Endocrine Abstracts

The physiological role of the orphan G-protein coupled receptor 35 (GPR35) is currently unknown. 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 a synthetic GPR35 ligand, rescues bone loss in rodent osteoporosis models indicating that GPR35 has an important role in bone. Our previous studies demonstrated GPR35 is highly expressed in human osteoclasts, and we sought to determine the receptor’s function in these cells. We differentiated human PBMCs to mature osteoclasts and assessed effects of GPR35 synthetic agonists, Zaprinast and TCG1001, on osteoclast activity and differentiation. Both agonists stimulated significant reductions in osteoclast bone resorption, 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. In contrast, GPR35 activation had no effect on the number of osteoclasts or nuclear translocation of NFAT, a transcription factor that drives osteoclast differentiation, suggesting GPR35 may not regulate osteoclast development. To understand the signalling pathways activated by GPR35 in osteoclasts, 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 and NFκB that drive transcription of genes required for bone resorption. Finally, we used chemical inhibitors and siRNA knockdown to show that GPR35 couples to G_i/o and G_12/13, but not to G_q/11 to stimulate these signalling pathways. Our findings demonstrate that GPR35 has an important inhibitory role in human osteoclast activity and have defined the signalling pathways that drive these processes. GPR35 represents a promising novel target to reduce osteoclast activity that could be exploited for osteoporosis treatments.