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.