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First published online August 9, 2007
doi: 10.1242/10.1242/jcs.004184

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Necessity of inositol (1,4,5)-trisphosphate receptor 1 and µ-calpain in NO-induced osteoclast motility

Departments of Pathology and of Cell Biology and Physiology, University of Pittsburgh, and Veteran's Affairs Medical Center, Pittsburgh, PA 15243, USA

View larger version (18K): [in this window] [in a new window]   Fig. 1. NO activates calpain in osteoclasts, antagonists of NO or cGMP inactivate it. (A) Fluorescent calpain substrate assays in living NO-inhibited or NO-activated osteoclasts. Cells were incubated 20 minutes in 50 µM of the cell-permeable calpain substrate BOC. Quenching BOC is separated from the fluorescent coumarin when proteinase activity cleaves the peptide linker (Leu-Met), increasing fluorescence intensity. Untreated cells are compared with cells incubated with the NO donor SNP (100 µM), the activating cGMP analog 8-pCPT-cGMP (100 µM), the cGMP-blocking analog Rp-cGMPS (50 µM) and the inhibitor of NO synthase L-NMMA (1 µM). Results from four experiments, each measuring a minimum of 16 cells, summarized as the mean ± s.d. of each experiment, normalized to untreated controls. The NO and cGMP agonists increased calpain activity relative to untreated controls (*P<0.05), and relative to the inhibitors Rp-cGMPS and L-NMMA (P<0.01). Inhibitors reduced substrate degradation relative to untreated control, but the difference was significant only for Rp-cGMPS (P<0.05). Note that, in the presence of Rp-cGMPS, the change in calpain activity after NO addition is small and not significant (fifth versus fourth bar). (B) Images of BOC fluorescence in cells with key treatments. SNP (right) increased fluorescence in essentially all of the cells with respect to control (left). Inhibitors such as L-NMMA (middle) reduce activity relative to untreated control, but the difference varies from experiment to experiment due to the variability in autocrine NO production in untreated cells. This is reflected also in the variability of response of osteoclasts to Rp-cGMPS and L-NMMA shown in A. (C) Calpain activity is suppressed by knockdown of VASP or PKG1. BOC fluorescence assays were carried out as in A but cells had been transfected with scrambled control siRNA or siRNAs targeting VASP and PKG1. The cells had been transfected with Cy3-labeled siRNAs 72 hours prior to the assay, and treated with 100 µM 8-pCPT-cGMP for 60 minutes prior to the assay. The assay scored BOC fluorescence over background in cells labeled with Cy3 (red). PKG1 suppression and VASP suppression reduced cGMP-stimulated calpain activity relative to the control (*P<0.05, in both cases; n=6, mean ± s.e.m.). Western blots for knockdown efficacy are shown in Fig. 6.

View larger version (26K): [in this window] [in a new window]   Fig. 2. Effects of calpain antagonists on on cGMP-induced motility and calpain activity. (A) Motility of NO-activated cells with and without 10 µM of the calpain inhibitor Calpeptin. There was very little cell movement of Calpeptin-treated osteoclasts relative to cGMP-activated cells (8-pCPT-cGMP). In cGMP-activated cells with Calpeptin added (middle bar), motility was reduced relative to cGMP-activated cells by 60%, with considerable variation between cells. However, the effect relative to 8-pCPT-cGMP-only treatment is significant (*P<0.05), averaging measurements over 20 cells ± s.d. (B) Effect of Calpeptin, calpastatin and calpastatin scrambled peptide on calpain activity in osteoclasts. Calpain activity was determined using the BOC assay as described for Fig. 1, in osteoclasts without stimulus or treated with the cGMP agonist 8-pCPT-cGMP or the NO donor SNP. Note that the scrambled calpastatin gives results essentially congruent with the controls (right two versus left two bars), whereas Calpeptin and calpastatin inhibited activity to below control cells and prevented calpain activation after cGMP or NO stimulation by more than 90%. Average of four experiments ± s.d. (left two bars) and of two experiments ± s.d. (right six bars) with each experiment measuring fluorescence in 40 cells. *P<0.05 relative to control unstimulated cells (first bar). P<0.01 relative to control stimulated cells (second bar). (C) Cell diameter decreased after cGMP was activated in Calpeptin-treated cells that did not show significant linear translocation. (Top panel) Group of three osteoclasts and a single smaller cell, probably an un-fused CD14 derived macrophage. Calpeptin and 8-pCPT-cGMP were added added at the time the top phase photograph was taken. (Middle and bottom panels) No linear translocation but the footprint of the cells shrank (compare encircled cell). Cell diameter decreased 15.7±5.1% at 15 minutes and 18.1±1.4% at 30 minutes (n=3, mean ± s.d.). After 30 minutes the mean diameter did not change significantly (not shown). Cell diameter also decreased with cGMP activation only (Yaroslavskiy et al., 2005), but not with Calpeptin treatment alone (not shown). Thus, NO-induced rearrangement of the cell attachment does not solely depend on calpain.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Calpain activity in osteoclast lysates and intact osteoclasts, and the effect of suppressing µ-calpain expression on NO-induced calpain activity in situ in osteoclasts. (A) The main active calpain in osteoclasts is µ-calpain. A zymogram is shown as a negative image (degradation is dark) with recombinant µ-calpain and m-calpain standards, and osteoclast lysate (10 µg). The zymogram was developed in Ca2+-EGTA buffers with 30 µM Ca2+ activity. Additional zymograms (not shown) were performed under the same conditions (30 µM Ca2+) or at 100 µM Ca2+ with lysates from osteoclasts treated with NO or cGMP. These studies showed no consistent differences between lysates from stimulated and unstimulated cells. m-calpain activity was not detected even when zymograms were developed at 100 µM Ca2+. (B) Only small amounts of processed µ-calpain or of talin (a calpain substrate) accumulate in cGMP-activated osteoclasts, although osteoclast lysates degrade calpain substrates at constantly elevated Ca2+ levels. (1) Western blot of talin (10% SDS-PAGE). A minor amount of degradation fragments accumulated in osteoclasts activated by 8-pCPT-cGMP for 1 hour, compared with cells treated with the calpain inhibitor Calpeptin (another lane from the same gel). Several other conditions were tested; the difference shown is the largest seen. Talin links actin to the integrin complex and is an established target for partial proteolysis by calpain. (2) Western blot of µ-calpain (10% SDS-PAGE). Osteoclast lysates showed small amounts of partially cleaved enzyme after cGMP activation. The large subunit of µ-calpain (80 kD) did not vary measurably after treating cells with several NO or cGMP activators and inhibitors (not shown), although partially cleaved enzyme (70 kD) was increased slightly at treatment with NO donor or cGMP activating analog (compare lower bands). Osteoclast lysates (30 µg) were prepared from osteoclasts treated 1 hour with 100 µM of the cGMP activator 8-pCPT-cGMP or 50 µM of the inhibitor Rp-cGMPS. (3) MAP2 degradation by osteoclast calpain in vitro (6% SDS-PAGE; silver stain). MAP2 is a structural protein (280 kDa) that is not expressed in osteoclasts and is a sensitive and well-studied calpain substrate. (Top panel) Comparisons show 2 µg of untreated MAP2, 3 µg of osteoclast lysate (Lysate), and MAP2 incubated for 15 minutes and 1 hour in buffer containing 100 µM Ca2+ with 3 µg of osteoclast lysate. Lysate was made from osteoclasts pre-treated 30 minutes with 8-pCPT-cGMP. (Bottom panel) Results with lysates from osteoclasts pre-treated with 10 µg Calpeptin. (C) siRNA inhibition of µ-calpain. Five days after transfection of siRNA targeting µ-calpain, the protein was reduced 85-90% as determined by western blotting. Micrograph shows efficiency of siRNA uptake was >95% (fluorescence microscopy of Cy3-labeled siRNA). Nuclei were labeled with Hoechst dye (blue) to demonstrate that very few cells were without siRNA uptake. (D) Specificity of intra-osteoclastic proteolysis for µ-calpain. The graph shows average BOC fluorescence of 20 cells (mean ± s.e.m.) normalized to unstimulated controls, for osteoclasts transfected with either non-coding siRNA or siRNA targeting µ-calpain, and treated or not with 200 µM of the NO-donor SNP for 10 minutes. Note that the NO donor fails to stimulate significant degradation of the fluorescent calpain substrate in cells transfected with the siRNA targeting µ-calpain. The reduction in NO-dependent calpain activity was comparable to the reduction in µ-calpain protein. Photomicrographs show sample fields of cells transfected with non-coding siRNA (top panels) or µ-calpain-targeting siRNA (bottom panels) without (left) or with (right) treatment with SNP. Note that the NO donor causes significant degradation of the fluorescent calpain substrate only in the control cells (top right panel).

View larger version (72K): [in this window] [in a new window]   Fig. 4. Ca2+ signals during movement of cGMP- or NO-activated osteoclasts. (A) Elevated Ca2+ in a moving osteoclast after cGMP activation. Four false-color Ca2+ images at 5-minute intervals are shown. Each frame depicts an average Fluo 3 signal from a confocal image exposed for 200 mseconds, showing Ca2+ activity as fluorescence at 525 nm. Sixty minutes before imaging, cells were treated with 100 µM 8-pCPT-cGMP, which induces motility in a large fraction of cells. Two cells are shown. One shows minimal changes in Ca2+ and did not move (right cell in each frame). The other (left cell in each frame) moved during the period shown. Movement occurred with large, localized, changes in Ca2+ (first and second frames) and was largely complete after 20 minutes. False color scale: red 1 µM > violet 100 nM > blue; black, no signal. (B) Large Ca2+ transients and motility were much less common in cells when the the cGMP pathway was inhibited. The number of moving cells and a Fluo3 signal two-fold above background was determined in four experiments, at 3-minute intervals over 1 hour in each experiment. Ca2+ fluxes with motility were uncommon in cells treated with the cGMP antagonist Rp-cGMPS (50 µM; left bar, n=43 cells) but frequent in cells treated with the cGMP analog 8-pCPT-cGMP (100 µM; right bar, n=77 cells) 30 minutes prior to analysis. (C) Mean intracellular Ca2+ activity from ratio imaging of osteoclasts with Oregon-Green-BAPTA and Fura Red. The average intracellular Ca2+ levels in unactivated cells was 74 nM; 15-30 minutes after treatment with 100 µM SNP the average intracellular Ca2+ level increased to 5 µM, although some measured values were indistinguishable from the maximum G:R ratio so, probably, some cells with higher intracellular Ca2+ levels occur (mean ± s.e.m.; n=10).

View larger version (91K): [in this window] [in a new window]   Fig. 5. Increased intracellular Ca2+ activity and increased motility in cells treated with cGMP agonists or NO donors compared with cGMP inhibitors, see Movies 1-3 in supplementary material for more detailed information. The false color scale is the same as that described in Fig. 4. (A) Increased motility and Ca2+ levels after preincubation with a cGMP agonist. After a 30-minute incubation with 100 µM 8-pCPT-cGMP, many of the cells show elevated Ca2+ levels using Fluo3 (left). These cells are moving, as shown by subtraction of a 30-minute image from a image taken at 0 minutes (right). (B) Low motility and low Ca2+ levels after preincubation with a cGMP antagonist. After a 30-minute preincubation with 50 µM 8-pCPT-cGMP only one cell with high Ca2+ levels is seen (left). This cell is moving, as shown by subtraction of a 30-minute image from a image taken at 0 minutes (right). The moving cell is an atypical, fusiform cell and might represent a macrophage derivative rather than an osteoclast (see text). (C) Increased motility and Ca2+ after preincubation with the NO donor SNP. After a 30-minute exposure to 100 µM SNP, many cells show elevated Ca2+ levels by ratio imaging (left) and are moving, as shown by subtraction of a 32-minute image from a image taken at 0 minutes (right).

View larger version (24K): [in this window] [in a new window]   Fig. 6. Src activity is required for calpain activation by cGMP, but levels of phosphorylated Akt are not increased. (A) Src is required for cGMP-mediated activation of calpain in osteoclasts. These assays used the BOC fluorescent calpain substrate as described in Fig. 1. In the absence of cGMP (untreated) there is weak basal activity, attributed to autocrine NO production. Following activation with 8-Br-cGMP (50 µM, 1 hour) calpain is strongly activated. The Src inhibitor PP2 reduced activity of calpain to below that of the control (PP2) and reduced 8-Br-cGMP (cGMP) activation by 85% (PP2+8-Br-cGMP). An inactive control, PP3, had no effect on 8-Br-cGMP (cGMP) activation (PP3+8-Br-cGMP). *P<0.01 relative to 8-Br-cGMP; n=25-60, mean ± s.e.m. (B) Activation of cGMP did not increase phosphorylation of Akt. Phosphorylated Akt (p-Akt) was identified in osteoclast lysates by antibody against Akt phosphorylated at Ser473. Lysates of cultures after treatment with the hydrolysis-resistant cGMP analog 8-pCPT-cGMP (100 µM) or the cGMP-blocking analog Rp-cGMPS (50 µM) for 30 minutes were compared with untreated cells. Density of antibody labeling was measured and expressed as a fraction of total Akt by stripping membranes and re-labeling for total Akt protein. Whereas no differences were significant, the largest p-AKT signal was in the control containing the cGMP-blocking analog (left bar). Since PI 3-kinase activates Akt phosphorylation, this suggests that PI 3-kinase is not a direct mediator of cGMP signaling in osteoclasts; n=3, mean ± s.e.m. (C) PKG1 and VASP are required for cGMP-dependent Src phosphorylation. Phosphorylation of Src (top) at Y416 after treatment with 100 µM 8-pCPT-cGMP was reduced by knockdown of PKG1 (left) or VASP (right). Protein loading controls (middle) and knockdown controls (bottom) are also shown. In either case, p-Src was reduced by 70-90%. siRNAs used for knockdown experiments have been described previously (Yaroslavskiy et al., 2005).

View larger version (54K): [in this window] [in a new window]   Fig. 7. Ins(1,4,5)P3R1 is required for normal osteoclast calpain activity. (A) Effect of Ins(1,4,5)P3R and RyR antagonists on calpain activity. BOC fluorescent calpain substrate assays as described in Fig. 1. Each culture was treated with 8-Br-cGMP. This produced strong control activity (Control). The RyR antagonist tetracaine (50 µM) did not affect activity. The Ins(1,4,5)P3R antagonist 2-APB (100 µM) reduced activity; *P<0.01, n=30, mean ± s.e.m. (B) Knockdown of Ins(1,4,5)P3R by using siRNA (IP3R siRNA). The protein (240 kDa) was visualized by western blotting after separation of 35-µg aliquots of cell lysates on 6% SDS-PAGE (top). The membrane was re-blotted for actin, which runs at the front on 6% PAGE. Because of this, the membrane was also stained for total protein using Ponceau Red S (bottom image). (C) Ins(1,4,5)P3R1 (IP3R) knockdown reduces calpain activity after cGMP analog or NO donor activation. BOC activity in transfected cells only, shown by Cy3-red labeling of siRNA, as shown in D. In cells shown on the left BOC-labeled cells were measured after control siRNA transfection, without treatment or with 8-Br-cGMP (100 µM) 40 minutes before BOC was added, or with SNP (100 µM) 10 minutes before BOC activity was assayed. The IP3R Knockdown Group was transfected with siRNA targeting Ins(1,4,5)P3R1 (IP3R1) but was otherwise the same. The activated knockdown cells are different from matched controls, *P<0. 05, n=20, mean ± s.d. (D) Appearance of transfected cells with and without cGMP activation. Top panels show cells transfected with control siRNA; bottom panels show cells transfected with siRNA targeting Ins(1,4,5)P3R1 (IP3R siRNA Knockdown). The left panel of each group shows phase-contrast (green) and Cy3-labeled siRNA (red). The Ins(1,4,5)P3R1-knockdown cells show a greater average diameter, although there is significant stochastic variation in cell size within both groups. Ins(1,4,5)P3R-knockdown cells have an average diameter 1.38±0.43 times that of control cells, n=10. The spread cells showed essentially no movement (not illustrated). The right panel of each group show BOC fluorescence (blue). Fluorescent BOC labeling was seen only in the control cells treated with nonsense siRNA after cGMP activation. Each image is 110 µm2.