Cell motility is a fundamental process required during normal embryonic development, wound healing, inflammatory responses, and tumor progression toward metastasis(1) . Hepatocyte growth factor/scatter factor (HGF/SF) ()is a multifunctional cytokine with activities on a wide variety of normal and neoplastic cells. HGF/SF is a mitogen, dissociation, and motility factor for many epithelial cells (2, 3, 4) and stimulates tubulogenesis of tubular epithelial cells (5) as well as the invasion of carcinoma cells(6) . In vivo, HGF/SF is a potent angiogenic factor (7, 8) and is involved in organ regeneration (9) and tumorigenesis(10, 11) . A high affinity receptor for HGF/SF has been identified as the product of the met proto-oncogene(12, 13) , which encodes a receptor tyrosine kinase originally isolated as the tpr-met oncogene(14, 15) . The Met receptor is predominantly expressed in epithelial cells in culture (15, 16, 17) and in epithelium in vivo (18, 19). ()The mature form of the Met receptor is a heterodimeric protein of 190 kDa, which consists of a 45-kDa extracellular -subunit linked by disulfide bonds to a 145-kDa -subunit that spans the membrane and contains the catalytic kinase domain(16, 20, 21, 22) . Binding of HGF/SF induces activation of the kinase and auto/transphosphorylation of the receptor (13) on specific tyrosine residues in the chain(12, 23) .

Phosphorylated tyrosine residues within receptor tyrosine kinases provide binding sites for molecules containing SH2 and PTB domains that act to transduce extracellular signals to the interior of the cell (24) . Following stimulation of cells with HGF/SF, several proteins are phosphorylated, activated, and/or associated with the Met receptor. These include p120 GTPase-activating protein, mitogen activated protein kinase, Src, phospholipase C, phosphatidylinositol 3-kinase (PI3-kinase), Grb2(25, 26, 27, 28, 29, 30) , Ras(31) , focal adhesion kinase(32) , -catenin, plakoglobin(33) , and the Shc adaptor protein(34) . The Met receptor tyrosine kinase is highly phosphorylated on two tyrosine residues (1234 and 1235) within the kinase domain (35, 36) that are essential for the catalytic activity of the receptor(29, 35) . In addition, tyrosine 1356 within the carboxyl terminus of the -subunit of the Met receptor is phosphorylated (29) and is essential for the recruitment of multiple substrates including the p85 subunit of PI3-kinase, phospholipase C, and Grb2(29, 30, 37, 38) . Although a role for many of these signal transduction pathways has been established in cell mitogenesis, their role in dissociation and scatter of epithelial cells is unknown.

To investigate the signaling pathways that are involved in these events, we have generated chimeric receptors containing the extracellular domain of the CSF-1 receptor fused to the transmembrane and intracellular domains of the Met receptor. Madin-Darby canine kidney (MDCK) epithelial cells dissociate, scatter, and form branching tubules in response to HGF/SF. MDCK cells expressing the CSF-MET chimera dissociate and scatter in response to CSF-1(39) ; however, MDCK cells expressing a mutant CSF-MET receptor containing a phenylalanine substitution for tyrosine 1356 (Y1356F) are unable to scatter or form branching tubules following stimulation with CSF-1(29) . This has suggested that one or more of the substrates that bind to Tyr are required for the induction of MDCK cell scatter following stimulation with HGF/SF. Our results demonstrate that following stimulation with HGF/SF, activation of PI3-kinase but not phospholipase C or pp70, a downstream target of PI3-kinase, is essential for MDCK cell scatter.

EXPERIMENTAL PROCEDURES

Cell Lines

Immunofluorescence Labeling

Phosphatidylinositol 3-Kinase Assay

S6 Kinase Immunoprecipitation, Western Blotting, and in Vitro Kinase Assay

RESULTS

A CSF-MET Mutant Chimera, Y1356F, Fails to Promote Cell Dissociation and Redistribution of Junctional Protein Complexes in Response to CSF-1

Figure 1: E-cadherin, desmoplakins I/II, and ZO-1 remain insoluble following CSF-1 stimulation of MDCK cells expressing the Y1356F CSF-MET mutant receptor. Colonies of MDCK cells expressing the Y1356F CSF-MET mutant receptor were serum-starved for 24 h and further incubated for 24 h in the same medium (a-d), in medium containing HGF/SF (5 units/ml) (e-h), or in medium containing CSF-1 (50 ng/ml) (i-l). Cells were extracted in situ with CSK buffer and fixed with 1.75% formaldehyde in PBS as described under ``Experimental Procedures.'' Cells were then processed for indirect immunofluorescence with antibodies against E-cadherin (b, f, and j), desmoplakins I/II (c, g, and k), and ZO-1 (d, h, and l) as primary antibodies, followed by rhodamine-coupled anti-mouse antibodies (for E-cadherin and ZO-1) and fluorescein isothiocyanate-coupled anti-rabbit antibodies (for desmoplakins I/II) as secondary antibodies.

Figure 3: The redistribution of E-cadherin and desmoplakins I/II, cell spreading, and scatter in HGF/SF-stimulated MDCK cells are inhibited by wortmannin. A, colonies of serum-starved MDCK cells were incubated for 3.5 h in medium containing 0.1% MeSO (a-d), preincubated in medium containing 0.1% MeSO for 30 min followed by stimulation with HGF/SF (10 units/ml) for 3 h (e-h), or preincubated in medium containing 500 nM wortmannin (in 0.1% MeSO) for 30 min followed by stimulation with HGF/SF for 3 h (i-l). Cells were extracted in situ with CSK buffer, fixed with 1.75% formaldehyde in PBS, and processed for indirect immunofluorescence with antibodies against E-cadherin (b, f, and j), desmoplakins I/II (c, g, and k), and ZO-1 (d, h, and l). B, colonies of serum-starved MDCK cells were incubated for 7.5 h in medium containing 0.1% MeSO (a), preincubated in medium containing 0.1% MeSO for 30 min followed by stimulation with HGF/SF (10 units/ml) for 7 h (b), preincubated in medium containing 1 µM wortmannin (in 0.1% MeSO) for 30 min followed by stimulation with HGF/SF for 7 h (c), preincubated in medium containing 5 nM staurosporine (in 0.1% MeSO) for 30 min followed by stimulation with HGF/SF for 7 h (d), preincubated in medium containing 2 µM U-73122 (e), or 2 µM U-73343 (f) (in 0.1% MeSO) for 30 min followed by stimulation with HGF/SF for 7 h. Cells were fixed in 0.2% glutaraldehyde in PBS.

MDCK cells expressing a CSF-MET chimera dissociate and scatter in a similar manner in response to CSF-1 or HGF/SF(39) . However, MDCK cells expressing a mutant CSF-MET receptor containing a phenylalanine substitution for tyrosine 1356 (Y1356F) fail to scatter following stimulation with CSF-1(29) . We show here that junctional complexes remained intact and in an insoluble form at cell-cell interfaces in CSF-1-stimulated MDCK cells expressing the Y1356F CSF-MET mutant (Fig. 1, i-l).

Wortmannin Inhibits PI3-Kinase Activity and Scatter of HGF/SF-stimulated MDCK Cells

Figure 2: PI3-kinase is activated following HGF/SF stimulation of MDCK cells and is inhibited by wortmannin in vitro and in vivo. A, MDCK cells starved for 48 h were stimulated with HGF/SF (10 units/ml) for different time intervals, and cell lysates were then immunoprecipitated with the anti-phosphotyrosine antibody PY20. Immune complexes were adsorbed using protein A-Sepharose and subjected to a PI3-kinase assay. The products of the reaction were analyzed by thin layer chromatography, visualized by autoradiography, and quantified by a PhosphorImager. B, phosphotyrosine-containing proteins from lysates of MDCK cells were immunoprecipitated with the PY20 antibody and assayed for PI3-kinase activity in vitro in the presence of either 0.1% MeSO or various concentrations of wortmannin for 10 min prior to the kinase reaction. C, serum-starved MDCK cells were preincubated with either 0.1% MeSO or wortmannin (10 nM to 10 µM) for 30 min at 37 °C, followed by stimulation with HGF/SF (10 units/ml) for 1 min at 37 °C. Cells were lysed, and phosphotyrosine-containing proteins were immunoprecipitated with the PY20 antibody. Immune complexes were then processed as described above. The position of phosphatidylinositol 3-phosphate (PI(3)P) is indicated.

To examine the role of PI3-kinase in vivo, serum-starved MDCK cells were pretreated for 30 min at 37 °C with either wortmannin or its solvent (MeSO 0.1%) and stimulated with HGF/SF, and PI3-kinase was immunoprecipitated with anti-phosphotyrosine antibodies and assayed in vitro. Wortmannin, at concentrations of 100 nM and 1 µM, reduced the level of PI3-kinase activity in HGF/SF-stimulated MDCK cells by 35 and 65% respectively (Fig. 2C). To evaluate the effect of wortmannin on HGF/SF-induced cell scatter, colonies of serum-starved MDCK cells were pretreated for 30 min at 37 °C with wortmannin (10 nM-1 µM) and then stimulated with HGF/SF. MDCK cells, following stimulation with HGF/SF, changed shape and flattened by 3 h (Fig. 3A, e), whereas MDCK cells in the presence of 500 nM wortmannin remained as tight colonies (Fig. 3A, i). Moreover, in the presence of 1 µM wortmannin, MDCK cells stimulated with HGF/SF for 7 h had flattened (Fig. 3B, c) but were inhibited in their ability to scatter when compared with control HGF/SF-stimulated MDCK cells (Fig. 3B, b). The ability of wortmannin to inhibit both cell spreading and scatter was concentration-dependent and correlated with its ability to inhibit PI3-kinase activity in vivo (data not shown).

To establish which processes were inhibited by wortmannin, we examined the effect of wortmannin on the redistribution of junctional complex proteins. The spreading and changes in morphology observed in MDCK cells, following HGF/SF stimulation for 3 h, were accompanied by a reduction in the amount of insoluble E-cadherin and desmoplakins I/II at cell-cell interfaces (Fig. 3A, f and g). At this time however, the concentration of insoluble ZO-1 at cell-cell interfaces (Fig. 3A, h) was comparable to that observed in control unstimulated MDCK cells (Fig. 3A, d). A redistribution of ZO-1 was only apparent when cells began to scatter (data not shown). However, in the presence of 500 nM wortmannin, all three proteins from junctional complexes were retained in an insoluble compartment at the plasma membrane (Fig. 3A, j-l). Thus, the ability of wortmannin to inhibit MDCK cell spreading and scatter correlated with the retention of insoluble junctional complexes and tight cell-cell interactions, suggesting that PI3-kinase activity is required for the redistribution of junctional complex proteins and cell dissociation induced by HGF/SF in MDCK cells.

Chemotaxis of some cell types transduced by the PDGF- and epidermal growth factor receptors requires phospholipase C(51, 56) ; however, the inhibition of protein kinase C, a downstream target of phospholipase C, did not inhibit MDCK cell scatter in response to HGF/SF(57) . To investigate the involvement of phospholipase C in HGF/SF-induced MDCK cell scatter, we used an inhibitor of phospholipase C (U-73122; IC of 1-2 µM) (56, 58, 59) and the protein kinase C inhibitor staurosporine (IC of 0.7 nM)(60) . Colonies of serum-starved MDCK cells were pretreated with MeSO (0.1%), staurosporine (1 nM-20 nM), U-73122 (0.25-2 µM), or an inactive analogue of U-73122 (U-73343; 0.25-2 µM) for 30 min at 37 °C. MDCK cells in the presence of staurosporine (Fig. 3B, d, and data not shown), the phospholipase C inhibitor U-73122 (Fig. 3B, e, and data not shown), the inactive analogue U-73343 (Fig. 3B, f, and data not shown), or MeSO (0.1%) (Fig. 3B, b) scattered following stimulation with HGF/SF for 7 h. Therefore, in contrast to PI3-kinase, phospholipase C and protein kinase C are not essential for MDCK cell spreading and scatter.

pp70Is Activated Following HGF/SF Stimulation of MDCK Cells But Is Not Required for Scatter

Figure 4: pp70 is activated following HGF/SF stimulation of MDCK cells and is inhibited by wortmannin or rapamycin. A, serum-starved MDCK cells were stimulated with HGF/SF (10 units/ml) for different time intervals, and proteins from cell lysates were immunoprecipitated with antibodies against p70. Immune complexes were adsorbed using protein A-Sepharose and subjected to SDS-polyacrylamide (8%) gel electrophoresis, transferred onto nitrocellulose and immunoblotted with an anti-p70 antibody (top), or subjected to an in vitro kinase assay using S6 peptide as a substrate as described under ``Experimental Procedures'' (bottom). The results are reported as counts incorporated into S6 peptide. B, serum-starved MDCK cells were preincubated with either 0.1% MeSO or various concentrations of wortmannin for 30 min at 37 °C, followed by further incubation in MeSO or by stimulation with HGF/SF (10 units/ml) for 20 min at 37 °C. Cells were lysed, pp70 was immunoprecipitated, and immune complexes were then processed as described above. C, serum-starved MDCK cells were preincubated with either 0.1% ETOH or various concentrations of rapamycin for 30 min at 37 °C, followed by further incubation in ETOH or by stimulation with HGF/SF (10 units/ml) for 20 min at 37 °C. Cells were lysed and pp70-immune complexes were processed as described above.

To establish whether pp70 is essential for MDCK cell dissociation and scatter induced by HGF/SF, serum-starved MDCK cells were pretreated with the macrolide antibiotic rapamycin, which inhibits the PI3-kinase-related protein RAFT1 (65) and as a consequence its downstream target pp70(66, 67, 68) . HGF/SF-induced pp70 phosphorylation (Fig. 4C, top) and in vitro kinase activity (Fig. 4C, bottom) were inhibited equally by rapamycin (1-50 ng/ml) or wortmannin (1-10 µM) (Fig. 4B). However, preincubation of MDCK cells with 20 ng/ml of rapamycin for 30 min followed by stimulation with HGF/SF did not inhibit scattering of MDCK cells (Fig. 5i). Moreover, rapamycin pretreatment did not inhibit the redistribution or solubilization of E-cadherin, desmoplakins I/II, and ZO-1 following stimulation of MDCK cells with HGF/SF (Fig. 5, j-l). Therefore, although pp70 is activated following stimulation with HGF/SF and is a possible downstream target of PI3-kinase in MDCK cells, it is not required for scatter of MDCK cells in response to HGF/SF.

Figure 5: Inhibition of pp70 activation in HGF/SF-stimulated MDCK cells by rapamycin does not block cell spreading and dissociation. Colonies of serum-starved MDCK cells were incubated for 24 h in medium containing 0.1% ETOH (a-d), preincubated in medium containing 0.1% ETOH for 30 min and then stimulated by HGF/SF (10 units/ml) for 24 h (e-h), or preincubated in medium containing 20 ng/ml of rapamycin for 30 min and then stimulated with HGF/SF for 24 h (i-l). Cells were extracted in situ with CSK buffer and fixed with 1.75% formaldehyde in PBS and then processed for indirect immunofluorescence with antibodies against E-cadherin (b, f, and j), desmoplakins I/II (c, g, and k), and ZO-1 (d, h, and l).

DISCUSSION

HGF/SF is a multifunctional cytokine that stimulates dissociation, scatter, and morphogenesis of epithelial cells(2, 4, 5) . The response of MDCK cells to HGF/SF can be visualized first as cell spreading (after 2-3 h), when cells are still associated and present within the colony (Fig. 3A), followed by cell dissociation (after 4-6 h) and cell scatter (from 6 h) (Fig. 1). Breakdown of cell contacts is a prerequisite for cell dissociation, and we have shown that dissociation of MDCK cells in response to HGF/SF is concomitant with the loss of stable insoluble junctional complexes at sites of cell contact. Interestingly, following HGF/SF stimulation of MDCK cells, a redistribution of E-cadherin and desmoplakins I/II, which are components of adherens junctions and desmosomes, is observed prior to the redistribution of ZO-1, a component of tight junctions ( Fig. 1and 3A). This may reflect the ability of HGF/SF to stimulate the phosphorylation of -catenin and plakoglobin(33) , which could contribute to the destabilization of adherens junctions and desmosomes but not tight junctions, in which these proteins are absent.

MDCK cells expressing a CSF-MET mutant receptor (Y1356F) fail to stimulate scatter (29) or redistribute junctional complexes (Fig. 1). Tyrosine 1356 in the carboxyl terminus of the Met receptor is essential for association with PI3-kinase, phospholipase C, and Grb2(29, 30, 37, 38) , suggesting that at least one of these signaling pathways is required for cell dissociation and scatter. However, a mutant CSF-MET receptor containing a substitution of a histidine residue for the asparagine downstream from tyrosine 1356 (N1358H), which failed to bind only the Grb2 adaptor protein, stimulated MDCK cell scatter in response to ligand, ()demonstrating that association of Grb2 with the Met receptor is not essential. Because binding sites for PI3-kinase (51, 52) or phospholipase C (51) in the PDGF receptor- are important for PDGF-BB-induced chemotaxis and because phospholipase C is involved in epidermal growth factor-mediated chemotaxis(56) , we have investigated the role of these signaling pathways in HGF/SF-induced cell dissociation and scatter of MDCK epithelial cells. We have shown that PI3-kinase plays an essential role in HGF/SF-mediated scatter of MDCK cells. In the presence of wortmannin, a potent inhibitor for PI3-kinase, MDCK cells stimulated with HGF/SF for 3 h remained as tight colonies and retained insoluble E-cadherin, desmoplakins I/II, and ZO-1 at cell-cell interfaces. Conversely, control MDCK cell colonies contained cells with a flattened appearance, which showed a redistribution of junctional complex proteins involving E-cadherin and desmoplakins I/II, whereas, as discussed above, ZO-1 from tight junctions was maintained while cells were in contact (Fig. 3A)(69) . Moreover, concurrent with the ability of wortmannin to inhibit the spreading of MDCK cells 3 h after HGF/SF stimulation, wortmannin also inhibited the scatter of MDCK cells at 7 h (Fig. 3B). In contrast, an inhibitor of phospholipase C (U-73122) or protein kinase C (staurosporine) at concentrations equal or higher than their respective IC did not inhibit scatter of MDCK cells in response to HGF/SF (Fig. 3B).

The ability of wortmannin to inhibit HGF/SF-induced spreading and scatter of MDCK cells correlated directly with the extent of PI3-kinase inhibition in vivo, thus supporting a crucial role for PI3-kinase activity in the dissociation and scatter of MDCK cells. The concentrations of wortmannin required for the inhibition of cell spreading, redistribution and solubilization of junctional complex proteins, and cell scatter were higher than that reported for the inhibition of diverse biological responses involving membrane ruffling (70) , histamine secretion(55) , respiratory burst(53, 71) , or glucose transport (72) (between 50-100 nM). Although high concentrations of wortmannin (in the µM range in vitro) have been reported to inhibit myosin light chain kinase(55, 73) , an enzyme thought to be involved in cell motility, treatment of MDCK cells with an inhibitor of myosin light chain kinase (ML-9) had no effect on HGF/SF-induced MDCK cell scatter (57) . Moreover, our data demonstrated that the canine PI3-kinase was sensitive to wortmannin in vitro, with an IC below 10 nM, which is comparable to other studies(53, 54, 55, 72) . In addition, wortmannin is a lipophilic compound and is expected to inactivate PI3-kinase localized at the plasma membrane. However, the Met receptor is localized to the basolateral surface of MDCK cells(74) . Thus the application of wortmannin to the apical surface of a colony of tightly associated MDCK cells may be unable to efficiently inactivate the Met-stimulated PI3-kinase localized to the basolateral compartment.

Wortmannin is also an unstable compound when maintained at 37 °C (54) . Thus, the ability of MDCK cells treated with wortmannin to begin to spread at 7 h post HGF/SF stimulation (Fig. 3B) may reflect the instability of wortmannin. Consistent with this possibility, MDCK cells stimulated with HGF/SF for 7 h remained as tight cell colonies when the medium containing wortmannin and HGF/SF was replaced at 2-h intervals (data not shown). Although MDCK cells showed some signs of toxicity under these conditions, this suggests that PI3-kinase is essential for cell spreading. We therefore conclude that PI3-kinase is required for MDCK cell dissociation and thus, scatter, following stimulation with HGF/SF, but due to the instability of wortmannin, we cannot rule out that other factors are involved in these events.

The pp70 has been described as a downstream target of PI3-kinase in various cell types(61, 62) . We show that pp70 is activated following stimulation of MDCK cells with HGF/SF and that this activity is also inhibited by wortmannin, suggesting that pp70 is a downstream target of PI3-kinase in MDCK cells (Fig. 4). pp70 is required for the progression through G1 in response to serum and growth factors in a variety of cells (66, 67, 68, 75) . However, although pretreatment of MDCK cells with rapamycin inhibited the activation of HGF/SF-induced pp70, this had no effect on MDCK cell spreading, redistribution of junctional complex proteins, or cell scatter (Fig. 5). These results suggest that although PI3-kinase is required for cell dissociation and scatter following stimulation of MDCK cells with HGF/SF, this is independent from the activation of pp70.

The requirement for a functional PI3-kinase has been implicated in actin reorganization at the plasma membrane (membrane ruffling) stimulated by PDGF (70, 76) and insulin(76) . Moreover, PI3-kinase is involved in the activation of the small GTP-binding protein Rac(77) , which is required for membrane ruffling in response to growth factors (76, 78) . Interestingly, microinjection of MDCK cells with a dominant negative mutant of Rac (N17Rac1) inhibits cell spreading and actin reorganization induced by HGF/SF(79) , thus supporting a crucial role for Rac in these events. In addition, Ras is also essential for the dissociation and scatter of MDCK cells(79, 80) . Expression of a dominant negative mutant Ras protein (N17Ras) (80) or the injection of a neutralizing antibody for Ras (Y13-259) (79) block HGF/SF-induced cell dissociation and scatter, whereas microinjection of an activated Ras (V12H-Ras) in MDCK cells promotes cell spreading in the absence of HGF/SF(79) . Furthermore, GTP-bound Ras interacts with PI3-kinase and may contribute to its activation(81, 82) . Thus, we propose that activation of PI3-kinase in MDCK cells following stimulation of the Met receptor by HGF/SF promotes cell dissociation, which is independent from the activation of pp70, but may involve the small GTP-binding proteins Rac and Ras. The relationship between PI3-kinase and these proteins in MDCK cells is currently under investigation.

FOOTNOTES

*

This research was supported by operating grants from the National Cancer Institute of Canada and the Medical Research Council of Canada (to M. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Recipient of a fellowship from the Fonds de la Recherche en Santé du Québec.

¶

Senior scholar of the National Cancer Institute of Canada. To whom correspondence should be addressed: Molecular Oncology Group, H5.21, 687 Pine Ave. West, Montreal, Quebec H3A 1A1, Canada. Tel.: 514-842-1231, ext. 5834; Fax: 514-843-1478.

()

The abbreviations used are: HGF/SF, hepatocyte growth factor/scatter factor; CSF, colony-stimulating factor; MDCK, Madin-Darby canine kidney; PI3-kinase, phosphatidylinositol 3-kinase; ETOH, ethanol; PBS, phosphate-buffered saline; PIPES, 1,4-piperazinediethanesulfonic acid; PDGF, platelet-derived growth factor; SH2, Src homology 2; PTB, phosphotyrosine binding.

()

X. M. Yang and M. Park(1995) Lab. Invest., in press.

()

T. Fournier and M. Park, manuscript in preparation.

ACKNOWLEDGEMENTS

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