18 research outputs found
Osteoclasts Control Osteoblast Chemotaxis via PDGF-BB/PDGF Receptor Beta Signaling
BACKGROUND: Bone remodeling relies on the tightly regulated interplay between bone forming osteoblasts and bone digesting osteoclasts. Several studies have now described the molecular mechanisms by which osteoblasts control osteoclastogenesis and bone degradation. It is currently unclear whether osteoclasts can influence bone rebuilding. METHODOLOGY/PRINCIPAL FINDINGS: Using in vitro cell systems, we show here that mature osteoclasts, but not their precursors, secrete chemotactic factors recognized by both mature osteoblasts and their precursors. Several growth factors whose expression is upregulated during osteoclastogenesis were identified by DNA microarrays as candidates mediating osteoblast chemotaxis. Our subsequent functional analyses demonstrate that mature osteoclasts, whose platelet-derived growth factor bb (PDGF-bb) expression is reduced by siRNAs, exhibit a reduced capability of attracting osteoblasts. Conversely, osteoblasts whose platelet-derived growth factor receptor beta (PDGFR-beta) expression is reduced by siRNAs exhibit a lower capability of responding to chemotactic factors secreted by osteoclasts. CONCLUSIONS/SIGNIFICANCE: We conclude that, in vitro mature osteoclasts control osteoblast chemotaxis via PDGF-bb/PDGFR-beta signaling. This may provide one key mechanism by which osteoclasts control bone formation in vivo
Transgenic Mice for a Tamoxifen-Induced, Conditional Expression of the Cre Recombinase in Osteoclasts
Background: Studies on osteoclasts, the bone resorbing cells, have remained limited due to the lack of transgenic mice allowing the conditional knockout of genes in osteoclasts at any time during development or adulthood. Methodology/Principal Finding: We report here on the generation of transgenic mice which specifically express a tamoxifen-inducible Cre recombinase in osteoclasts. These mice, generated on C57BL/6 and FVB background, express a fusion Cre recombinase-ERT2 protein whose expression is driven by the promoter of cathepsin K (CtsK), a gene highly expressed in osteoclasts. We tested the cellular specificity of Cre activity in CtsKCreERT2 strains by breeding with Rosa26LacZ reporter mice. PCR and histological analyses of the CtsKCreERT2LacZ positive adult mice and E17.5 embryos show that Cre activity is restricted largely to bone tissue. In vitro, primary osteoclasts derived from the bone marrow of CtsKCreERT2+/2LacZ+/2 adult mice show a Cre-dependent b-galactosidase activity after tamoxifen stimulation
Src-dependent repression of ARF6 is required to maintain podosome-rich sealing zones in bone-digesting osteoclasts
Bone digestion occurs when osteoclasts adhere onto bone surfaces and polarize to form acidic, hydrolase-rich resorption lacunae. For this process, they condense their actin-rich podosomes in tight belts to establish sealing zones, which segregate their basal membranes from those facing resorption lacunae. This polarization process remains poorly understood. Here, we combined quantitative proteomics and gene silencing to identify new substrates of the Src tyrosine kinase, a key regulator of osteoclast function. We now report that a depletion of the ARF GTPase-activating protein GIT2, which localizes to sealing zones upon Src phosphorylation, or a lack of GTP hydrolysis on ARF6 impairs sealing zone formation and polarized membrane traffic. Surprisingly, the Rho guanine nucleotide exchange factors α and β PIX, which usually coordinate ARF and Rho signaling, were found to be dispensable. We conclude that the Src-dependent localization of GIT2 is essential for down-regulating ARF6 activity at sealing zones, and thus for maintaining osteoclast polarity
BAR Proteins PSTPIP1/2 Regulate Podosome Dynamics and the Resorption Activity of Osteoclasts
<div><p>Bone resorption in vertebrates relies on the ability of osteoclasts to assemble F-actin-rich podosomes that condense into podosomal belts, forming sealing zones. Sealing zones segregate bone-facing ruffled membranes from other membrane domains, and disassemble when osteoclasts migrate to new areas. How podosome/sealing zone dynamics is regulated remains unknown. We illustrate the essential role of the membrane scaffolding F-BAR-Proline-Serine-Threonine Phosphatase Interacting Proteins (PSTPIP) 1 and 2 in this process. Whereas PSTPIP2 regulates podosome assembly, PSTPIP1 regulates their disassembly. PSTPIP1 recruits, through its F-BAR domain, the protein tyrosine phosphatase non-receptor type 6 (PTPN6) that de-phosphophorylates the phosphatidylinositol 5-phosphatases SHIP1/2 bound to the SH3 domain of PSTPIP1. Depletion of any component of this complex prevents sealing zone disassembly and increases osteoclast activity. Thus, our results illustrate the importance of BAR domain proteins in podosome structure and dynamics, and identify a new PSTPIP1/PTPN6/SHIP1/2-dependent negative feedback mechanism that counterbalances Src and PI(3,4,5)P3 signalling to control osteoclast cell polarity and activity during bone resorption.</p></div
Sealing zone dynamics and osteoclast activity in digestion.
<p><b>A</b> Osteoclasts were treated with siRNAs targeting the indicated genes and then plated onto osteological discs. After 48 hours, the digested areas of osteological discs (seen in white whereas the surface appears in grey) were visualized by microscopy. <b>B</b> Bone marrow osteoclast precursors isolated from long bones of Cre-ERT2+/+,PSTPIP1+/+ and Cre-ERT2-/-,PSTPIP1+/+ mice were treated with M-CSF and RANKL and the resulting osteoclasts were further treated with tamoxifen as indicated in Materials and Methods. Sealing zones of osteoclasts plated on osteological discs were stained with phalloidin and observed by confocal microscopy. <b>C</b> PSTPIP1 and PSTPIP2 expression was determined by western blotting. <b>D</b> The activity of these osteoclasts in resorption was determined as indicated above and as described in Materials and Methods. Scale bars 50 μm. The figures presented are representative of at least 3 independent experiments. <b>E</b> Quantification of resorption pit assays were perfomed as indicated in materials and methods. Quantifications of areas from 3 different experiments are plotted in chart (mean ± SD * represents p<0.05 and ** p<0.01, significance was calculated using <i>t</i>-test). Knockdown efficiencies (>90%) determined by western blotting were as presented in previous figures. <b>F</b> The change in relative sealing zone diameter per minute was plotted for each condition; controls of individual experiments were taken together. Statistical significance was tested using students t-test for each KD. (Mean ± SD, **** p< 0.0001).</p
PSTPIP1/2 localization and role in sealing zone dynamics.
<p><b>A, B</b> Osteoclasts grown on osteological discs were fixed and stained with antibody against PSTPIP1 (green) (<b>A</b>) or PSTPIP2 (green) (<b>B</b>) and phalloidin (red). (scale bars: 20μm) and images were analyzed using the Fiji software. Fluorescence intensities across the indicated white lanes are indicated and Pearson’s coefficients were calculated (0.65 for PSTPIP1 and 0.25 for PSTPIP2 from datasets of three different experiments N = 3, and n = 68 measurements). <b>C</b> Effect of siRNA-mediated depletion of PSTPIP1 or PSTPIP2 or both PSTPIP1 and PSTPIP2 on sealing zone assembly. Osteoclasts were treated with siRNA targeting the indicated genes and then grown for 48 hours on osteological discs as indicated in Materials and Methods. Cells were then fixed and stained with phalloidin. Scale bars 20 μm. <b>D</b> Sealing zone dynamics in PSTPIP1-depleted osteoclasts. Osteoclasts were treated with siRNAs targeting PSTPIP1 and then plated on osteological discs. After 24 hours, they were infected with a recombinant adenovirus encoding the mRFP-Ezrin actin-binding domain. After 32 hours, osteoclasts were observed by time-lapse videomicroscopy (300 msec. per frame, 1 frame per 1 min., see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164829#pone.0164829.s006" target="_blank">S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164829#pone.0164829.s007" target="_blank">S2</a> Movies 1, 2). <b>E</b> The knockdown efficiencies were determined by western blotting and quantified. The figures presented are representative of at least 3 independent experiments (mean ± SD). <b>F</b> Sealing zone diameter was measured using the Fiji software. The relative sealing zone diameter (biggest sealing zone as reference) was plotted for each sealing zone assessed (n = 3 independent experiments). <b>G</b> The change of relative sealing zone diameter per minute was plotted and tested using students t-test. (mean ± SD * represents p<0.05 and ** p<0.01, *** p< 0.001).</p
Effect of PAO on tyrosine phosphorylation.
<p>Effect of PAO on tyrosine phosphorylation.</p
SHIP1/2 localization and effect of their depletion on sealing zone dynamics.
<p><b>A, B</b> Non treated osteoclasts or osteoclasts treated with siRNAs targeting PSTPIP1 were grown on osteological discs, then fixed and stained with anti SHIP1 <b>(A)</b> or SHIP2 antibodies (<b>B)</b> (green) and phalloidin (red) (scale bars: 20 μm) Images were analyzed using the Fiji software. Fluorescence intensities across the indicated white lanes are indicated, magnifications of encircled regions were inserted and Pearson’s coefficients were calculated (0.49 for SHIP1, 0.24 for SHIP2 from datasets of three different experiments N = 3, and n = 103 for SHIP1, and n = 130 for SHIP2 measurements). <b>C</b> Sealing zone dynamics in SHIP1/2 depleted osteoclasts. Osteoclasts were treated with siRNAs targeting SHIP1 or SHIP2 and then plated on osteological discs. After 24 hours, they were infected with a recombinant adenovirus encoding the mRFP-Ezrin actin-binding domain. After 32 hours, osteoclasts were observed by time-lapse videomicroscopy (100 msec. per frame, 1 frame 1 min., see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164829#pone.0164829.s016" target="_blank">S11</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164829#pone.0164829.s018" target="_blank">S13</a> Movies). <b>D</b> Sealing zone diameter was measured using the Fiji software. The relative sealing zone diameter (biggest sealing zone as reference) was plotted for each sealing zone assessed. <b>E</b> The knockdown efficiencies were determined and quantified by western blotting. The figures presented are representative of at least 3 independent experiments (mean ± SD). Scale bars 20 μm. <b>F</b> The change of relative sealing zone diameter per minute was plotted and tested using students t-test. (mean ± SD * represents p<0.05 and ** p<0.01, *** p< 0.001)</p
PSTPIP1/2 dynamics at podosomes.
<p>Osteoclasts were grown on glass coverslips and transfected with constructs to express mRFP-tagged actin-binding domain of Ezrin (mRFP-ABDE) together with the GFP-PI3,4,5 P3 binding domain of Akt kinase or mRFP-Ezrin actin-binding domain and GFP-PSTPIP1 or GFP-PSTPIP1 and mRFP-PSTPIP2. <b>A</b> Individual podosomes were then observed by time-lapse videomicroscopy 48 hours after transfection (100 msec. per frame, 1 frame per 2 sec., <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164829#pone.0164829.s008" target="_blank">S3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164829#pone.0164829.s009" target="_blank">S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0164829#pone.0164829.s010" target="_blank">S5</a> Movies). <b>B</b> The fluorescence intensities represent the variations of mean fluorescence intensity associated to 35 podosomes in three independent experiments.</p