Essential Role of Neural Wiskott-Aldrich Syndrome Protein in Podosome Formation and Degradation of Extracellular Matrix in src-transformed Fibroblasts

INTRODUCTION

Attachment of cells to the ECM 3 is a critical event that controls their survival, growth, and migration. For instance, detachment of normal epithelial cells, but not transformed cells, from the ECM leads to an apoptosis called as anoikis (1) . Cells use several types of adhesive structures that are classified primarily according to morphological criteria. Podosomes are known to occur specifically in monocyte-derived hematopoietic cells including macrophages and osteoclasts (2) . Podosomes contain an adhesive receptor, integrin (3) , and several actin-regulating proteins such as cortactin (4) , talin (3) , and vinculin (3) that link integrin to the actin cytoskeleton. One important characteristic of podosomes is their dynamic nature. They are rapidly constructed and destroyed, and therefore, they have been thought suitable for transient adhesions during cellular motility but not for generating intimate and stable attachments to the extracellular environment.

Several recently published reports have documented the importance of the WASP for formation of podosomes in macrophages, although many other actin-regulating proteins are also known to accumulate in 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 immunodeficiency (5) . WASP is expressed exclusively in hematopoietic cells (5) . We subsequently identified a ubiquitously expressed WASP-homologous protein, N-WASP (6) , and the WASP/N-WASP-related proteins WAVE/Scars (WAVE1, WAVE2, and WAVE3; Refs. 7 , 8 ). This family of proteins possesses a common domain for activating the Arp2/3 complex, which induces rapid polymerization of actin in vitro and in vivo (9, 10, 11) . Macrophages obtained from Wiskott-Aldrich syndrome patients show a specific defect in podosome formation (12) and directed movement induced by chemoattractant (13) . In addition, Cdc42, an upstream regulator of WASP, and the Arp2/3 complex, an effector molecule of WASP for inducing rapid actin polymerization, are reported to play critical roles in podosome formation (13 , 14) . Therefore, macrophages appear to use the Cdc42/WASP pathway to activate the Arp2/3 complex and induce rapid actin polymerization in podosomes.

Podosome formation has also been observed in cells transformed by v-src oncogenes (15) . One of the primary substrates for activated Src family tyrosine kinases is cortactin (16) , which bundles actin filaments (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 CortBP1 that contain Pro-rich motifs (18) , although the precise physiological role of their interaction still remains to be clarified. In addition, cortactin associates directly with and activates, though weakly, the Arp2/3 complex via its NH₂-terminal acidic region, which is similar to regions found in WASP family proteins (19) . Therefore, a similar mechanism to activate the Arp2/3 complex appears to exist both in podosomes of src-transformed cells and in macrophages. It remains unclear, however, whether cortactin alone is sufficient to activate the Arp2/3 complex and cause formation of podosomes in src-transformed cells.

We hypothesized that some WASP family proteins may play a role in podosome formation in src-transformed cells. In the present study, we found that N-WASP accumulates in podosomes in rat 3Y1 fibroblasts transformed with v-src (3Y1-src). Expression of dominant-negative N-WASP mutants that cannot activate the Arp2/3 complex suppresses podosome formation. In addition, we found that 3Y1-src cells degrade fibronectin primarily at the podosomes and that suppression of podosome formation by N-WASP mutants abolishes fibronectin degradation.

MATERIALS AND METHODS

Antibodies and Recombinant Proteins.

The polyclonal anti-N-WASP antibody and anti-WAVE antibody were made as described previously (7 , 20) . The anti-β-galactosidase and the anti-GST polyclonal antibodies were purchased from Chemicon and Santa Cruz, respectively. The anti-p80/85 (cortactin) and the anti-Myc monoclonal antibody were from Upstate Biotechnology and Santa Cruz Biotechnology, respectively. Secondary antibodies conjugated to alkaline phosphatase (used in Western blotting) and fluorescein (used in immunofluorescence microscopy) were from Promega and Cappel, respectively. GST-SH3 cortactin, GST-SH3 IRSp53, and GST-SH3 p85 were prepared as described previously (21 , 22) .

Binding Assay.

GST-fusion proteins were immobilized on 20 μl of glutathione-Sepharose 4B beads (Amersham Pharmacia) and mixed with 3Y1-src cell lysates. After being washed in lysis buffer, the beads were suspended in SDS sample buffer and subjected to SDS-PAGE, followed by Coomassie brilliant blue staining or Western blot analysis.

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 at 4°C with rotation. Agarose beads conjugated with protein A (for anti-N-WASP) or protein G (for anti-Myc; Pierce) were then added, and the mixture was incubated for an additional 2 h. The beads were washed with lysis buffer and suspended in SDS sample buffer.

Transient Expression in 3Y1-src Cells.

WT, Δcof, and ΔVPH N-WASP-expressing plasmids were constructed as described previously (6 , 23 , 24) . As a control, Lac-Z-expressing plasmid was also constructed. Two μg 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 ΔSH3 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 needles. 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

Localization of N-WASP in Podosomes in 3Y1-src Cells.

We first determined which members of the WASP family were present in 3Y1-src cells by Western blotting with anti-WASP, anti-N-WASP, and pan anti-WAVE antibodies (this pan anti-WAVE antibody recognizes all isoforms of WAVEs; data not shown). We found that the anti-N-WASP and anti-WAVE antibodies specifically recognized endogenous N-WASP and WAVEs, respectively, in both 3Y1 and 3Y1-src cells (data not shown). As expected, we observed no positive signal with the anti-WASP antibody (data not shown). We then used immunofluorescence microscopic analyses to determine whether N-WASP and/or WAVEs were localized to podosomes. As mentioned earlier, podosomes are rich in F-actin and can be visualized clearly with phalloidin staining as dot like. As shown in Fig. 1A ⇓ , most 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 Δcof and ΔVPH N-WASP.

We next examined whether N-WASP is required for podosome formation. We used the Δcof and the ΔVPH 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 Δcof or ΔVPH 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-, Δcof N-WASP-, and ΔVPH N-WASP-expressing cells. Eighty-four % of Lac-Z-expressing control cells showed podosome formation. In Δcof- and ΔVPH 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.

Interaction of N-WASP and Cortactin.

An SH3 domain is present in the COOH-terminal region of cortactin, and N-WASP has a proline-rich region to which several SH3 proteins bind. We speculated that the SH3 domain of cortactin may interact with N-WASP and participate in N-WASP localization in podosomes. Indeed, pull-down assays with GST-fusion proteins containing the cortactin SH3 domain revealed specific binding to N-WASP but not to WAVEs (Fig. 3A) ⇓ . To investigate further whether N-WASP can interact directly with cortactin, we performed a Far-Western blot assay. Recombinant His-tagged N-WASP proteins were blotted onto a membrane filter, and the membrane filter was overlaid with GST-SH3 cortactin. As shown in Fig. 3B ⇓ , cortactin SH3 domain binds directly to N-WASP, but control GST does not.

We then examined whether endogenous cortactin and N-WASP form a protein complex in 3Y1-src cells. 3Y1-src cell lysates were immunoprecipitated with anti-N-WASP antibody, and the precipitates were subjected to Western blot analysis with anti-cortactin antibody. As shown in Fig. 3C ⇓ , a significant amount of cortactin was coimmunoprecipitated with anti-N-WASP antibody, indicating in vivo formation of cortactin/N-WASP complexes. To investigate whether the in vivo interaction occurs via the SH3 domain of cortactin, we prepared Myc-tagged cortactin expression constructs of WT and a mutant lacking the SH3 domain (ΔSH3). We then expressed N-WASP alone or with Myc-cortactin (WT or ΔSH3) and performed immunoprecipitation with anti-Myc antibody. Examination of the immunoprecipitates with anti-N-WASP antibody revealed that N-WASP was coimmunoprecipitated with Myc-cortactin (WT) but not with Myc-cortactin (ΔSH3; Fig. 3D ⇓ ), indicating that the SH3 domain is essential for in vivo binding between N-WASP and cortactin.

If cortactin through its SH3 domain recruits N-WASP in podosomes, inhibition of binding between cortactin and N-WASP should suppress podosome formation. Indeed, microinjection of a GST-fusion protein containing the SH3 domain of cortactin inhibited podosome formation in 3Y1-src cells, but microinjection of other SH3 domain proteins that do not interact with N-WASP, such as those of IRSp53 or p85 regulatory subunit of phosphatidylinositol 3-kinase (22) , did not interfere with podosome formation (Fig. 4) ⇓ . These data suggest that the interaction between N-WASP and cortactin is important for podosome formation.

Suppression of ECM Degradation by Δcof N-WASP.

During tumor metastasis, degradation of the ECM is considered a key process for malignant cells to escape from the primary tumor and to invade into other organs. In some tumor cell lines, proteolytic activity has been observed at protrusive cellular sites that are structurally similar 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 FITC-conjugated fibronectin and then seeded 3Y1-src cells onto the coverslips. ECM degradation was visualized as black areas against the uniformly fluorescent fibronectin. The results of these experiments are shown in Fig. 5A ⇓ . ECM degradation occurred in a dot-like manner, and most degraded areas colocalized with F-actin-rich podosomes. Incomplete overlap may be attributable to cell movement during the ECM degradation assay. This result strongly suggests that 3Y1-src cells degrade the ECM at podosomes.

We then examined whether podosomes are essential for inducing ECM degradation. Because expression of Δcof N-WASP suppressed podosome formation and expression of WT N-WASP induced unusually large accumulation of actin filaments (Fig. 2A) ⇓ , we expressed WT or Δcof N-WASP in 3Y1-src cells and subjected the cells to the in vitro ECM degradation assay. As shown in Fig. 5B ⇓ , 3Y1-src cells expressing control Lac-Z showed normal podosome formation. In contrast, WT N-WASP-expressing cells formed large accumulation of actin filaments, where ECM degradation occurred. On the other hand, 3Y1-src cells expressing Δcof N-WASP did not form podosomes, and ECM degradation was also suppressed. The percentages of degraded area in Lac-Z-, WT N-WASP-, and Δcof N-WASP-expressing cells were 3.6, 11, and 0.9%, respectively (Fig. 5C) ⇓ . These data confirm the importance of podosomes in ECM degradation and suggest that N-WASP may play an important role in tumor metastasis through regulation of podosomes that degrade ECM.

DISCUSSION

In the present study, we found that N-WASP, a ubiquitously expressed WASP homologue, plays an essential role in podosome formation in 3Y1-src cells. Our results are similar to those of a recent report on the role of WASP in macrophages (12) . We also found that cortactin binds to N-WASP through its COOH-terminal SH3 domain. In this context, it would be of interest to note that HS1, a cortactin-related protein expressed exclusively in hematopoietic cells (21) , is also localized in podosomes of macrophages, binds WASP through its COOH-terminal SH3 domain, and microinjection of the HS1 SH3 domain strongly blocks the formation of podosomes, 4 suggesting the general role of both cortactin family and WASP family of actin-binding proteins in podosome formation in rapidly migrating cells.

A previous report noted that cortactin binds directly to Arp2/3 complex via its acidic NH₂-terminal region (19) . It is likely that cortactin links N-WASP to the effector Arp2/3 complex. Although N-WASP can bind and activate the Arp2/3 complex directly (10) , indirect association through cortactin should facilitate the activation of Arp2/3 complex by N-WASP. Therefore, it is reasonable that cortactin recruits N-WASP to the site where the Arp2/3 complex is localized and induces a strong activation of the Arp2/3 complex by N-WASP. Weed et al. (19) reported that cortactin is recruited via its NH₂-terminal region to lamellipodia through an interaction with the Arp2/3 complex, which supports the idea that cortactin plays an important role in determining the localization of N-WASP in vivo. It may also be that cortactin activates N-WASP, because several SH3 domain-containing proteins, such as Grb2/Ash, WISH, and Nck, have been shown to activate N-WASP (28, 29, 30) . We, however, found that cortactin has little, if any, ability to activate N-WASP (data not shown).

We showed that podosomes in 3Y1-src cells possess proteolytic activity for degradation of the ECM. Chen (25) reported previously that ECM-degrading activity is concentrated in podosome-like protrusive structures that the author termed “invadopodia.” Because Chen used v-src-transformed cells to observe the invadopodia, we believe that invadopodia are podosomes. One important question is whether podosome-like structures are essential for degradation of the ECM. In the present study, we addressed this question by suppressing podosome formation with an N-WASP dominant-negative mutant. Expression of Δcof N-WASP abolishes both formation of podosomes and degradation of fibronectin that normally occurs at the locations of podosomes. In addition, expression of WT N-WASP induces large podosome-like accumulation of actin filaments and enhances significantly the degradation of ECM. These results strongly suggest the importance of podosomes in ECM degradation. It remains unclear why inhibition of podosomes also blocks fibronectin degradation. It is possible that matrix proteases must be concentrated in the podosomes to function. Some matrix metalloproteinases are reported to accumulate at significant levels in podosomes (31) .

Podosomes have long been thought to mediate adhesion to the ECM. A recent detailed electron microscopic study indicated that the central areas of podosomes invaginate into cells, and Ochoa et al. (32) suggested that these may be sites where dynamic membrane trafficking, including endocytosis, occurs. These possible functions are not contradictory to each other. Podosomes may first attach to the ECM and then “sense” the environment by sampling the surrounding material through endocytosis. Because cells are normally surrounded by the ECM, it seems reasonable that podosomes may have multiple functions and that the inhibition of podosomes leads to inability of ECM degradation.