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Originally published In Press as doi:10.1074/jbc.M201743200 on July 23, 2002

J. Biol. Chem., Vol. 277, Issue 40, 37904-37911, October 4, 2002
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Crk Synergizes with Epidermal Growth Factor for Epithelial Invasion and Morphogenesis and Is Required for the Met Morphogenic Program*

Louie LamorteDagger §, Sonia RodriguesDagger , Monica Naujokas||, and Morag ParkDagger ||**DaggerDagger

From the Departments of Dagger  Biochemistry, || Medicine, and ** Oncology, Molecular Oncology Group, McGill University Hospital Center, McGill University, Montreal, Quebec H3A 1A1, Canada

Received for publication, February 20, 2002, and in revised form, July 19, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Activation of the Met receptor tyrosine kinase through its ligand, hepatocyte growth factor, stimulates cell spreading, cell dispersal, and the inherent morphogenic program of various epithelial cell lines. Although both hepatocyte growth factor and epidermal growth factor (EGF) can activate downstream signaling pathways in Madin-Darby canine kidney epithelial cells, EGF fails to promote the breakdown of cell-cell junctional complexes and initiate an invasive morphogenic program. We have undertaken a strategy to identify signals that synergize with EGF in this process. We provide evidence that the overexpression of the CrkII adapter protein complements EGF-stimulated pathways to induce cell dispersal in two-dimensional cultures and cell invasion and branching morphogenesis in three-dimensional collagen gels. This finding correlates with the ability of CrkII to promote the breakdown of adherens junctions in stable cell lines and the ability of EGF to stimulate enhanced Rac activity in cells overexpressing CrkII. We have previously shown that the Gab1-docking protein is required for branching morphogenesis downstream of the Met receptor. Consistent with a role for CrkII in promoting EGF-dependent branching morphogenesis, the binding of Gab1 to CrkII is required for the branching morphogenic program downstream of Met. Together, our data support a role for the CrkII adapter protein in epithelial invasion and morphogenesis and underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Epithelial morphogenesis is essential for normal embryonic development and involves proliferation, migration, cellular invasion, turnover of surrounding extracellular matrix, and the deposition of newly synthesized extracellular matrix (1). Several growth factors stimulate the morphogenic program of epithelial cells. One of the most potent inducers of a morphogenic program in epithelial cells is hepatocyte growth factor (HGF)1 (2). HGF is a mesenchymal derived growth factor that promotes several distinct biological responses through activation of the Met receptor tyrosine kinase (3). HGF was originally identified as a potent mitogen for primary rat hepatocytes (4), and HGF serum levels increase following damage to the liver, kidney, stomach, or lung (5). HGF was independently isolated as "scatter factor" as it stimulates epithelial cell dissociation and migration (6). HGF is also a potent morphogen for Madin-Darby canine kidney (MDCK) cells (7) and promotes the inherent morphogenic program of kidney, breast, and lung epithelium grown in matrix cultures (2, 9). Importantly, HGF and Met are deregulated in several human tumors (10) and can promote tumor metastasis and angiogenesis (11, 12).

Epidermal growth factor (EGF) is an important regulator of embryonic development and cell growth. In addition, EGF receptor knock-out mice exhibit impaired ductal growth and branching morphogenesis (13), implicating EGF or other EGF receptor ligands in the morphogenic process. EGF can stimulate branching morphogenesis in some mammary and kidney epithelial cells (13-15) and promotes cell dispersal and invasion in several carcinoma cell lines (17-20). However, the EGF-dependent signals required for these processes are poorly understood.

Whereas HGF promotes a branching morphogenic program in MDCK and primary renal proximal tubular epithelial cells, EGF and other growth factors fail to do so (2, 7, 21). However, treatment of primary renal proximal tubular epithelial cells with a combination of growth factors promotes a similar morphogenic response as HGF (21), suggesting that the co-coordinated activation of multiple signaling pathways must be achieved to undergo an invasive morphogenic program. Hence, MDCK cells provide an experimental system to examine the signals that cooperate with EGF to promote epithelial cell dispersal and morphogenesis.

Using chimeric Met receptors, we have undertaken structure function studies to define Met-dependent signals required for the morphogenic program. These demonstrated that a single tyrosine residue (Tyr-1356) and, in particular, the recruitment of the Grb2 adapter protein to this tyrosine is critical for the morphogenic process (22, 23). Tyrosine 1356 forms a multisubstrate binding site, coupling the Met receptor directly with the Grb2 and Shc adapter proteins and indirectly with Gab1-docking protein (23-28). The morphogenic program of Met receptor mutants is rescued following overexpression of the Gab1-docking protein (29). This identifies Gab1 as a critical modulator of the morphogenic response downstream from the Met receptor and allows a structure function approach to define the Gab1-dependent signals required.

Gab1 is a member of a family of docking proteins: Gab1, Gab2, and Gab3, which contain a conserved pleckstrin homology (PH) domain and multiple tyrosine residues that provide binding sites for Src homology 2 (SH2) domain containing proteins (30-34). Gab1 acts to integrate signals downstream from the Met receptor. Following tyrosine phosphorylation, Gab1 associates with multiple signaling proteins including the p85 subunit of PI3K, phospholipase Cgamma , CrkII/L, and the SHP-2 tyrosine phosphatase (29, 35-40). The Gab1 PH domain has specificity for phosphatidylinositol 3,4,5-trisphosphate and is required for the morphogenic response downstream from the Met receptor (29, 41) as is the recruitment of the SHP-2 phosphatase to Gab1 (39).

CrkII and CrkL are composed of a single SH2 and two SH3 domains (SH2-SH3-SH3) (43, 44). The Crk SH2 domain binds a number of tyrosine-phosphorylated proteins including p130Cas, paxillin, Cbl, and Gab1, whereas the amino-terminal SH3 domain binds C3G, DOCK180, and Abl (45). The overexpression of CrkII or CrkL enhances cell migration (46-50). However, the role of Crk adapter proteins in epithelial morphogenesis has not been addressed. We demonstrate that the coupling of Crk with Gab1 is required for the invasive morphogenic program downstream from the Met receptor. Moreover, the overexpression of CrkII in MDCK cells synergizes with EGF-stimulated signaling pathways to promote the dispersal of colonies of MDCK epithelial cells, invasion, and branching morphogenesis, whereas each alone is insufficient.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials and Antibodies-- Dr. George Vande Woude (Van Andel Research Institute, Grand Rapids, MI) provided HGF, and CSF-1 was provided by the Genetics Institute (Boston, MA). EGF was purchased from Roche Diagnostics (Laval, Quebec, Canada). CrkII and Rac antibodies were purchased from BD Transduction Laboratories (Missisauga, Ontario, Canada). HA.11 antibodies were obtained from Berkley Antibody Company (Berkley, CA). Antibodies recognizing the phosphorylated form of ERK1/2 were purchased from New England BioLabs (Mississauga, Ontario, Canada). Dr. John Blenis (Harvard Medical School, Boston, MA) provided an ERK1/2 antibody (C2) that recognizes total ERK1/2. Met (144) antibodies were described previously (51). pcDNA1.1-Gab1Delta Crk expression plasmids were described previously (38).

Cell Culture-- MDCK cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) and gentamicin (Invitrogen). The generation of stable cell lines overexpressing CrkII was described previously (52). For the generation of stable cell lines expressing Gab1Delta Crk, MDCK cells expressing CSF-Met N1358H (CSF-MetDelta Grb2) (23) were co-transfected with pcDNA1.1-Gab1Delta Crk and pLXSH, which confers resistance to Hygromycin B, using GenePorter (Gene Therapy Systems, San Diego, CA). Cell lines were selected in Hygromycin B (300 ng/ml, Roche Diagnostics) for 10-14 days, and stable clones were isolated and screened by Western blotting.

Indirect Immunofluorescence-- Cells were fixed in 3.7% formaldehyde diluted in phosphate-buffered saline and processed for indirect immunofluorescence as described previously (52).

Collagen Assays-- The ability of MDCK cells to form branching tubules was assayed as described previously (22). 5 × 103 cells were resuspended in 500 µl of a collagen solution composed of 95-98% Type I collagen with the remainder composed of Type III collagen (Cohesion Technologies, Inc., Palo Alto, CA) and layered over 350 µl of the same collagen solution in a 24-well plate. The cells were maintained in Liebowitz medium containing 5% FBS and allowed to form cysts for 5-7 days. For stimulations, HGF (15 units/ml), CSF-1 (5 units/ml), or EGF (20 or 100 ng/ml) was added to Liebowitz medium containing 3% FBS. Fresh growth factor and medium were added every 5-6 days. The tubules were photographed 10-14 days later using a Retiga 1300 digital camera (QIMAGING, Burnaby, British Columbia, Canada) and a Zeiss Axiovert 135 microscope with a ×10 or 32 objective (Carl Zeiss Canada Ltd., Toronto, Ontario, Canada). Image analysis was carried out using Northern Eclipse version 6.0 (Empix Imaging, Missisauga, Ontario, Canada). Each assay was quantitated by counting the number of cysts and branched tubules in 4-6 independent fields for each cell line using a dissecting microscope. The results from 4-5 independent experiments were pooled and are represented graphically. The invasion assays were carried out in the same fashion as described above with the exception that 104 cells were seeded and allowed to form small colonies for 2 days prior to stimulation with growth factor. Cells were photographed 2 days later using a ×32 objective.

Growth Factor Stimulations-- MDCK and MDCK cells overexpressing CrkII were plated at 6 × 105/100-mm dish and were serum-starved the next day for 20 h in Dulbecco's modified Eagle's medium containing 0.02% FBS. Cells were stimulated with 70 units/ml HGF or 70 ng/ml EGF for 5 and 180 min, respectively. Cells were lysed in 1.0% Triton X-100 lysis buffer containing 50 mM HEPES, pH 7.5, 150 mM NaCl, 2 mM EGTA, 1.5 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3VO4, 50 mM NaF, 10 µg/ml aprotinin, and 10 µg/ml leupeptin. 30 µg of cell lysate was used for Western blotting with antibodies recognizing the phosphorylated forms of ERK1/2. The membranes were stripped and reprobed with ERK1/2 antibodies.

Rac Pulldown Assays-- MDCK and MDCK cells overexpressing CrkII were grown for 2 days in Dulbecco's modified Eagle's medium containing 10% FBS and serum-starved for 4 h in Dulbecco's modified Eagle's medium containing 0.02% FBS. Cells were then stimulated with 70 units/ml HGF or 70 ng/ml EGF for the indicated times and lysed in Rac lysis buffer (53). 700 µg of cell lysate was used for pull down assays with the CRIB domain of PAK1 fused to glutathione S-transferase as described previously (53).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Overexpression of CrkII Promotes EGF-dependent Cell Dispersal and Invasion-- HGF induces the dispersal and morphogenesis of MDCK cells. Other growth factors, such as EGF, fail to induce this response (29, 54), even though MDCK cells express the EGF receptor and downstream signaling pathways including Gab1 are activated following EGF stimulation (29). Thus, MDCK cells provide an experimental system to examine the signals that cooperate with EGF to promote cell dispersal and morphogenesis. Cell dispersal in response to HGF (Fig. 1A, d-f) occurs in a stepwise process, whereby cells in the colony spread initially lose their cell-cell adherens junctions and then adopt a fibroblastic cell morphology and disperse (53, 55-57). To examine why EGF fails to induce the dispersal of colonies of MDCK cells, we examined the response of MDCK cells to EGF. Although colonies of MDCK cells show some morphological changes in response to EGF (20 and 100 ng/ml), they failed to disperse (Fig. 1A, g-l). Moreover, in response to EGF, cells retain adherens junctions and tight junctions as indicated by the presence of beta -catenin and ZO-1 at cell-cell junctions (Fig. 1A, g-l). In contrast, in response to HGF, adherens-based cell-cell junctions are decreased in spread cells and lost in dispersed cells as demonstrated by the loss of beta -catenin and ZO-1 at the cell membrane (Fig. 1A, d-f) (55, 57).


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  		Fig. 1.   The inability of EGF to promote cell dispersal correlates with its inability to promote the breakdown of adherens junctions and tight junctions. A, MDCK cells (a-c) were stimulated for 24 h with 2.5 units/ml HGF (d-f), 20 ng/ml EGF (g-i), or 100 ng/ml EGF (j-l) and fixed. Cells were stained with beta -catenin/alpha -mouse-CY3 antibodies (a, d, g, and j) and ZO-1/alpha -rabbit-Alexa488 antibodies (b, e, h, and k). Corresponding phase-contrast images are shown (c, f, i, and l). B, MDCK (a-e) and MDCK cells overexpressing CrkII (f-j) were left unstimulated (a and f) or stimulated for 24 h with 0.5 units/ml HGF (b and g), 2.5 units/ml HGF (c and h), 20 ng/ml EGF (d and i), or 100 ng/ml EGF (e and j), fixed, and photographed.

Cell spreading and loss of adherens junctions in response to HGF requires activation of PI3K, MEK1, and the small GTPase Rac (53, 55-57). In a search for other HGF-dependent signals that play a role in this process, we have established that overexpression of the CrkII adapter protein in MDCK cells promotes cell spreading and loss of adherens junctions in the absence of HGF (52). CrkII overexpression mimics the early stages following HGF stimulation, and the cells remain as colonies (Fig. 1B, f). Consistent with this finding, MDCK cells overexpressing CrkII dispersed in response to suboptimal levels of HGF (Fig. 1B, b and g, 0.5 units/ml), demonstrating that CrkII can synergize with HGF for epithelial cell dispersal.

These results prompted us to examine whether CrkII would synergize with a growth factor such as EGF that fails to promote the dispersal and invasion of MDCK cells. Unlike parental MDCK cells (Fig. 1B, d and e), MDCK cells overexpressing CrkII dispersed in response to EGF (Fig. 1B, i and j, 20 or 100 ng/ml). Hence, CrkII overexpression synergizes with EGF to promote the dispersal of colonies of MDCK cells, suggesting that CrkII may synergize with EGF to promote an invasive and morphogenic response. Cell invasiveness in response to EGF or HGF was examined by stimulating MDCK and MDCK cells overexpressing CrkII seeded in three-dimensional collagen gels (Fig. 2). In the absence of growth factor stimulation, both MDCK and MDCK cells overexpressing CrkII formed small spherical colonies (Fig. 2, a and e). HGF stimulation of MDCK and MDCK cells overexpressing CrkII promoted the dispersal and invasion of cells (Fig. 2, b and f). In contrast, EGF stimulation of MDCK cells promoted the formation of small cellular extensions, but the cells failed to detach and invade the collagen gel (Fig. 2, c and d). In contrast, in the presence of EGF, MDCK cells overexpressing CrkII dispersed and invaded the collagen gel where 80-90% of the colonies underwent invasion in response to EGF (Fig. 2, g and h). 80-100 colonies were scored for each condition. The invasive response to EGF in CrkII-overexpressing cells (Fig. 2, g and h) was similar to cells stimulated with HGF (Fig. 2, b and f). Similar responses were observed in several independent clones of MDCK cells overexpressing CrkII (data not shown). Hence, although the overexpression of CrkII or the activation of EGF-dependent signaling pathways is not sufficient for the dispersal of epithelial colonies or the invasion of MDCK cells plated in three-dimensional collagen gels, together they cooperate to promote both cell dispersal and invasion.


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  		Fig. 2.   CrkII overexpression in MDCK cells promotes invasion following EGF stimulation. MDCK (a-d) and MDCK cells overexpressing CrkII (e-h) were plated in three-dimensional collagen gels. 48 h later, cells were left untreated (a and e) or treated with 15 units/ml HGF (b and f), 20 ng/ml EGF (c and g), or 100 ng/ml EGF (d and h). Cells were photographed 48 h later.

MEK1-dependent Signals Synergize with CrkII to Promote the Loss of Tight Junctions and Cell Dispersal-- The ability of EGF to promote cell dispersal and invasion in MDCK cells overexpressing CrkII but not parental MDCK cells suggested that CrkII-dependent signals synergize with EGF. In response to HGF, the loss of adherens junctions is blocked by pharmacological inhibitors of MEK1 and PI3K (55, 57). To investigate the EGF-dependent signals required for the dispersal of CrkII-overexpressing cells, cells were pretreated with pharmacological inhibitors of MEK1 (U0126) and PI3K (LY294002). Whereas LY294002 inhibited HGF-stimulated dispersal of MDCK cells and loss of the tight junction marker, ZO-1 (Fig. 3A, c and d), LY294002 pretreatment failed to inhibit EGF- or HGF-induced dispersal of CrkII-overexpressing cells (Fig. 3B, e, f, k, and l). In contrast, the pretreatment of CrkII-overexpressing cells with UO126 blocked both the HGF- and EGF-dependent loss of ZO-1 from cell-cell junctions and cell scatter (Fig. 3B, g, h, m, and n), indicating that MEK1-dependent pathways are absolutely required.


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  		Fig. 3.   PI3K but not MEK1 activity is dispensable for HGF- and EGF-stimulated cell dispersal in MDCK cells overexpressing CrkII. A, MDCK cells were pretreated with Me2SO (a and b), 25 µM LY294002 (c and d), or 5 µM UO126 (e and f) for 60 min prior to stimulation with 10 units/ml HGF for 24 h. Cells were fixed and co-stained with beta -catenin/alpha -mouse-CY3 antibodies (a, c, and e) and ZO-1/alpha -rabbit-Alexa488 antibodies (b, d, and f). B, MDCK cells overexpressing CrkII were pretreated with Me2SO (a-d, i, and j), 25 µM LY294002 (e, f, k, and l), or 5 µM UO126 (g, h, m, and n) for 60 min prior to stimulation with 10 units/ml HGF (c-h) or 20 ng/ml EGF (i-n) for 24 h. Cells were fixed and co-stained with beta -catenin/alpha -mouse-CY3 antibodies (a, c, e, g, i, k, and m) and ZO-1/alpha -rabbit-Alexa488 antibodies (b, d, f, h, j, l, and n).

Sustained ERK1/2 activation in response to HGF correlates with a Met-dependent morphogenic response (39), and the pharmacological inhibition of MEK1 blocked both cell dispersal (57) and the morphogenic program (54) in response to HGF. Hence, the inability of EGF to stimulate cell dispersal and branching morphogenesis in MDCK cells may reflect the ability of HGF but not EGF to promote sustained ERK1/2 activation. To examine this possibility, we established whether the overexpression of CrkII altered the temporal activation of ERK1/2 in response to EGF. Lysates were prepared from MDCK and MDCK cells overexpressing CrkII stimulated with HGF or EGF for 5 and 180 min and immunoblotted with a phosphorylation-specific ERK1/2 antibody raised against the active site. EGF- and HGF-stimulated ERK1/2 phosphorylation was increased and similar in MDCK cells stimulated for 5 min (Fig. 4A, upper panel). Although HGF stimulation promoted sustained ERK1/2 phosphorylation, up to 180 min, ERK1/2 phosphorylation returned to basal levels in both CrkII-overexpressing and in MDCK cells stimulated with EGF (Fig. 4A, upper panel). All samples contained similar levels of ERK1/2 (Fig. 4A, lower panel). Although the overexpression of CrkII did not promote sustained ERK1/2 phosphorylation in response to EGF, the ability of EGF to promote dispersal in MDCK cells overexpressing CrkII is MEK1-dependent.


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  		Fig. 4.   Overexpression of CrkII promotes elevated Rac activation but fails to promote sustained ERK1/2 activation in response to EGF. A, MDCK and MDCK cells overexpressing CrkII were stimulated with 70 units/ml HGF (H) or 70 ng/ml EGF (E) for the indicated times. 30 µg of whole cell lysate was subjected to SDS-polyacrylamide gel electrophoresis, and proteins on the gel were transferred to nitrocellulose membranes. Western blotting was performed with alpha pERK1/2 (upper panel), and the membranes were stripped and reprobed with alpha ERK1/2 (lower panel). B, MDCK or MDCK cells overexpressing CrkII was stimulated with 70 units/ml HGF or 70 ng/ml EGF for the indicated times. Cells were lysed, and 700 µg of protein lysate was incubated for 60 min with glutathione S-transferase-CRIB fusion proteins bound to glutathione-Sepharose beads. The beads were washed extensively, and bound proteins together with 20 µg of whole cell lysate were resolved on a 12% SDS-polyacrylamide gel. Proteins on the gel were transferred to a nitrocellulose membrane and immunoblotted with alpha Rac. Fold induction is expressed relative to unstimulated MDCK cells. WB, Western blotting.

CrkII-overexpressing Cells Exhibit Elevated Rac Activation-- HGF-dependent breakdown of cell-cell junctions and cell spreading requires the activity of members of the Rho GTPase family, Rac and Cdc42 (53, 56). In MDCK cells, HGF stimulation leads to the activation of Rac and Cdc42 (53), and HGF-induced cell spreading is inhibited by the expression of dominant negative mutants of Rac1 (N17Rac1) that fail to bind GTP (53, 56). Similarly, CrkII fails to promote cell spreading when microinjected together with N17Rac1 (52). To establish whether the ability of EGF to stimulate the spreading of colonies of MDCK cells overexpressing CrkII reflects elevated Rac activity. MDCK and MDCK cells overexpressing CrkII were stimulated or not with HGF or EGF, and GTP-bound Rac levels were assayed in vitro using a glutathione S-transferase fusion protein containing the PAK1 CRIB domain. As shown previously (53), the stimulation of MDCK cells with HGF induced a modest activation of Rac (Fig. 4B, upper panel). EGF stimulation of MDCK cells also induced the activation of Rac, but Rac activation was consistently lower than that observed following HGF stimulation (Fig. 4B, upper panel). In contrast, MDCK cells overexpressing CrkII showed elevated levels of GTP-bound Rac (Fig. 4B, upper panel) (52), and Rac activation was greatly enhanced following stimulation with HGF or EGF when compared with parental MDCK cells (Fig. 4B, upper panel). Similar levels of Rac were detected in whole cell lysates (Fig. 4B, lower panel).

CrkII Synergizes with EGF to Promote a Morphogenic Program-- As EGF does not promote invasion (Fig. 2) or branching morphogenesis of MDCK cells (29) but promotes the invasion of cells overexpressing CrkII (Fig. 2), we determined whether EGF could also promote a morphogenic program in CrkII-overexpressing cells. Cells were seeded in three-dimensional collagen gels and allowed to form cysts (a hollow sphere of polarized epithelia) for 5 days. Cysts were then stimulated with HGF or EGF, and the appearance of branching tubules was monitored over the course of 10-14 days. As described previously (2, 23), HGF stimulation of MDCK results in the formation of branched tubules (Fig. 5A, b), structures whose length is five times greater than their width. The overexpression of CrkII (Fig. 5A, e) or stimulation of MDCK cells with EGF (Fig. 5A, c and d) failed to promote branching morphogenesis. Rather, CrkII overexpression or EGF stimulation each promoted cell growth as displayed by the larger size of the cysts (Fig. 5A, c-e) when compared with unstimulated MDCK cells (Fig. 5A, a). Consistent with the ability of EGF stimulation to promote the invasion of MDCK cells overexpressing CrkII in three-dimensional collagen gels (Fig. 2), EGF promoted the formation of branching tubules in MDCK cells overexpressing CrkII (Fig. 5A, g and h). For each cell line, the results from four independent experiments were quantified and pooled to represent the percentage of cells that form branching tubules (Fig. 5B). Whereas vector-transfected MDCK cells remained as cysts in response to 20 or 100 ng/ml EGF (98%), CrkII-overexpressing cell lines responded to 20 and 100 ng/ml EGF with only 18-20% of the original cysts showing no response (Fig. 5B). EGF stimulation of MDCK cells overexpressing CrkII generated branched tubules (35-47%) as well as structures too short to be considered tubules (32-40%, referred to as Partial Response in Fig. 5B). Interestingly, the tubules obtained following HGF stimulation of MDCK cells overexpressing CrkII appeared more branched (Fig. 5A, b and f), further supporting a role for CrkII in branching morphogenesis.


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  		Fig. 5.   MDCK cells overexpressing CrkII form branching tubules in response to EGF, whereas control cells form large cysts. A, MDCK (a-d) and MDCK cells overexpressing CrkII (e-h) were plated in three-dimensional collagen gels and allowed to form cysts for 5-7 days. Cells were then left untreated (a and e) or treated with 15 units/ml HGF (b and f), 20 ng/ml EGF (c and g), or 100 ng/ml EGF (d and h). Branched tubules appeared 10-14 days later and were photographed. B, quantitation of the morphogenic response following stimulation with EGF (20 and 100 ng/ml) was performed as described under "Experimental Procedures." Results from four independent experiments were pooled and plotted as the percentage of cysts that have undergone branching morphogenesis.

A Gab1-Crk Complex Is Required for a Met-dependent Morphogenic Program-- In a search for signals required for the Met-dependent morphogenic program, we had previously generated a chimeric CSF-Met receptor, allowing a structure function analysis of Met-dependent signals in response to CSF in MDCK cells that express endogenous Met receptors. The Gab1-docking protein was found to rescue the morphogenic defect of a Met receptor mutant that fails to recruit the Grb2 adapter protein and has a reduced ability to recruit Gab1 (CSF-MetDelta Grb2) (29, 39, 41, 58). Following activation of the Met receptor, Gab1 is tyrosine-phosphorylated and recruits multiple signaling proteins including the Crk adapter proteins (35-38). We determined whether the binding of Crk to Gab1 was required for HGF-dependent branching morphogenesis in MDCK cells as Gab1 binds to CrkII in MDCK cells stimulated with HGF (52). We have previously shown that a Gab1 mutant in which five tyrosine residues contained within a YXXP motif were substituted with phenylalanine (Gab1Delta Crk) failed to bind CrkII (38). This mutant was overexpressed in MDCK cells expressing CSF-MetDelta Grb2, and several independent clones expressing HA-tagged Gab1Delta Crk at equivalent levels or greater than HA-tagged wild type Gab1 were selected (Fig. 6A). Similar levels of CSF-MetDelta Grb2 were expressed in each cell line (Fig. 6B). All cell lines formed cysts when plated in collagen gels (Fig. 6C, a, d, and g). As reported previously, MDCK cells expressing a chimeric CSF-MetDelta Grb2 receptor failed to undergo branching morphogenesis (Fig. 6C, c) (23, 29), whereas the overexpression of wild type Gab1 in these cells promoted a morphogenic program in response to CSF (Fig. 6C, f) (29). In contrast, the overexpression of Gab1Delta Crk mutant did not efficiently rescue the branching morphogenesis phenotype of MDCK cells expressing a chimeric CSF-MetDelta Grb2 (Fig. 6C, i). The majority of cells expressing Gab1Delta Crk remained as cysts or formed stunted tubule-like structures, which failed to branch in response to CSF (Fig. 6C, i). The results from six independent experiments were quantified and pooled together (Fig. 6D). Stunted tubule-like structures that failed to branch in cells expressing Gab1Delta Crk were scored as a "partial response." Whereas 56% of cells overexpressing a wild type Gab1 protein efficiently formed branching tubules in response to CSF-Met activation, <6% of all cells overexpressing Gab1Delta Crk formed branching tubules (Fig. 6D). In contrast, 50% of cells expressing Gab1Delta Crk formed unbranched tubule-like structures in response to CSF stimulation (Fig. 6D). Importantly, 70% of all cells expressing vector, Gab1, or Gab1Delta Crk formed branching tubules in response to HGF activation of the endogenous Met receptor, indicating that the morphogenic program was not impaired (Fig. 6C, b, e, and h and data not shown). Hence, the recruitment of Crk to the Gab1-docking protein is critical for the epithelial branching morphogenic program induced by the Met receptor.


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  		Fig. 6.   The expression of Gab1Delta Crk fails to restore branching morphogenesis in MDCK cells expressing CSF-MetDelta Grb2. A and B, proteins from lysates of MDCK cells expressing CSF-MetDelta Grb2 and vector, HA-Gab1 or HA-Gab1Delta Crk, were subjected to Western blotting with alpha -HA (A) or alpha -Met (B). C, MDCK cells expressing CSF-MetDelta Grb2 and vector, Gab1 or Gab1Delta Crk, were seeded in collagen and allowed to form cysts for 7 days. Cysts were left unstimulated (a, d, and g) or stimulated with 15 units/ml HGF (b, e, and h) or 5 units/ml CSF-1 (c, f, and i). Branching tubule formation was visualized 10-14 days later, and structures were photographed. The large bar represents 50 µm, and the small bar represents 100 µm. D, quantitation of the morphogenic response following stimulation with HGF or CSF-1 was performed as described under "Experimental Procedures." Results from six independent experiments were pooled together and plotted as the percentage of cysts that have undergone branching morphogenesis. Partial responses represent tubule-like structures, which failed to branch.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

HGF but not EGF promotes cell dispersal and branching morphogenesis in MDCK cells. This provides an experimental system to identify HGF-dependent signals and to dissect signals that synergize with EGF in mediating the dispersal of epithelial sheets, epithelial remodeling, invasion, and morphogenesis. We show that the recruitment of the Crk adapter protein to the Gab1-docking protein is required for the morphogenic response downstream from Met. Moreover, the overexpression of the CrkII adapter protein converts an EGF-dependent signal in MDCK cells from noninvasive to invasive, promoting the dispersal of epithelial cell sheets, invasion, and branching morphogenesis. EGF-dependent MEK1 activity is required for the breakdown of tight junctions and dispersal of CrkII-overexpressing cells. Although, CrkII overexpression does not alter EGF-dependent ERK1/2 activation, CrkII overexpression synergizes with EGF to promote a robust activation of Rac. Together, our data support a role for the CrkII adapter protein in the integration of upstream signals to promote epithelial cell dispersal, invasion, and morphogenesis.

The dispersal of epithelial cells in response to HGF occurs in a stepwise progression, which involves cell spreading and the breakdown of epithelial adherens junctions and tight junctions. The inability of EGF to stimulate cell dispersal and invasion correlates with the inability of a EGF-dependent signal to stimulate the breakdown of adherens and tight junctions in MDCK cells (Fig. 1A). In a search for signals that could synergize with EGF to induce an invasive response in MDCK cells, we established that the overexpression of CrkII promoted the early stages of an HGF response including cell spreading and breakdown of adherens junctions (52). However, in the absence of HGF stimulation, MDCK cells overexpressing CrkII failed to scatter or invade collagen gels (Fig. 2), consistent with the inability of these cells to breakdown ZO-1 containing tight junctions and disperse in two-dimensional cultures (Fig. 1B) (52). The addition of EGF to cells overexpressing CrkII promoted the dispersal of epithelial colonies in two-dimensional cultures and the invasion of cells in three-dimensional collagen gels (Figs. 1B and 2). This finding suggests that although the activation of EGF-stimulated or CrkII-dependent signaling pathways alone is not sufficient, together they can cooperate to activate signals required for cell dispersal and invasion.

Our work and the work of others (55, 57) have identified several of the signals required for cell dispersal in response to HGF. HGF-dependent cell spreading and loss of adherens junctions are blocked by inhibitors of MEK1 and PI3K (55, 57) and require the activation of the Rho GTPases, Rac and Cdc42 (53, 56). Consistent with the synergy between EGF and CrkII signals, MDCK cells overexpressing CrkII displayed a robust activation of Rac in response to HGF or EGF compared with MDCK cells stimulated with HGF or EGF (Fig. 4B). Hence CrkII overexpression synergizes with HGF and EGF in promoting the activation of Rac. The involvement of Rac in cell invasion has been established in several experimental systems (59-62). The overexpression of activated Rac1 promotes the invasion of some carcinoma cell lines in collagen (59, 63), whereas the dominant negative forms of Rac1 inhibit leptin-stimulated cell invasion in collagen gels (60), implicating Rac1-dependent pathways in epithelial cell invasion. However, activated Rac also promotes enhanced cell-cell junction assembly in MDCK cells (64), inhibiting their dispersal in response to HGF (65, 66). The differences observed with activated Rac and CrkII overexpression in MDCK cells may reflect the ability of Rac activity to turn over in MDCK cells overexpressing CrkII, in contrast with cells expressing activated Rac where Rac is constantly GTP-bound. Whereas MDCK cells overexpressing wild type Rac1 are more polarized than vector-transfected MDCK cells, they are able to disperse in response to HGF (data not shown). Moreover, the overexpression of wild type Rac1 in MDCK cells was not sufficient to promote an invasive response to EGF (data not shown), implicating additional Crk-dependent signals in the invasive response.

EGF-induced loss of ZO-1 at tight junctions and dispersal of CrkII-overexpressing cells were blocked by pharmacological inhibitors of MEK1 but not PI3K (Fig. 3B). This finding is consistent with a requirement for MEK1 activity in the loss of ZO-1 from tight junctions and for the maintenance of the dispersed phenotype in epithelial cells expressing activated Ha-Ras or Raf-1 (67, 68). This finding also supports previous data indicating a requirement for PI3K activity in the breakdown of adherens junctions (55, 57). Because CrkII overexpressing cells show decreased adherens junctions as visualized through reduced beta -catenin staining at cell-cell junctions (Fig. 3B), CrkII overexpression may have overcome the requirement for PI3K in the breakdown of adherens junctions.

As an adapter protein, Crk contains an SH2 domain that binds tyrosine-phosphorylated proteins (45) and an SH3 domain that binds proteins containing PXLPXK motifs commonly found in exchange factors for members of the Ras superfamily of GTP-binding proteins (69). The mechanism through which CrkII promotes elevated basal Rac activity and elevated Rac activity in response to HGF and EGF is currently unknown. CrkII can activate Rac through an interaction with DOCK180, an exchange factor for Rac1 (70, 71). However, an association of CrkII with DOCK180 was not observed in CrkII-overexpressing MDCK cells, although DOCK180 is expressed in these cells (data not shown), suggesting that other Rac exchange factors may be involved. Alternatively, CrkII may activate Rac indirectly through an alternate mechanism.

The observation that CrkII overexpression did not alter EGF-dependent ERK1/2 activation is in contrast to previous data where the overexpression of v-Crk in PC12 cells promoted sustained ERK1/2 activity and neurite outgrowth in response to EGF (72). This may represent differences in v-Crk versus CrkII in addition to differences in cellular context and the possible presence of different Crk-binding proteins in these two cell types. Nevertheless, the observation that a loss of tight junctions requires EGF-dependent MEK1 activation supports a synergistic interaction between an EGF signal and CrkII for cell dispersal and invasion. This finding is consistent with the ability of EGF to stimulate the dispersal and invasion of several carcinoma cell lines (17-20), each of which may have undergone several genetic changes such as the loss of adherens junctions, the consequence of which is similar to MDCK cells overexpressing CrkII.

Epithelial morphogenesis requires cell invasion as well as the ability to reorganize and reform cellular junctions (42). The formation of a cyst of polarized MDCK epithelial cells requires 5 days in collagen. Once polarized, cells are subjected to additional signals from surrounding matrix that act to promote epithelial organization (16, 42). We have previously demonstrated that the docking protein Gab1 is required for the morphogenic program in response to HGF stimulation (29). In the absence of any catalytic activity, Gab1 functions as a docking protein that when phosphorylated by Met or other receptors recruits multiple signaling proteins including PI3K and SHP-2 (29, 36, 39) as well as CrkII and CrkL (35-38). The significance of Gab1/Crk coupling in branching morphogenesis had not been previously addressed. We provide evidence supporting a role for Gab1/Crk coupling in branching morphogenesis downstream from a Met receptor tyrosine kinase unable to bind Grb2 (Fig. 6C). Although the precise role for Crk in this process is unknown, CrkII overexpression enhances HGF-dependent activation of Rac (Fig. 4B) and Rac1 is involved in epithelial remodeling (8). Furthermore, this finding is consistent with the observation that overexpression of CrkII in MDCK cells synergizes with EGF to promote epithelial remodeling and branching morphogenesis (Fig. 5). In conclusion, we have demonstrated that the coupling of Gab1 with Crk is required for branching morphogenesis following activation of the Met receptor tyrosine kinase. These results emphasize the potential importance of Crk-dependent signaling pathways in epithelial morphogenesis and invasion. The ability of CrkII overexpression to switch an EGF signal in MDCK cells from non-invasive to invasive underscores the importance of considering the synergistic actions of signaling pathways in cancer progression.

    ACKNOWLEDGEMENTS

We thank George Vande Woude, John Blenis, Alan Hall, Bruce Mayer, John Collard, and the Genetics Institute for reagents provided in this study.

    FOOTNOTES

* This research was supported by an operating grant from the Canadian Breast Cancer Research Initiative with money from the Canadian Cancer Society (to M. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a Canadian Institutes of Health Research studentship.

Recipient of a MUHC Research Institute studentship.

Dagger Dagger Recipient of a Canadian Institutes of Health Research scientist award. To whom correspondence should be addressed: Molecular Oncology Group, McGill University Hospital Centre, Rm. H510, 687 Pine Ave., W., Montreal, Quebec H3A 1A1, Canada. Tel.: 514-842-1231, ext. 35834; Fax: 514-843-1478.

Published, JBC Papers in Press, July 23, 2002, DOI 10.1074/jbc.M201743200

    ABBREVIATIONS

The abbreviations used are: HGF, hepatocyte growth factor; MDCK, Madin-Darby canine kidney; PH, pleckstrin homology; EGF, epidermal growth factor; SH2 or SH3, Src homology 2 or 3, respectively; FBS, fetal bovine serum; PI3K, phosphatidylinositol 3'-kinase; HA, hemagglutinin; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; CSF, colony-stimulating factor; CRIB, Cdc42/Rac interactive binding.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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 All ASBMB Journals   Molecular and Cellular Proteomics 
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Copyright © 2002 by the American Society for Biochemistry and Molecular Biology.
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