Gsα enhances commitment of mesenchymal progenitors to the osteoblast lineage but restrains osteoblast differentiation in mice

Abstract

The heterotrimeric G protein subunit Gsα stimulates cAMP-dependent signaling downstream of G protein-coupled receptors. In this study, we set out to determine the role of Gsα signaling in cells of the early osteoblast lineage in vivo by conditionally deleting Gsα from osterix-expressing cells. This led to severe osteoporosis with fractures at birth, a phenotype that was found to be the consequence of impaired bone formation rather than increased resorption. Osteoblast number was markedly decreased and osteogenic differentiation was accelerated, resulting in the formation of woven bone. Rapid differentiation of mature osteoblasts into matrix-embedded osteocytes likely contributed to depletion of the osteoblast pool. In addition, the number of committed osteoblast progenitors was diminished in both bone marrow stromal cells (BMSCs) and calvarial cells of mutant mice. In the absence of Gsα, expression of sclerostin and dickkopf1 (Dkk1), inhibitors of canonical Wnt signaling, was markedly increased; this was accompanied by reduced Wnt signaling in the osteoblast lineage. In summary, we have shown that Gsα regulates bone formation by at least two distinct mechanisms: facilitating the commitment of mesenchymal progenitors to the osteoblast lineage in association with enhanced Wnt signaling; and restraining the differentiation of committed osteoblasts to enable production of bone of optimal mass, quality, and strength.

Figure 1. Deletion of G_sα in the osteoblast lineage leads to severe osteoporosis.

(A and B) Skeletal preparations of mice at P1 in WT (A) and G_sα^OsxKO (KO) mice (B). (C and D) H&E-stained sections of primary spongiosa from WT (C) and KO (D) tibiae at E18.5. (E and F) Von Kossa staining of WT (E) and KO (F) proximal tibiae at 1 week. (G and H) H&E-stained sections of WT (G) and KO (H) mid-diaphyseal tibiae at 2 weeks. (I) Bone volume over trabecular volume (BV/TV), trabecular thickness (TbTh), trabecular number (TbN), and trabecular spacing (TbSp) in WT (white) and KO (black) mice at 1 week. *P < 0.01 (n = 7 [WT] and n = 5 [KO]). Original magnification, ×200 (C and D), ×100 (E–H).

Figure 2. Decreased bone mass in the absence of G_sα is due to inadequate bone formation.

(A–D) Micro-CT of WT (A) and KO (B) femurs at 1 week. Cortical bone area (Ct.Ar) (C) and cortical area fraction (Ct.Ar/Tt.Ar) (D) at 1 week. *P < 0.05 (n = 5 [WT] and n = 3 [KO]). (E–H) H&E- (E and F) and TRAP-stained (G and H) sections of cortical bone from WT (E and G) and KO (F and H) tibiae at E18.5. Dashed yellow line denotes the endocortical edge of cortical bone. (I) Osteoclast surface per bone surface (OcS/BS, n = 7 [WT] and n = 5 [KO]). (J) Serum TRACP 5b levels (n = 6 per genotype). (K) Osteoblast number per bone perimeter (NOb/BPm). *P < 0.001 (n = 7 [WT] and n = 5 [KO]). (L) Serum osteocalcin levels. *P < 0.01 (n = 6 per genotype). (M and N) Double calcein labeling in WT (M) and KO (N) cortical bone at week 1. (O and P) Cortical bone from WT (O) and KO (P) mice at 2 weeks stained with Sirius red and photographed under polarized light. Scale bars: 100 μm (A and B). Original magnification, ×200 (E–H, M, and N), ×400 (O and P).

Figure 3. Intramembranous bone formation is defective in G_sα^OsxKO mice.

(A and B) Skeletal preparations of calvariae of WT (A) and KO (B) mice at P1. p, parietal bone; s, sagittal suture; l, lambdoid suture. (C–F) H&E-stained calvarial sections at P4 (C and D) and 2 weeks (E and F) in WT (C and E) and KO (D and F) mice. (G and H) Sirius red–stained sections of WT (G) and KO (H) calvarial bone at 2 weeks photographed under polarized light. (I) Cortical porosity void area (VD.Ar/T.Ar) in WT and KO mice at 2 weeks of age. *P < 0.05 (n = 4 per genotype). Original magnification, ×400 (C–H).

Figure 4. Osteogenic differentiation is not impaired in G_sα^OsxKO mice.

(A) In situ hybridization for osteogenic markers ALP, collagen Iα1 (Col1α1), and osteocalcin (OC). (B and C) In situ hybridization for MMP-13 expression in WT (B) and KO (C) proximal tibiae at P7. (D) Mmp13 mRNA levels in G_sα^fl/fl calvarial osteoblasts infected with control adenovirus (β-gal) or Cre recombinase. *P < 0.05 (n = 5). (E) ALP staining and (F) alizarin staining of calvarial osteoblast cells undergoing osteogenic differentiation. Results are representative of 5 experiments. (G and H) Calvarial osteoblasts were harvested from control or KO mice crossed to R26 reporter mice, then subjected to osteogenic differentiation. After 21 days in culture, wells were stained with alizarin to highlight mineral deposition and X-gal to identify cells descended from Osx⁺ precursors in control (G) and KO (H) mice. Original magnification, ×100 (A, E15.5 and P1), ×40 (A, P9), ×400 (G and H).

Figure 5. Osteogenic differentiation is accelerated in the absence of G_sα.

(A–C) Alizarin staining (A and B) and frequency of Col2.3::GFP^hi cells in Col2.3::GFP;G_sα^fl/fl calvarial cells infected with adeno-Cre or adeno–β-gal and subjected to osteogenic differentiation (C). *P < 0.05 (n = 3). (D) Frequency of Col2.3::GFP⁺ cells in sorted ALP^– cells from Col2.3::GFP;G_sα^fl/fl mice subjected to osteogenic differentiation. *P < 0.05 (n = 4). (E) G_sα^fl/fl BMSCs infected with adeno-β-gal or adeno-Cre and induced to undergo osteogenic differentiation, then stained with ALP or alizarin after 2 weeks in culture. (F) Frequency of Col2.3::GFP⁺ cells in BMSCs from Col2.3::GFP;G_sα^fl/fl mice after 2 weeks of osteogenic differentiation. *P < 0.02 (n = 3). (G) mRNA levels for G_sα, ALP, collagen Iα1 (Col1a1), Runx2, Osx, and Atf4 in G_sα^fl/fl calvarial osteoblasts infected with adeno-Cre or adeno–β-gal and induced to undergo osteogenic differentiation for 6 days. *P < 0.05 (n = 3–5 experiments).

Figure 6. Accelerated osteogenic differentiation occurs in vivo in G_sα^OsxKO mice.

(A and B) X-gal staining of cortical bone at P7 from WT (A) and KO (B) mice crossed to R26 reporter mice. (C and D) H&E-stained sections of cortical bone from tibiae of WT (C) and KO (D) mice at P7. (E) Osteocyte density in cortical bone of tibiae at P7. *P < 0.005 (n = 3). (F) BrdU pulse-chase in WT and KO trabecular bone of tibiae at P4. White arrows indicate BrdU-negative osteocytes; red arrows indicate BrdU-positive osteocytes. (G) Frequency of BrdU⁺ osteocytes and osteoblasts 4 and 24 hours after injection. *P < 0.05 (n = 3). (H and I) mRNA levels of osteocyte markers Dmp1 and Phex in G_sα^fl/fl calvarial osteoblasts (H) (n = 6) and bone marrow stromal cells (I) (n = 3) infected with adeno-Cre (black) or adeno–β-gal (white) and induced to undergo osteogenic differentiation for 1 (H) or 2 (I) weeks. *P < 0.05. Original magnification, ×400 (A–D), ×1,000 (F).

Figure 7. Accelerated osteoblast maturation in G_sα^OsxKO mice is associated with woven bone and abnormal osteocytes.

(A and B) Anti-E11 immunohistochemical staining in cortical bone of WT (A) and KO (B) tibiae at week 3. (C and D) Bodian staining of the osteocyte lacuno-canalicular network in WT (C) and KO (D) mice at week 3. (E and F) Toluidine blue staining of osteocytes in WT (E) and KO (F) cortical bone at week 2. (G and H) Transmission electron microscopy of WT (G) and KO (H) osteocytes. (I) mRNA level of BSP in differentiating G_sα^fl/fl calvarial osteoblasts. *P < 0.05 (n = 7). Original magnification, ×400 (A, B, E, and F), ×1,000 (C and D), ×3,400 (G and H).

Figure 8. G_sα^OsxKO mice have reduced Wnt signaling in the osteoblast lineage and a depleted osteoprogenitor pool.

(A and B) CFU-F and CFU-ALP assays in WT and KO BMSCs (A) and calvarial cells (B). *P < 0.01 (n = 6–9). (C) Sost mRNA levels in calvariae of WT and KO mice. *P < 0.01 (n = 6). (D and E) Immunohistochemical staining for sclerostin protein in WT (D) and KO (E) tibia at week 3. (F) mRNA levels of Sost and Dkk1 and (G) Wnt target genes Axin2 and Lef1 in G_sα^fl/fl calvarial osteoblasts infected with adeno-Cre (black) or adeno–β-gal (white) and induced to undergo osteogenic differentiation. *P < 0.05 (n = 3). (H) TopGal mRNA levels in calvariae of WT and KO mice crossed to TopGal reporter mice. *P < 0.05 (n = 3). Original magnification, ×400 (D and E).