P2X7 receptor and its splice variants in osteosarcoma

To P2X7 receptor a role in bone formation and resorption has been assigned since the first studies on receptor knock out mice. Both osteoblast proliferation and osteoclast activation and fusion have been demonstrated to be related to P2X7 receptor. These data have been further supported by findings correlating P2X7 single nucleotide polymorphisms (SNPs) and osteoporosis. P2X7 has been shown to be involved also in tumour formation, growth and neovascularisation, and expressed in different tumour types (Adinolfi E. et al. Cancer Res.72(12):2957–69. 2012). Although P2X7 function in bonerelated cancer has been recently reviewed (Adinolfi E. et al. J. Osteoporosis; 637863, 2012), further insights into P2X7 contribution to osteosarcoma biology are still requested. Nine different naturally occurring splice variants of the receptor are actually known. Among them, P2X7A is the wellcharacterized full-length isoform. Depending on the dose of the agonist, P2X7A is able to form both a plasma membrane channel for small cations, and a larger pore, permeable to solutes of molecular weight up to 900 kDa. Conversely, P2X7B is a shorter isoform, widely expressed in human tissues, only capable to generate the membrane channel. The present study was dedicated to investigate a possible role of P2X7 and its main splice variants in human osteosarcoma and in related osteoblastic activity. A tissue array containing 59 different osteosarcoma samples was tested at immunohistochemistry using an anti-P2X7 antibody: all tumours stained positively with some difference depending on the osteosarcoma type. Moreover, the human osteosarcoma cell line Te85, not expressing the receptor, has been transfected with the two main hP2X7 splice variants, either separately (Te85-P2X7Aand Te85-P2X7B), or jointly (Te85-P2X7A + B). P2X7 expression and function in transfected cells have been assessed at different levels including mRNA, plasma membrane and cytosolic Ca2+ increase. As previously shown in another cell model, (Adinolfi E. Et al. FASEB J. 24(9):3393–404. 2010) co-transfection with P2X7A + B resulted in potentiated responses. P2X7 expression conferred a proliferative advantage in absence of serum, the highest proliferative activity being that of Te85-P2X7B cells. NFATc1 (Nuclear factor of activated T lymphocytes) activity, that is a marker of osteoblast proliferation, resulted increased in all P2X7-transfected cells. Addressing our attention to osteoblast activity, we found that Te85-P2X7B cells showed reduced osteodeposition. Accordingly, also alkaline phosphatase activity in these cells was the lowest among all P2X7-transfected cells. Finally, a significant reduction ofmRNA RANK-L levels was found in all P2X7-transfected cells, particularly in the Te85-P2X7B cells. These results show that osteosarcomas express P2X7 receptor at high levels and that the receptor might contribute to bone mass increase in osteoblastic malignant lesions.