Abstract
Osteoporosis is a common bone disease that has a strong genetic component. Genome-wide linkage studies have identified the chromosomal region 3p14-p22 as a quantitative trait locus for bone mineral density (BMD). We have previously identified associations between variation in two related genes located in 3p14-p22, ARHGEF3 and RHOA, and BMD in women. In this study we performed knockdown of these genes using small interfering RNA (siRNA) in human osteoblast-like and osteoclast-like cells in culture, with subsequent microarray analysis to identify genes differentially regulated from a list of 264 candidate genes. Validation of selected findings was then carried out in additional human cell lines/cultures using quantitative real-time PCR (qRT-PCR). The qRT-PCR results showed significant down-regulation of the ACTA2 gene, encoding the cytoskeletal protein alpha 2 actin, in response to RHOA knockdown in both osteoblast-like (P<0.001) and osteoclast-like cells (P = 0.002). RHOA knockdown also caused up-regulation of the PTH1R gene, encoding the parathyroid hormone 1 receptor, in Saos-2 osteoblast-like cells (P<0.001). Other findings included down-regulation of the TNFRSF11B gene, encoding osteoprotegerin, in response to ARHGEF3 knockdown in the Saos-2 and hFOB 1.19 osteoblast-like cells (P = 0.003–0.02), and down-regulation of ARHGDIA, encoding the Rho GDP dissociation inhibitor alpha, in response to RHOA knockdown in osteoclast-like cells (P<0.001). These studies identify ARHGEF3 and RHOA as potential regulators of a number of genes in bone cells, including TNFRSF11B, ARHGDIA, PTH1R and ACTA2, with influences on the latter evident in both osteoblast-like and osteoclast-like cells. This adds further evidence to previous studies suggesting a role for the ARHGEF3 and RHOA genes in bone metabolism.
Highlights
Osteoporosis is a common and debilitating bone disease that is characterised by a low bone mineral density (BMD), which leads to an increased risk of fracture [1]
Given the associations that we have previously identified between the RHOA and ARHGEF3 genes and BMD, coupled with the evidence in the literature suggesting a role for RhoA in osteoblasts and osteoclasts, we decided to further investigate the role of these genes in these particular cell types
Osteoclast Microarray Results The osteoclastic phenotype of the cells was confirmed by expression of the genes encoding the osteoclastic biochemical markers tartrate resistant acid phosphatase (TRAP) (ACP5), cathepsin K (CTSK) and calcitonin receptor (CALCR) from the microarray output
Summary
Osteoporosis is a common and debilitating bone disease that is characterised by a low bone mineral density (BMD), which leads to an increased risk of fracture [1]. The disease is prevalent in postmenopausal women due to a reduction in oestrogen production, with subsequent effects on bone as well as intestinal and renal calcium handling [2]. We have previously identified significant associations between variation in the RHOA and ARHGEF3 genes, which are both located within the 3p14-p22 region, and BMD in women [15,16]. The functions of these genes are related, with the product of the ARHGEF3 gene (the Rho guanine nucleotide exchange factor (GEF) 3) activating two members of the RhoGTPase family: RhoA (encoded for by the RHOA gene) and RhoB [17]. RhoA is involved with regulating cytoskeletal dynamics and actin polymerisation [18] and has been shown to have a role in osteoblast differentiation [19,20] and osteoclastic bone resorption [21]
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