Activation of Cdc42, Rac, PAK, and Rho-Kinase in Response to Hepatocyte Growth Factor Differentially Regulates Epithelial Cell Colony Spreading and Dissociation

Abstract

Hepatocyte growth factor (HGF), the ligand for the Met receptor tyrosine kinase, is a potent modulator of epithelial-mesenchymal transition and dispersal of epithelial cells, processes that play crucial roles in tumor development, invasion, and metastasis. Little is known about the Met-dependent proximal signals that regulate these events. We show that HGF stimulation of epithelial cells leads to activation of the Rho GTPases, Cdc42 and Rac, concomitant with the formation of filopodia and lamellipodia. Notably, HGF-dependent activation of Rac but not Cdc42 is dependent on phosphatidylinositol 3-kinase. Moreover, HGF-induced lamellipodia formation and cell spreading require phosphatidylinositol 3-kinase and are inhibited by dominant negative Cdc42 or Rac. HGF induces activation of the Cdc42/Rac-regulated p21-activated kinase (PAK) and c-Jun N-terminal kinase, and translocation of Rac, PAK, and Rho-dependent Rho-kinase to membrane ruffles. Use of dominant negative and activated mutants reveals an essential role for PAK but not Rho-kinase in HGF-induced epithelial cell spreading, whereas Rho-kinase activity is required for the formation of focal adhesions and stress fibers in response to HGF. We conclude that PAK and Rho-kinase play opposing roles in epithelial-mesenchymal transition induced by HGF, and provide new insight regarding the role of Cdc42 in these events. INTRODUCTIONEpithelial-mesenchymal transition and cell migration are required during normal embryonic development and during pathological situations such as the dispersal of tumor cells. Hepatocyte growth factor (HGF) is a multifunctional factor that, in addition to promoting epithelial cell growth and survival, has the ability in vitro to stimulate epithelial cell dissociation, motility, invasion, and the endogenous morphogenic program of epithelial cells in three-dimensional matrix or organ culture Nakamura, 1996, 1997;Montesano et al., 1997). Genetic studies have shown that HGF/Met signaling is essential for normal murine embryological development (Birchmeier and Gherardi, 1998). In addition, HGF/Met signaling was also shown to be involved in angiogenesis (Bussolino et al., 1992;Grant et al., 1993) and has been implicated in the dissociation and migration of muscle precursor cells of the dermomyotome, in the guidance and survival of motor neurons, and in the development and survival of sensory neurons (Birchmeier and Gherardi, 1998). HGF and Met were also shown to play a role in pathological conditions, including tissue regeneration (Matsumoto and Nakamura, 1997), tumorigenesis, and metastasis (Jeffers et al., 1996;Bardelli et al., 1997).The conversion from a sessile to a migratory phenotype requires an extensive remodeling of the actin cytoskeleton (Mitchison and Cramer, 1996). Members of the Rho family of small GTP-binding proteins, including Cdc42, Rac, and Rho, are involved in regulating the organization of the actin cytoskeleton and the assembly of associated integrin com-** Corresponding author. E-mail address: morag@lan1.molonc.mcgill.ca. ...

p120 Catenin Is Required for Growth Factor–dependent Cell Motility and Scattering in Epithelial Cells

“…Moreover, some of the signaling pathways used by HGF/Met to induce scattering have been delineated in these cells (Ponzetto et al, 1994;Ridley et al, 1995;Royal et al, 2000;Zondag et al, 2000). To confirm the growth factor dependence of p120-induced cell migration observed in keratinocytes ( Figure 1C), and to dissect the biochemical steps downstream of p120, we introduced p120 and ⌬N-p120 into MDCK cells by either adenoviral infection or transfection and isolation of cell clones.…”

“…It was shown previously that HGF-induced dissociation of MDCK epithelial cells is dependent on phosphatidylinositol 3-kinase (PI3K) activity (Potempa and Ridley, 1998). It was also suggested that whereas HGF-induced Ras activation increases Rac-GTP in a PI3K-dependent manner (Ridley et al, 1995;Scita et al, 1999;Royal et al, 2000;Zondag et al, 2000), RhoA might be activated through a PI3K-independent pathway (Zondag et al, 2000). In contrast to the latter report, we found that both RhoA ( Figure 7B) and Rac activation by HGF were strictly dependent on PI3K activity, being inhibited by treatment of the cultures with the PI3K inhibitor LY294002 (2 h, 20 M; Figure 7B).…”

“…Growth factors like HGF, EGF, and PDGF have been shown to stimulate Rac (Ridley et al, 1995;Scita et al, 1999;Royal et al, 2000;Zondag et al, 2000) and RhoA activities (Zondag et al, 2000) both in fibroblasts and epithelial cells. Accordingly, HGF stimulation of control MDCK cells resulted in a rapid and transient increase (between 2 and 10 min after addition of the growth factor) of Rac-GTP (our unpublished results).…”

From Tpr-Met to Met, tumorigenesis and tubes

“…The association of Gab1 with the SHP-2 phosphatase is necessary for the sustained activation of MAPK required for branching morphogenesis (Maroun et al, 2000;Schaeper et al, 2000;Lamorte et al, 2002b). Met also activates pathways that modulate the actin cytoskeleton, cell adhesion and migration, including Src, Rho, Rac and PAK (Rahimi et al, 1998;Royal et al, 2000). The activation of some of these pathways is mediated through the interaction between Gab1 and Crk, whereby Crk can couple Gab1 to Rap1 and Rac (Lamorte et al, 2002a) and is required for the breakdown of cell-cell junctions and cell dispersal, in addition to branching morphogenesis in response to HGF/SF (Lamorte et al, 2002b;Rodrigues et al, 2005).…”

Le récepteur Met fête ses 30 ans

“…Par exemple, l'activation des petites protéines G Ras, Rac et PAK (p21-activated kinase) contrôle le réarrangement du cytosquelette et la motilité [17]. La voie PI3K, via l'activation d'Akt (protéine kinase B), joue aussi un rôle central dans la survie cellulaire induite par Met [18], tout comme la régulation de p53 par Abl et p38 [19].…”