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First published online 9 December 2008
doi: 10.1242/jcs.031534

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Activation of P2X7 receptors causes isoform-specific translocation of protein kinase C in osteoclasts

CIHR Group in Skeletal Development and Remodeling, and Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, The University of Western Ontario, London, Ontario, Canada N6A 5C1

View larger version (56K): [in this window] [in a new window]   Fig. 1. Translocation of PKC-EGFP upon stimulation of P2X7 receptors. RAW 264.7 cells were seeded onto glass-bottomed dishes, cultured for 2 days in the presence of RANKL and transfected with plasmids encoding PKC-EGFP. At 2 days following transfection, cells were bathed in HEPES-buffered, bicarbonate-free medium and observed using confocal microscopy. (A) In the absence of BzATP, PKC-EGFP was uniformly distributed throughout the cytosol. Notably, there was no fluorescence in the nuclei. (B) Treatment of the same osteoclast-like cell with the P2X7-receptor agonist BzATP (150 µM) caused redistribution of PKC-EGFP to the plasma membrane (representative of 68 out of 77 cells examined). The image was obtained 30 seconds following the addition of BzATP. The lower panels in A and B are reconstructed z-stack images of the osteoclast-like cell shown above. PKC translocated exclusively to the upper (basolateral) membrane. Blue horizontal lines indicate the location of the confocal images shown above. (C,D) PKC translocation was quantified by using line profiles of fluorescence intensity, indicated by white diagonal lines in A and B. Fluorescence within 5 µm of the edge of the cell was considered to be membrane associated and the intervening region was considered cytosolic. Lines were positioned to avoid nuclei. Localization was quantified from the average pixel intensity in the membrane-associated regions (Fm, outlined by pink rectangles) and the average pixel intensity in the cytosol (Fc, outlined by blue rectangle), with values of the ratio Fm:Fc exceeding 1 taken as indicating membrane localization. For the line scans shown, the membrane-localization ratio was 0.43 in the absence and 4.8 in the presence of BzATP. Data shown are representative of responses of cells from 14 independent transfections (as summarized in Table 1).

View larger version (40K): [in this window] [in a new window]   Fig. 2. BzATP-induced translocation of PKC is transient. (A) An osteoclast-like cell expressing PKC-EGFP was stimulated with BzATP (150 µM) at 60 seconds, causing transient redistribution of the fluorescence from the cytosol to the periphery of the cell. (B) Plot represents the time course of the membrane localization, with the time of addition of BzATP indicated in this and subsequent figures by the vertical broken line. Translocation of PKC to the membrane was transient, with recovery by 600 seconds. Some photobleaching of EGFP occurred at longer recording times. Duration at half peak of responses was 258±26 seconds (mean ± s.e.m., n=16 cells).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Ability of P2-receptor agonists to induce translocation of PKC-EGFP. Because, in addition to activating P2X7, BzATP activates other P2 receptors, we investigated the effects of other P2-receptor agonists. UTP (150 µM, which activates P2Y2 receptors) or ATP (10 µM, which activates P2X4 and P2Y2 receptors) did not induce translocation of PKC-EGFP. By contrast, ATP (3 mM) or BzATP (150 µM), both of which activate P2X7 receptors, caused significant translocation. Brilliant Blue G (BBG, 10 µM, which blocks P2X7 receptors) inhibited the translocation of PKC that was induced by BzATP. Data are means ± s.e.m. for at least three independent experiments. *P<0.05 compared with control as assessed by one-way ANOVA followed by Bonferroni post hoc test.

View larger version (70K): [in this window] [in a new window]   Fig. 4. BzATP fails to cause translocation of PKC in osteoclasts derived from the bone marrow of P2rx7–/– mice. (A) PKC-EGFP was located in the cytosol of osteoclasts generated from wild-type (WT) mice, and addition of vehicle alone had no effect. Addition of BzATP (150 µM), however, did cause the translocation of PKC to the membrane in wild-type osteoclasts. (B) Addition of BzATP (150 µM) to osteoclasts that were generated from P2rx7–/– mice failed to elicit translocation of PKC. However, addition of PMA (10 µM) did induce translocation to the membrane. Images were obtained from cells that were fixed 1-2 minutes after addition of the agonist. Red horizontal lines indicate the location of the reconstructed z-stacks shown below. Blue horizontal lines indicate the location of the x-y confocal images shown above. Data are representative of three independent experiments (Table 1).

View larger version (47K): [in this window] [in a new window]   Fig. 5. Control experiments reveal the specificity of PKC translocation. (Ai) Treatment of osteoclast-like cells with vehicle alone had no effect on the distribution of PKC-EGFP, with unchanged localization in the cytosol. Plot at right illustrates the time course of membrane localization, with no change in response to vehicle. Test agents were applied at the time indicated by the vertical broken lines. Data shown are representative of six independent experiments. (Aii) An osteoclast-like cell expressing EGFP alone exhibited fluorescent label in both the cytosol and nuclei, in contrast to the localization of EGFP-tagged PKC (Ai). BzATP caused no change in distribution of EGFP, as quantified on the right. Data shown are representative of three independent experiments. (Aiii) Treatment with PMA (10 µM) caused persistent translocation of PKC-EGFP from the cytosol to the membrane (data shown are representative of eight independent experiments). (B) Images show an x-y section and reconstructed z-stack of an osteoclast-like cell, revealing that PMA induced PKC translocation to the basolateral membrane, similar to that seen in response to BzATP. Images labeled `After PMA' were obtained at 120 seconds following the addition of PMA. This osteoclast also exhibited a prominent vacuole in the centre of the cell. Notably, PKC did not translocate to the vacuolar membrane, as both the x-y and x-z projections show.

View larger version (26K): [in this window] [in a new window]   Fig. 6. Close temporal relationship between the BzATP-induced rise of [Ca2+]i and PKC translocation. (A) Simultaneous imaging of [Ca2+]i using the fluorescent Ca2+ indicator dye Fura-red (red) and PKC-EGFP (green). Fura-red intensity (F) decreases with the rise of [Ca2+]i, so data are expressed as the ratio of initial intensity (F0) to F for a region of interest in the cytosol. BzATP was added at 50 seconds (vertical broken line) and PKC translocation follows the rise in [Ca2+]i. Data are representative of seven out of ten cells tested. (B,C) BzATP induced a repetitive translocation of PKC-EGFP in some osteoclast-like cells. An osteoclast-like cell was stimulated with BzATP (150 µM, applied at 25 seconds) as indicated, inducing repetitive translocation of PKC between the cytosol and membrane. A confocal slice through the cell is shown in B, with membrane localization plotted in C for the same cell. Responses are representative of repetitive PKC translocation seen in 18 out of 68 osteoclast-like cells that responded to BzATP.

View larger version (32K): [in this window] [in a new window]   Fig. 7. BzATP-induced translocation of PKC is dependent on extracellular Ca2+. Osteoclast-like cells were bathed in medium containing EGTA (4 mM) to chelate extracellular Ca2+. (A) BzATP (150 µM) was applied at 60 seconds, but no translocation was observed (representative of responses in 14 cells). Subsequently, PMA (10 µM) was added to the bath at 830 seconds as a positive control, and did induce translocation of PKC-EGFP to the membrane. (B) Membrane localization plotted for cell shown in A reveals no effect of BzATP in the absence of Ca2+ (BzATP added to bath at the time indicated by the initial broken line). However, translocation was induced by PMA added at the time indicated by the second broken line. Data are representative of more than five independent experiments.

View larger version (62K): [in this window] [in a new window]   Fig. 8. BzATP causes translocation of PKCβI from the cytosol to membrane. (A) PKCβI-EGFP was distributed throughout the cytosol of an osteoclast-like cell under control conditions, as seen at 0 seconds. Addition of BzATP at 120 seconds caused prompt translocation of fluorescence from the cytosol to the periphery of the cell, with recovery at later times. The lower panels are reconstructed z-stack images of the cell shown above at 0 and 250 seconds, showing that PKCβI-EGFP translocated exclusively to the upper (basolateral) membrane. Blue horizontal lines indicate the location of the confocal images shown above. Data are representative of five independent experiments. (B) Membrane localization plotted for cell shown in A, revealing that translocation of PKCβI to the membrane was transient and recovered. BzATP was added at the time indicated by the vertical broken line. Mean duration at half peak of the response was 101±15 seconds (determined in 12 osteoclast-like cells). The bar graph compares the response duration for PKC and PKCβI isozymes. *P<0.0001, analyzed by Student's t-test.

View larger version (46K): [in this window] [in a new window]   Fig. 9. BzATP does not induce translocation of the novel isozyme PKC. (A) PKC-EGFP was located largely in the cytosol, but to some extent in the nuclei in unstimulated osteoclast-like cells (control). Addition of BzATP (150 µM, at 250 seconds) did not cause redistribution of PKC (`After BzATP'). However, addition of PMA (10 µM, at 2120 seconds) to the same cell induced translocation to the membrane (`After PMA'). Data are representative of eight experiments. (B) Time course of membrane localization plotted for the cell shown in A, illustrating the failure of BzATP to cause translocation of PKC, whereas PMA resulted in a transient translocation to the membrane. (C) Images show x-y and reconstructed z-stacks of an osteoclast-like cell, revealing that PMA induces translocation of PKC primarily to the basolateral membrane. Images labeled `After PMA' were obtained 240 seconds following the addition of PMA.

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