Fig 1.
ATP-induced Ca2+ response characteristics are conserved across murine osteoblast lines.
Fura2-loaded BMP2-transfected C2C12 osteoblastic cells (C2-OB), bone-marrow-derived osteoblasts (BM-OB), or compact-bone-derived osteoblasts (CB-OB) were stimulated with ATP (10−8 to 10−3 M), changes in [Ca2+]i were recorded, and characteristic parameters of individual cell-level Ca2+ responses were quantified. (A) Representative ATP-induced Ca2+ signature responses for different osteoblastic lines. Recording duration: 120 s. (B) Activation time, magnitude and oscillatory characteristics of Ca2+ responses in different osteoblastic cells were aligned to obtain consensus on dose-dependency behaviours (see S1 Fig for intermediary alignment steps). Data are response means, normalized to peak dose-response. Solid curves: Loess curves fit to normalized response means. Vertical solid lines: peak magnitude; Vertical dashed lines: peak oscillatory activity; CI: confidence interval, M: molar concentration.
Fig 2.
Functional P2Y2 and P2X7 are expressed in osteoblastic cells.
(A) P2 expression determined by RT-qPCR in C2-OB, BM-OB, and CB-OB. Relative transcript expression was calculated by ΔΔCT method, and P2ry2 and P2rx4 were used as calibrators for P2Y and P2X receptors, respectively. Data are means ± SEM, n = 3 independent cultures per cell line. (B) Fura2-loaded C2-OB cells were stimulated by ATP, UTP, or BzATP and [Ca2+]i response magnitudes were measured. Data are normalized means ± SEM (markers) fitted with hill functions (curves) for their dose-response curves. (C) Representative Ca2+ responses observed in C2-OB stimulated by 10−6 M ATP or UTP, and 10−3 M ATP or BzATP. (D) P2Y2 and P2X7 protein expression assessed by immunoblot in WT, P2ry2Δ and P2rx7Δ C2-OB whole cell lysates. Histone H3 was used as a loading control.
Fig 3.
Schematic of the mathematical model describing P2 receptor-mediated Ca2+ responses.
(A) ATP activates P2X7 and P2Y2 receptors on the plasma membrane, stimulating Ca2+ entry and IP3 production, respectively. IP3 production leads to Ca2+ release from the endoplasmic reticulum (ER) through IP3Rs. Sarco/endoplasmic Ca2+ ATPase (SERCA) activity replenishes the ER and allows for Ca2+ oscillations when combined with the biphasic response of IP3Rs to Ca2+ due to Ca2+-induced Ca2+-release (CICR). Ca2+ is removed from the cell by plasma membrane Ca2+ ATPases (PMCA). A constant inward Ca2+ leak ensures positive [Ca2+]i in the absence of ATP. EC: extracellular space. (B) Schematic of the P2X7R Markov Model. Middle, lower and upper rows: Fraction of P2X7Rs in naïve, sensitized and desensitized states, respectively. Open and solid circles: Sites unoccupied and occupied by ATP, respectively. Receptors in the closed (Ci) and desensitized (Di) states have closed channel pores, whereas receptors in the open (Qi) states, have open channel pores with identical conductance, i = 1−4. Model parameter values are listed in Table 1.
Fig 4.
Comparison of simulated and experimental dynamics of ATP-induced [Ca2+]i responses.
(A) Time series simulations of [Ca2+]i responses generated by the complete model of P2Y2 and P2X7 (full model, blue), P2X7 submodel (grey) and P2Y2 submodel (yellow). Parameter values are provided in Table 1. (B) Experimental recordings of [Ca2+]i responses in WT (blue), P2ry2Δ (grey) and P2rx7Δ (yellow) C2-OB cells in response to varying [ATP].
Table 1.
Mathematical Model Parameters.
Fig 5.
Ca2+ oscillatory response dynamics defined by the P2Y2-induced changes in [Ca2+]ER and [IP3].
The model defined by Eqs (1)–(3), was examined with JP2X7R = 0. (A) The slow variables representing [IP3] and [Ca2+]ER were set to be independent parameters and the continuation method in XPPATU was applied to track two supercritical Hopf bifurcation points that enclose the oscillatory region (grey region). Arrows indicate the three possible scenarios that describe the changes in [IP3] and [Ca2+]ER during Ca2+ responses: 10−7 M ATP response trajectory remains outside the oscillatory region (red), 10−5 M ATP response trajectory spends an extended period of time inside the oscillatory region (green) and 10−3 M ATP response trajectory briefly crosses through the oscillatory region (blue). (B-D) Simulated changes in IP3 (B), [Ca2+]ER (C) and [Ca2+]i (D) following the application of 10−7 M ATP (red) 10−5 M ATP (green) or 10−3 M ATP (blue).
Fig 6.
Interaction between P2Y2R and P2X7R underlies the non-monotonic magnitude dose-dependency.
(A) The magnitude dose-responses of ATP-induced [Ca2+]i elevations. Markers indicate experimental means ± SEM in wild-type (WT; blue) and P2rx7Δ (red) C2-OB cells. Curves indicate simulated data generated by the full model (blue) and P2Y2 submodel (red). (B) P2X7R-mediated Ca2+ entry estimated from simulated area under the P2X7R flux curve (0–10 s). (C) Simulated maximal flux through IP3Rs in the full model (blue) and P2Y2 only submodel (red). (D) Rate of P2X7R desensitization estimated from simulated time required for P2X7R flux to decay to half of its maximum. (E) Simulated maximum flux through P2X7Rs. Shaded regions in all panels: characteristic first (light grey) and second (dark grey) magnitude troughs observed in WT cells that disappear in P2rx7Δ cells.
Fig 7.
P2Y2 alters P2X7-mediated [Ca2+]i response to high [ATP].
(A) The magnitude dose-responses of ATP-induced [Ca2+]i elevations. Markers indicate experimental means ± SEM in wild-type (WT; blue) and P2ry2Δ (red) C2-OB cells. Curves indicate simulated data generated by the full model (blue), a P2X7 submodel initiated from the naïve closed state C1 (solid red curve), or a P2X7 submodel initiated from the sensitized closed state C4 (dashed red curve). (B) Density distributions of experimental Ca2+ response magnitudes to 10−2 M [ATP] in WT cells (blue density; unimodal) and P2ry2Δ cells (red density; bimodal). Vertical lines show simulated response magnitudes (10−2 M [ATP]) obtained by the full model (solid blue line), naïve P2X7 submodel (solid red line) and sensitized P2X7 submodel (dashed red line).
Fig 8.
P2Y2R and P2X7R contribution to mechanically stimulated signals in bone cells.
(A) Fura2-loaded C2-OB cells were plated on a glass-bottom dish and individual cell (primary; 1°) was mechanically stimulated with a glass-micropipette inducing ATP release into the extracellular space, which subsequently stimulated P2 responses in neighbouring cells (secondary; 2°). (B) Representative images of [Ca2+]i (pseudocolor of 340/380 ratio) in C2-OB parental (top) and P2ry2Δ (bottom) cultures, in which a single cell was mechanically stimulated at t = 0 s (white arrows). The snapshot at 15 s demonstrate secondary responses in neighboring cells. Red traces: primary responses; Black traces: secondary responses. (C) Time-series recordings in WT (top panel), P2ry2Δ (middle panel), and P2rx7Δ (lower panel) cells. (D) Quantification of primary and secondary [Ca2+]i response parameters, including signaling radius, fractions of responding cells, response magnitudes and areas under the curves in WT (blue), P2ry2Δ (yellow), and P2rx7Δ (grey) cells. Data are means ± SEM, *p<0.05, **p<0.01 and ***p<0.001 indicate comparisons between WT and P2ry2Δ or P2rx7Δ cells assessed by ANOVA and Bonferroni-corrected t-test. AU: arbitrary units.