl littermates and differentiated into osteoblast. ALP staining, von Kossa staining, and Alizarin red staining of BMSCs were performed after 7, 14, and 21 days of differentiation, respectively, while treated with or without PTH(1–34) (80 μg/kg). Scale bars, 5 mm.

  • Fig. 5 COLIV is an activating ligand of Gpr126 to regulate osteoblast differentiation and mineralization.

    (A) COLIV stimulated cAMP production in Ctrl osteoblast but not in Gpr126 deletion osteoblasts. COLIV (1 μM) was coated on 24-well plates, and then the BMSCs were seeded for differentiation. After 14 days, the cells were harvested and subjected to cAMP ELISA assay. **P < 0.01, ***P < 0.001. ns, no significant difference. n = 3. (B) COLIV-induced ALP enzyme activity (n = 3) and OCN mRNA expression (n = 2) in Ctrl osteoblasts but not in Gpr126 deletion osteoblasts. ns, not significant; *P < 0.05; ***P < 0.001. (C) COLIV-stimulated osteoblast differentiation and mineralization in Osx-Cre Ctrl (Ctrl) osteoblasts but not in Gpr126 deletion osteoblasts. ALP staining, von Kossa staining, and Alizarin red staining of BMSCs were performed after 7, 14, and 21 days of differentiation, respectively, while treated with or without COLIV. Scale bars, 5 mm.

  • Fig. 6 The reduction of body length, bone mass, and bone strength in Osx-Cre;Gpr126fl/fl mice was partly rescued by PTH treatment.

    Osx-Cre;Gpr126fl/fl (CKO) mice and Ctrl littermates (n = 6 mice per group) were injected daily with PBS or PTH(1–34) (80 μg/kg) from P5 to P30. The mice were then sacrificed for body length, bone length, bone mass, and bone strength analysis. (A and B) Representative images of Osx-Cre;Gpr126fl/fl (CKO) mice and Ctrl littermates treated with PBS or PTH(1–34) (80 μg/kg) (A). The body length was measured (B). Scale bars, 2 cm. *P < 0.05; **P < 0.01. n = 6. (C and D) Representative femur bone images of Osx-Cre;Gpr126fl/fl (CKO) mice and Ctrl littermates treated with or without PTH(1–34) (80 μg/kg) (C). The femur bone length was measured (D). Scale bars, 2 cm. *P < 0.05. n = 6. (E and F) Bone mass was restored in Osx-Cre;Gpr126fl/fl mice when treated with PTH. Representative μCT images of femurs from 1-month-old Osx-Cre;Gpr126fl/fl mice and Ctrl littermates treated with PTH(1–34). The proximal femur (top) and trabecular bone of the femur metaphysis (bottom) are presented (E). Quantitative μCT analysis of femoral trabecular bone parameters (F). Scale bars, 500 μm (E, top) and 200 μm (E, bottom). *P < 0.05; **P < 0.01. n = 6. (G) Maximal loading of humeral diaphysis from 1-month-old mice by three-point bending assay. *P < 0.05; **P < 0.01. n = 6. Photo credit: Liang He, East China Normal University.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/6/12/eaaz0368/DC1

    Fig. S1. Strategy of targeting the Gpr126flox allele.

    Fig. S2. The expression of Gpr126 in different bone cells and genotyping.

    Fig. S3. Body length and embryonic bone formation in Lysm-Cre;Gpr126fl/fl and Col2-Cre;Gpr126fl/fl was not different compared to their control littermates.

    Fig. S4. Deletion of Gpr126 in osteoblast lineage (Osx-Cre) had little effect on osteoclastogenesis and osteoclast activity in vivo and in vitro.

    Fig. S5. Deletion of Gpr126 in osteoblast lineage (Osx-Cre) had little effect on chondrocyte differentiation and hypertrophy.

    Fig. S6. The expression of COLIV and Laminin-211 in osteoblast, osteoclast, and chondrocyte cells.

    Fig. S7. Laminin-211 was not an activating ligand of Gpr126 to regulate osteoblast differentiation and mineralization under static conditions.

    Fig. S8. The selective Wnt/β-catenin inhibitor KYA1797K had little effect on COLIV-induced osteoblast differentiation and mineralization.

    Fig. S9. Administration of FSK had little effect on the body length, femur bone length, bone mass, and bone strength of Osx-Cre;Gpr126fl/fl mice.

    Fig. S10. The expression of IL-6 was increased in chondrocytes treated by FSK.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Strategy of targeting the Gpr126flox allele.
    • Fig. S2. The expression of Gpr126 in different bone cells and genotyping.
    • Fig. S3. Body length and embryonic bone formation in Lysm-Cre;Gpr126fl/fl and Col2-Cre;Gpr126fl/fl was not different compared to their control littermates.
    • Fig. S4. Deletion of Gpr126 in osteoblast lineage (Osx-Cre) had little effect on osteoclastogenesis and osteoclast activity in vivo and in vitro.
    • Fig. S5. Deletion of Gpr126 in osteoblast lineage (Osx-Cre) had little effect on chondrocyte differentiation and hypertrophy.
    • Fig. S6. The expression of COLIV and Laminin-211 in osteoblast, osteoclast, and chondrocyte cells.
    • Fig. S7. Laminin-211 was not an activating ligand of Gpr126 to regulate osteoblast differentiation and mineralization under static conditions.
    • Fig. S8. The selective Wnt/β-catenin inhibitor KYA1797K had little effect on COLIV-induced osteoblast differentiation and mineralization.
    • Fig. S9. Administration of FSK had little effect on the body length, femur bone length, bone mass, and bone strength of Osx-Cre;Gpr126fl/fl mice.
    • Fig. S10. The expression of IL-6 was increased in chondrocytes treated by FSK.

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