Post-translational allosteric activation of the P2X7 receptor through glycosaminoglycan chains of CD44 proteoglycans

Abstract

Here, we present evidence for the positive allosteric modulation of the P2X7 receptor through glycosaminoglycans (GAGs) in CHO (cell line derived from the ovary of the Chinese hamster) cells. The marked potentiation of P2X7 activity through GAGs in the presence of non-saturating agonists concentrations was evident with the endogenous expression of the receptor in CHO cells. The presence of GAGs on the surface of CHO cells greatly increased the sensitivity to adenosine 5'-triphosphate and changed the main P2X7 receptor kinetic parameters EC50, Hill coefficient and E max. GAGs decreased the allosteric inhibition of P2X7 receptor through Mg(2+). GAGs activated P2X7 receptor-mediated cytoplasmic Ca(2+) influx and pore formation. Consequently, wild-type CHO-K1 cells were 2.5-fold more sensitive to cell death induced through P2X7 agonists than mutant CHO-745 cells defective in GAGs biosynthesis. In the present study, we provide the first evidence that the P2X7 receptor interacts with CD44 on the CHO-K1 cell surface. Thus, these data demonstrated that GAGs positively modulate the P2X7 receptor, and sCD44 is a part of a regulatory positive feedback loop linking P2X7 receptor activation for the intracellular response mediated through P2X7 receptor stimulation.

Figure 1

Concentration–response curves for the ability of adenosine, ATP, ADP, BzATP, UDP or UTP to stimulate Ca²⁺ influx to cytoplasm of the CHO cells. Cytoplasmic Ca²⁺ influx, [Ca²⁺]_cyt, measurements were monitored through changes of the Fluo-4 fluorescence intensity in real time using the Flex Station 3 microplate reader system. CHO-K1 (●-●) and CHO-745 (□-□) cells were seeded onto black 96-well plates (10⁴ cells/well). Then, the cells were incubated with Fluo-4 for 1 h at 37 °C. The samples were monitored for 200 s, and the amplitude of the basal and maximum fluorescence value, after agonist stimulus with adenosine (a), ATP (b), ADP (c), BzATP (d), UDP (e) or UTP (f) to obtain the corresponding concentration–response curves. The data represent the mean±S.E.M. (N=6).

Figure 2

Characterization of the P2X₇ receptor in CHO cells. Cytoplasmic Ca²⁺ influx, [Ca²⁺]_cyt, measurements were monitored through changes in the Fluo-4 fluorescence intensity in real time using the Flex Station 3 microplate reader system. Time course curves of the Fluo-4 fluorescence intensity, referent to transient Ca²⁺ influx in CHO-K1 (a) and CHO-745 (b) cells after stimulation with 100 μM BzATP, in the absence (black symbols) or presence of 300 μM oxATP (white symbols). The cytoplasmic Ca²⁺ increase in the absence (black bars) or presence of oxATP (white bars) in CHO cells is shown in c. Time course curves of PI accumulation in CHO-K1 (●-●) and CHO-745 (□-□) cells in response to 4 mM ATP (d). Concentration–response curves for the ability of BzATP to stimulate Ca²⁺ influx in CHO-K1 (circles) and CHO-745 (squares) cells in the absence (black) or presence of 10 mM MgCl₂ (white) (e). Concentration–response curves for the ability of Cu²⁺ to block Ca²⁺ influx mediated through 100 μM BzATP in CHO-K1 (●-●) and CHO-745 (□-□) cells (f). The data represent the mean±S.E.M. (N=6). *P<0.05.

Figure 3

ATP-gated P2X₇ receptor is expressed on the CHO cells surface. The P2X₇ receptor expression was determined through flow cytometry analysis. CHO cells were labeled with antibody anti-P2X₇ conjugated with Alexa Fluor 488, and the data were collected using a FACSCalibur flow cytometer (Becton–Dickinson) and analyzed using FlowJo software (Tree Star). The boundary between positive and negative cells labeled for the P2X₇ receptor was determined according to the fluorescence distribution of positive cells relative to unlabeled control samples. (a) The amount of P2X₇ receptor expressed in whole CHO cells. (b) P2X₇ receptor expressed at the surface of CHO cells. (c) Immunofluorescence labeling of P2X₇ in CHO-K1 and CHO-745 cells. Cells were stained with DAPI (blue) and immunolabelled with anti-P2X₇ (green) and Alexa Fluor 594 conjugated to WGA (red) at left column; ER-Tracker (red) at central column; and with CellLight Golgi Fluorescent Protein (red) at right column. The histograms and images are representative of the results of three experiments. Scale bars, 20 μm.

Figure 4

P2X₇-mediated cell death is dependent of GAGs/Proteoglycans in CHO cells. (a) CHO-K1 and CHO-745 cells were incubated with ATP (1 and 4 mM) or BzATP (0.5 and 1 mM) for 24 h, at 37 ºC, and the viability of the CHO cell lines was determined using the MTT assay. (b, c) P2X₇-mediated cell death in CHO-K1 and CHO-745 cells was also investigated using annexin V-APC/7-AAD double staining and analyzed through flow cytometry. The data represent the mean±S.E.M. (N=6), *P<0.05. (d) CHO cells were stimulated with 4 mM ATP or 1 mM BzATP for 24 h. ATP stimulation induces morphological changes and decreases the CHO cell number as observed in phase contrast microscopy.

Figure 5

The enhancement in P2X₇-mediated cytoplasmic Ca²⁺ influx is dependent on GAGs/proteoglycans in CHO cells. Concentration–effect curves for the ability of heparin (0–100 μM) to modulate Ca²⁺ influx stimulated through ATP at concentrations of 10 μM and 1 mM (a and b) or BzATP at concentrations of 3, 10 and 100 μM (c and d) in CHO-K1 (a and c) and CHO-745 (b and d) cells. CHO-K1 and CHO-745 cells were pre-incubated with sodium chlorate, an inhibitor of PAPS, at concentrations of 20 and 50 mM for 48 h (e and f), or with 2 mM o-nitrophenyl-β-D-xylopyranoside, an inhibitor of endogenous proteoglycan biosynthesis, for 120 h (g and h); the CHO cells were subsequently stimulated with ATP (e and g) or BzATP (f and h). Cytoplasmic Ca²⁺ influx measurements in CHO cells were monitored through changes in the Fluo-4 fluorescence intensity in real time using the Flex Station 3 microplate reader system. The data represent the mean±S.E.M. (N=6). *P<0.05.

Figure 6

P2X₇ and CD44 colocalize and closely interact in wild-type CHO-K1 cells. (a) Syndecan 1, (b) syndecan 2, (c) glypican 6 and (d) CD44 mRNA constitutive expression in CHO-K1 (black columns) and CHO-745 (white columns) cells, normalized over hypoxanthine–guanine phosphoribosyltransferase mRNA levels, and expressed as fold increases over controls (CTR, n=4). The data are represented as the means±S.E.M. (N=6). *P<0.05. (e) Immunofluorescence labeling with anti-P2X₇ (green, first column) and anti-CD44 (red, second column) of CHO-K1 and CHO-745 cells. The nuclei were stained with DAPI (blue). Merge, third column. Scale bars, 20 μm. (f) P2X₇ immunoprecipitation of whole-CHO-K1 cell lysate. Aliquots (500 μg) of cell lysates were incubated with polyclonal anti-P2X₇ antibody overnight at 4 °C, followed by the addition of 20 μl of protein A/G PLUS-Agarose for 4 h at 4 °C. The proteins were resolved through 10% SDS-PAGE and transferred onto polyvinylidene fluoride membranes. The immunoprecipitated proteins were detected using anti-P2X₇ and anti-CD44 antibodies, followed by incubation with the respective secondary antibodies conjugated to horseradish peroxidase. The bands were revealed using the SuperSignal West Pico Chemiluminescent Substrate.

Figure 7

P2X₇ molecular dynamics is altered through heparin binding. (a) P2X₇ apo trimer, (b) P2X₇ bound to ATP and (c) P2X₇ bound to ATP and heparin (pink sticks). The overall shape of the P2X₇ pore calculated as hollow under all conditions is shown in red. (d–f) Top view from a–c, respectively. (g–i) Ca RMSD for each individual subunit during the 50-ns dynamics simulations. Each ribbon color represents one chain from the representation above, ATP (yellow sticks) and heparin (pink sticks). The images were generated using VMD.