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Vol. 20, Issue 20, 4324-4334, October 15, 2009
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Department of *Molecular Genetics, The Weizmann Institute of Science, Rehovot 76100, Israel and Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel
Submitted December 1, 2008;
Revised July 13, 2009;
Accepted August 11, 2009
Monitoring Editor: Richard K. Assoian
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Gimona and Buccione, 2006; Linder, 2007; Teitelbaum, 2007). Podosomes are punctate structures that contain an actin-rich core surrounded by a ring of associated proteins, which convey the signal generated by contact with matrix to the actin core and the actin cytoskeleton. Proteins present in the podosomal ring include integrins and associated molecules such as vinculin, paxillin, Cbl, Cas, Src, Pyk2, and the small GTPases Rho, Rac, and CDC42 (Linder and Aepfelbacher, 2003; Gimona, 2008).
In addition to osteoclasts podosomes are found in, among other cell types, macrophages, dendritic cells, and in endothelial and epithelial cells (Linder, 2007; Gimona et al., 2008). In osteoclasts, however, the organization of podosomes is linked to the ability of the cell to fulfill its physiological role. In cultured osteoclasts that are not actively resorbing bone, podosomes are scattered at random. Podosomes can assemble into clusters that grow and transform into dynamic rings, which further expand to form a large superstructure at the cell periphery that is characteristic of mature, bone-resorbing cells. In osteoclasts grown on degradable matrix, this peripheral, belt-like superstructure, referred to as the sealing zone, contains densely packed podosomes that are usually not individually discernible. In osteoclasts grown on nondegradable surface, podosomes are arranged in a less-crowded sealing zone-like structure (SZL), in which individual podosomes are visible (Destaing et al., 2003; Luxenburg et al., 2006a, 2007; Gimona et al., 2008; Saltel et al., 2008). Examination of osteoclast structure by scanning electron microscopy revealed that SZL podosomes are made of actin pillars and are interconnected by a dense array of radial actin fibers (Luxenburg et al., 2007). Podosomes are dynamic and short-lived structures with an average life span of 1–5 min (Destaing et al., 2003, 2008; Luxenburg et al., 2006b). Interestingly, as podosomes become associated with rings and the SZL, which are structures typical of active resorption, their life span is reduced and they become further destabilized. The reason for this is not known (Luxenburg et al., 2006a,b). Because osteoclast activity is dependent upon podosome-mediated adhesion to bone, pathways that regulate podosomal behavior are of importance in understanding how physiological bone resorption is regulated.
Tyrosine phosphorylation participates in osteoclast signaling pathways downstream of integrins, receptor activator of nuclear factor-
B ligand (RANKL) and macrophage colony-stimulating factor (M-CSF; CSF-1) (Bruzzaniti and Baron, 2006; Ross, 2006; Wada et al., 2006), all of which play key roles in the differentiation and activity of osteoclasts. Disruptions in tyrosine phosphorylation can therefore significantly affect osteoclast activity in vivo. Accordingly, mice lacking Src contain increased amounts of dysfunctional osteoclasts that contain disorganized podosomes and exhibit significantly increased bone mass (Soriano et al., 1991; Lowe et al., 1993). The formation, structure, and life span of podosomes in osteoclasts is affected significantly by Src activity (Sanjay et al., 2001; Miyazaki et al., 2004; Luxenburg et al., 2006b; Destaing et al., 2008), although the noncatalytic domains of Src also seem important in this respect (Schwartzberg et al., 1997; Destaing et al., 2008). Aberrant podosomal organization, reduced osteoclast function, and increased bone mass also have been described in mice lacking Pyk2 or Syk (Gil-Henn et al., 2007; Zou et al., 2007).
In contrast to the tyrosine kinases, little is known about how tyrosine phosphatases (PTPs) regulate osteoclasts (Granot-Attas and Elson, 2008). The Src homology (SH)2 domain-containing PTP SHP1 inhibits osteoclast activity because mice lacking this phosphatase exhibit increased bone resorption and reduced bone mass (Aoki et al., 1999; Umeda et al., 1999). This effect may be due to SHP1 negatively regulating RANKL signaling in precursor cells (Zhang et al., 2003). In contrast, the receptor-type PTP CD45 supports osteoclast activity; osteoclasts from CD45-deficient mice display aberrant morphology and reduced activity, which may be caused by reduced Src kinase activity in these cells (Shivtiel et al., 2008). The receptor-type PTPRO (PTP-oc) is also believed to support osteoclast function by affecting Src activity (Amoui et al., 2007). Targeted deletion of this phosphatase in RAW264.7 cells inhibited their osteoclastic differentiation in vitro, whereas osteoclast-directed overexpression of PTP-oc in transgenic mice increased bone resorption (Yang et al., 2007; Sheng et al., 2009). In vitro cell studies have suggested that PTP-PEST is an additional positive regulator of osteoclasts (Chellaiah and Schaller, 2009).
Another positive regulator of osteoclast activity is PTP epsilon (PTPe). Two major protein forms of PTPe exist—these are the receptor-type form (RPTPe) and the nonreceptor form (cyt-PTPe), which are produced by use of alternative promoters from the single Ptpre gene (Krueger et al., 1990; Elson and Leder, 1995a,b; Tanuma et al., 1999). The expression patterns of RPTPe and cyt-PTPe among cells and tissues are virtually nonoverlapping (Elson and Leder, 1995a); cyt-PTPe is expressed strongly in osteoclasts (Chiusaroli et al., 2004) and is at the focus of this study. Our previous studies have shown that cyt-PTPe supports osteoclast activity by affecting the adherence of these cells to bone. Female mice lacking PTPe (EKO mice, which lack all forms of PTPe) exhibit increased bone mass and reduced osteoclast activity in vivo and in vitro, which coincides with reduced adhesion of these cells to bone in vivo (Chiusaroli et al., 2004). EKO osteoclasts are also defective in recruiting hematopoietic precursor cells from the bone marrow into the general circulation (Kollet et al., 2006). In agreement with the above-mentioned functional data, podosomes of osteoclast-like cells (OCLs) grown from bone marrow of EKO mice are typically disorganized and often lack a clear core-ring structure (Chiusaroli et al., 2004). The present study broadens the scope of the abnormalities in the structure and dynamics of podosomes in EKO osteoclasts, and characterizes the molecular role of cyt-PTPe in osteoclasts and its regulation by physiological signaling.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), GFP cyt-PTPe (Kraut et al., 2002). C277,572S cyt-PTPe was constructed from wild-type cyt-PTPe by site-directed mutagenesis followed by sequence verification. PTPe cDNAs were cloned in the pcDNA3 expression vector (Invitrogen, Carlsbad, CA) and contained a FLAG tag at their C termini. Also used were cDNAs for Chicken Y527F Src (a gift from Dr. S. Courtneidge, The Burnham Institute for Medical Research, LaJolla, CA), wild-type Src (from Dr. J. den Hertog, Netherlands Institute of Developmental Biology, Utrecht, The Netherlands), monomeric red fluorescent protein-labeled actin (from Dr. B. Geiger, The Weizmann Institute, Rehovot, Israel), and Pyk2 (from Dr. S. Lev, The Weizmann Institute). Polyclonal antibodies used included anti-PTPe (Elson and Leder, 1995b), anti-Y402 Pyk2 and anti-Y416 Src (Cell Signaling Technology, Danvers, MA), and anti phospho-PTPe. The latter antibody was shown previously to react specifically with PTPe phosphorylated at its C-terminal tyrosine residue (Y638 in cyt-PTPe=Y695 in RPTPe; Berman-Golan and Elson, 2007). Monoclonal antibodies used included anti-v-Src (clone 327; Calbiochem, San Diego, CA), anti-Pyk2 (clone 11; BD Biosciences, San Jose, CA), anti-phosphotyrosine (clone PY99; Santa Cruz Biotechnologies, Santa Cruz, CA), anti-Rho (BD Biosciences), and anti-Rac (Cytoskeleton, Denver, CO). Also used were anti-FLAG monoclonal antibodies coupled to agarose beads (clone M2; Sigma-Aldrich., St. Louis, MO). Actin was stained with phalloidin Alexa-488 (Invitrogen). PP1 and Y27632 were from Calbiochem. Adenoviruses for expressing red fluorescent protein (RFP) or Src were purchased from Capital Biosciences (Rockville, MD). Adenoviruses for expressing wild-type (WT) or Y638F cyt-PTPe were prepared by cloning the relevant mouse cDNAs into the viral genome by using the Adeno-1 system (Capital Biosciences). Cell-permeable C3 exoenzyme (a generous gift from Dr. Alexander Bershadsky of The Weizmann Institute) was from Cytoskeleton.
Mice
Gene targeted mice lacking all forms of PTPe (EKO mice, C57BL/6 x 129 genetic background) were described previously (Peretz et al., 2000). Mice transgenic for an actin-green fluorescent protein (GFP) fusion protein were kindly provided by Dr. Andrew Matus (Friedrich Miescher Institute, Basel, Switzerland) (Fischer et al., 2000) and were crossed with EKO mice. All experiments were approved by the Institutional Animal Care and Use Committee of The Weizmann Institute in accordance with Israeli law.
Cell Culture
Osteoclasts: Marrow was extracted from femora and tibiae of 5- to 10-wk-old mice. Bone marrow cells were cultured in OCL medium (
-minimal essential medium; Sigma-Aldrich) containing 10% fetal calf serum (FCS; Invitrogen), 4 mM glutamine, 50 U/ml penicillin, 50 µg/ml streptomycin, 20 ng/ml M-CSF, and 20 ng/ml RANKL (both from R&D Systems, Minneapolis, MN)). Cells were incubated at 37°C and 5% CO2 for 5–6 d. RAW264.7 cells were purchased from the American Type Culture Collection (Manassas, VA) and grown in complete DMEM medium (Invitrogen) supplemented with 10% FCS (HyClone Laboratories, Logan, UT), glutamine, and antibiotics as described above, at 37°C and 7% CO2. RAW264.7 cells express negligible amounts of endogenous PTPe (data not shown). Cells were differentiated in OCL medium as described above at 37°C in 5% CO2 for 4 d. Chinese hamster ovary (CHO) cells stably expressing the β3 integrin subunit (CHOβ3 cells; a generous gift from Dr. M. Ginsberg, University of California, San Diego, LaJolla, CA) were grown in Ham's-F12 medium (Biological Industries, Beit Haemek, Israel) supplemented with 10% FCS (Invitrogen), glutamine, and antibiotics as described above. SYF cells were maintained as described in Gil-Henn and Elson (2003). RAW264.7 and CHOβ3 cells were transfected using Lipofectamine 2000 or Lipofectamine (Invitrogen), respectively, according to the manufacturer's instructions.
Replating Experiments
After overnight starvation in medium containing 0.1% FCS, CHOβ3 cells were trypsinized, suspended in DMEM containing 20 mM HEPES, and 1 mg/ml bovine serum albumin, and incubated at 37°C for an hour with gentle rotation. RAW264.7 preosteoclasts and primary bone marrow preosteoclasts were starved on their third and fourth days of differentiation, respectively, for 4 h in OCL medium containing 1% serum and no cytokines. Cells were detached by a short treatment with 10 mM EDTA and incubated as described above. In some cases, cells were replated on plates precoated with 20 µg/ml fibronectin (Sigma-Aldrich), 10 µg/ml vitronectin (Biological Industries), 10% serum, or 20 mg/ml poly-L-lysine. For replating of mature primary OCLs, bone marrow cells were induced to differentiate on plastic dishes covered with rat tail collagen gel (Roche, Basel, Switzerland) for 5 d. The collagen gel was digested with 0.1% collagenase (Sigma-Aldrich) at 37°C for 20 min with gentle shaking, after which the cells were replated on glass slides and fixed for analysis.
Adenoviral Infection of OCLs
Bone marrow from mice was cultured in OCL medium as described above in 35-mm glass-bottomed tissue culture plates (3–4 plates/mouse). Three days after seeding, medium was replaced with 1 ml of OCL medium containing adeno-RFP or a mixture of adeno-RFP and adenoviruses for expression Src or cyt-PTPe (WT or Y638F). After overnight incubation the medium was changed and the cells were fed daily with fresh OCL medium (containing cytokines). Cells were processed for live-cell imaging 6–8 d after seeding. Most cells in these cultures were infected and expressed RFP.
Immunoprecipitation, Src Activity, and Protein Blotting
FLAG-tagged WT and Y638F cyt-PTPe were expressed in 293 cells and purified by immunoprecipitation with anti-FLAG antibodies as described previously (Tiran et al., 2006). The purity and amounts of eluted material were determined by gel electrophoresis and silver staining. For pull-down of Src and Pyk2 with cyt-PTPe, 2 mg of cell lysates were incubated with 5 µg of purified WT or Y638F cyt-PTPe bound to M2 FLAG beads for 1 h. The PTP inhibitor sodium iodoacetate (5 mM) was present during all stages of this study, including the initial precipitation of FLAG-PTPe. Src kinase activity assay was performed as described previously (Gil-Henn and Elson, 2003) by allowing precipitated Src to phosphorylate enolase with [
-32P]ATP. SDS-polyacrylamide gel electrophoresis (PAGE), blotting, and antibody hybridization were done as described previously (Gil-Henn et al., 2000).
Rac and Rho Activity Assays
Activities were determined using the RhoA and Rac1 Activation Assay Biochem kits (Cytoskeleton) according to manufacturer's instructions. In brief, 2 mg of total protein from lysates of primary OCLs from bone marrow were used to pull down guanosine triphosphate (GTP)-bound Rac or Rho. Amounts of activated, precipitated Rac/Rho were determined by protein blotting with antibodies against Rac or Rho, followed by normalizing to total Rac/Rho amounts detected in the crude lysates.
Fluorescence Microscopy and Live-Cell Imaging
Cells were fixed for 2 min in warm 3% paraformaldehyde (PFA) (Merck, Darmstadt, Germany) containing 0.5% Triton X-100 (Sigma-Aldrich) and then in 3% PFA alone for an additional 20 min. After incubations with primary and secondary antibodies, cells were mounted with Fluoromount-G solution (Southern Biotechnology Associates, Birmingham, AL). Images were collected on a confocal Radiance 2100 laser scanning system (Bio-Rad Laboratories, Hercules, CA) or on a deconvolution DeltaVision system (Applied Precision, Issaquah, WA), including an inverted microscope (IX70) equipped with a UPlanSApo 100x/1.4 numerical aperture objective (Olympus, Tokyo, Japan). Images were acquired and deconvoluted using Resolve3D software (Applied Precision). The latter system was also used for live-cell imaging, in which cells were examined in glass-bottomed plates in a temperature- and atmosphere-controlled chamber. Cells were examined in DMEM containing 25 mM HEPES (Biological Industries) and 10% FCS. Life spans of podosomes were measured by following individual podosomes in consecutive images of cells as described in Luxenburg et al. (2006b). After adenoviral infection, only infected (RFP-expressing) cells were analyzed.
Scanning Electron Microscopy
Osteoclasts were prepared using the ventral membrane preparation (VMP) technique as described in Luxenburg et al. (2007). In brief, bone marrow cells were cultured on electron microscope grids and induced to differentiate into mature osteoclasts. The osteoclast cell body was mechanically removed by short incubation in hypotonic solution followed by mechanical peeling of the cells. The samples were processed for electron microscopy and visualized using the Ultra 55 high-resolution scanning electron microscope system (Carl Zeiss, Oberkochen, Germany).
Statistical Analysis
Except where noted, statistical analysis was performed by a two-tailed, unpaired Student's t test, with the significance level set at p = 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). To study podosomal structures in greater detail, we examined OCLs by scanning electron microscopy. In agreement with previous studies (Luxenburg et al., 2007), podosomes in the SZL of OCLs from WT mice occurred as individual, evenly distributed concentrations of actin that were interconnected by a radial network of fine actin filaments (Figure 1, A and B). In contrast, the periphery of EKO OCLs contained a complex network of disorganized fibers with very few podosomal structures (Figure 1, C and D). Qualitatively similar observations were made when OCLs were analyzed by immunostaining for the podosomal core protein actin and for vinculin, which normally surrounds the actin-rich core. Podosomal cores in the SZL of WT OCLs were of similar size and regular distribution and were surrounded by a uniform vinculin-rich region (Figure 1A, inset). In contrast, the SZL of OCLs from PTPe-deficient mice was severely disorganized and contained large irregular concentrations of actin (Figure 1C, inset; also see Chiusaroli et al., 2004). We conclude that loss of cyt-PTPe severely disrupts the internal structure of podosomes in the SZL.
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). Podosomes in EKO OCLs are therefore unable to organize properly in response to an acute stimulus in the form of replating.
Podosomes are short-lived structures, whose life span is further shortened upon associating with structures typical of active osteoclasts, such as rings or the SZL (Luxenburg et al., 2006b). To determine whether lack of cyt-PTPe affects podosomal life span, we examined OCLs from WT or EKO mice that expressed an actin-GFP transgene that fluorescently labeled their podosomal cores. The time that elapsed between appearance and disappearance of individual cores in these cells was measured using live-cell imaging. Our results confirm that in WT OCLs the life span of podosomal cores that are arranged in clusters, which are typical of nonresorbing cells, is significantly longer than cores arranged in rings or in the SZL. In contrast, the life span of podosomal cores from EKO OCLs is significantly longer irrespective of their organization within the cells; this is especially noticeable in the rings and the SZL (Figure 1I and Supplemental Table 1). We conclude that cyt-PTPe participates in regulating podosomal structure and function in OCLs, and that lack of this phosphatase affects the internal structure, stability, and cellular organization of podosomes.
Dysregulation of Src, Pyk2, Rho, and Rac in OCLs That Lack cyt-PTPe
The structure and organization of podosomes in OCLs are regulated by integrin-mediated mechanical contact with matrix. Major effectors of integrin signaling in OCLs include the tyrosine kinases Src and Pyk2 and their downstream effectors, such as the small GTPases Rho and Rac (Fukuda et al., 2005; Jurdic et al., 2006). Examination of EKO OCLs indicated that the kinase activity of Src was reduced by 
40% (Figure 2A). In agreement, autophosphorylation of Src at Y416, which qualitatively correlates with Src activity, was reduced to a greater extent. Autophosphorylation of Pyk2 at Y402, which roughly correlates with activity of this kinase, was reduced as well in EKO osteoclasts (Figure 2B). In separate experiments, we precipitated the active forms of Rho and Rac by using glutathione transferase (GST) fusion proteins that bound the GTP-bound forms of these GTPases. EKO OCLs exhibited a doubling in activated Rho and a drop of 39% in activated Rac (Figures 2, C and D). The studies presented here were performed in relatively pure and homogeneous cultures of OCLs differentiated from bone marrow by using purified M-CSF and RANKL. Previous studies used EKO OCLs produced by coculturing bone marrow with WT osteoblasts. The less-homogeneous nature of these cultures and partial contamination by osteoblasts may have masked the differences in Src and Pyk2, which were not detected previously. In all, the data indicate that lack of cyt-PTPe strongly affects the activity of molecular effectors of integrin signaling in OCLs.
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), are not localized exclusively to podosomes in OCLs.
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) and promotes inhibitory association of cyt-PTPe with microtubules (Sines et al., 2007). To determine whether integrin signaling affects phosphorylation of cyt-PTPe at Y638, we examined CHOβ3 cells, which express endogenous v and exogenous β3 integrin subunits; vβ3 integrin is the major functional integrin in osteoclasts. Exogenous cyt-PTPe was phosphorylated in adherent CHOβ3 cells, was not phosphorylated in cells held in suspension, and was rephosphorylated in cells replated on dishes coated with the integrin ligand fibronectin (Figure 4A). cyt-PTPe was not phosphorylated in cells that were replated on plates coated with polylysine, indicating that it is phosphorylated in response to constitutive or acute activation of integrins. Y638F cyt-PTPe was not phosphorylated in adherent or in replated CHOβ3 cells (Figure 4A), indicating that integrin activation induces phosphorylation of cyt-PTPe exclusively at Y638. Immunofluorescence studies indicated that the subcellular distribution patterns of Y638F and of WT cyt-PTPe are identical (data not shown); hence, lack of phosphorylation of Y638F cyt-PTPe was not due to its mislocalization. Similar adhesion-linked phosphorylation of cyt-PTPe at Y638 was observed when the phosphatase was examined in RAW264.7 OCLs plated on the integrin ligand vitronectin (Figure 4B), by using an antibody specific for C-terminally phosphorylated PTPe. Most importantly, similar phosphorylation at Y638 was observed when endogenous cyt-PTPe was examined in primary WT OCLs (Figure 4C), indicating that phosphorylation of cyt-PTPe at Y638 is triggered by integrins also in vivo. Adhesion-dependent phosphorylation of the same C-terminal tyrosine residue was observed also in the receptor-type RPTPe (Figure 4D), suggesting that this form, which is absent from OCLs, may participate in integrin signaling in other cell types.
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), raising the possibility that it might be indirect or require the presence of a third molecule.
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) (C277,572S) cyt-PTPe (CCSS cyt-PTPe) was not phosphorylated (Figure 5C, lanes 10–12), suggesting that catalytic activity of cyt-PTPe is needed for its own phosphorylation. Similar results were obtained with another inactive trapping mutant of cyt-PTPe, D245A cyt-PTPe (data not shown). Y527F Src did induce phosphorylation of CCSS cyt-PTPe at Y638 (Figure 5C, lanes 13–15), indicating that lack of phosphorylation of CCSS cyt-PTPe is not due to inaccessibility of Y638 due to aberrant folding of CCSS cyt-PTPe or its possible interactions with other molecules. Increasing Src activity can then overcome the requirement for active cyt-PTPe for phosphorylation of the phosphatase.
C-Terminal Phosphorylation Is Required for cyt-PTPe to Activate Src, to Associate with Src and Pyk2, and to Shorten Podosomal Life Span in OCLs
Src is a known substrate of PTPe in mammary tumor cells and in fibroblasts; the phosphatase activates Src by removing its inhibitory phosphorylation at Y527 (Gil-Henn and Elson, 2003). Decreased Src activity in EKO OCLs (Figure 2A) and the ability of constitutively active Src to bypass the need for active cyt-PTPe in PTPe phosphorylation (Figure 5C) suggest that PTPe activates Src in OCLs as well. To investigate this possibility, we expressed wild-type cyt-PTPe in OCLs from EKO mice by adenoviral infection. The nonphosphorylatable Y638F cyt-PTPe was also included in this study to examine the functional effects of phosphorylation at this site; expression levels of both forms of cyt-PTPe were similar to those of endogenous cyt-PTPe in WT OCLs (Figure 6A). Endogenous Src exhibited basal activity in EKO OCLs in the absence of cyt-PTPe due to its partial activation by other PTPs. Src activity was increased more than twofold upon expression of WT cyt-PTPe (Figure 6B); in contrast, expression of Y638F resulted in a trend for increased Src activity relative to mock-infected cells, which did not reach statistical significance (Figure 6B). As indicated previously, this result is not due to mislocalization of the Y638F cyt-PTPe protein. Similar changes in Src activity were observed when exogenous WT and Y638F cyt-PTPe were expressed in RAW264.7 OCLs (Figure 6, C and D). Src activity in RAW264.7 cells held in suspension was inconsistent between experiments (data not shown), most likely the result of dysregulation of multiple processes that regulate Src activity in these normally adherent cells. Src activity returned to its normal values (including its dependence on phosphorylation of cyt-PTPe; Figure 4C) in readherent cells, a finding that is consistent with these effects being influenced by integrin-mediated cell adhesion. Further studies revealed that upon its addition to lysates of primary mouse OCLs, WT cyt-PTPe formed stable complexes with Src and Pyk2. In contrast, Y638F cyt-PTPe did not associate with either kinase (Figure 6E).
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cyt-PTPe Controls Podosomal Stability in OCLs by Regulating Src and Rho
Absence of cyt-PTPe from OCLs stabilizes podosomes in correlation with reduced Src activity (Figures 1I and 2A), whereas expressing cyt-PTPe in EKO OCLs destabilizes podosomes and increases Src activity (Figure 6). Together with previous reports that reduced activity of Src lengthens podosomal life span (Luxenburg et al., 2006b; Destaing et al., 2008), these results suggest a causative link between these events, namely that lack of cyt-PTPe stabilizes podosomes by reducing Src activity. According to this mechanism increasing Src activity in EKO OCLs should shorten the life span of podosomes, thus correcting the aberrant podosomal life span phenotype of these cells. To examine this possibility, we used adenoviral vectors to express WT Src in OCLs prepared from WT or EKO mice that carried the actin-GFP transgene; podosomal stability in the SZL was examined by live-cell imaging. In agreement with results presented in Figures 1 and 6, podosomes from EKO OCLs that were not infected with Src adenovirus were more stable than their counterparts from WT cells (Figure 7). Added expression of Src in WT and EKO OCLs shortened significantly the life spans of podosomes; the differences in podosomal life span between the two genotypes disappeared (Figure 7 and Supplemental Table 3). Addition of Src therefore corrects the podosome stability phenotype of EKO OCLs, strongly supporting the conclusion that this phenotype is caused by reduced biological activity of Src that is, in turn, caused by loss of cyt-PTPe.
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; Ory et al., 2008). Rho activity is doubled in EKO OCLs (Figure 2C), suggesting that increased Rho activity contributes to increased podosome stability in the absence of cyt-PTPe. To examine this possibility, we determined whether inhibition of Rho or of Rho kinase (ROCK), a downstream effector of Rho, would shorten podosome life span in the SZL of EKO OCLs. WT and EKO OCLs were treated with cell-permeable C3 exoenzyme, an inhibitor of Rho, or with the ROCK inhibitor Y27632, and SZL podosomes were examined using live-cell imaging. Inhibition of Rho by the C3 exoenzyme significantly shortened podosomal life span in both WT and EKO OCLs (Figure 7 and Supplemental Table 3). Inhibition of ROCK had little effect on WT OCLs, but significantly shortened the life span of podosomes in the SZL of EKO OCLs to levels similar to those observed in WT OCLs (Figure 7 and Supplemental Table 3). The weaker effect of ROCK inhibition on podosomal life spans in WT OCLs in comparison to direct inhibition of Rho may be due to Rho acting via additional downstream effectors. The fact that inhibition of Rho corrected the podosomal stability phenotype of EKO osteoclasts leads us to conclude that Rho functions downstream of cyt-PTPe this respect. We note that the absolute life span of podosomal cores is affected also by the exact conditions the cells are grown in and, for example, ranged between 88 and 116 s for SZL podosomes in WT OCLs and between 113 and 212 s for similar podosomes in EKO OCLs. Nevertheless, in each experiment podosomes of WT OCLs were always clearly and significantly shorter lived than podosomes of EKO OCLs that were organized in the same type of structure (Figures 1, 6, and 7 and Supplemental Tables 1–3). |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Adhesion of osteoclasts and organization of their podosomes are ultimately regulated by integrins (e.g., McHugh et al., 2000), which suggests that cyt-PTPe participates in integrin signaling in OCLs. The present study establishes that integrin activation induces phosphorylation of cyt-PTPe exclusively at its C-terminal residue Y638 (Figure 4). The functional consequences of this phosphorylation event are significant and include enabling cyt-PTPe to associate with Src, to further activate this kinase and to shorten the life span of podosomal cores (Figure 6). The approximate twofold increase in Src activity in EKO OCLs upon cyt-PTPe expression (Figure 6B) is physiologically relevant because Src activity is reduced by approximately half in EKO OCLs (Figure 2A). Expression of cyt-PTPe in these cells then increases Src activity to levels found in WT OCLs and generates a clear physiological effect by altering podosomal stability (Figure 6F). Furthermore, the magnitude of this increase in Src activity is similar to that observed when RPTPe activates Src in fibroblasts and in mouse mammary tumor cells induced by Neu, in which it affects cellular morphology (Gil-Henn and Elson, 2003; Berman-Golan and Elson, 2007). C-Terminal phosphorylation does not seem to alter the specific activity of PTPe (Berman-Golan and Elson, 2007); its effect may be mediated by affecting the ability of PTPe to bind other molecules. The latter suggestion is consistent with the phospho-displacement model (Zheng et al., 2000), according to which the phosphorylated C-terminal of PTPe binds the SH2 domain of Src, thereby displacing pY527 of the kinase and promoting its dephosphorylation. The findings that cyt-PTPe must be phosphorylated to activate Src in osteoclasts and that this phosphorylation is induced after activation of integrins suggest that cyt-PTPe participates in linking Src activation with integrins. This conclusion is supported also by the finding that the stability of podosomes, which are highly responsive to integrin signaling, can be rescued in EKO OCLs by expressing cyt-PTPe, by expressing Src, or by inhibiting the downstream Src effector molecule Rho. It is important to note that lack of PTPe reduces, but does not abolish, activity of Src (Figure 6B; Gil-Henn and Elson, 2003; Berman-Golan and Elson, 2007) because other PTPs, such as PTP alpha (Zheng et al., 2000) and PTP-oc (Lau et al., 2006; Sheng et al., 2009) can activate Src. The exogenous Src added to EKO OCLs is therefore active.
Phosphorylation of cyt-PTPe subsequent to integrin activation is mediated at least in part by Src itself. Accordingly, phosphorylation of cyt-PTPe is increased when exogenous Src is added to cells and is decreased when Src is inhibited (Figure 5). Importantly, catalytic activity of cyt-PTPe is required for its own phosphorylation; this requirement can be overcome by constitutively active Src, whose activity is independent of cyt-PTPe. Collectively, these data suggest that cyt-PTPe participates in integrin signaling both upstream and downstream of Src and serves as a molecular amplifier of Src activity. According to this model (Figure 8), integrin activation partially activates Src. Src then induces phosphorylation of cyt-PTPe at Y638, either directly or indirectly; pY638 cyt-PTPe further activates the kinase, which affects podosomes by regulating Rho and other downstream effectors, such as ROCK. Because phosphorylation of cyt-PTPe at Y638 is critical for the above-mentioned activities, C-terminal dephosphorylation of cyt-PTPe may shut down the ability of cyt-PTPe affect integrin–Src–podosome signaling in OCLs. PTPe undergoes potent autodephosphorylation at its C-terminal tyrosine (Berman-Golan and Elson, 2007), suggesting a mechanism by which this may occur.
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vβ3 integrin to the Src SH3 domain (Obergfell et al., 2002; Arias-Salgado et al., 2003). In addition, interaction between phospho-Y402 of Pyk2 and the Src SH2 domain counters the inhibitory intramolecular interaction between the SH2 domain and pY527 of Src (Lakkakorpi et al., 2003; Horne et al., 2005); phospholipase C has also been suggested to link integrins with Src (Epple et al., 2008). Src may also be partially activated by other PTPs in OCLs in a phosphorylation-independent manner. The related receptor-type PTP alpha (RPTPa) may fulfill such a role because it is expressed in OCLs (Chiusaroli et al., 2004) and is reported to activate Src/Fyn downstream of integrins in fibroblasts in a manner that is independent of C-terminal phosphorylation (von Wichert et al., 2003; Zeng et al., 2003; Chen et al., 2006; Abreu et al., 2008). The respective roles of cyt-PTPe and RPTPa in integrin signaling may then differ in their timing: RPTPa may contribute to activation of Src initially upon integrin activation, whereas cyt-PTPe may participate in subsequent amplification and maintenance of Src activity. These distinct roles may arise from RPTPa being an integral receptor-type protein; cyt-PTPe is predominantly cytosolic and associates with the cell membrane only in part (Elson and Leder, 1995a). This single distinction accounts for the different modes of functional interaction between either phosphatase and the delayed rectifier, voltage-gated potassium channel Kv2.1 (Tiran et al., 2006). One role of cyt-PTPe in osteoclasts is then to amplify activation of Src by integrins, which occurs initially via mechanisms that do not require cyt-PTPe.
Our results agree with those of Destaing et al. (2008), who showed that Src regulates the formation, structure, life span, and rate of actin polymerization in podosomes of primary OCLs. The kinase activity and either the SH2 or SH3 domains of Src are required to restore normal podosome organization and dynamics in Src-deficient OCLs (Destaing et al., 2008); this last issue has not been addressed in EKO cells. The association between lack of cyt-PTPe, decreased Src activity and increased Rho activity also agrees with studies that document a Src-dependent decrease in Rho activity upon integrin activation (Arthur et al., 2000). Reduced Src activity in the absence of cyt-PTPe should then prevent the expected reduction in Rho activity, as we have observed. The functional significance of the reductions observed in Pyk2 phosphorylation and Rac activity in EKO OCLs remain to be determined.
Nevertheless, Src and Pyk2 associate with each other in OCLs, and both kinases are pulled down with cyt-PTPe in these cells (Figure 6E). We believe Src is the main partner of cyt-PTPe in this system because Src is significantly more effective than Pyk2 in inducing phosphorylation of cyt-PTPe (Figure 5A) and because, in contrast with Pyk2, Src is activated by tyrosine dephosphorylation. Because Src can phosphorylate Pyk2 in OCLs, reduced phosphorylation of Pyk2 in EKO OCLs (Figure 2B) may be an indirect consequence of reduced Src activity in the absence of cyt-PTPe. Further studies are required to determine whether cyt-PTPe can regulate Pyk2 directly.
Our findings concerning the small GTPases Rho and Rac agree with the established roles of these molecules in regulating podosomes in osteoclasts downstream of integrin activation (Zhang et al., 1995; Chellaiah et al., 2000; Ory et al., 2000, 2008; Fukuda et al., 2005). Increased Rho activity destabilizes the SZL in OCLs (Ory et al., 2008), whereas inhibition of Rho stabilizes the SZL in correlation with acetylation and stabilization of the microtubule network in these cells (Destaing et al., 2005). In agreement, OCLs from mice lacking Pyk2 exhibit increased Rho activity, reduced stability of the SZL, and reduced microtubule acetylation (Gil-Henn et al., 2007). Acetylation of tubulin is not reduced in EKO OCLs (data not shown), possibly because these cells are not null for Pyk2. Rac and Rho perform antagonistic functions in several systems, including cytoskeletal organization in neurite formation (Leeuwen et al., 1997), Swiss 3T3 cells (Rottner et al., 1999), and avian multinucleated cells (Ory et al., 2000, 2002), in correlation with the opposite effects lack of cyt-PTPe has on both proteins in OCLs.
Finally, other PTPs activate Src in various physiological systems, and some PTPs have been shown to activate Src in OCLs (Yang et al., 2007; Shivtiel et al., 2008); activation of Src in OCLs is then not the exclusive domain of cyt-PTPe. Nevertheless, the existence of a clear integrin-related podosomal phenotype in EKO osteoclasts indicates that cyt-PTPe plays a role that cannot be compensated for by other PTPs. cyt-PTPe may play a qualitatively unique role, for example, by acting in a unique signaling context, or its contribution to the total PTP activity that targets Src may be significant enough such that its absence reduces Src activity below a minimal threshold needed for proper cellular function. In view of the central role integrins play in many diverse cell types and because the receptor-type form of PTPe is also responsive to integrin signaling, PTPe may participate in integrin signaling in additional cell types as well.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: Laboratory of Mammalian Cell Biology and Development, The Rockefeller University, New York, NY 10065. 
Address correspondence to: Ari Elson (ari.elson{at}weizmann.ac.il).
Abbreviations used: cyt-PTPe, nonreceptor isoform of PTPe; EKO, PTPe-deficient; M-CSF, macrophage colony-stimulating factor; OCL, osteoclast-like cell; PTP, protein tyrosine phosphatase; RANKL, receptor activator of nuclear factor-B; RPTPe, receptor-type isoform of PTPe; SZL, sealing zone-like structure; WT, wild type.
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REFERENCES |
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0.04, by paired two-tailed Student's t test; N = 7. (D) Expression of endogenous cyt-PTPe (Mock) or of exogenous (+ endogenous) cyt-PTPe (WT, Y638F lanes) in adherent (A) or replated (Rp) RAW264.7 cells. *, nonspecific bands. (E) WT, but not Y638F, cyt-PTPe associates with Src and Pyk2 in OCLs. Crude lysates of primary OCLs from WT mice were subjected to an in vitro pull-down assay using purified FLAG-tagged WT or Y638F cyt-PTPe. Mass markers are in kilodaltons. (F). WT, but not Y638F, cyt-PTPe shortens the life span of SZL podosomes from EKO OCLs. WT and EKO OCLs prepared from actin-GFP mice were grown on glass, infected with adenoviral vectors expressing WT or Y638F cyt-PTPe, and examined by live-cell imaging. All four cultures were also infected with adenovirus-RFP to identify infected cells. Shown is the average time between appearance and disappearance of individual GFP-labeled actin podosomal cores. *, p 
