No abstract

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.

Prior studies demonstrated that deletion of the protein phosphatase Phlpp1 in Ctsk-Cre expressing cells enhances bone mass, characterized by diminished osteoclast activity and increased coupling to bone formation. Due to non-specific expression of Ctsk-Cre, the definitive mechanism for this observation was unclear. To further define the role of bone resorbing osteoclasts, we performed ovariectomy (Ovx) and Sham surgeries on Phlpp1 cKOCtsk and WT mice. Micro-CT analyses confirmed enhanced bone mass of Phlpp1 cKOCtsk Sham females. In contrast, Ovx induced bone loss in both groups, with no difference between Phlpp1 cKOCtsk and WT mice. Histomorphometry demonstrated that Ovx mice lacked differences in osteoclasts per bone surface, suggesting that estradiol (E2) is required for Phlpp1 deficiency to have an effect. We performed high throughput unbiased transcriptional profiling of Phlpp1 cKOCtsk osteoclasts and identified 290 differentially expressed genes. By cross-referencing these differentially expressed genes with all estrogen response element (ERE) containing genes, we identified IGFBP4 as potential estrogen-dependent target of Phlpp1. E2 induced PHLPP1 expression, but reduced IGFBP4 levels. Moreover, genetic deletion or chemical inhibition of Phlpp1 was correlated with IGFBP4 levels. We then assessed IGFBP4 expression by osteoclasts in vivo within intact 12-week-old females. Modest IGFBP4 immunohistochemical staining of TRAP+ osteoclasts within WT females was observed. In contrast, TRAP+ bone lining cells within intact Phlpp1 cKOCtsk females robustly expressed IGFBP4, but levels were diminished within TRAP+ bone lining cells following Ovx. These results demonstrate that effects of Phlpp1 conditional deficiency are lost following Ovx, potentially due to estrogen-dependent regulation of IGFBP4.

No abstract

Osteoporosis and sarcopenia are maladies of aging that negatively affect more women than men. In recent years, it has become apparent that bone and muscle are coupled not only mechanically as muscle pulls on bone, but also at a higher level with myokines, biochemical and molecular signaling occurring between cells of the two tissues. However, how estrogen deficiency in females impacts the chemical crosstalk between bone and muscle cells is not understood. We hypothesize that changes in estrogen signaling alters myokine expression and intensifies bone loss in women. In our present study, we demonstrate that conditioned media from ovariectomized or skeletal muscle deficient in estrogen receptor α (ERα) expression enhances osteoclast differentiation and activity. Using a cytokine array, we identified myokines that have altered expressions in response to loss of estrogen signaling in muscle. Lastly, we demonstrate that conditional deletion of ERα in skeletal muscle results in osteopenia due to an increase in the osteoclast surface per bone surface. Our results suggest that estrogen signaling modulates expression of myokines that regulate osteoclast differentiation and activity.

Previous studies examining the role of the histone deacetylase Hdac3 within myeloid cells demonstrated that Hdac3 promotes M2 activation and tissue healing in inflammatory conditions. Since myeloid lineage cells are required for proper bone formation and regeneration, in this study we examined the functions of Hdac3 during bone healing. Conditional deletion of Hdac3 within myeloid progenitors accelerates healing of cortical bone defects. Moreover, reduced osteoclast numbers within the defect site are correlated with Hdac3 suppression. Ex vivo osteoclastogenesis assays further demonstrate that Hdac3 deficiency limits osteoclastogenesis, the number of nuclei per cell and bone resorption, suggesting a defect in cell fusion. High throughput RNA sequencing identified the transmembrane protein Pmepa1 as a differentially expressed gene within osteoclast progenitor cells. Knockdown of Pmepa1 partially restores defects in osteoclastogenesis induced by Hdac3 deficiency. These results show that Hdac3 is required for optimal bone healing and osteoclast fusion, potentially via its regulation of Pmepa1 expression.

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.

Myocyte enhancement factor 2C (MEF2C) is a transcription factor studied in the development of skeletal and smooth muscles. Bone resorption studies have exhibited that the reduced expression of MEF2C contributes to osteopetrosis and the dysregulation of pathological bone remodeling. Our current study aims to determine how MEF2C contributes to osteoclast differentiation and to analyze the skeletal phenotype of Mef2c-cKO mice (Cfms-cre; Mef2cfl/fl). qRT-PCR and Western blot demonstrated that Mef2c expression is highest during the early days of osteoclast differentiation. Osteoclast genes, including c-Fos, c-Jun, Dc-stamp, Cathepsin K, and Nfatc1, had a significant reduction in expression, along with a reduction in osteoclast size. Despite reduced CTX activity, female Mef2c cKO mice were osteopenic, with decreased bone formation as determined via a P1NP ELISA, and a reduced number of osteoblasts. There was no difference between male WT and Mef2c-cKO mice. Our results suggest that Mef2c is critical for osteoclastogenesis, and that its dysregulation leads to a sex-specific osteopenic phenotype.