Histone deacetylases (HDACs) are negative regulators of transcription and have been shown to regulate specific changes in gene expression. In vertebrates, eighteen HDACs have thus far been identified and subdivided into four classes (I-IV). Key roles for several HDACs in bone development and biology have been elucidated through in vitro and in vivo models. By comparison, there is a paucity of data on the roles of individual HDACs in osteoclast formation and function. In this study, we investigated the gene expression patterns and the effects of suppressing individual class II (Hdac4, 5, 6, 9, and 10) and class IV (Hdac11) HDACs during osteoclast differentiation. We demonstrated that HDAC class II and IV members are differentially expressed during osteoclast differentiation. Additionally, individual shRNA-mediated suppression of Hdac4, 5, 9, 10 and 11 expression resulted in increased multinucleated osteoclast size and demineralization activity, with little to no change in the overall number of multinucleated osteoclasts formed compared with control shRNA-treated cells. We also detected increased expression of genes highly expressed in osteoclasts, including c-Fos, Nfatc1, Dc-stamp and Cathepsin K. These observations indicate that HDACs cooperatively regulate shared targets in a non-redundant manner.

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.

Fibroblast growth factors (FGFs) and their receptors (FGFRs) have been implicated in promoting breast cancer growth and progression. While the autocrine effects of FGFR activation in tumor cells have been extensively studied, little is known about the effects of tumor cell-derived FGFs on cells in the microenvironment. Because FGF signaling has been implicated in the regulation of bone formation and osteoclast differentiation, we hypothesized that tumor cell-derived FGFs are capable of modulating osteoclast function and contributing to growth of metastatic lesions in the bone. Initial studies examining FGFR expression during osteoclast differentiation revealed increased expression of FGFR1 in osteoclasts during differentiation. Therefore, studies were performed to determine whether tumor cell-derived FGFs are capable of promoting osteoclast differentiation and activity. Using both non-transformed and transformed cell lines, we demonstrate that breast cancer cells express a number of FGF ligands that are known to activate FGFR1. Furthermore our results demonstrate that inhibition of FGFR activity using the clinically relevant inhibitor BGJ398 leads to reduced osteoclast differentiation and activity in vitro. Treatment of mice injected with tumor cells into the femurs with BGJ398 leads to reduced osteoclast activity and bone destruction. Together, these studies demonstrate that tumor cell-derived FGFs enhance osteoclast function and contribute to the formation of metastatic lesions in breast cancer.

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The actions of selective estrogen receptor modulators are tissue dependent. The primary objective of the current study was to determine the tissue selective effects of bazedoxifene (BZA) on the musculoskeletal system of ovariectomized (OVX) female mice, focusing on strengths of muscle-bone pairs in the lower hindlimb. Treatment with BZA after ovariectomy (OVX+BZA) did not prevent body or fat mass gains (p<0.05). In vivo plantarflexor muscle isometric torque was not affected by treatment with BZA (p=0.522). Soleus muscle peak isometric, concentric and eccentric tetanic force production were greater in OVX+BZA mice compared to OVX+E2 mice (p≤0.048) with no effect on maximal isometric specific force (p=0.228). Tibia from OVX+BZA mice had greater cortical cross-sectional area and moment of inertia than OVX mice treated with placebo (p<0.001), but there was no impact of BZA treatment on cortical bone mineral density, cortical thickness, tibial bone ultimate load or stiffness (p≥0.086). Overall, these results indicate that BZA may be an estrogen receptor agonist in skeletal muscle, as it has previously been shown in bone, providing minor benefits to the musculoskeletal system.

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