G protein-coupled receptor 35 (GPR35) stimulation reduces osteoclast activity in primary human bone cells
G protein-coupled receptor 35 (GPR35) is an orphan receptor that is widely expressed in tissues including human osteoblasts and osteoclasts. Expression of the GPR35 gene and protein are downregulated in osteoporosis patients and in mouse models of the disease. Gpr35-knockout mice have reduced bone mass, while GPR35 agonism rescues bone loss in rodent osteoporosis models indicating that GPR35 has an important role in bone. Our previous studies demonstrated GPR35 is expressed in human osteoclasts, and we sought to determine the receptors function in these cells. We differentiated human peripheral blood mononuclear cells to mature osteoclasts and assessed effects of the GPR35 synthetic agonists, TCG1001 and Zaprinast on osteoclast activity and differentiation. Both agonists stimulated significant reductions in osteoclast bone resorption and TRAP activity, and downregulated expression of MMP9, a gene that regulates osteoclast bone resorption. These effects were prevented by pre-incubation of cells with a GPR35-specific antagonist. To understand GPR35 signaling pathways, we measured the phosphorylation of secondary messengers known to have important roles in osteoclast activity using AlphaLISA assays. Upon GPR35 stimulation, we observed reduced phosphorylation of cSrc, which stimulates actin ring formation necessary for bone resorption, and decreased phosphorylation of Akt, CREB and NF{kappa}B that drive transcription of genes required for bone resorption. Additionally, we used chemical inhibitors and siRNA knockdown to show that GPR35 couples to Gi/o and G12/13 to stimulate these signaling pathways. Finally, we compared the ability of GPR35 agonists to suppress osteoclast activity to that of current osteoporosis drugs, denosumab and alendronic acid, and showed TRAP activity was similar suppressed under all conditions. Our findings demonstrate that GPR35 has an important inhibitory role in human osteoclast activity and have defined the signaling pathways that drive these processes. GPR35 represents a promising novel target to reduce osteoclast activity that could be exploited for osteoporosis treatments. Lay summaryExpression of G protein-coupled receptor 35 (GPR35) is reduced in osteoporosis and Gpr35-knockout mice have reduced bone mass. Here we showed stimulation of GPR35 activates Gi/o and G12/13 signaling pathways to reduce bone resorption in human osteoclast. The anti-resorptive activity of GPR35 agonists was comparable to current osteoporosis drugs, denosumab and alendronic acid. Our findings demonstrate that GPR35 has an important inhibitory role in human osteoclast activity and have defined the signaling pathways that drive these processes. GPR35 represents a promising novel target to reduce osteoclast activity that could be exploited for osteoporosis treatments.