Loss of Gsα in the Postnatal Skeleton Leads to Low Bone Mass and a Blunted Response to Anabolic Parathyroid Hormone Therapy

Abstract

Parathyroid hormone (PTH) is an important regulator of osteoblast function and is the only anabolic therapy currently approved for treatment of osteoporosis. The PTH receptor (PTH1R) is a G protein-coupled receptor that signals via multiple G proteins including Gsα. Mice expressing a constitutively active mutant PTH1R exhibited a dramatic increase in trabecular bone that was dependent upon expression of Gsα in the osteoblast lineage. Postnatal removal of Gsα in the osteoblast lineage (P-Gsα(OsxKO) mice) yielded markedly reduced trabecular and cortical bone mass. Treatment with anabolic PTH(1-34) (80 μg/kg/day) for 4 weeks failed to increase trabecular bone volume or cortical thickness in male and female P-Gsα(OsxKO) mice. Surprisingly, in both male and female mice, PTH administration significantly increased osteoblast numbers and bone formation rate in both control and P-Gsα(OsxKO) mice. In mice that express a mutated PTH1R that activates adenylyl cyclase and protein kinase A (PKA) via Gsα but not phospholipase C via Gq/11 (D/D mice), PTH significantly enhanced bone formation, indicating that phospholipase C activation is not required for increased bone turnover in response to PTH. Therefore, although the anabolic effect of intermittent PTH treatment on trabecular bone volume is blunted by deletion of Gsα in osteoblasts, PTH can stimulate osteoblast differentiation and bone formation. Together these findings suggest that alternative signaling pathways beyond Gsα and Gq/11 act downstream of PTH on osteoblast differentiation.

Keywords: G protein; G protein-coupled receptor (GPCR); PTH/PTH-related peptide receptor; osteoblast; osteoporosis; parathyroid hormone.

FIGURE 1.

G_sα is required for increase of trabecular bone by constitutively active PTH1R. Hematoxylin- and eosin-stained sections of proximal tibiae (A) and skeletal preparations of rib cages of 7-day-old control (WT), G_sα^OsxKO (KO), caPTH1R, or double mutant (caPTH1R; KO) mice are shown. C, H&E-stained sections of proximal tibiae of caPTH1R or double mutant (caPTH1R; KO) mice treated with doxycycline (doxy) from plug until 1 week of age. D, H&E-stained sections of proximal tibiae of caPTH1R or double mutant (caPTH1R; KO) mice treated with doxycycline from plug until 1 week of age and analyzed at 3 weeks of age. Images are representative of at least three mice per genotype.

FIGURE 2.

Postnatal deletion of G_sα leads to osteopenia and increased sclerostin expression. A, X-Gal staining for β-galactosidase activity in 6-week-old G_sα^OsxKO mice treated with doxycycline (Doxy) from plug until delivery (100× magnification). B, X-Gal staining of 6-week-old P-G_sα^OsxKO mice treated with doxycycline until 6 weeks of age (100×). C, survival frequency in control (WT no doxy; white bar; n = 18) and G_sα^OsxKO (KO no doxy; hatched bar; n = 8) mice without doxycycline treatment or in control (WT + doxy; gray bar; n = 7) or P-G_sα^OsxKO mice (KO + doxy; black bar; n = 10) on 10 μg/ml doxycycline from plug until birth. D, pregnant dams were administered 10 μg/ml doxycycline in drinking water from plug until delivery. The resulting mice were analyzed at 6 weeks of age. E, H&E-stained sections of proximal tibia from female 6-week-old mice treated with 10 μg/ml doxycycline until birth. F, bone mineral density (BMD) is significantly reduced in the humerus of P-G_sα^OsxKO mice. gm, gram. G, Gnas mRNA levels in femurs of WT and P-G_sα^OsxKO mice at 6 weeks. H, Sost mRNA levels in calvariae of 6-week-old mice. I, sclerostin immunostaining in cortical bone of WT and P-G_sα^OsxKO tibiae at 6 weeks of age. n = 5, WT; n = 6, P-G_sα^OsxKO. *, p < 0.05. Error bars represent S.E.

FIGURE 3.

Intermittent PTH does not increase trabecular bone mass in P-G_sα^OsxKO male and female mice. A, mice were treated with 10 μg (mcg)/ml doxycycline from plug until delivery. At 8 weeks of age, control (WT) and P-G_sα^OsxKO (KO) mice were injected with 80 μg/kg/day (d) PTH(1–34) or PBS 5 days/week (wk). H&E-stained sections of proximal humerus (B), μCT analysis of distal femur trabecular bone (C), BV/TV (D), Tb.N (E), Tb.Th (F), and Tb.Sp (G) of male (left) and female (right) mice are shown. n = 6–9 for each group of male mice, and n = 5–8 for each group of female mice. *, p < 0.05 versus respective PBS control; #, p < 0.05 versus respective WT control. Error bars represent S.E.

FIGURE 4.

Cortical thickness is decreased in P-G_sα^OsxKO male and female mice. μCT analysis of femoral midshaft cortical bone (A) and cortical thickness (C.Th) in WT and P-G_sα^OsxKO (KO) male (left) and female (right) mice treated with PBS and PTH is shown. n = 6–9 for each group of male mice, and n = 5–8 for each group of female mice. Serum levels of P1NP (C) and CTX (D) from male and female WT and P-G_sα^OsxKO mice treated with PBS and PTH are shown. n = 8–11 for each group of male mice, and n = 6–9 for each group of female mice. *, p < 0.05 versus respective PBS control; #, p < 0.05 versus respective WT control. Error bars represent S.E.

FIGURE 5.

PTH enhances osteogenic differentiation of G_sα-deficient osteoblasts. A, Alizarin red staining of G_sα(fl/fl) calvarial osteoblasts infected with adeno-β-gal (control) or adeno-Cre, then treated with PBS or PTH for 6 h of every 48 h, and subjected to osteogenic differentiation for 7 and 14 days. B, quantitation of mineralized nodule area at day 14. C, numbers of cells per well of G_sα(fl/fl) calvarial osteoblasts treated with adeno-β-gal or adeno-Cre and then with PBS or PTH. n = 3 experiments. D, mRNA expression levels of Runx2, Osx, ColIα1, Opn, and Bglap at day 7 in cells described above. Mean results from three replicate experiments are shown. *, p < 0.05 versus respective PBS control; #, p < 0.05 versus respective WT control. Error bars represent S.E.

FIGURE 6.

Intermittent PTH increases bone formation in DSEL mice. von Kossa-stained plastic sections (A) and H&E stained paraffin sections (B) from proximal tibiae of 16-week old WT and DSEL (D/D) mice treated with vehicle (VEH) or intermittent PTH (80 μg/kg/day) from 12 weeks of age. n = 7–9 for each group. C–F, μCT analysis of 16-week-old L5 vertebrae from WT and DSEL (D/D) mice treated with vehicle (PBS) or intermittent PTH (80 μg/kg/day) for the last 4 weeks. Tb.BV/TV (%) (C), Tb.N (1/mm) (D), Tb.Th (mm) (E), and Tb.Sp (1/mm³) (F) were measured in eight to nine animals of each group. Serum P1NP (G) and CTX (H) were measured in 16-week-old WT and DSEL (D/D) mice treated with vehicle (VEH) or intermittent PTH (80 μg/kg/day) for the last 4 weeks. n = 8 per group. *, p < 0.05 versus respective PBS control; #, p < 0.05 versus respective WT control. Error bars represent S.E.