Abstract Background: Both osteoporosis and sarcopenia are musculoskeletal diseases associated with estrogen deficiency and aging. Previous studies suggest that the relationship between muscle and bone is more than purely mechanical, as there is reciprocal paracrine and endocrine signaling (Tagliaferri, Wittrant et al. 2015). Muscle can secrete myokines which can impact bone (Harry, Sandison et al. 2008, Reginster, Beaudart et al. 2016). It is unknown how muscle specific estrogen deficiency impacts bone. Understanding how estrogen deficiency impacts this muscle-bone crosstalk may lead to a clearer mechanism of how musculoskeletal diseases form. Objective: To determine whether estrogen receptor alpha (ERalpha) signaling in muscle can lead to differential expression of myokines, which can impact osteoclastogenesis. Methods: In order to study the crosstalk between the muscle-bone unit as a potential mechanism for osteoporosis, skeletal muscle specific estrogen receptor alpha deficient mice (ERalphaKO) (n=17) and wild type (WT) (n=7) mice were used. In the first experiment, osteoclasts from ERalphaKO and WT mice were evaluated for osteoclast differentiation gene expression using qRT-PCR. Tartrate resistant acid phosphatase staining (Raue, Slivka et al.) and Osteo-assays (resorption assays) were used to determine osteoclast size, number, resorption capabilities. Shapiro-Wilk test was used to evaluate normality. Non-parametric T tests were performed. In a separate experiment, extensor digitorum longus muscle was isolated and contraction experiments were performed in order to isolate myokines from ERalphaKO and WT mice. WT Osteoclasts were cultured with myokines from either ERalphaKO or WT mice and osteoclast differentiation genes were measured by qRT-PCR. Shapiro-Wilk test was used to evaluate normality. ANOVA and Welche’s T tests were performed. Finally, to determine how estrogen receptor alpha signaling affects the myokine profile, muscle was isolated from ERalphaKO and WT mice. Muscle was homogenized with zirconium oxide beads and RNA was isolated. cDNA was made using iScript. PCR array was used in order to compare myokine expression in ERalphaKO and WT mice. Results: ERalphaKO and WT osteoclasts had no difference in size, number, resorption capability, and osteoclast differentiation gene expression. ERalphaKO and WT osteoclasts demonstrated no difference in expression of Nfatc1, DC-stamp, and Cathepsin k. There was a trend towards increased C-Fos expression in the osteoclasts cultured with myokines from ERalphaKO compared to WT (p=0.0692). Several myokines were downregulated in the muscle cells of ERalphaKO compared to WT including: Bmp2 (530-fold), Osm (226-fold), and Hc (66-fold). Conclusions: ERalpha deficiency specific to muscle does change the myokine profile which is secreted by muscle cells. However, there is no difference in osteoclast gene expression when osteoclasts are cultured with myokines from ERalphaKO compared to WT. There is also no difference in size, number, and resorption pits of osteoclasts from ERalphaKO and WT mice. Therefore, absence of ERalpha signaling specific to muscle does not affect osteoclasts, as expression of osteoclast differentiation genes, osteoclast size, number, and resorption activity is unchanged. Bmp2, Osm, Hc are downregulated in the absence of ERalpha signaling in muscle cells. These genes are associated with osteoblast activity and suggest that future experiments should focus on osteoblasts in the absence of ERalpha signaling in muscle in order to explore potential mechanisms for osteoporosis.
University of Minnesota M.S. thesis. June 2020. Major: Dentistry. Advisor: Massimo Costalonga. 1 computer file (PDF); ix, 68 pages.
Estrogen Receptor Alpha Deficiency In Skeletal Muscle Leads To Differential Expression of Myokines, But Does Not Affect Osteoclastogenesis.
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