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. 2010 Oct;90(10):1533-42.
doi: 10.1038/labinvest.2010.120. Epub 2010 Jun 21.

Functional Osteoclast Attachment Requires inositol-1,4,5-trisphosphate Receptor-Associated cGMP-dependent Kinase Substrate

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Functional Osteoclast Attachment Requires inositol-1,4,5-trisphosphate Receptor-Associated cGMP-dependent Kinase Substrate

Beatrice B Yaroslavskiy et al. Lab Invest. .
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Abstract

Osteoclast activity is central to balanced bone turnover to maintain normal bone mass. A specialized osteoclast attachment to bone localizes acid secretion to remove bone mineral; in some cases, attachment is functionally impaired despite normal attachment proteins. The inositol-1,4,5-trisphosphate receptor-1 (IP3R1) is an intracellular calcium channel required for regulation of reversible osteoclast attachment by nitric oxide (NO), an important regulator of both normal and pathological bone degradation. In studies using human osteoclasts produced in vitro, we found that IP3R1 binds an endosomal isoform of the IP3R-associated cGMP-dependent kinase substrate (IRAG). IRAG is a substrate of cGMP-dependent kinase-1 (PKG1) and binds the PKG1 isoform PKG1β, which was the predominant form of PKG1 in human osteoclasts. Western blots of IRAG were consistent with NO-dependent serine phosphorylation of IRAG. An additional effect of PKG1β activity in osteoclasts was disassociation of IP3R1-IRAG complexes, as shown by analysis of IP3R1 complexes and by localization of the proteins within cells. IP3R1-IRAG complexes were stabilized by PKG or Src antagonists, Src activity being a requirement for IP3R1 calcium release downstream of PKG. IP3R1-mediated calcium release regulates cellular detachment in part through the calcium-dependent proteinase μ-calpain. In osteoclasts with IRAG suppressed by siRNA, activity of μ-calpain was increased relative to cells with normal IRAG, and regulation of μ-calpain by NO was lost. Furthermore, cells deficient in IRAG detached easily from substrate and had smaller attached diameters and randomly distributed podosomes, although IRAG knockdown did not affect cell viability. Our results indicate that IRAG is required for PKG1β-regulated cyclic calcium release during motility, and that disruption of the IP3R1-IRAG calcium regulation system is a novel cause of dysfunctional osteoclasts unrelated to defects in attachment proteins or acid secretion.

Figures

Figure 1
Figure 1. Isolation of CD14 osteoclast precursors, differentiation of osteoclasts in vitro, and expression of IP3R1 and 2 in CD14 cells and osteoclasts
A. Peripheral blood mononuclear cells (left) and CD14-selected cells (right) were evaluated for CD14, CD3 and CD19 expression by flow cytometry. Affinity purified CD14 cells (right column) contained 1-2% CD19 or CD3 lymphocytes, with marked enrichment for monocytes. B. CD14 purified cells appeared by phase microscopy as monotonous mononuclear cells. The field is 180 μm square. C. Osteoclasts, stained for TRAP activity, from CD14 cells after two weeks in 40 ng/ml RANKL and 10 ng/ml CSF-1. Nearly all show TRAP positivity and the majority of the cell nuclei are fused into giant cells. Transmitted light photograph, the field is 350 μm square. D. Expression of IP3R1 and IP3R2 in CD14-selected cells and osteoclasts by quantitative PCR. Messenger RNA for both IP3R1 and IP3R2 were present. IP3R1 was the predominant transcript in differentiated cells, ~ 10 fold greater signal than IP3R2 (* p <0.05).
Figure 2
Figure 2. The IP3R-associated cGMP-dependent kinase substrate (IRAG) and the IRAG-binding isoform of PKG1 in osteoclasts
A. Western blotting for IRAG was performed on anti-IRAG immunoprecipitates from osteoclasts that were untreated (Ctl) or treated with the NO donor sodium nitroprusside (SNP) at 100 μM for 5-10 minutes, or with the inhibitory cGMP analog 8-(Rp-4-chlorophenylthio)guanosine-3′,5′-cyclic phosphorothioate (Rp-cGMPS), 50 μM, for 30 minutes with or without subsequent treatment with SNP (100 μM for 10 minutes). Western blotting showed similar amounts of the large and small forms of IRAG, with Mr of ~ 150 and 100 kDa, under all conditions. The last lane shows an isotype control (nonimmune serum). B. Anti-phosphoserine blot (upper panel) of anti-IRAG immunoprecipitates from untreated osteoclasts (Ctl) or osteoclasts treated with 100 μM SNP for 5-10 minutes, or with 50 μM Rp-cGMPS for 30 minutes. The blots were then stripped and reprobed with anti-IRAG antibody to confirm similar recovery of IRAG (lower panel). IRAG phosphoserine was increased after treatment with SNP, but reduced by the PKG antagonist. C. Relative expression of PKG1 isoforms in osteoclasts. Quantitative PCR using primers specific to PKG1β or PKG1α or recognizing both forms (Common) was performed. The amount of product for each primer set is shown relative to GAPDH as an internal control. The amount of PKG1β was indistinguishable from total PKG1. PKG1α mRNA was present, but at levels significantly lower than PKG1β (* p < 0.05; n.s., not significant).
Figure 3
Figure 3. Localization of IRAG at endosomal sites
Photomicrographs of human osteoclasts on glass coverslips with immune labeling as indicated. To determine colocalization of IP3R1 without subjectivity, pixels labeling both for IRAG and IP3R1, were calculated by digital coincidence selection (Methods), the results shown in black in columns labeled Coincidence. Colocalization was more prominent when PKG was inhibited by Rp-cGMPS, than when PKG was activated by NO, and this difference was accentuated when Ca2+ was low. All fields shown are 50 μm square A. IP3R1 and IRAG in human osteoclasts on glass coverslips with PKG inhibited by 50 μM Rp-cGMPS (30 minute incubation; top row of panels) or activated by 100 μM sodium nitroprusside (10 minute incubation, bottom row of panels). B. The same experiment was performed after pre-treatment with the cell-permeant Ca2+ chelator-donor, 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetate (BAPTA, 70 μM, as acetoxymethyl ester) added 40 minutes before the PKG effectors.
Figure 4
Figure 4. Effects of NO donors on distribution of IRAG at non-endosomal sites
A. At the plane of cell attachment to glass substrate, antibody reacting with long and short forms of IRAG (red) did not localize with punctate cellular attachments (top, phalloidin, green; arrows). However, after NO donor activation IRAG localized in cellular attachments (bottom, yellow; arrows). Fields are 30 μm square. IP3R1 precipitates from osteoclasts treated or not with NO donor or PKG inhibitor and IRAG precipitates from supernatants after IP3R1 precipitation were blotted for migfilin and VASP (bottom panels). In IP3R1 precipitates (lanes 1-3) there was no significant migfilin. After depletion of IP3R1, precipitates of supernatant IRAG included migfilin. This association was increased by sodium nitroprusside. This fraction also included VASP, which also increased with sodium nitroprusside. VASP in IP3R1 precipitates was not determined. B. In cells treated with NO donors, IRAG nuclear localization was increased relative to cells in which PKG was blocked. Similar effects were seen using activating cGMP analogs (not illustrated). Fields are 50 μm square. C. Effect of PKG knockdown on redistribution of IRAG to nuclei. PKG siRNA reduced PKG expression by approximately 80% (top); the amount of PKG was not affected by sodium nitroprusside (100 μM treatment, 30 minutes before cell lysis). In PKG silenced cells (siRNA shown by green label), following sodium nitroprusside treatment (100 μM, 10 minutes) IRAG no longer was redistributed to nuclei (bottom left), while in cells with control siRNA, also after sodium nitroprusside treatment, nuclear localization was strong (bottom right).
Figure 5
Figure 5. Inhibition of IRAG expression by siRNA and effects on calpain activity and relative intracellular Ca2+
A. Suppression of expression of the large form of IRAG by 72 hour incubation after transfection of a mixture of two siRNAs; this reduced the amount of the 150 kDa form of IRAG to ~30% of control by Western blot for total IRAG protein. Changes in the small form of IRAG were not seen, which is expected since the siRNAs targeted the long transcript and the target sequences are not present in the short isoform. This was intentional in that the long form only localizes to the endosomal membranes (see text). B. When IRAG is suppressed, basal Ca2+/calpain is increased, but it is no longer sensitive to NO. Suppression of IRAG increased average cellular μ-calpain activity in the absence of stimulation of PKG (bar (1) versus (4), *p< 0.05). Preparations incubated 45 minutes with the strong PKG antagonist Rp-CPT-cGMPS, 50 μM, showed low calpain activity in both control and IRAG-suppressed cells. When sodium nitroprusside was added to IRAG suppressed or control cells, differences in calpain activity were not significant (second versus fifth bars). Calpain activity was assayed using t-butoxycarbonyl-Leu-Met-chloromethylaminocoumarin (BOC), which when cleaved by calpain generates a fluorescent signal. C. Suppressing IRAG increased average Ca2+ in osteoclasts. Calcium was determined using Fluo-3, measuring fluorescence at 520 nM. Fluorescent signal were measured relative to no-cell background at 0, 30, and 45 minutes, all of which gave similar results. Differences were consistent, with p <0.01 by analysis of variance for control versus IRAG suppressed. Mean ± SEM, n=7 (control, black), n=12 (IRAG suppressed, grey).
Figure 6
Figure 6. Transfection of siRNA to reduce IRAG expression reduced cell spreading
A. Phase photograph of osteoclasts (green) after transfection with fluorescently tagged siRNA (red). Approximately 70% of cells are labeled, indicating the transfection efficiency. B. Cells with inhibited IRAG production had mean cell diameter reduced by ~30%; the difference is significant (p< 0.05); n=10. C. Phase photomicrographs of osteoclast cultures after transfection with IRAG-specific siRNA (1, left) or control siRNA (2, right). No increase in floating or apparently apoptotic cells were seen IRAG suppression, although cell spreading was reduced. Photographs in A and C are of 220 μm square fields.
Figure 7
Figure 7. Association of IP3R with the 150 kDa form of IRAG and effects of PKG or Src activity; IRAG not precipitated with IP3R1 is binds cytoskeletal proteins
A. Immune precipitation of IP3R1 followed by Western analysis for IRAG. The ~150 kDa form of IRAG binds IP3R1, consistent with other reports. In keeping with immune localization (Fig 3), the IRAG-IP3R1 complex was inhibited by NO donors. The Src antagonist PP2, but not its inactive congener PP3, also stabilized the complex, even when sodium nitroprusside (SNP) was added. A control Western blot from total lysate confirmed similar quantities of large and small forms of IRAG in this preparation (lower panel). B. Studies precipitating IP3R1, then blotting using antibody to IP3R1 (top panel), stripping it and re-blotting for IRAG (third panel). Separate aliquots of the lysates were blotted directly for IP3R1 phosphotyrosine353 (second panel), followed by stripping and re-blotting for actin (bottom panel). Two cGMP inhibitors preserve IRAG-IP3R association (left two lanes) in preparations where cell Ca2+ held at a low level by BAPTA. In the NO donor sodium nitroprusside (SNP) and with the cGMP analog 8-Br-cGMP complexes were dissipated. With the NO donor or the cGMP analog, tyrosine phosphorylation of IP3R1 was seen (this is dependent on Src; see text and (C) below). C. Immune precipitation of IP3R1 with Western analysis for IP3R1, followed by stripping membranes and re-blotting for IRAG (top two panels). Separate aliquots of the supernatants were analyzed by Western blot for phospho-Src tyrosine415; this blot was stripped and blotted for total Src and actin (bottom three panels). Strong Src phosphorylation occurred after sodium nitroprusside (100 μm), but the Src antagonist PP2 eliminated this and, as in (A), IRAG-IP3R1 association was retained in NO donor cells when Src activity was eliminated.

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