Expression and role of P2X7 receptor splice variants in osteosarcoma

The P2X7 receptor for extracellular ATP was recently indicated as determinant in tumour formation, growth, vascularization and metastatization in different experimental cancer models. Despite the fact that P2X7 promotes osteogenesis affecting osteoblast proliferation and osteodeposition studie investigating P2X7 involvement in a primary bone tumor, such as osteosarcoma, were lacking. For these reasons, we examined P2X7 expression in human osteosarcomas. The receptor was detected by immunohistochemistry on an osteosarcoma tissue array using two difefrent anti-P2X7 antibodies: a monoclonal antibody directed against the extracellular loop of the molecule, and a polyclonal antibody recognizing the intracellular C-terminal tail. These two antibodies allow recognition of two different P2X7 splice variants: the full length P2X7A isoform and the C-terminal truncated P2X7B. Both these isoforms were related to a trophic activity in difefrent cell types. We showed a greater number of tmour specimens labelled by the monoclonal antibody (85%) with respect to the polyclonal one that labelled the 65% of the osteosarcoma samples, suggesting that P2X7B will be the most represented in osteosarcoma. These data prompted us to investigate the role of P2X7A and B in Te85 osteosarcoma cell line that does not endogenously express the receptor. Te85 were stably transfected with P2X7A, B or both. P2X7 transfected cells showed higher proliferation in absence of serum and more elevated NFATc1 levels than Te85 wt cells. In accordance to what suggested by osteosarcoma expression Te85 P2X7B transfected cells were those showing the highest proliferation rate. Moreover mRNA for RANK-L, the main regulator of osteoblast-osteoclast cross talk, was significantly decreased in all P2X7 transfected clones. Fianlly, osteodeposition was significantly decreased in Te85 P2X7B cells and increased in Te85 P2X7A+B clones. Taken together our data strongly suggets that both P2X7 isoforms have a role in osteosarcoma biology affecting tumour cell growth and interaction with osteoclasts together with osteodeposition.