Osteoclastogenic activity and RANKL expression are inhibited in osteoblastic cells expressing constitutively active Gα₁₂ or constitutively active RhoA

Gα₁₂-RhoA signaling is a parathyroid hormone (PTH)-stimulated pathway that mediates effects in bone and may influence genetic susceptibility to osteoporosis. To further elucidate effects of the pathway in osteoblasts, UMR-106 osteoblastic cells were stably transfected with constitutively active (ca) Gα₁₂ or caRhoA or dominant negative (dn) RhoA and co-cultured with RAW 264.7 cells to determine effects on hormone-stimulated osteoclastogenesis. Whereas PTH and calcitriol-stimulated osteoclastogenesis in co-cultures with UMR-106 cells expressing pcDNA or dominant negative RhoA, the osteoclastogenic effects of PTH and calcitriol were significantly attenuated when the UMR-106 cells expressed either caRhoA or caGα₁₂. These inhibitory effects were partially reversed by the Rho kinase inhibitor Y27632. None of the constructs affected osteoclastogenesis in untreated co-cultures, and the constructs did not inhibit the osteoclastogenic responses to receptor activator of NFκB ligand (RANKL). To investigate the mechanism of the inhibitory effects of caGα ₁₂ and caRhoA, expression of RANKL, osteoprotegerin (OPG), osteopontin (OPN), and intercellular adhesion molecule-1 (ICAM) in response to PTH or calcitriol was examined in the UMR-106 cells. In the cells expressing pcDNA or dnRhoA, PTH and calcitriol increased RANKL mRNA and decreased OPG mRNA, whereas these effects were absent in the cells expressing caGα ₁₂ or caRhoA. Basal expression of RANKL and OPG was unaffected by the constructs. The results suggest that Gα₁₂-RhoA signaling can inhibit hormone-stimulated osteoclastogenesis by effects on expression of RANKL and OPG. Since PTH can stimulate the Gα₁₂-RhoA pathway, the current findings could represent a homeostatic mechanism for regulating osteoclastogenic action.