Sclerostin activity plays a key role in the negative effect of glucocorticoid signaling on osteoblast function in mice

Abstract

Stress during prenatal development is correlated with detrimental cognitive and behavioral outcomes in offspring. However, the long-term impact of prenatal stress (PS) and disrupted glucocorticoid signaling on bone mass and strength is not understood. In contrast, the detrimental effect of lead (Pb) on skeletal health is well documented. As stress and Pb act on common biological targets via glucocorticoid signaling pathways and co-occur in the environment, this study first sought to assess the combined effect of stress and Pb on bone quality in association with alterations in glucocorticoid signaling. Bone parameters were evaluated using microCT, histomorphometry, and strength determination in 8-month-old male mouse offspring subjected to PS on gestational days 16 and 17, lifetime Pb exposure (100 p.p.m. Pb in drinking water), or to both. Pb reduced trabecular bone mass and, when combined with PS, Pb unmasked an exaggerated decrement in bone mass and tensile strength. Next, to characterize a mechanism of glucocorticoid effect on bone, prednisolone was implanted subcutaneously (controlled-release pellet, 5 mg·kg^-1 per day) in 5-month-old mice that decreased osteoblastic activity and increased sclerostin and leptin levels. Furthermore, the synthetic glucocorticoid dexamethasone alters the anabolic Wnt signaling pathway. The Wnt pathway inhibitor sclerostin has several glucocorticoid response elements, and dexamethasone administration to osteoblastic cells induces sclerostin expression. Dexamethasone treatment of isolated bone marrow cells decreased bone nodule formation, whereas removal of sclerostin protected against this decrement in mineralization. Collectively, these findings suggest that bone loss associated with steroid-induced osteoporosis is a consequence of sclerostin-mediated restriction of Wnt signaling, which may mechanistically facilitate glucocorticoid toxicity in bone.

Figure 1

Effect of prenatal stress (PS) on trabecular bone structure. Dams were exposed to stress by restraint chambers on days 16 and 17 of gestation. Bone quality parameters were measured by microCT in 8-month-old male offspring. 3D images (left) are representative transverse sections in (a) third lumbar vertebrae (LV), (b) femur (F), and (c) tibia (T). (d) Bone parameter values are presented in the graphs (right). Bar=500 μm. Data are mean±s.e.m. for six mice per group, *P<0.05 for effect of stress. BV/TV, trabecular bone volume/total volume; SMI, structural model index; Tb.N, trabecular number; Tb.Sp, trabecular spacing; Tb.Th, trabecular thickness.

Figure 2

Prenatal stress disposes the skeleton to enhanced deficits in bone quality as a consequence of lead (Pb) exposure. Lifelong treatment of 0 or 100 p.p.m. Pb in drinking water in no stress (NS) and prenatal stress (PS) mice influence trabecular bone properties at 8 months. 3D images (top) are representative microCT sections. Trabecular bone parameters in distal femurs were analyzed (bottom). Modest decrements as a consequence of PS were exacerbated by co-exposure with Pb. Bar=500 μm. Data are mean±s.e.m. for six mice per group, *P<0.05 for effect of stress or Pb, ^#P<0.05 for differences in means using post-hoc multiple comparisons.

Figure 3

The effects of PS and Pb exposure on adipocyte, osteoblast, and osteoclast formation parameters in trabecular bone. Images are representative alcian blue hematoxylin stains (top) of medial tibial sections from water and Pb-exposed NS and PS mice. Trabecular bone area was measured for histologic parameters and presented in the graphs (bottom). Bar: 100 μm. Data are mean±s.e.m. of four mice per group. *P<0.05 for effect of stress or Pb, ^#P<0.05 for differences in means using post-hoc multiple comparisons. Ad size, adipocyte size; AV/TV, adipocyte volume/total volume; BV/TV, trabecular bone volume/total volume; N.Ob/Tb.Ar, osteoblast number/trabecular area; N.Oc/Tb.Ar, osteoclast number/trabecular area; Oc.S/BS, osteoclast surface/bone surface.

Figure 4

GC treatment decreased osteoblastic parameters while only transiently increased osteoclast parameters. Images are representative alcian blue hematoxylin stains (top) of LV2 from placebo and GC-treated mice at 14 and 42 days post implantation. Trabecular bone area was measured for histologic parameters and presented in the graphs (bottom). Bar: 100 μm. Data are mean±s.e.m. of four mice per group. *P<0.05 for effect of prednisolone, ^#P<0.05 for differences in means using post-hoc multiple comparisons. AV/TV, adipocyte volume/total volume; BV/TV, trabecular bone volume/total volume; GC, glucocorticoid; LV, lumbar vertebrae; N.Ob/Tb Ar, osteoblast number/trabecular area; N.Oc/Tb Ar, osteoclast number/trabecular area; Oc.S/BS, osteoclast surface/bone surface.

Figure 5

Sclerostin levels are elevated in mice treated with prednisolone. Levels of type 1 procollagen (a), leptin (b), and sclerostin (c) were measured using standard ELISA methods in serum 14, 28, and 42 days after implantation with prednisolone or placebo tablet. (d) Images are representative immunohistochemical stains of sclerostin protein in the second lumbar vertebrae after 28 days. Arrows indicate the areas of contrasting sclerostin osteoblastic positive staining. Bar: 500 μm. Data are mean±s.e.m. for four mice per group, *P<0.05 for effect of prednisolone.

Figure 6

Effect of dexamethasone on Wnt signaling in primary cells. Isolated mouse calvarial osteoblasts were treated with dexamethasone. (a) Expression of Wnt signaling proteins was determined by Western blotting after 24 and 48 h. (b) Blots of interest were quantified using ImageJ relative to Actin levels. (c–e) Expression profiles of SOST, Runx2, ALP, C/EBP-α, and PPAR-γ were assessed over 24 h by quantitative PCR following exposure to dexamethasone. Data are mean±s.e.m. for three trials, *P<0.05 for effect of dexamethasone, ^#P<0.05 for multiple comparisons of means.

Figure 7

Osteoblasts deficient of sclerostin are resistant to dexamethasone-induced decrease in bone mineralization. Bone nodule formation was assessed by (a) alkaline phosphatase and (b) alizarin red staining from isolated bone marrow cells of wild-type and SOST-KO mice following 10 and 21 days in osteogenic media and dexamethasone. (c) Adipocyte formation was measured by Oil Red O staining after 5 days in adipogenic media plus dexamethasone. Representative stains of cell cultures from each group (left) with quantification (right) are presented. Data are mean±s.e.m. for 3 trials, *P<0.05 for effect of dexamethasone or SOST-KO, ^#P<0.05 for multiple comparisons of means.