The effect of P2X7 receptor polymorphisms in osteoblast cells

The P2X7 receptor (P2X7R) gene is highly polymorphic with at least six non-synonymous single-nucleotide polymorphisms (SNPs) being previously described as having either loss-of-function (LOF) or gain-of-function (GOF) effects.We have previously demonstrated expression of functional P2X7R on both osteoblasts and osteoclasts and found an association of LOF P2RX7 polymorphisms and LS-BMD. The exact mechanism behind this observed association is currently unknown. We provide evidence here that it is the effects of P2XR7 SNPs on osteoblast cells that may be driving the observed association with lower BMD in individuals with P2XR7 SNPs. To determine the effect of P2XR7 SNPs on osteoblast cell function, Te85 cells were transfected with P2XR7 wild type (WT) or SNPs cDNA. P2X7R activation, intracellular calcium levels, cell proliferation, alkaline phosphatase (ALP) activity and in-vitro mineralisation were measured. Transfected WT or P2XR7 SNPs did not function in Te85 cells unless co-transfected with a naturally occurring truncated isoform, splice variant B (P2X7B). Upon ATP stimulation, pore formation was observed in theWT and GOF SNP, 155Y; but not in any of the three LOF SNPs; 496A, 568 N, or 307Q. Increased intracellular calcium levels were found in all co-transfected Te85 cells, compared to the naïve or single P2X7B transfected cells. As expected, cell proliferation increased in Te85 cells transfected with the trophic P2X7B isoform, but co-transfection ofWT or P2XR7 SNPs reduced cell proliferation. Transfection of P2X7B alone significantly decreased ALP activity by 23 % compared to naïve cells, whilst co-transfection of P2X7B and WT significantly increased ALP activity by 36 %. Co-transfection with the LOF 496A and 568 N P2XR7 SNPs gave significant decreases in ALP of 15 % and 13 % respectively. In-vitro mineralisation was also significantly increased with co-transfection of P2X7B andWT or 307Q SNP compared to naïve cells, but this increased mineralisation was not detected with other P2XR7 SNPs. The results of this study demonstrate that the P2X7B isoform is necessary for P2X7R-mediated osteogenesis in Te85 osteoblasts cells and that P2XR7 SNPs significantly alter osteoblast cell function which may account for the reduced BMD previously observed in women with P2X7R polymorphisms. The results of this study extend our knowledge into the role of the P2X7R in maintaining bone homeostasis, and may help identify people at risk of developing osteoporosis.