Influence of ARHGEF3 and RHOA knockdown on ACTA2 and other genes in osteoblasts and osteoclasts.
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.
Dept. of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia; School of Biomedical Sciences and CHIRI Biosciences Research Precinct, Faculty of Health Sciences, Curtin University, Bentley, Western Australia, Australia; School of Medicine and Pharmacology, The University of Western Australia, Nedlands, Western Australia, Australia.
Benjamin H
BH
Mullin
School of Biomedical Sciences and CHIRI Biosciences Research Precinct, Faculty of Health Sciences, Curtin University, Bentley, Western Australia, Australia.
Cyril
C
Mamotte
Dept. of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia; School of Medicine and Pharmacology, The University of Western Australia, Nedlands, Western Australia, Australia.
Richard L
RL
Prince
Dept. of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Western Australia, Australia; School of Medicine and Pharmacology, The University of Western Australia, Nedlands, Western Australia, Australia; Twin and Genetic Epidemiology Research Unit, St Thomas' Hospital Campus, King's College London, London, United Kingdom.
Scott G
SG
Wilson
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D015519:Bone Density / Q000235:genetics; D002893:Chromosomes, Human, Pair 3 / Q000235:genetics*; D005260:Female; D005786:Gene Expression Regulation / Q000235:genetics*; D055785:Gene Knockdown Techniques; D006801:Humans; D046228:Microarray Analysis; D010006:Osteoblasts / Q000378:metabolism; D010010:Osteoclasts / Q000378:metabolism; D010024:Osteoporosis / Q000235:genetics*; D034741:RNA, Small Interfering / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D044168:Receptor, Parathyroid Hormone, Type 1 / Q000235:genetics / Q000378:metabolism; D064067:Rho Guanine Nucleotide Exchange Factors / Q000235:genetics*; D013053:Spectrophotometry; D020742:rhoA GTP-Binding Protein / Q000235:genetics*
C541116:ACTA2 protein, human; C576601:ARHGEF3 protein, human; D000199:Actins; C473449:PTH1R protein, human; D034741:RNA, Small Interfering; D044168:Receptor, Parathyroid Hormone, Type 1; D064067:Rho Guanine Nucleotide Exchange Factors; C066737:RHOA protein, human; D020742:rhoA GTP-Binding Protein