Essential Role of Neural Wiskott-Aldrich Syndrome Protein in Podosome Formation and Degradation of Extracellular Matrix in Src-Transformed Fibroblasts1

[CANCER RESEARCH 62, 669–674, February 1, 2002] Essential Role of Neural Wiskott-Aldrich Syndrome Protein in Podosome Formation and Degradation of Extracellular Matrix in src-transformed Fibroblasts1

Kiyohito Mizutani, Hiroaki Miki, Hong He, Hiroshi Maruta, and Tadaomi Takenawa2 Department of Biochemistry, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan [K. M., H. M., T. T.]; PRESTO [H. M.] and CREST [T. T.], Japan Science and Technology Corporation, Saitama 332-0012 Japan; and Ludwig Institute for Cancer Research, PO Royal Melbourne Hospital, Victoria 3050, Australia [H. H., H. Ma.]

ABSTRACT etic cells (5). We subsequently identified a ubiquitously expressed WASP-homologous protein, N-WASP (6), and the WASP/N-WASP- Transformation of cells by the src oncogene causes dramatic changes in related proteins WAVE/Scars (WAVE1, WAVE2, and WAVE3; adhesive structures. In v-src-transformed 3Y1 rat fibroblasts (3Y1-src), Refs. 7, 8). This family of proteins possesses a common domain for there are actin-rich protrusive structures called podosomes by which attachment to the extracellular matrix is thought to occur. In this study, activating the Arp2/3 complex, which induces rapid polymerization of we found that neural Wiskott-Aldrich syndrome protein (N-WASP) colo- actin in vitro and in vivo (9–11). Macrophages obtained from Wiskott- calizes with filamentous actin (F-actin) in podosomes. Expression of dom- Aldrich syndrome patients show a specific defect in podosome for- inant-negative mutants of N-WASP, ⌬cof N-WASP and ⌬VPH N-WASP, mation (12) and directed movement induced by chemoattractant (13). both of which are incapable of activating the Arp2/3 complex, suppressed In addition, Cdc42, an upstream regulator of WASP, and the Arp2/3 podosome formation, suggesting that N-WASP is essential in this process. complex, an effector molecule of WASP for inducing rapid actin Localization of N-WASP in podosomes appears to be attributable to polymerization, are reported to play critical roles in podosome for- interaction between N-WASP and the SH3 domain of cortactin. Indeed, mation (13, 14). Therefore, macrophages appear to use the Cdc42/ microinjection of the cortactin SH3 domain suppressed podosome forma- WASP pathway to activate the Arp2/3 complex and induce rapid actin tion. We also observed that 3Y1-src cells cultured on fibronectin degrade polymerization in podosomes. the fibronectin primarily at the podosomes and that the inhibition of podosome formation by ⌬cof N-WASP abolishes the fibronectin degrada- Podosome formation has also been observed in cells transformed by tion. These results suggest the importance of N-WASP in podosome v-src oncogenes (15). One of the primary substrates for activated Src formation and extracellular matrix degradation, which are processes family tyrosine kinases is cortactin (16), which bundles actin fila- thought to underlie the invasive phenotype of 3Y1-src cells. ments (F-actin; Ref. 17). As mentioned above, cortactin is concentrated in podosomes (4). Cortactin contains an SH3 domain at its COOH terminus, which binds a few proteins such as the neuronal INTRODUCTION CortBP1 that contain Pro-rich motifs (18), although the precise phys- Attachment of cells to the ECM3 is a critical event that controls iological role of their interaction still remains to be clarified. In their survival, growth, and migration. For instance, detachment of addition, cortactin associates directly with and activates, though normal epithelial cells, but not transformed cells, from the ECM leads weakly, the Arp2/3 complex via its NH2-terminal acidic region, which to an apoptosis called as anoikis (1). Cells use several types of is similar to regions found in WASP family proteins (19). Therefore, adhesive structures that are classified primarily according to morpho- a similar mechanism to activate the Arp2/3 complex appears to exist logical criteria. Podosomes are known to occur specifically in mono- both in podosomes of src-transformed cells and in macrophages. It cyte-derived hematopoietic cells including macrophages and oste- remains unclear, however, whether cortactin alone is sufficient to oclasts (2). Podosomes contain an adhesive receptor, integrin (3), and activate the Arp2/3 complex and cause formation of podosomes in several actin-regulating proteins such as cortactin (4), talin (3), and src-transformed cells. vinculin (3) that link integrin to the actin cytoskeleton. One important We hypothesized that some WASP family proteins may play a role characteristic of podosomes is their dynamic nature. They are rapidly in podosome formation in src-transformed cells. In the present study, constructed and destroyed, and therefore, they have been thought we found that N-WASP accumulates in podosomes in rat 3Y1 fibro- suitable for transient adhesions during cellular motility but not for blasts transformed with v-src (3Y1-src). Expression of dominant- generating intimate and stable attachments to the extracellular envi- negative N-WASP mutants that cannot activate the Arp2/3 complex ronment. suppresses podosome formation. In addition, we found that 3Y1-src Several recently published reports have documented the importance cells degrade fibronectin primarily at the podosomes and that sup- of the WASP for formation of podosomes in macrophages, although pression of podosome formation by N-WASP mutants abolishes many other actin-regulating proteins are also known to accumulate in fibronectin degradation. podosomes. WASP was originally identified as the causative gene product for the hereditary X-linked disease Wiskott-Aldrich syndrome, which is characterized by thrombocytopenia, eczema, and MATERIALS AND METHODS immunodeficiency (5). WASP is expressed exclusively in hematopoi- Antibodies and Recombinant Proteins. The polyclonal anti-N-WASP antibody and anti-WAVE antibody were made as described previously (7, 20). Received 3/23/01; accepted 12/4/01. The anti-␤-galactosidase and the anti-GST polyclonal antibodies were pur- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with chased from Chemicon and Santa Cruz, respectively. The anti-p80/85 (cortac- 18 U.S.C. Section 1734 solely to indicate this fact. tin) and the anti-Myc monoclonal antibody were from Upstate Biotechnology 1 This study was supported in part by a Grant-in-Aid for Cancer Research from the and Santa Cruz Biotechnology, respectively. Secondary antibodies conjugated Ministry of Education, Science, Sports and Culture of Japan, and in part by a Grant-in-Aid to alkaline phosphatase (used in Western blotting) and fluorescein (used in Research for the Future Program from the Japan Society for the Promotion of Sciences. 2 To whom requests for reprints should be addressed, at Department of Biochemistry, immunofluorescence microscopy) were from Promega and Cappel, respec- Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo tively. GST-SH3 cortactin, GST-SH3 IRSp53, and GST-SH3 p85 were pre- 108-8639, Japan. Phone: 81-3-5449-5510; Fax: 81-3-5449-5417; E-mail: takenawa@ims. pared as described previously (21, 22). u-tokyo.ac.jp. Binding Assay. GST-fusion proteins were immobilized on 20 ␮l of gluta- 3 The abbreviations used are: ECM, extracellular matrix; F-actin, filamentous actin; GST, glutathione S-transferase; WASP, Wiskott-Aldrich syndrome protein; N-WASP, thione-Sepharose 4B beads (Amersham Pharmacia) and mixed with 3Y1-src neural WASP; WT, wild type. cell lysates. After being washed in lysis buffer, the beads were suspended in 669

Transient Expression in 3Y1-src Cells. WT, ⌬cof, and ⌬VPH N-WASP- expressing plasmids were constructed as described previously (6, 23, 24). As a control, Lac-Z-expressing plasmid was also constructed. Two ␮g of each recombinant plasmid were transfected into 3Y1-src cells with Lipofectamine 2000 (Life Technologies, Inc.) reagents. Twenty-four h after transfection, the cells were fixed with formaldehyde. For immunoprecipitation assay, WT and ⌬SH3 Myc-tagged, cortactin-expressing plasmids were constructed in pEF- BOS plasmid vector and transfected into COS7 cells with Lipofectamine 2000 reagents. Microinjection of 3Y1-src Cells. GST-fusion proteins (3.0 mg/ml) were microinjected with a Micromanipulator 5171 (Eppendorf) with Femtotip nee- dles. After injection, the cells were cultured for an additional 1 h and then fixed. In Vitro ECM Degradation Assay. 3Y1-src cells were seeded on FITC- fibronectin-coated glass coverslips for in vitro ECM degradation assay as described previously (25). To quantify the degraded area of FITC-fibronectin, we used NIH-image 1.62f and calculated the percentage of degraded area/cell area. Immunofluorescence Microscopy. Cells cultured on coverslips were fixed in 3.7% formaldehyde in PBS for 20 min and permeabilized with 0.2% Triton X-100 in PBS. The cells were then incubated with primary antibody, followed by appropriate secondary antibodies. To visualize actin filaments, rhodamine-conjugated phalloidin (Molecular Probes) was used. To observe stained cells, a laser scanning confocal imaging system (Bio-Rad) was used.

RESULTS Fig. 1. Localization of N-WASP in podosomes of 3Y1-src cells. A, 3Y1 and 3Y1-src Localization of N-WASP in Podosomes in 3Y1-src Cells. We cells were stained with anti-N-WASP antibody and anti-WAVE antibody, respectively. To visualize actin filaments, the cells were double-stained with rhodamine-phalloidin. B, 3Y1 first determined which members of the WASP family were present in and 3Y1-src cells were stained with anti-N-WASP antibody, anti-cortactin antibody, and 3Y1-src cells by Western blotting with anti-WASP, anti-N-WASP, phalloidin. and pan anti-WAVE antibodies (this pan anti-WAVE antibody rec- ognizes all isoforms of WAVEs; data not shown). We found that the anti-N-WASP and anti-WAVE antibodies specifically recognized en- SDS sample buffer and subjected to SDS-PAGE, followed by Coomassie dogenous N-WASP and WAVEs, respectively, in both 3Y1 and brilliant blue staining or Western blot analysis. 3Y1-src cells (data not shown). As expected, we observed no positive Immunoprecipitation. Anti-N-WASP antibody or anti-Myc antibody was added to cell lysates from 3Y1-src cells or COS7 cells and incubated for 2 h signal with the anti-WASP antibody (data not shown). We then used at 4°C with rotation. Agarose beads conjugated with protein A (for anti-N- immunofluorescence microscopic analyses to determine whether N- WASP) or protein G (for anti-Myc; Pierce) were then added, and the mixture WASP and/or WAVEs were localized to podosomes. As mentioned was incubated for an additional 2 h. The beads were washed with lysis buffer earlier, podosomes are rich in F-actin and can be visualized clearly and suspended in SDS sample buffer. with phalloidin staining as dot like. As shown in Fig. 1A, most

Fig. 2. Ectopic expression of the N-WASP mutants in 3Y1-src cells. A, WT, ⌬cof, and ⌬VPH N-WASP were expressed transiently in 3Y1-src cells. Twenty-four h after transfection, the cells were fixed and stained with anti-N-WASP antibody and phalloidin. B, cells expressing N-WASP were counted and classified according to the mor- phology of their podosomes as normal, large, or no podosome. Bars, SD.

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Downloaded from cancerres.aacrjournals.org on October 1, 2021. © 2002 American Association for Cancer Research. ESSENTIAL ROLE OF N-WASP IN PODOSOME FORMATION endogenous N-WASP was colocalized with dot-like accumulations of F-actin in 3Y1-src cells, whereas WAVEs did not show similar colocalization. This was not observed in parental 3Y1 cells. To confirm that the dot-like structures containing N-WASP and F-actin were podosomes, we stained 3Y1-src cells with anti-cortactin antibody. Cortactin has been shown to be concentrated in podosomes (4), and therefore, many investigators have used cortactin as a podosome marker. Triple staining with anti-N-WASP antibody, anti-cortactin antibody, and phalloidin showed that a significant amount of endogenous N-WASP had accumulated in cortactin-positive podosomes (Fig. 1B). These results indicate that N-WASP is localized specifically to podosomes and suggest that N-WASP may play an important role in the formation of podosomes. Inhibition of Podosome Formation by Overexpression of ⌬cof and ⌬VPH N-WASP. We next examined whether N-WASP is re- quired for podosome formation. We used the ⌬cof and the ⌬VPH mutant forms of N-WASP, which lack regions essential to induce Arp2/3 complex-mediated rapid actin polymerization (10). Previous studies have shown that the ectopic expression of these mutants suppresses various N-WASP-dependent cell biological events including Cdc42-induced formation of filopodia (23), intracellular motility of Shigella flexneri (26), epidermal growth factor-induced formation of filopodia (24), and nerve growth factor-induced neurite extension (27). As shown in Fig. 2A, expression of either ⌬cof or ⌬VPH N-WASP disrupted formation of podosomes. This disruption was not an artifact attributable to ectopic expression because podosomes were still observed in Lac-Z-expressing control cells. The proportions of podosome-forming cells were determined for control (Lac-Z), WT N-WASP-, ⌬cof N-WASP-, and ⌬VPH N-WASP-expressing cells. Eighty-four % of Lac-Z-expressing control cells showed podosome formation. In ⌬cof- and ⌬VPH N-WASP-expressing cells, the proportions were reduced significantly to 22 and 23%, respectively. In contrast, expression of WT N-WASP in 3Y1-src cells resulted in large podosome-like accumulations of F-actin in 75% of cells (Fig. 2B), whereas expression of N-WASP in 3Y1 cells did not cause any significant change in accumulation of F-actin. It is unclear if the unusually large accumulations of F-actin we observed function in the same manner as that of normal-sized podosomes. However, cortactin, a podosome marker, colocalized with the large F-actin accumulations (data not shown), suggesting that these F-actin accumulations may be large podosomes. The data with the mutants strongly suggest that N-WASP is involved in podosome formation by regulating actin polymerization through the Arp2/3 complex. Fig. 3. Association of N-WASP with the SH3 domain of cortactin. A, 3Y1-src cell Interaction of N-WASP and Cortactin. An SH3 domain is pres- lysates were mixed with GST-SH3 cortactin protein immobilized on beads, and bound ent in the COOH-terminal region of cortactin, and N-WASP has a proteins were analyzed by Western blotting with anti-N-WASP and anti-WAVE antibod- proline-rich region to which several SH3 proteins bind. We speculated ies. B, recombinant His-tagged N-WASP proteins (right lane) were analyzed by far- Western blot with GST (100 nM), GST cortactin SH3 (10 and 100 nM) or without proteins that the SH3 domain of cortactin may interact with N-WASP and (Ϫ). Detection was by an anti-GST antibody. C, 3Y1-src cell lysates were mixed with participate in N-WASP localization in podosomes. Indeed, pull-down anti-N-WASP antibody (or control preimmune rabbit serum) and incubated for 2 h with assays with GST-fusion proteins containing the cortactin SH3 domain rotation. Protein A-conjugated agarose beads were then added and incubated for an additional 2 h. Proteins bound to the beads were analyzed by Western blotting with revealed specific binding to N-WASP but not to WAVEs (Fig. 3A). anti-cortactin and anti-N-WASP antibodies. D, N-WASP alone or with Myc-cortactin To investigate further whether N-WASP can interact directly with (WT or ⌬SH3) were coexpressed in COS7 cells and then immunoprecipitated with cortactin, we performed a Far-Western blot assay. Recombinant His- anti-Myc antibody. The precipitates were analyzed by immunoblotting. tagged N-WASP proteins were blotted onto a membrane filter, and the membrane filter was overlaid with GST-SH3 cortactin. As shown in occurs via the SH3 domain of cortactin, we prepared Myc-tagged cor- Fig. 3B, cortactin SH3 domain binds directly to N-WASP, but control tactin expression constructs of WT and a mutant lacking the SH3 domain GST does not. (⌬SH3). We then expressed N-WASP alone or with Myc-cortactin (WT We then examined whether endogenous cortactin and N-WASP form or ⌬SH3) and performed immunoprecipitation with anti-Myc antibody. a protein complex in 3Y1-src cells. 3Y1-src cell lysates were immuno- Examination of the immunoprecipitates with anti-N-WASP antibody precipitated with anti-N-WASP antibody, and the precipitates were sub- revealed that N-WASP was coimmunoprecipitated with Myc-cortactin jected to Western blot analysis with anti-cortactin antibody. As shown in (WT) but not with Myc-cortactin (⌬SH3; Fig. 3D), indicating that the Fig. 3C, a significant amount of cortactin was coimmunoprecipitated with SH3 domain is essential for in vivo binding between N-WASP and anti-N-WASP antibody, indicating in vivo formation of cortactin/ cortactin. N-WASP complexes. To investigate whether the in vivo interaction If cortactin through its SH3 domain recruits N-WASP in podo- 671

Fig. 4. Inhibition of podosome formation by microinjection of GST-SH3 cortactin. A, immunofluorescence staining of cells injected with GST or GST-SH3 proteins. GST-fusion proteins were detected with anti-GST antibody. Actin filaments were detected with phalloidin. B, GST-fusion proteins used in this experiment. C, percentage of podosome-forming 3Y1-src cells among cells injected with GST-fusion proteins. Bars, SD.

somes, inhibition of binding between cortactin and N-WASP should podosome formation and expression of WT N-WASP induced unusu- suppress podosome formation. Indeed, microinjection of a GST- ally large accumulation of actin filaments (Fig. 2A), we expressed WT fusion protein containing the SH3 domain of cortactin inhibited po- or ⌬cof N-WASP in 3Y1-src cells and subjected the cells to the in dosome formation in 3Y1-src cells, but microinjection of other SH3 vitro ECM degradation assay. As shown in Fig. 5B, 3Y1-src cells domain proteins that do not interact with N-WASP, such as those of expressing control Lac-Z showed normal podosome formation. In IRSp53 or p85 regulatory subunit of phosphatidylinositol 3-kinase contrast, WT N-WASP-expressing cells formed large accumulation of (22), did not interfere with podosome formation (Fig. 4). These data actin filaments, where ECM degradation occurred. On the other hand, suggest that the interaction between N-WASP and cortactin is impor- 3Y1-src cells expressing ⌬cof N-WASP did not form podosomes, and tant for podosome formation. ECM degradation was also suppressed. The percentages of degraded Suppression of ECM Degradation by ⌬cof N-WASP. During area in Lac-Z-, WT N-WASP-, and ⌬cof N-WASP-expressing cells tumor metastasis, degradation of the ECM is considered a key process were 3.6, 11, and 0.9%, respectively (Fig. 5C). These data confirm the for malignant cells to escape from the primary tumor and to invade importance of podosomes in ECM degradation and suggest that N- into other organs. In some tumor cell lines, proteolytic activity has WASP may play an important role in tumor metastasis through been observed at protrusive cellular sites that are structurally similar regulation of podosomes that degrade ECM. to podosomes (25). To determine whether podosomes in 3Y1-src cells possess localized ECM degradation activity, we performed an in vitro ECM degradation assay in which we first coated glass coverslips with DISCUSSION FITC-conjugated fibronectin and then seeded 3Y1-src cells onto the coverslips. ECM degradation was visualized as black areas against the In the present study, we found that N-WASP, a ubiquitously ex- uniformly fluorescent fibronectin. The results of these experiments are pressed WASP homologue, plays an essential role in podosome for- shown in Fig. 5A. ECM degradation occurred in a dot-like manner, mation in 3Y1-src cells. Our results are similar to those of a recent and most degraded areas colocalized with F-actin-rich podosomes. report on the role of WASP in macrophages (12). We also found that Incomplete overlap may be attributable to cell movement during the cortactin binds to N-WASP through its COOH-terminal SH3 domain. ECM degradation assay. This result strongly suggests that 3Y1-src In this context, it would be of interest to note that HS1, a cortactin- cells degrade the ECM at podosomes. related protein expressed exclusively in hematopoietic cells (21), is We then examined whether podosomes are essential for inducing also localized in podosomes of macrophages, binds WASP through its ECM degradation. Because expression of ⌬cof N-WASP suppressed COOH-terminal SH3 domain, and microinjection of the HS1 SH3 672

Fig. 5. Proteolytic activity of 3Y1-src cell podosomes. A, 3Y1 and 3Y1-src cells were seeded on FITC-fibronectin-coated coverslips. The cells were cultured for 1 day and then fixed. To visualize actin filaments, the cells were stained with phalloidin. Arrowheads, areas of degraded fibronectin that overlap with podosomes. B, WT and ⌬cof N-WASP-expressing plasmids (or control Lac-Z- expressing plasmids) were transfected into 3Y1-src cells. Twenty-four h after transfection, the cells were subjected to the in vitro ECM degradation assay. C, percentage of degraded area/cell area. By using NIH-image 1.62f, the degraded areas of FITC-fibronectin were quantified. Bars, SD.

domain strongly blocks the formation of podosomes,4 suggesting the Arp2/3 complex, which supports the idea that cortactin plays an general role of both cortactin family and WASP family of actin- important role in determining the localization of N-WASP in vivo.It binding proteins in podosome formation in rapidly migrating cells. may also be that cortactin activates N-WASP, because several SH3 A previous report noted that cortactin binds directly to Arp2/3 domain-containing proteins, such as Grb2/Ash, WISH, and Nck, have complex via its acidic NH2-terminal region (19). It is likely that been shown to activate N-WASP (28–30). We, however, found that cortactin links N-WASP to the effector Arp2/3 complex. Although cortactin has little, if any, ability to activate N-WASP (data not N-WASP can bind and activate the Arp2/3 complex directly (10), shown). indirect association through cortactin should facilitate the activation We showed that podosomes in 3Y1-src cells possess proteolytic of Arp2/3 complex by N-WASP. Therefore, it is reasonable that activity for degradation of the ECM. Chen (25) reported previously cortactin recruits N-WASP to the site where the Arp2/3 complex is that ECM-degrading activity is concentrated in podosome-like pro- localized and induces a strong activation of the Arp2/3 complex by trusive structures that the author termed “invadopodia.” Because Chen N-WASP. Weed et al. (19) reported that cortactin is recruited via its used v-src-transformed cells to observe the invadopodia, we believe NH -terminal region to lamellipodia through an interaction with the 2 that invadopodia are podosomes. One important question is whether podosome-like structures are essential for degradation of the ECM. In 4 S. Linder, H. He, T. Watanabe, A. Abo, M. Aepfelbacher, and H. Maruta. HS1 forms a complex with WASP to organize podosomes in macrophages, manuscript in the present study, we addressed this question by suppressing podo- preparation. some formation with an N-WASP dominant-negative mutant. Expres- 673