Abstract
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 Ca2+ responses to ATP, including a transition of Ca2+ 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 (IP3) mediated Ca2+ 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 Ca2+ responses due to the biphasic nature of IP3Rs and the interaction of SERCA pumps with IP3Rs. At high [ATP], activation of P2X7R modulated the response magnitudes through an interplay between the biphasic nature of IP3Rs 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 Ca2+ response signatures, which are important in mediating bone mechanoadaptation.
Highlights
Extracellular ATP has long been implicated in diverse physiological functions [1], including neurotransmission [2], mechanical adaptation [3] and the regulation of inflammation [4]
We used experimental and computational modeling approaches to determine the underlying dynamics of ATP-mediated calcium signaling in osteoblasts
The latter was done by developing a mathematical model that was comprised of a subset of low- (P2X7) and high- (P2Y2) affinity P2 receptors, reflecting the conserved P2 expression observed across different osteoblast models
Summary
Extracellular ATP has long been implicated in diverse physiological functions [1], including neurotransmission [2], mechanical adaptation [3] and the regulation of inflammation [4]. ATP is the physiological agonist for all P2X receptors (P2XRs) as well as the P2Y2 and P2Y11 receptors (P2Y2R and P2Y11R, respectively) [6]. Together they cover a range of extracellular ATP concentrations ([ATP]) spanning six orders of magnitude (10−8 M to 10−2 M) [7]. The mechanism by which P2XRs and P2YRs alter [Ca2+]i differs: P2XR activation increases Ca2+ influx across the plasma membrane [14] while P2YR activation enhances Ca2+ release from the endoplasmic reticulum (ER) by stimulating the Gq protein signaling pathway, leading to the production of inositol triphosphate (IP3) and the activation of IP3 receptors (IP3Rs) [15]. While it was proposed that specific interactions between the high-affinity, mid-range and low-affinity P2Rs may explain the [ATP]-dependence, no mechanistic studies at the level of cellular signaling has yet been performed
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
