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[Cancer Research 62, 2503-2509, May 1, 2002]
© 2002 American Association for Cancer Research

Biochemistry and Biophysics

Neural Wiskott-Aldrich Syndrome Protein Is Involved in Hepatocyte Growth Factor-induced Migration, Invasion, and Tubulogenesis of Epithelial Cells1

Hideki Yamaguchi, Hiroaki Miki and Tadaomi Takenawa2

Division of Biochemistry [H. Y., T. T.] and Cancer Genomics [H. M.], Institute of Medical Science, University of Tokyo, Tokyo 108-8639, and CREST [H. Y., T. T.] and PRESTO [H. M.], Japan Science and Technology Corp., Saitama 332-0012, Japan


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neural Wiskott-Aldrich syndrome protein (N-WASP), a member of the WASPfamily, regulates reorganization of the actin cytoskeleton through activation of the Arp2/3 complex. To date, most studies of N-WASP have focused on intracellular and morphological phenomena, such as vesicle transport and filopodium formation. We investigated the importance of N-WASP in epithelial morphogenesis, using Madin-Darby canine kidney epithelial cells, which form branching tubules when cultured with hepatocyte growth factor (HGF) in collagen gel. We established MDCK cell lines that overexpress wild-type N-WASP (WT-NW) or a dominant-negative form of N-WASP (DN-NW). WT-NW and parental Madin-Darby canine kidney cells formed branching tubules in collagen gel in response to HGF. However, formation of branching tubules was suppressed in DN-NW cells. During tubulogenesis, endogenous N-WASP accumulated at cell extensions protruding from the walls of the cysts and at the tips of the extending tubules. Gross cell morphology, cell-cell adhesion, cell polarity, and scattering in response to HGF were unaffected in WT-NW and DN-NW cells. In contrast, directed cell migration and HGF-induced invasion were significantly repressed in DN-NW cells. These results indicate that N-WASP regulates HGF-induced cell migration and invasion, which are required for epithelial tubulogenesis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In multicellular organisms, movement of cells plays an essential role in the normal development of many organs and tissues. Epithelial tubulogenesis, which is necessary for formation of many organs, including kidney, lung, and mammary gland, requires complex cell rearrangements involving cell-cell adhesion, cell polarity, migration, and invasion (1) . The reorganization of the actin cytoskeleton is fundamental to these processes. Therefore, various changes in actin cytoskeletal structures underlie epithelial tubulogenesis.

MDCK3 epithelial cells have been used for an in vitro model of the kidney tubulogenesis system. When cultured in a collagen gel matrix, MDCK cells form fluid-filled cysts that comprise a monolayer of polarized cells. When exposed to HGF, MDCK cells form branching tubular structures that resemble renal tubules (2) . Because this process requires coordinated invasion and migration of cells through the collagen gel, the experimental system is useful for examining morphogenetic cell movement of epithelial cells.

HGF-induced tubulogenesis is mediated by the cell surface receptor c-Met, which is a transmembrane tyrosine kinase encoded by the c-met proto-oncogene. Several signaling molecules, including Gab1, Grb2, PI3-kinase, Stat3, SHP-2, and SHIP-1, have been reported to function downstream of c-Met in tubulogenesis of MDCK cells (3, 4, 5, 6, 7, 8) . These molecules bind directly or indirectly to phosphorylated c-Met and transmit diverse signals required for rearrangement of cells. The Rho family of small GTPases, Rho, Rac, and Cdc42, has also been implicated in HGF-stimulated reorganization of the actin cytoskeleton of MDCK cells (9 , 10) . Rho family proteins appear to function downstream of PI3-kinase (11) , and Rho is also reported to act downstream of SHP-2 in MDCK cells (12) . Additionally, HGF signaling mediated by these molecules is also strongly linked to increased invasion and metastasis of tumor cells (13) . However, the molecular mechanisms that directly control actin cytoskeleton and cell rearrangements downstream of these molecules are not well understood.

WASP family proteins control changes in cell morphology in response to various external stimuli by regulating reorganization of the actin cytoskeleton (14 , 15) . Mammalian WASP family proteins identified to date include WASP, N-WASP, and WAVE1, -2, and -3 (16, 17, 18, 19) . N-WASP is closely related to WASP, but its tissue distribution is different: WASP is expressed exclusively in hematopoietic cells, whereas N-WASP is expressed ubiquitously (17) . Accumulating evidence suggests that WASP and N-WASP are downstream targets of Cdc42 (20 , 21) , whereas WAVE1, -2, and -3 function downstream of Rac (18 , 22) . All WASP family proteins induce rapid actin polymerization through activation of the Arp2/3 complex, a stimulator of actin nucleation (23) . N-WASP has been shown to function in filopodium formation (21) , neurite extension (24) , vesicle transport (25) , and intracellular motility of several bacterial and viral pathogens (26 , 27) . Although the importance of N-WASP is clear in intracellular events and changes in cell morphology that occur through reorganization of the actin cytoskeleton, its function in dynamic cell rearrangements during organization of multicellular structures remains unknown.

We examined the role of N-WASP in the morphogenetic movement of MDCK cells during epithelial tubulogenesis in collagen gel. We found that N-WASP plays an important role in epithelial tubulogenesis, probably through regulation of directed cell migration and invasion induced by HGF.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Antibodies.
The following antibodies were used: anti-N-WASP antibody (17) ; antiactin antibody (Chemicon, Temecula, CA); anti-GFP antibody (Molecular Probes, Eugene, OR); anti-ß-catenin antibody (Transduction Laboratories, Lexington, KY); and anti-gp135 antibody (a generous gift from Dr. George Ojakian, SUNY Health Science Center, Brooklyn, NY).

Generation of Stable MDCK Cell Lines.
The MDCK Tet-Off cell line (Clontech, Palo Alto, CA), which expresses a tetracycline-controlled transactivator, was used to generate stable cell lines. The construct for DN-N-WASP, which lacks the verprolin homology domains, has been described previously (28) . MDCK Tet-Off cells were cotransfected with pTK-Hyg and pTRE2, which encode GFP, WT-N-WASP, or DN-N-WASP, with Lipofectamine (Life Technologies, Inc., Grand Island, NY) according to the manufacturer’s instructions. Single colonies were isolated after 2–3 weeks of hygromycin selection (300 µg/ml). Protein expression was assessed by immunoblotting of cell lysates with appropriate antibodies. To suppress expression of exogenous genes, DOX (Clontech) was added to culture medium at concentrations of 0.1–1 µg/ml at least 1 day before each assay. All cells were routinely maintained in complete growth medium without DOX: DMEM supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin.

Cell-scattering Assay.
Cell scattering was assessed by seeding 1 x 104 cells/well in 6-well tissue culture plates. After 16–24 h, the culture medium was replaced with fresh culture medium with or without 20 ng/ml HGF (Sigma Chemical Co., St. Louis, MO). After an additional 16 h, the cultures were examined by phase-contrast microscopy and photographed.

Tubulogenesis Assay.
The collagen mixture was made with 8 volumes of type I collagen stock solution (Nitta Gelatin, Osaka, Japan), 1 volume of 10x concentrated MEM, 1 volume of 0.05 N NaOH containing 2.2% NaHCO3, and 200 mM HEPES. Six-well tissue culture plates were coated with the collagen mixture at 1 ml/well and then incubated at 37°C until the collagen solidified. Cells (5 x 103 ) were added to collagen mixture (1 ml) and transferred gently to the coated 6-well plates. The cultures were incubated at 37°C to allow the type I collagen mixture to gel and then overlaid with 1 ml of growth medium. After 4 days, the medium was replaced with fresh growth medium with or without 20 ng/ml HGF. After an additional 4 days, cells were observed by phase-contrast microscopy and photographed. For quantification of tubulogenesis, brightfield images at the midline focal plane of individual cysts were obtained with an inverted microscope equipped with a CCD camera. The area occupied by cysts and tubules was calculated with NIH Image software.

Migration Assay.
Migration assays were carried out with Cell Culture Inserts (6.5-mm diameter, 8 µm pore size; Becton Dickinson, Franklin Lakes, NJ). Cells (2 x 105) were added to the upper chambers, and the chambers were placed in 24-well dishes containing medium with or without HGF (20 ng/ml). Migration assays were carried out for 15 h, and membranes were then fixed with 3.7% formaldehyde in PBS. Nonmigrated cells on the upper side of the membrane were removed with a cotton swab, and the migrated cells were stained with 0.4% crystal violet in 10% ethanol for 30 min. The cells were photographed with an inverted phase-contrast microscope (x10 objective), and the migrated cells were counted for quantitation of cell migration. For each determination, photographs of nine randomly selected fields on three independent filters were analyzed.

Immunofluorescence Microscopy.
Cells cultured on coverslips and filters were fixed in 3.7% formaldehyde in PBS. Cells were permeabilized with 0.2% Triton X-100 for 5 min and then incubated with primary antibodies for 60 min. After being washed with PBS, cells were incubated with secondary antibodies conjugated with fluorescein or Cy5 for 30 min. For visualization of actin filaments, rhodamine-conjugated phalloidin (Molecular Probes) was added during the incubation with secondary antibodies. Coverslips and filters were then washed and mounted on glass slides.

Staining of MDCK cells in collagen gel was carried out as described previously (29) . Immunostained cells and collagen gels were observed with Radiance 2000 (Bio-Rad, Hercules, CA) and TCS SP2 (Leica, Wetzlar, Germany) confocal laser-scanning microscopes.

Invasion Assay.
Invasion of MDCK cells into the collagen gel was analyzed as described previously (30) . Type I collagen solution containing 40% Vitrogen 100 (3 mg/ml; Cohesion Technologies, Palo Alto, CA), 1x MEM, 0.5% NaHCO3, and 20 mM HEPES (pH 7.5) was dispensed into 24-well tissue culture wells (500 µl/well) and allowed to gel at 37°C for 1 h. Cells (1 x 104 /well) were seeded onto gels in 500 µl of growth medium with or without HGF (10–20 ng/ml). After 4 days, cells were photographed, and foci of invasive cells in at least three randomly selected fields were counted microscopically with a x10 phase-contrast objective.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Establishment of Stable MDCK Cell Lines.
To assess the function of N-WASP in epithelial morphogenesis, we first established stable lines of MDCK cells that overexpressed either WT-N-WASP or DN-N-WASP lacking the two verprolin homology domains, which are binding sites for monomeric actin and essential for Arp2/3 complex-mediated actin polymerization by N-WASP. This mutant cannot induce reorganization of the actin cytoskeleton, whereas it maintains other functional domains, including binding sites for upstream molecules (28) . Thus, this mutant is thought to function as a dominant-negative form against endogenous N-WASP proteins. For a control, we also generated cell lines that express GFP. Expression of exogenous genes was under the control of the DOX-repressible transactivator; in the presence of DOX, expression of the transgene was inhibited. Expression of GFP, WT-N-WASP, and DN-N-WASP was confirmed by immunoblotting (Fig. 1A)Citation . Levels of WT- and DN-N-WASP proteins were several times higher than the level of endogenous N-WASP, and expression was repressed by the addition of DOX to the culture medium (Fig. 1A)Citation . Additionally, there was no significant difference in the expression levels of N-WASP proteins between cells grown in normal culture dishes and those grown on collagen gel (Fig. 1B)Citation . The cell lines were named as follows: WT-NW overexpressed WT-N-WASP, and DN-NW overexpressed DN-N-WASP. We used these cell lines for further analyses.



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  		Fig. 1. Establishment of cell lines stably overexpressing WT- and DN-N-WASP. A, MDCK clones overexpressing GFP or WT-N-WASP (WT), or DN-N-WASP (DN) were cultured in the presence or absence of 1 µg/ml DOX for 1 day. Cell lysates were subjected to SDS-PAGE and immunoblotted with indicated antibodies. Arrow indicates endogenous and WT-N-WASP; arrowhead indicates DN-N-WASP. B, MDCK clones were cultured in normal culture dishes (N) or on collagen gels (C) for 2 or 4 days. Cell lysates were subjected to immunoblotting.

 
Stable Expression of DN-N-WASP Suppresses Tubulogenesis of MDCK Cells.
When cultured in collagen gels, MDCK cells form spherical cysts with fluid-filled lumens. In the presence of HGF, MDCK cysts are stimulated to form branching tubules. WT-NW cells as well as control cells overexpressing GFP formed branching tubules in response to HGF stimulation (Fig. 2A)Citation . However, formation of branching tubules was suppressed in DN-NW cells (Fig. 2A)Citation . This suppression of tubulogenesis was abolished when DOX, which inhibits expression of DN-N-WASP, was added to the culture medium. The morphology and size of the cysts formed by DN-NW cells were comparable to those of control cells, suggesting that cyst formation and cell growth in collagen gel were not affected by overexpression of DN-N-WASP. Similar results were obtained with an independent DN-N-WASP transfectant (data not shown). For quantitation, we calculated the area of cell cysts and tubules from photographs (Fig. 2B)Citation and constructed a representative scatter plot (Fig. 2C)Citation . The differences in tubule formation between DN-NW cells and control GFP cells were statistically significant (P < 0.005), and the differences were abrogated by the addition of DOX to the culture medium. These results suggest that N-WASP is involved in tubulogenesis of MDCK cells in collagen gels.



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  		Fig. 2. Tubulogenesis is blocked by overexpression of DN-N-WASP. A, stable transfectants grown in collagen gel were stimulated with 20 ng/ml HGF. After 4 days, cells were examined by phase-contrast microscopy. Bar, 100 µm. B, quantification of tubulogenesis. The area occupied by cysts and tubules was calculated from the phase-contrast images. Top and bottom panels show phase-contrast and traced images, respectively. The number denotes the area index (square pixels). C, scatter diagrams represent the areas of the cysts and tubules calculated as in B. Numbers at the top represent the total number of cysts examined for each clone. *, P < 0.005, Student’s t test.

 
Subcellular Localization of N-WASP in Tubulogenesis.
To examine localization of endogenous N-WASP during tubulogenesis, we examined cysts formed from parental MDCK cells in collagen gels by immunofluorescence microscopy (Fig. 3A)Citation . In cysts not treated with HGF, N-WASP was localized uniformly throughout the cytoplasm. After stimulation with HGF for 12 h, cells in the cyst wall had sent out small extensions containing actin filaments into the surrounding collagen gel matrix. N-WASP had accumulated in these extensions. To assess localization of expressed N-WASP proteins, we examined cysts formed by NW-WT and NW-DN cells. Strong signals that represented expressed N-WASP proteins were also detected at cell extensions protruding from these cysts (Fig. 3B)Citation . In parental MDCK cysts treated with HGF for 4 days, strong, punctate signals for endogenous N-WASP were observed at the tips of the extending tubules (Fig. 3C)Citation . These findings suggest that N-WASP is involved in the protrusion of cell extensions and elongation of branched tubules induced by HGF.



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  		Fig. 3. N-WASP accumulates at extensions protruding from cysts and at the tips of extending tubules during tubulogenesis. A, parental MDCK cysts grown in collagen gels were incubated with or without 20 ng/ml HGF for 12 h. The cysts were stained with anti-N-WASP antibody and rhodamine-phalloidin to visualize localization of N-WASP and actin filaments, respectively. Arrowheads denote cell extensions observed after stimulation with HGF. The punctate signals present outside the cysts in N-WASP staining are artifacts caused by the collagen gel. Bar, 20 µm. B, WT- and DN-NW cysts were treated with 20 ng/ml HGF for 12 h. The cysts were stained with anti-N-WASP antibody. Arrowheads denote cell extensions with accumulations of expressed N-WASP proteins. C, parental MDCK cysts were stimulated with 20 ng/ml HGF for 4 days. Cryosections of the cysts were prepared and stained with anti-N-WASP antibody and rhodamine-phalloidin. N-WASP is present at high levels at the tips of extending tubules (arrowheads). Bar, 20 µm.

 
Expression of DN-N-WASP Does Not Affect Cell Scattering, Adherence Junctions, or Cell Polarity Induced by HGF.
Tubulogenesis requires strict control of cell-cell adhesion, cell polarity, and changes in cell morphology. Therefore, we examined the possible involvement of N-WASP in these processes.

We first examined cell scattering stimulated by HGF treatment. The gross cell morphology of WT-NW and DN-NW cells in monolayer culture was indistinguishable from that of control cells expressing GFP. In response to HGF stimulation, WT-NW and DN-NW cells scattered, as did control cells (Fig. 4A)Citation . In scattering cells, endogenous N-WASP predominantly accumulated in the nucleus and perinuclear region (Fig. 4B)Citation . Only part of N-WASP proteins was localized at protruding lamellipodia. We then examined formation of adherence junctions. MDCK transfectants cultured on coverslips were stained with phalloidin and an antibody against ß-catenin, a component of adherence junctions. Actin filaments and ß-catenin were localized exclusively to cell-cell adhesion sites in all MDCK transfectants, and we did not observe any abnormalities in the adherence junctions (Fig. 4C)Citation . We then examined the cell polarity of these transfectants. We stained cells grown on permeable filters, which maintain epithelial cell polarity, with anti-ß-catenin and anti-gp135 antibodies as markers of the basolateral and apical membranes, respectively. In all transfectants, ß-catenin and gp135 were restricted to the basolateral and apical membranes, respectively (Fig. 4D)Citation . We also stained cell cysts formed by DN-NW cells in collagen gels with anti-ß-catenin and anti-gp135 antibodies and found no abnormalities in cell polarity (data not shown). These results show that cell scattering, cell-cell adhesion, and cell polarity are not affected by overexpression of DN-N-WASP.



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  		Fig. 4. Cell scattering, adherence junctions, and cell polarity are not affected by overexpression of WT- or DN-N-WASP. A, cells grown in tissue culture dishes were incubated with or without HGF (20 ng/ml) for 16 h, and the scattering response was examined by phase-contrast microscopy. Bar, 100 µm. B, parental MDCK cells grown on coverslips were treated with HGF (20 ng/ml) for 16 h. The cells were stained with anti-N-WASP antibody and rhodamine-phalloidin. Arrowheads denote lamellipodia. Bar, 40 µm. C, cells grown on coverslips were stained with anti-ß-catenin antibody and rhodamine-phalloidin and observed by fluorescence microscopy. Bar, 40 µm. D, cells grown on filters were stained with anti-ß-catenin and anti-gp135 antibodies. Images are XZ sections collected with a confocal microscope. Bar, 20 µm.

 
Directed Migration toward HGF Is Reduced by Overexpression of DN-N-WASP.
We then examined directed migration of MDCK cells toward HGF with a modified Boyden chamber assay. Control GFP cells and WT-NW cells migrated toward HGF, and there was no significant difference in cell migration between these two cell lines. However, the number of cells that migrated toward HGF was decreased modestly in DN-NW cells (Fig. 5A)Citation . We quantified the migration by counting the migrated cells in photographs. This analysis revealed a statistically significant difference (P < 0.03) in cell migration between DN-NW and control GFP cells; the difference was abrogated by the addition of DOX to the culture medium. (Fig. 5B)Citation . We found no additional differences in cell migration at earlier time points (data not shown). To assess localization of endogenous N-WASP in migrating cells, we stained cells migrating through the pores of membrane filters toward HGF. N-WASP signals were clearly concentrated in the cell extensions that protruded from the basal surfaces of migrating cells (Fig. 5C)Citation . Actin filaments also accumulated in these extensions. In WT- and DN-NW cells, strong N-WASP signals were also seen in the cell extensions (Fig. 5D)Citation .



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  		Fig. 5. Directed migration toward HGF is reduced in cells overexpressing DN-N-WASP. A, cells were plated on Transwell culture inserts and allowed to migrate to the underside of the filters for 15 h. HGF (20 ng/ml) was added to the lower chamber. Migrated cells were stained with crystal violet and photographed with an inverted phase-contrast microscope. Bar, 100 µm. B, quantification of cell migration. The number of migrated cells was calculated from the photographs. Error bars, SD. *, P < 0.03, Student’s t test. C, parental MDCK cells were plated on filters and allowed to migrate for 9 h. Cells were stained with rhodamine-phalloidin and anti-N-WASP antibody. Dashed boxes show the regions of membrane pores. Images are XZ sections collected with a confocal microscope. Bar, 10 µm. D, WT-NW and DN-NW cells migrating through filters were stained with anti-N-WASP antibody as in C.

 
These results suggest that N-WASP functions in directed cell migration toward HGF. However, the reduction in migration of DN-NW cells was relatively small compared with that in tubulogenesis, suggesting that N-WASP has another important function in tubulogenesis.

Overexpression of DN-N-WASP Suppresses HGF-induced Invasion of MDCK Cells.
Because formation of branching tubules in a collagen gel matrix requires cell invasion through the collagen gel, we next tested whether N-WASP is involved in HGF-induced invasiveness of MDCK cells. When cultured on collagen gel, MDCK cells grow as a monolayer that remains confined to the surface of the gel. In the presence of HGF, however, MDCK cells invade the underlying collagen gel and form foci in the gel (30 , 31) . WT-NW cells and control GFP cells invaded and formed foci in collagen gels in response to HGF (Fig. 6, A and B)Citation . In contrast, DN-NW cells showed impairment of the invasive phenotype; the number of invasive foci was reduced in these cells (Fig. 6, A and B)Citation . We then examined localization of endogenous N-WASP protein in invading MDCK cells. Parental MDCK cells cultured on collagen gels in the presence of HGF were subjected to immunofluorescence microscopy. N-WASP accumulated at the cell projections of the basal cell membrane that extended into the collagen gel (Fig. 6C)Citation . Strong signals for filamentous actin were also observed at these projections and were clearly colocalized with those of N-WASP. In WT- and DN-NW cells, N-WASP proteins were also observed at the projections, although a large part of the N-WASP proteins was distributed throughout the cytoplasm (Fig. 6D)Citation . These results suggest that N-WASP functions in HGF-induced cell invasion by regulating protrusion of the membrane projections that invade the collagen gel.



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  		Fig. 6. HGF-induced invasion is suppressed in cells overexpressing DN-N-WASP. A, MDCK cells cultured on collagen gels were incubated with or without HGF (10 ng/ml) for 4 days and photographed with a phase-contrast microscope. Arrowheads denote foci formed by cells that invaded the collagen gels. Bar, 100 µm. B, quantification of invasion induced by HGF. Foci formed by cells that invaded the collagen gels were counted. Error bars, SD. C, parental MDCK cells were cultured on collagen gels in the presence of HGF (20 ng/ml) for 4 days and stained with rhodamine-phalloidin and anti-N-WASP antibody. N-WASP is localized at the cell projections, which contain actin filaments and extend from the cells into the collagen gel (arrowheads). The double-headed arrow marks the boundaries of the collagen gel. Images are XZ sections collected with a confocal microscope. Bar, 20 µm. D, WT-NW and DN-NW cells invading collagen gels were stained with anti-N-WASP antibody as in C. Arrowheads denote cell projections.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Epithelial tubulogenesis involves complex cell rearrangements that require strict control of cell-cell adhesion, cell polarity, and migration (1) . We showed that overexpression of DN-N-WASP represses HGF-induced tubulogenesis of MDCK cells without affecting cell scattering, cell-cell adhesion, or cell polarity. Thus, N-WASP may specifically regulate tubulogenesis among various functions of epithelial cells. The phenotype elicited by overexpression of DN-N-WASP is unique because most of the molecules implicated in tubulogenesis, including Ras, PI3-kinase, and Grb2, also function in cell scattering and/or cell-cell adhesion (5 , 9 , 30 , 32) . Only MT1-MMP and Stat-3 were reported to function specifically in tubulogenesis (6 , 31 , 33) . The possible functional relationship between N-WASP and MT1-MMP in tubulogenesis is discussed below. To our knowledge, there is no report of a functional connection between N-WASP and Stat3, which appears to regulate expression of genes required for tubulogenesis (6) .

Tubulogenesis also requires extensive remodeling of the extracellular matrix and cell invasion through the matrix. We observed a significant decrease in invasion by DN-NW cells. This is the first evidence that N-WASP functions in cell invasion. As described above, MT1-MMP, a membrane-anchored matrix metalloproteinase that degrades extracellular matrix, is necessary for tubulogenesis of MDCK cells (33) . MT1-MMP is overexpressed in various tumor cells and is thought to play important roles in invasion and metastasis of tumor cells. MT1-MMP localizes at the invasive membrane protrusions of tumor cell surface, called invadopodia (34) . Interestingly, N-WASP also localizes at the invadopodia of invasive tumor cells.4 Because N-WASP accumulates at cell protrusions extending into collagen gel during tubulogenesis and invasion, it may be involved in degradation of the extracellular matrix by regulating the formation of invadopodia. Because HGF and its receptor, c-Met, play crucial roles in enhanced invasiveness and metastasis of many types of tumor cells (13) , N-WASP may also function in these phenomena in tumor cells.

Downstream signaling of c-Met is mediated by many signaling molecules, including adapter proteins such as Shc, Gab1, and Grb2/Ash (35) . The small GTPases of the Rho family, Cdc42, Rac, and Rho, act downstream of c-Met and mediate cytoskeletal rearrangements (9 , 10) . N-WASP has binding sites for many signaling molecules, including Grb2/Ash and Cdc42 (17 , 21) . These molecules both bind to and activate N-WASP by releasing it from an autoinhibited conformation (36 , 37) . Grb2/Ash interacts with phosphorylated c-Met, and this interaction appears necessary for tubulogenesis of MDCK cells (4 , 30) . Cdc42 is activated downstream of c-Met and mediates rearrangement of the actin cytoskeleton in MDCK cells (10 , 38) . Thus, these molecules may function in the signaling pathway that connects c-Met receptor to N-WASP.

In conclusion, we found that N-WASP is important in morphogenic migration during epithelial tubulogenesis of MDCK cells. We showed that N-WASP functions in directed cell migration and invasion induced by HGF. These findings will provide important insights into the mechanism of cell migration and invasion, which are necessary for many types of physiological and pathological phenomena, including metastasis of tumor cells.


   ACKNOWLEDGMENTS
 
We thank Dr. George Ojakian (SUNY Health Science Center, Brooklyn, NY) for providing antibody against gp135.


   FOOTNOTES
 
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 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science, and Culture of Japan. Back

2 To whom requests for reprints should be addressed, at Division of Biochemistry, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Fax 81-3-5449-5417; E-mail: takenawa{at}ims.u-tokyo.ac.jp Back

3 The abbreviations used are: MDCK, Madin-Darby canine kidney; HGF, hepatocyte growth factor; PI3-kinase, phosphatidylinositol 3'-kinase; GFP, green fluorescent protein; WASP, Wiskott-Aldrich syndrome protein; N-WASP, neural WASP; DN, dominant-negative; WT, wild type; DOX, doxycycline; MT1-MMP, membrane type 1 matrix metalloproteinase. Back

4 Our unpublished observation. Back

Received 11/14/01. Accepted 2/28/02.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Pollack A. L., Runyan R. B., Mostov K. E. Morphogenetic mechanisms of epithelial tubulogenesis: MDCK cell polarity is transiently rearranged without loss of cell-cell contact during scatter factor/hepatocyte growth factor-induced tubulogenesis. Dev. Biol., 204: 64-79, 1998.[Medline]
  2. Montesano R., Matsumoto K., Nakamura T., Orci L. Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell, 67: 901-908, 1991.[Medline]
  3. Weidner K. M., Di Cesare S., Sachs M., Brinkmann V., Behrens J., Birchmeier W. Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature (Lond.), 384: 173-176, 1996.[Medline]
  4. Royal I., Fournier T. M., Park M. Differential requirement of Grb2 and PI3-kinase in HGF/SF-induced cell motility and tubulogenesis. J. Cell Physiol., 173: 196-201, 1997.[Medline]
  5. Khwaja A., Lehmann K., Marte B. M., Downward J. Phosphoinositide 3-kinase induces scattering and tubulogenesis in epithelial cells through a novel pathway. J. Biol. Chem., 273: 18793-18801, 1998.[Abstract/Free Full Text]
  6. Boccaccio C., Ando M., Tamagnone L., Bardelli A., Michieli P., Battistini C., Comoglio P. M. Induction of epithelial tubules by growth factor HGF depends on the STAT pathway. Nature (Lond.), 391: 285-288, 1998.[Medline]
  7. Maroun C. R., Naujokas M. A., Holgado-Madruga M., Wong A. J., Park M. The tyrosine phosphatase SHP-2 is required for sustained activation of extracellular signal-regulated kinase and epithelial morphogenesis downstream from the met receptor tyrosine kinase. Mol. Cell. Biol., 20: 8513-8525, 2000.[Abstract/Free Full Text]
  8. Stefan M., Koch A., Mancini A., Mohr A., Weidner K. M., Niemann H., Tamura T. Src homology 2-containing inositol 5-phosphatase 1 binds to the multifunctional docking site of c-Met and potentiates hepatocyte growth factor-induced branching tubulogenesis. J. Biol. Chem., 276: 3017-3023, 2001.[Abstract/Free Full Text]
  9. Ridley A. J., Comoglio P. M., Hall A. Regulation of scatter factor/hepatocyte growth factor responses by Ras, Rac, and Rho in MDCK cells. Mol. Cell. Biol., 15: 1110-1122, 1995.[Abstract]
  10. Kodama A., Takaishi K., Nakano K., Nishioka H., Takai Y. Involvement of Cdc42 small G protein in cell-cell adhesion, migration and morphology of MDCK cells. Oncogene, 18: 3996-4006, 1999.[Medline]
  11. Cantrell D. A. Phosphoinositide 3-kinase signalling pathways. J. Cell Sci., 114: 1439-1445, 2001.[Abstract]
  12. Kodama A., Matozaki T., Fukuhara A., Kikyo M., Ichihashi M., Takai Y. Involvement of an SHP-2-Rho small G protein pathway in hepatocyte growth factor/scatter factor-induced cell scattering. Mol. Biol. Cell, 11: 2565-2575, 2000.[Abstract/Free Full Text]
  13. Bardelli A., Comoglio P. M. Scatter factor receptors are key players in a unique multistep program leading to invasive growth. Ciba Found. Symp., 212: 133-147, 1997.[Medline]
  14. Higgs H. N., Pollard T. D. Regulation of actin filament network formation through Arp2/3 complex: activation by a diverse array of proteins. Annu. Rev. Biochem., 70: 649-676, 2001.[Medline]
  15. Takenawa T., Miki H. WASP and WAVE family proteins: key molecules for rapid rearrangement of cortical actin filaments and cell movement. J. Cell Sci., 114: 1801-1809, 2001.[Abstract]
  16. Derry J. M., Ochs H. D., Francke U. Isolation of a novel gene mutated in Wiskott-Aldrich syndrome. Cell, 78: 635-644, 1994.[Medline]
  17. Miki H., Miura K., Takenawa T. N-WASP, a novel actin-depolymerizing protein, regulates the cortical cytoskeletal rearrangement in a PIP2-dependent manner downstream of tyrosine kinases. EMBO J., 15: 5326-5335, 1996.[Medline]
  18. Miki H., Suetsugu S., Takenawa T. WAVE, a novel WASP-family protein involved in actin reorganization induced by Rac. EMBO J., 17: 6932-6941, 1998.[Medline]
  19. Suetsugu S., Miki H., Takenawa T. Identification of two human WAVE/SCAR homologues as general actin regulatory molecules which associate with the Arp2/3 complex. Biochem. Biophys. Res. Commun., 260: 296-302, 1999.[Medline]
  20. Symons M., Derry JM, Karlak B., Jiang S., Lemahieu V., McCormick F., Francke U., Abo A. Wiskott-Aldrich syndrome protein, a novel effector for the GTPase CDC42Hs, is implicated in actin polymerization. Cell, 84: 723-734, 1996.[Medline]
  21. Miki H., Sasaki T., Takai Y., Takenawa T. Induction of filopodium formation by a WASP-related actin-depolymerizing protein N-WASP. Nature (Lond.), 391: 93-96, 1998.[Medline]
  22. Miki H., Yamaguchi H., Suetsugu S., Takenawa T. IRSp53 is an essential intermediate between Rac and WAVE in the regulation of membrane ruffling. Nature (Lond.), 408: 732-735, 2000.[Medline]
  23. Yamaguchi H., Miki H., Suetsugu S., Ma L., Kirschner M. W., Takenawa T. Two tandem verprolin homology domains are necessary for a strong activation of Arp2/3 complex-induced actin polymerization and induction of microspike formation by N-WASP. Proc. Natl. Acad. Sci. USA, 97: 12631-12636, 2000.[Abstract/Free Full Text]
  24. Banzai Y., Miki H., Yamaguchi H., Takenawa T. Essential role of neural Wiskott-Aldrich syndrome protein in neurite extension in PC12 cells and rat hippocampal primary culture cells. J. Biol. Chem., 275: 11987-11992, 2000.[Abstract/Free Full Text]
  25. Taunton J., Rowning B. A., Coughlin M. L., Wu M., Moon R. T., Mitchison T. J., Larabell C. A. Actin-dependent propulsion of endosomes and lysosomes by recruitment of N-WASP. J. Cell Biol., 148: 519-530, 2000.[Abstract/Free Full Text]
  26. Suzuki T., Sasakawa C. N-WASP is an important protein for the actin-based motility of Shigella flexneri in the infected epithelial cells. Jpn. J. Med. Sci. Biol., 51 (Suppl.): S63-S68, 1998.
  27. Frischknecht F., Moreau V., Rottger S., Gonfloni S., Reckmann I., Superti-Furga G., Way M. Actin-based motility of vaccinia virus mimics receptor tyrosine kinase signalling. Nature (Lond.), 401: 926-929, 1999.[Medline]
  28. Miki H., Takenawa T. Direct binding of the verprolin-homology domain in N-WASP to actin is essential for cytoskeletal reorganization. Biochem. Biophys. Res. Commun., 243: 73-78, 1998.[Medline]
  29. Pollack A. L., Barth A. I., Altschuler Y., Nelson W. J., Mostov K. E. Dynamics of ß-catenin interactions with APC protein regulate epithelial tubulogenesis. J. Cell Biol., 137: 1651-1662, 1997.[Abstract/Free Full Text]
  30. Atabey N., Gao Y., Yao Z. J., Breckenridge D., Soon L., Soriano J. V., Burke T. R., Jr., Bottaro D. P. Potent blockade of hepatocyte growth factor-stimulated cell motility, matrix invasion and branching morphogenesis by antagonists of Grb2 Src homology 2 domain interactions. J. Biol. Chem., 276: 14308-14314, 2001.[Abstract/Free Full Text]
  31. Hotary K., Allen E., Punturieri A., Yana I., Weiss S. J. Regulation of cell invasion and morphogenesis in a three-dimensional type I collagen matrix by membrane-type matrix metalloproteinases 1, 2, and 3. J. Cell Biol., 149: 1309-1323, 2000.[Abstract/Free Full Text]
  32. Royal I., Park M. Hepatocyte growth factor-induced scatter of Madin-Darby canine kidney cells requires phosphatidylinositol 3-kinase. J. Biol. Chem., 270: 27780-27787, 1995.[Abstract/Free Full Text]
  33. Kadono Y., Shibahara K., Namiki M., Watanabe Y., Seiki M., Sato H. Membrane type 1-matrix metalloproteinase is involved in the formation of hepatocyte growth factor/scatter factor-induced branching tubules in Madin-Darby canine kidney epithelial cells. Biochem. Biophys. Res. Commun., 251: 681-687, 1998.[Medline]
  34. Nagase H. Cell surface activation of progelatinase A (proMMP-2) and cell migration. Cell Res., 8: 179-186, 1998.[Medline]
  35. Furge K. A., Zhang Y. W., Vande Woude G. F. Met receptor tyrosine kinase: enhanced signaling through adapter proteins. Oncogene, 19: 5582-5589, 2000.[Medline]
  36. Carlier M. F., Nioche P., Broutin-L’Hermite I., Boujemaa R., Le Clainche C., Egile C., Garbay C., Ducruix A., Sansonetti P., Pantaloni D. GRB2 links signaling to actin assembly by enhancing interaction of neural Wiskott-Aldrich syndrome protein (N-WASp) with actin-related protein (ARP2/3) complex. J. Biol. Chem., 275: 21946-21952, 2000.[Abstract/Free Full Text]
  37. Rohatgi R., Ho H. Y., Kirschner M. W. Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4,5-bisphosphate. J. Cell Biol., 150: 1299-1310, 2000.[Abstract/Free Full Text]
  38. Royal I., Lamarche-Vane N., Lamorte L., Kaibuchi K., Park M. Activation of cdc42, rac, PAK, and rho-kinase in response to hepatocyte growth factor differentially regulates epithelial cell colony spreading and dissociation. Mol. Biol. Cell, 11: 1709-1725, 2000.[Abstract/Free Full Text]



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