Protection From Glucocorticoid-Induced Osteoporosis by Anti-Catabolic Signaling in the Absence of Sost/Sclerostin

Abstract

Excess of glucocorticoids, either due to disease or iatrogenic, increases bone resorption and decreases bone formation and is a leading cause of osteoporosis and bone fractures worldwide. Improved therapeutic strategies are sorely needed. We investigated whether activating Wnt/β-catenin signaling protects against the skeletal actions of glucocorticoids, using female mice lacking the Wnt/β-catenin antagonist and bone formation inhibitor Sost. Glucocorticoids decreased the mass, deteriorated the microarchitecture, and reduced the structural and material strength of bone in wild-type (WT), but not in Sost^-/- mice. The high bone mass exhibited by Sost^-/- mice is due to increased bone formation with unchanged resorption. However, unexpectedly, preservation of bone mass and strength in Sost^-/- mice was due to prevention of glucocorticoid-induced bone resorption and not to restoration of bone formation. In WT mice, glucocorticoids increased the expression of Sost and the number of sclerostin-positive osteocytes, and altered the molecular signature of the Wnt/β-catenin pathway by decreasing the expression of genes associated with both anti-catabolism, including osteoprotegerin (OPG), and anabolism/survival, such as cyclin D1. In contrast in Sost^-/- mice, glucocorticoids did not decrease OPG but still reduced cyclin D1. Thus, in the context of glucocorticoid excess, activation of Wnt/β-catenin signaling by Sost/sclerostin deficiency sustains bone integrity by opposing bone catabolism despite markedly reduced bone formation and increased apoptosis. This crosstalk between glucocorticoids and Wnt/β-catenin signaling could be exploited therapeutically to halt resorption and bone loss induced by glucocorticoids and to inhibit the exaggerated bone formation in diseases of unwanted hyperactivation of Wnt/β-catenin signaling. © 2016 American Society for Bone and Mineral Research.

Keywords: CORTICOSTEROIDS; GENETIC ANIMAL MODELS; MOLECULAR PATHWAYS - REMODELING; OSTEOPOROSIS; WNT/β-CATENIN/LRPS.

Fig. 1.

Sost^−/− mice are protected from bone loss and decreased bone strength induced by glucocorticoids. (A) Percent change in BMD for WT or Sost^−/− mice treated with placebo, 1.4 mg/kg/day prednisolone (GC1), or 2.1 mg/kg/day prednisolone (GC2) for 28 days, measured by DXA. n = 7–10. (B) Representative reconstructed μCT images of L₆ vertebral cancellous bone (3D, top row) and cortical bone (2D, lower row). The dorsal surface of cortical bone for which thickness was also quantified is indicated by the gray boxes. (C) BV/TV, Tb.Th, Tb.Sp, and Tb.N, cortical BA/TA, total Ct.Th, and thickness of the dorsal surface of cortical bone (dorsal Ct.Th) are shown. n = 7–10. (D) Biomechanical properties were measured in vertebral bone (L₆) by axial compression testing; n = 6–9. Bars represent means ± SD. *p < 0.05 versus corresponding placebo-treated mice and #p < 0.05 versus placebo-treated WT mice, by two-way ANOVA; §p < 0.05 versus corresponding placebo-treated mice by one-way ANOVA, Tukey post hoc test. BV/TV = bone volume/tissue volume; Tb.Th = trabecular thickness; Tb.Sp = trabecular separation; Tb.N = trabecular number; BA/TA = bone area/total area; Ct.Th = thickness of cortical bone.

Fig. 2.

Sost/sclerostin deficiency does not prevent glucocorticoid-induced sarcopenia. Final and percent change in lean body mass (A), the mass of the indicated muscles (B), and final and percent change in total body weight (C) are shown; n = 8–10. Bars represent means ± SD. *p < 0.05 versus corresponding placebo-treated mice and #p < 0.05 versus placebo-treated WT mice by two-way ANOVA, Tukey post hoc test.

Fig. 3.

Sost/sclerostin deficiency does not prevent glucocorticoid-induced decrease in bone formation or increase in osteoblast/osteocyte apoptosis. (A) Representative images of fluorochrome incorporation and dynamic histomorphometric data measured in longitudinal sections of lumbar vertebra (L₁–L₃). MS/BS, MAR, and BFR/BS are shown. Scale bars = 50 μm, n = 8–10. P1NP, n = 6–10 (B), and alkaline phosphatase, n = 7–10 (C) were measured in blood collected 2 or 4 weeks after pellet implantation. (D) OCN gene expression was quantified by qPCR in L₅ lumbar vertebra and femur. n = 7–10 for WT and n = 6–8 for Sost^−/− mice. mRNA levels were normalized to the housekeeping gene Rplp2. (E) Apoptosis of Ob and Ot was quantified in cancellous and cortical bone in longitudinal sections of lumbar vertebrae (L₁–L₃) stained for TUNEL. n = 6. Representative images of cortical bone are shown. Scale bar = 20 μm. (F) Quantification of mineralization in cultures of calvaria-derived primary osteoblastic cells from WT or Sost^−/− mice treated with veh or dex for 1 or 2 weeks stained using the OsteoImage Mineralization Assay Kit (RFU 492 nm/520 nm excitation/emission fluorescence). Scale bars = 400 μm. n = 7–8. Bars represent means ± SD. (A–E) *p < 0.05 versus corresponding placebo-treated mice and #p < 0.05 versus placebo-treated WT mice by two-way ANOVA, Tukey post-hoc test. (F) *p < 0.05 versus corresponding vehicle-treated primary osteoblasts by Student’s t test. MS/BS = mineralizing bone surface per bone surface; MAR = mineral apposition rate; BFR/BS = bone formation rate per bone surface; OCN = Osteocalcin; Rplp2 = ribosomal protein large P2; Ob = osteoblasts; Ot = osteocytes; veh = vehicle; dex = dexamethasone.

Fig. 4.

Sost^−/− mice are protected from the increase in bone resorption induced by glucocorticoid. (A) Representative microscopy images of osteoclasts on cancellous bone surface in lumbar vertebra (L₂) stained for TRAPase. N.Oc/BS and Oc.S/BS normalized to bone surface were measured. Scale bars = 30 μm, n = 5. CTX, n = 6–11 (B) and TRAP 5b, n = 6–7 (C) were measured in blood collected 2 and 4 weeks after pellet implantation. Bars represent means ± SD. *p < 0.05 versus corresponding placebo-treated mice and #p < 0.05 versus placebo-treated WT mice by two-way ANOVA, Tukey post hoc test. N.Oc/BS = osteoclast number; Oc.S/BS = osteoclast surface.

Fig. 5.

Glucocorticoids differentially alter Wnt/β-catenin signaling in a Sost/sclerostin–dependent manner. (A) Sost gene expression was quantified by qPCR in L₅ lumbar vertebra. n = 7–10 for WT and n = 6–8 for Sost^−/−. (B) The percentage of sclerostin-positive osteocytes was quantified in longitudinal sections of femoral bone stained with an anti-sclerostin antibody and is shown as percentage ± SD, n = 3–4. Scale bars = 50 μm. (C) Expression of the indicated genes normalized to the housekeeping gene Rplp2 in femur and lumbar vertebra (L₅). n = 7–10 for WT and n = 6–8 for Sost^−/−. Bars represent means ± SD. *p < 0.05 versus corresponding placebo-treated mice and #p < 0.05 versus placebo-treated WT mice by two-way ANOVA and Tukey post hoc test. §p < 0.05 versus placebo-treated WT mice, by one-way ANOVA and the Tukey post hoc test.

Fig. 6.

Genetic or pharmacologic inhibition of Sost/sclerostin blocks downregulation of OPG expression and the increase in RANKL/OPG ratio induced by glucocorticoid. (A) Expression of the indicated genes normalized to the housekeeping gene Rplp2 in femur and lumbar vertebra (L₅). n = 7–10 for WT and n = 6–8 for Sost^−/−. *p < 0.05 versus corresponding placebo-treated mice and #p < 0.05 versus WT mice treated with the same glucocorticoid dose by two-way ANOVA, Tukey post hoc test. (B, C) Gene expression in bones from WT or Sost^−/− mice, n = 6 (B) or from WT mice cultured with or without anti-sclerostin antibody (Scl-Ab), n = 16 (C) were treated ex vivo with vehicle or dexamethasone for 6 hours. *p < 0.05 versus corresponding vehicle-treated bones, by paired t test. OPG = osteoprotegerin.

Fig. 7.

Sost/sclerostin deficiency prevents GC-induced bone loss. GC-induced osteoporosis is characterized by decreased bone formation, increased apoptosis of Ot and Ob, and increased Oc and bone resorption, which combined induced bone loss and fragility. Activation of Wnt/β-catenin signaling resulting from Sost/sclerostin deletion protects from glucocorticoid-osteoporosis by inhibiting bone resorption through sustained anti-catabolic signaling driven by OPG. GC = glucocorticoid; Ot = osteocytes; Ob = osteoblasts; Oc = osteoclasts; OPG = osteoprotegerin.