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Abstract
Bone remodeling occurs via coupling between bone resorption by osteoclasts and bone formation by osteoblasts. The mechanisms that regulate osteoclast signals to osteoblasts are not well understood. Published studies have reported that BMP signaling in osteoclasts regulate osteoclast coupling targets. To investigate the necessity of canonical BMP signaling on osteoclast differentiation and coupling, we mated Smad1fl/fl; Smad5fl/fl mice to c-Fms-Cre mice. We analyzed male mice at 3 months of age to determine the skeletal phenotype of the Smad1fl/fl; Smad5fl/fl;c-Fms-Cre (SMAD1/5 cKO) mice. There was a 1.2-fold decrease in trabecular BV/TV in SMAD1/5 cKO. Analyses of osteoclast serum markers in SMAD1/5 cKO mice, showed a significant increase in CTX-1 (1.5 fold) and TRAP ELISA (3 fold) compared to control mice. In these same mice, there was a 1.3-fold increase in cortical thickness. Consistent with the increase in cortical thickness, we found a 3-fold increase in osteoblast activity as measured by P1NIP ELISA assay from SMAD1/5 cKO mice. To explain the changes in cortical thickness and P1NP activity, we determined conditioned media from SMAD1/5 cKO osteoclast cultures enhanced mineralization of an osteoblast cell line and coupling factors expressed by osteoclasts that regulate osteoblast activity Wnt1 (4.5-fold increase), Gja1 (3-fold increase) and Sphk1 (1.5-fold increase) were all upregulated in osteoclasts from SMAD1/5 cKO compared to control osteoclasts. Lastly osteoclasts treated with dorsomorphin, a chemical inhibitor of SMAD1/5 signaling, demonstrates an increase in Wnt1 and Gja1 expression similar to the SMAD1/5 cKO mice. Previous studies demonstrated that TGF-β signaling in osteoclasts leads to increases in WNT1 expression by osteoclasts. Therefore, our data suggest that TGF-β and BMP signaling pathways in osteoclasts could act in an antagonistic fashion to regulate osteoblast activity through WNT1 and other coupling factors.
Highlights
We reported that osteoclasts deficient for either SMAD1/5 or SMAD4 expression were tartrate resistant acid phosphatase (TRAP) positive but smaller and less active compared to bone marrow macrophages (BMMs) infected with control adenovirus [4]
On day three the WT population is similar in number compared to SMAD1/5 cKO population; there is a slight decrease in the size of the SMAD1/5 cKO osteoclasts
Histological sections from WT and SMAD1/5 cKO distal femurs were analyzed with TRAP staining and we measured a significant increase in number of osteoclasts per bone surface in SMAD1/5 cKO (Fig 2G)
Summary
In vivo loss of function and in vitro overexpression studies revealed that the BMP antagonist Twisted gastrulation (Twsg1) acts as an inhibitor of osteoclast differentiation [1, 5]. These works established the important role for BMPs in osteoclast differentiation but did not address their mechanism of action. We confirmed the in vivo significance of BMPs in osteoclastogenesis by showing increased bone mass due to reduced osteoclast differentiation in a mouse model with a conditional deletion of type II BMP receptor (BMPRII) that primarily disrupted non-canonical MAP kinase pathways while leaving SMADs intact [2]
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