High-affinity P2Y(2) and low-affinity P2X(7) receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts

The P2 purinergic receptor family implicated in many physiological processes, including neurotransmission, mechanical adaptation and inflammation, consists of ATP-gated non-specific cation channels P2XRs and G-protein coupled receptors P2YRs. Different cells, including bone forming osteoblasts, express multiple P2 receptors; however, how P2X and P2Y receptors interact in generating cellular responses to various doses of [ATP] remains poorly understood. Using primary bone marrow and compact bone derived osteoblasts and BMP2-expressing C2C12 osteoblastic cells, we demonstrated conserved features in the P2-mediated Ca(2+) responses to ATP, including a transition of Ca(2+) response signatures from transient at low [ATP] to oscillatory at moderate [ATP], and back to transient at high [ATP], and a non-monotonic changes in the response magnitudes which exhibited two troughs at 10(−4) and 10(−2) M [ATP]. We identified P2Y2 and P2X7 receptors as predominantly contributing to these responses and constructed a mathematical model of P2Y2R-induced inositol trisphosphate (IP(3)) mediated Ca(2+) release coupled to a Markov model of P2X7R dynamics to study this system. Model predictions were validated using parental and CRISPR/Cas9-generated P2Y2 and P2Y7 knockouts in osteoblastic C2C12-BMP cells. Activation of P2Y2 by progressively increasing [ATP] induced a transition from transient to oscillatory to transient Ca(2+) responses due to the biphasic nature of IP(3)Rs and the interaction of SERCA pumps with IP(3)Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP(3)Rs and the desensitization kinetics of P2X7Rs. Moreover, we found that P2Y2 activity may alter the kinetics of P2X7 towards favouring naïve state activation. Finally, we demonstrated the functional consequences of lacking P2Y2 or P2X7 in osteoblast mechanotransduction. This study thus provides important insights into the biophysical mechanisms underlying ATP-dependent Ca(2+) response signatures, which are important in mediating bone mechanoadaptation.