Epigenetic Regulators Involved in Osteoclast Differentiation.

Age related changes to the skeleton, such as osteoporosis, increase the risk of fracture and morbidity in the elderly population. In osteoporosis, bone remodeling becomes unbalanced with an increase in bone resorption and a decrease in bone formation. Osteoclasts are large multinucleated cells that secrete acid and proteases to degrade and resorb bone. Understanding the molecular mechanisms that regulate osteoclast differentiation and activity will provide insight as to how hyper-active osteoclasts lead to pathological bone loss, contributing to diseases such as osteoporosis. Reversible modifications to the DNA such as histone acetylation, methylation, phosphorylation and ubiquitylation alters the access of transcriptional machinery to DNA and regulates gene expression and osteoclast differentiation and activity. It is critical for the management of bone related diseases to understand the role of these chromatin modifying proteins during osteoclast differentiation, as potential therapies targeting these proteins are currently under development.

Regulation of Osteoclast Differentiation and Skeletal Maintenance by Histone Deacetylases.

Bone is a dynamic tissue that must respond to developmental, repair, and remodeling cues in a rapid manner with changes in gene expression. Carefully-coordinated cycles of bone resorption and formation are essential for healthy skeletal growth and maintenance. Osteoclasts are large, multinucleated cells that are responsible for breaking down bone by secreting acids to dissolve the bone mineral and proteolytic enzymes that degrade the bone extracellular matrix. Increased osteoclast activity has a severe impact on skeletal health, and therefore, osteoclasts represent an important therapeutic target in skeletal diseases, such as osteoporosis. Progression from multipotent progenitors into specialized, terminally-differentiated cells involves carefully-regulated patterns of gene expression to control lineage specification and emergence of the cellular phenotype. This process requires coordinated action of transcription factors with co-activators and co-repressors to bring about proper activation and inhibition of gene expression. Histone deacetylases (HDACs) are an important group of transcriptional co-repressors best known for reducing gene expression via removal of acetyl modifications from histones at HDAC target genes. This review will cover the progress that has been made recently to understand the role of HDACs and their targets in regulating osteoclast differentiation and activity and, thus, serve as potential therapeutic target.

SMAD1/5 signaling in osteoclasts regulates bone formation via coupling factors.

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-ß signaling in osteoclasts leads to increases in WNT1 expression by osteoclasts. Therefore, our data suggest that TGF-ß and BMP signaling pathways in osteoclasts could act in an antagonistic fashion to regulate osteoblast activity through WNT1 and other coupling factors.

Breast cancer cell-derived fibroblast growth factors enhance osteoclast activity and contribute to the formation of metastatic lesions.

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.

Class II and IV HDACs function as inhibitors of osteoclast differentiation.

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.

Regulation of Osteoclast Differentiation by Myosin X.

Osteoclasts begin as mononuclear cells that fuse to form multinuclear cells able to resorb bone. The mechanisms that regulate all the steps of osteoclast differentiation are not entirely known. MYO10, an unconventional myosin, has previously been shown in mature osteoclasts to play a role in attachment and podosome positioning. We determined that MYO10 is also expressed early during osteoclast differentiation. Loss of MYO10 expression in osteoclast precursors inhibits the ability of mononuclear osteoclasts to fuse into multinuclear osteoclasts. Expression of Nfatc1, Dc-stamp, Ctsk, and ß 3 integrin is reduced in the osteoclasts with reduced MYO10 expression. A slight reduction in the osteoclasts ability to migrate, as well as a reduction in SMAD 1/5/8 phosphorylation are also noted with reduced MYO10 expression. Interestingly we also detected a change in the ability of the osteoclast precursors to form tunneling nanotubes (TNTs), which suggests that MYO10 may regulate the presence of TNTs through its interaction with the cytoskeletal proteins.