High-affinity P2Y2 and low-affinity P2X7 receptor interaction modulates ATP-mediated calcium signaling in murine osteoblasts

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.

Astrocyte arborization enhances Ca2+ but not cAMP signaling plasticity

“…Calcium oscillations were linked to the activity of protein kinase C (Nash et al, 2002 ) and its modulatory effect (Bartlett et al, 2015 ; Morita et al 2015b ) via increased SERCA expression that upregulated Ca 2+ oscillations (Morita & Kudo, 2010 ). A recent article (Mikolajewicz et al, 2021 ) also describes a complex interplay between receptors in cells exposed to increasing ATP concentration, resulting in shift between transient and oscillatory type of [Ca 2+ ] i responses. At 100 μM ATP appears to elicit oscillatory, transient and sustained responses ( Figure S7 ).…”

Extracellular ATP and its derivatives provide spatiotemporal guidance for bone adaptation to wide spectrum of physical forces

“…3 ). Importantly, many P2 receptors interact, either by directly forming heterodimers ( Brown et al, 2002 ; Nakata et al, 2005 ; Palygin et al, 2010 ), or indirectly through downstream signaling ( Mikolajewicz et al, 2021 ). One of the most prominent P2 downstream signals is a transient elevation of [Ca 2+ ] i , which has a complex [ATP] dependence ( Grol et al, 2013 ; Mikolajewicz et al, 2021 ).…”

“…Importantly, many P2 receptors interact, either by directly forming heterodimers ( Brown et al, 2002 ; Nakata et al, 2005 ; Palygin et al, 2010 ), or indirectly through downstream signaling ( Mikolajewicz et al, 2021 ). One of the most prominent P2 downstream signals is a transient elevation of [Ca 2+ ] i , which has a complex [ATP] dependence ( Grol et al, 2013 ; Mikolajewicz et al, 2021 ). The amplitude of [Ca 2+ ] i elevation has a non-monotonic dependence on [ATP], which was attributed to the interactions between high affinity and intermediate or low-affinity receptors ( Mikolajewicz et al, 2021 ; Xing et al, 2016 ).…”

“…One of the most prominent P2 downstream signals is a transient elevation of [Ca 2+ ] i , which has a complex [ATP] dependence ( Grol et al, 2013 ; Mikolajewicz et al, 2021 ). The amplitude of [Ca 2+ ] i elevation has a non-monotonic dependence on [ATP], which was attributed to the interactions between high affinity and intermediate or low-affinity receptors ( Mikolajewicz et al, 2021 ; Xing et al, 2016 ). In addition, the duration of [Ca 2+ ] i elevation exhibits a sigmoidal [ATP] dependence, due to P2X 7 activation ( Grol et al, 2013 ; Xing et al, 2016 ).…”

“…Consequently, there are two ranges of [ATP] where P2 stimulation results in pronounced increases in the cumulative amount of [Ca 2+ ] i during the signaling event (i.e., the area under the calcium response): first at low [ATP], which can be considered a sensitivity threshold, and second at high [ATP] corresponding to tissue damage – an injury threshold ( Grol et al, 2013 ; Xing et al, 2016 ). Moreover, P2-mediated responses to intermediate 0.1–10 μM [ATP] exhibit highly oscillatory dynamics, that changes with an increase in [ATP] ( Mikolajewicz et al, 2021 ). Cyclic AMP (cAMP) is another prominent secondary messenger downstream of P2Y receptors, however, it has been less studied in the context of bone cell mechanotransduction.…”