Caveolin-1 regulates P2X7 receptor signaling in osteoblasts

Abstract

The synthesis of new bone in response to a novel applied mechanical load requires a complex series of cellular signaling events in osteoblasts and osteocytes. The activation of the purinergic receptor P2X(7)R is central to this mechanotransduction signaling cascade. Recently, P2X(7)R have been found to be associated with caveolae, a subset of lipid microdomains found in several cell types. Deletion of caveolin-1 (CAV1), the primary protein constituent of caveolae in osteoblasts, results in increased bone mass, leading us to hypothesize that the P2X(7)R is scaffolded to caveolae in osteoblasts. Thus, upon activation of the P2X(7)R, we postulate that caveolae are endocytosed, thereby modulating the downstream signal. Sucrose gradient fractionation of MC3T3-E1 preosteoblasts showed that CAV1 was translocated to the denser cytosolic fractions upon stimulation with ATP. Both ATP and the more specific P2X(7)R agonist 2'(3')-O-(4-benzoylbenzoyl)ATP (BzATP) induced endocytosis of CAV1, which was inhibited when MC3T3-E1 cells were pretreated with the specific P2X7R antagonist A-839977. The P2X7R cofractionated with CAV1, but, using superresolution structured illumination microscopy, we found only a subpopulation of P2X(7)R in these lipid microdomains on the membrane of MC3T3-E1 cells. Suppression of CAV1 enhanced the intracellular Ca(2+) response to BzATP, suggesting that caveolae regulate P2X(7)R signaling. This proposed mechanism is supported by increased mineralization in CAV1 knockdown MC3T3-E1 cells treated with BzATP. These data suggest that caveolae regulate P2X(7)R signaling upon activation by undergoing endocytosis and potentially carrying with it other signaling proteins, hence controlling the spatiotemporal signaling of P2X(7)R in osteoblasts.

Keywords: P2X7R; caveolin-1; endocytosis; osteoblasts; purinergic signaling.

Fig. 1.

Activation of the purinergic receptor P2X₇R induces CAV1 endocytosis. A: MC3T3-E1 cells were subjected to sucrose gradient centrifugation, and 1-ml fractions were collected from the top of the gradient and immunoblotted for caveolin 1 (CAV1). Addition of 250 μM ATP caused a translocation of CAV1 from the lipid fractions (fractions 3–6) to the denser cytosolic fractions (fractions 9–12), indicating loss of CAV1 from the plasma membrane microdomains. B: quantification of CAV1 translocation with ATP treatment shows a significant loss of CAV1 from the buoyant lipid microdomain fractions (fractions 3–6). ^aP < 0.01. C: CAV1 is present on the plasma membrane of MC3T3-E1 cells under control conditions (i, arrows), but the staining pattern was strongly reduced when the cells were stimulated for 10 min with 250 μM ATP (ii) or 150 μM 2′(3′)-O-(4-benzoylbenzoyl)ATP (BzATP, iii). CAV1 remained on the plasma membrane when the cells were pretreated with 500 nM A-839977 before addition of 150 μM BzATP (iv). D: percentage of control and ATP-, BzATP-, and A-839977 + BzATP-treated cells in which CAV1 is present on the plasma membrane (PM). There was a significant reduction in the number of cells with CAV1 on the plasma membrane within 10 min of ATP or BzATP treatment and a significant difference between BzATP-treated cells and cells pretreated with A-839977. ^aP < 0.05 vs. control; ^bP < 0.05 vs. BzATP (by Tukey-Kramer test). E and F: line profile showing intensity of CAV1 staining across the cell, as indicated by the red line across the cell. In control cells, intensity peaks on the membrane region and tapers down toward the inside of the cell. When the cell is treated with ATP or BzATP, the intensity profile is distributed across the cytoplasm of the cell, and the profile of A-839977 + BzATP is similar to the control.

Fig. 2.

A subpopulation of P2X₇R is present in caveolae of osteoblasts. A: P2X₇R cofractionated with CAV1 in cells subjected to sucrose gradient centrifugation. One-milliliter fractions were collected from the top of the gradient and an equal volume of each fraction was immunoblotted for P2X₇R and CAV1. B: representative Western blot showing P2X₇R, along with CAV1, in the Triton X-100-insoluble (IS) fractions. C: superresolution structured illumination microscopy photomicrograph (i) illustrates colocalization of P2X₇R (green) with cholera toxin B (CTX-B, red), seen as yellow regions. Magnified images (ii–iv) show subpopulation of P2X₇R in caveolae (arrows). D: colocalization coefficients from the superresolution structured illumination microscopy images after appropriate threshold values are set show Mander's correlation coefficient of 0.45 for the P2X₇R with CTX-B. This indicates that up to 45% of P2X₇R in a cell is present in the CTX-B-enriched caveolae of MC3T3-E1 cells.

Fig. 3.

Suppression of CAV1 enhanced P2X₇R-mediated intracellular Ca²⁺ ([Ca²⁺]_i) signaling. A: Western blot showing suppression of CAV1 in MC3T3-E1 cells treated with siRNA (siCAV1). B: representative [Ca²⁺]_i trace showing change in fluorescence intensity with 150 μM BzATP treatment over time. Fluo 4 was used to measure the change in [Ca²⁺]_i. C: mean fold change in peak Ca²⁺ response to BzATP in CAV1 knockdown (siCAV1) cells showed a significant increase compared with control (P < 0.05).

Fig. 4.

Mineralization due to P2X₇R activation is amplified in CAV1 KD cells. A: immunoblot showing extent of CAV1 suppression in stable MC3T3-E1 CAV1 KD cells generated using shRNA. B: mineralization nodules observed after von Kossa staining on MC3T3-E1 cells after 21 days. Addition of 150 μM BzATP clearly increased mineralization in CAV1 KD cells, as seen by the nodules. C: extent of mineralization shows a significant increase CAV1 KD cells with BzATP treatment. *P < 0.001. D: Alizarin red staining shows a strong increase in mineralization in CAV1 KD cells treated with BzATP compared with untreated (vehicle) cells. E: extent of mineralization was quantified using ImageJ. ^aP < 0.05 vs. control.