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K-Ras mediates cytokine-induced formation of E-cadherin-based adherens junctions during liver development

2002; Springer Nature; Volume: 21; Issue: 5 Linguagem: Inglês

10.1093/emboj/21.5.1021

1460-2075

Takaaki Matsui, Taisei Kinoshita, Yoshihiro Morikawa, Kazuo Tohya, Motoya Katsuki, Yoshiaki Ito, Akihide Kamiya, Atsushi Miyajima,

Protein Kinase Regulation and GTPase Signaling

Article1 March 2002free access K-Ras mediates cytokine-induced formation of E-cadherin-based adherens junctions during liver development Takaaki Matsui Takaaki Matsui Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan Search for more papers by this author Taisei Kinoshita Taisei Kinoshita Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan Search for more papers by this author Yoshihiro Morikawa Yoshihiro Morikawa Department of Anatomy and Neurobiology, Wakayama Medical School, Wakayama, 640-8155 Japan Search for more papers by this author Kazuo Tohya Kazuo Tohya Department of Anatomy, Kansai College of Oriental Medicine, 2-11-1 Wakaba, Kumatori-Cho, Sennan-Gun, Osaka, 590-0433 Japan Search for more papers by this author Motoya Katsuki Motoya Katsuki Institute of Medical Science, University of Tokyo, 4-6-1 Sirokanedai, Minato-ku, Tokyo, 118-8639 Japan Search for more papers by this author Yoshiaki Ito Yoshiaki Ito Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka-shi, Iwate, 020-8550 Japan Search for more papers by this author Akihide Kamiya Akihide Kamiya Kanagawa Academy of Science and Technology (KAST), Teikyo University Biotechnology Research Center, 907 Nogawa, Miyamae-ku, Kawasaki, 216-0001 Japan Search for more papers by this author Atsushi Miyajima Corresponding Author Atsushi Miyajima Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan Kanagawa Academy of Science and Technology (KAST), Teikyo University Biotechnology Research Center, 907 Nogawa, Miyamae-ku, Kawasaki, 216-0001 Japan Search for more papers by this author Takaaki Matsui Takaaki Matsui Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan Search for more papers by this author Taisei Kinoshita Taisei Kinoshita Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan Search for more papers by this author Yoshihiro Morikawa Yoshihiro Morikawa Department of Anatomy and Neurobiology, Wakayama Medical School, Wakayama, 640-8155 Japan Search for more papers by this author Kazuo Tohya Kazuo Tohya Department of Anatomy, Kansai College of Oriental Medicine, 2-11-1 Wakaba, Kumatori-Cho, Sennan-Gun, Osaka, 590-0433 Japan Search for more papers by this author Motoya Katsuki Motoya Katsuki Institute of Medical Science, University of Tokyo, 4-6-1 Sirokanedai, Minato-ku, Tokyo, 118-8639 Japan Search for more papers by this author Yoshiaki Ito Yoshiaki Ito Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka-shi, Iwate, 020-8550 Japan Search for more papers by this author Akihide Kamiya Akihide Kamiya Kanagawa Academy of Science and Technology (KAST), Teikyo University Biotechnology Research Center, 907 Nogawa, Miyamae-ku, Kawasaki, 216-0001 Japan Search for more papers by this author Atsushi Miyajima Corresponding Author Atsushi Miyajima Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan Kanagawa Academy of Science and Technology (KAST), Teikyo University Biotechnology Research Center, 907 Nogawa, Miyamae-ku, Kawasaki, 216-0001 Japan Search for more papers by this author Author Information Takaaki Matsui1, Taisei Kinoshita1, Yoshihiro Morikawa2, Kazuo Tohya3, Motoya Katsuki4, Yoshiaki Ito5, Akihide Kamiya6 and Atsushi Miyajima 1,6 1Institute of Molecular and Cellular Biosciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-0032 Japan 2Department of Anatomy and Neurobiology, Wakayama Medical School, Wakayama, 640-8155 Japan 3Department of Anatomy, Kansai College of Oriental Medicine, 2-11-1 Wakaba, Kumatori-Cho, Sennan-Gun, Osaka, 590-0433 Japan 4Institute of Medical Science, University of Tokyo, 4-6-1 Sirokanedai, Minato-ku, Tokyo, 118-8639 Japan 5Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka-shi, Iwate, 020-8550 Japan 6Kanagawa Academy of Science and Technology (KAST), Teikyo University Biotechnology Research Center, 907 Nogawa, Miyamae-ku, Kawasaki, 216-0001 Japan *Corresponding author. E-mail: mi[email protected] The EMBO Journal (2002)21:1021-1030https://doi.org/10.1093/emboj/21.5.1021 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The E-cadherin-based adherens junction (AJ) is essential for organogenesis of epithelial tissues including the liver, although the regulatory mechanism of AJ formation during development remains unknown. Using a primary culture system of fetal hepatocytes in which oncostatin M (OSM) induces differentiation, we show here that OSM induces AJ formation by altering the subcellular localization of AJ components including E-cadherin and catenins. By retroviral expression of dominant-negative forms of signaling molecules, Ras was shown to be required for the OSM-induced AJ formation. Fetal hepatocytes derived from K-Ras knockout (K-Ras−/−) mice failed to form AJs in response to OSM, whereas AJ formation was induced normally by OSM in mutant hepatocytes lacking both H-Ras and N-Ras. Moreover, the defective phenotype of K-Ras−/− hepatocytes was restored by expression of K-Ras, but not by H-Ras and N-Ras. Finally, pull-down assays using the Ras-binding domain of Raf1 demonstrated that OSM directly activates K-Ras in fetal hepatocytes. These results indicate that K-Ras specifically mediates cytokine signaling for formation of AJs during liver development. Introduction Organogenesis involves massive cell proliferation and a series of differentiation and maturation processes. While these processes are cell autonomous to some extent, they are influenced profoundly by extracellular signals such as soluble factors and membrane-bound proteins. These signals activate multiple intracellular signaling pathways and induce the expression of genes that affect cell proliferation and differentiation. A number of soluble factors including hormones and cytokines are known to play a crucial role in the development of various tissues. One example is the transforming growth factor-β (TGF-β) family, whose members are involved in the determination of cell fate during a very early stage of development (Conlon et al., 1994; Winnier et al., 1995). In contrast, direct cell–cell interaction, particularly homophilic adhesion, becomes more important during later stages of development, as cells start organizing the tissue architecture. At least three types of intercellular architecture connect adjacent cells; adherens junctions (AJs), tight junctions and desmosomes. AJs, predominant in epithelial tissues and neurons, are composed of cadherin and catenins (Nose et al., 1988; Takeichi, 1995; Drubin and Nelson, 1996; Gumbiner, 1996). Cadherin is a transmembrane protein responsible for Ca2+-dependent homophilic interaction through its extracellular domain (Nose et al., 1988; Takeichi, 1995). Since forced expression of E-cadherin, the epithelial prototype, in E-cadherin-negative cells such as fibroblasts results in cell aggregation, expression of a specific cadherin molecule is likely to be a key determinant of direct cell–cell interaction (Nagafuchi and Takeichi, 1988; Takeichi, 1988). The cytoplasmic domain of cadherin binds to β-catenin, which in turn associates with α-catenin. β-catenin acts as a bridge connecting cadherin to α-catenin, which is required for linkage between AJs and the actin cytoskeleton (Nagafuchi et al., 1994; Drubin and Nelson, 1996; Gumbiner, 1996). The association of the cadherin–catenin complex with the actin cytoskeleton is necessary for tight AJ structure (Nagafuchi et al., 1994; Takeichi, 1995). By using epithelial cell lines, evidence is accumulating that a number of intracellular molecules participate in the regulation of cell adhesion. The Rho family proteins, Rho, Rac and Cdc42, are known to regulate cell adhesion and cytoskeletal organization (Hall, 1998). Rho is involved in the association of AJ with the actin cytoskeleton through control of actin reorganization (Braga et al., 1997; Takaishi et al., 1997). On the other hand, Rac and Cdc42 regulate formation of the cadherin–catenin complex by acting on α- and β-catenins, and also modulate association of the complex with the actin cytoskeleton (Braga et al., 1997; Kuroda et al., 1997; Takaishi et al., 1997). The complex also associates with protein kinases and phosphatases that regulate phosphorylation of β-catenin (Kypta et al., 1996; Aicher et al., 1997; Ozawa and Kemler, 1998; Rosato et al., 1998; Roura et al., 1999), implicating these molecules in the regulation of AJ formation. However, many questions regarding AJ formation remain unexplored. A typical example is the formation of AJs during liver development (Stamatoglou et al., 1992). In adult liver, E-cadherin and β-catenin are expressed abundantly and localized at cell–cell junctions, indicating that E-cadherin-based AJs tightly connect adjacent hepatocytes. In contrast, despite the fact that fetal hepatocytes at late gestation express these proteins at a level comparable with adult hepatocytes, they are distributed diffusely throughout the cell membrane (Stamatoglou et al., 1992; Kamiya et al., 1999; our unpublished observations). These observations suggest that fetal hepatocytes are not connected tightly to each other by AJs and that there is a mechanism that regulates formation of AJs during liver development. However, it has not been studied how AJ formation is regulated developmentally and what extracellular and intracellular molecules are involved in this process. While established cell lines have been used for the study of AJ formation, they do not provide a model to study the developmental regulation of AJ formation. An in vitro system that recapitulates the developmental process is required to address such questions. Liver development proceeds through multiple stages and is influenced by extracellular signals. In mice, liver primodium appears at embryonic day 8–9 (E8–9) (Gualdi et al., 1996). At E11, the liver accepts hematopoietic cells originating from the yolk sac and the aorta–gonad–mesonephros (AGM) region and supports embryonic hematopoiesis until birth (Medvinsky et al., 1996; Mukouyama et al., 1998). After birth, the liver acquires many metabolic functions and starts forming the architecture of liver lobules. We previously showed that liver development is stimulated by oncostatin M (OSM), an interleukin-6 (IL-6) family cytokine (Kamiya et al., 1999). In fetal liver at mid-gestation, OSM is expressed in CD45+ hematopoietic cells, whereas the OSM receptor is expressed in fetal hepatocytes, suggesting that OSM is a paracrine factor in fetal liver (Kamiya et al., 1999). In a primary culture of fetal hepatocytes from E14.5 murine embryos, OSM at a physiological concentration in the presence of dexamethasone (Dex) promotes functional maturation of fetal hepatocytes to neonatal liver cells as evidenced by expression of various liver enzymes, glycogen accumulation, ammonia clearance and lipid accumulation (Kamiya et al., 1999; Kojima et al., 2000). These results suggest that this in vitro system mimics the process of in vivo liver development. OSM manifests its functions through the OSM receptor, which consists of the OSM-specific subunit (OSMRβ) (Lindberg et al., 1998; Tanaka et al., 1999) and gp130, the common signal transducer of IL-6 family cytokines (Hirano et al., 1997), and it activates multiple intracellular signaling molecules including STAT3, Ras, PI3K and MAPK in various cell types including fetal hepatocytes (Taga and Kishimoto, 1997; Ito et al., 2000). Our recent studies using a retrovirus-mediated gene transfer system demonstrated that STAT3 plays major roles in the expression of liver-specific genes as well as the accumulation of glycogen induced by OSM (Ito et al., 2000). In contrast, while Ras was shown to regulate expression of OSM-responsive genes negatively, its precise function in liver development remains unknown (Ito et al., 2000). Interestingly, we also noted that hepatic differentiation in response to OSM caused dramatic changes in the morphology of fetal hepatocytes (Kamiya et al., 1999). In particular, homophilic cell–cell contact became more apparent in a manner similar to that seen in differentiated hepatocytes. These observations indicate that OSM may also regulate cell adhesion(s) in fetal hepatocytes to organize cellular architecture. In this study, by taking advantages of our in vitro system that recapitulates hepatic differentiation, we investigated the extracellular and intracellular signaling mechanism of AJ formation during liver development. Here we show that Dex up-regulates protein levels of AJ components including E-cadherin and catenins, while OSM promotes their localization at cell–cell junctions of lateral membranes. Interestingly, OSM-triggered E-cadherin localization depends specifically on K-Ras, but not other Ras or Rho family proteins. Thus, our results not only demonstrate a novel biological function of K-Ras but also shed light on the regulatory mechanism of E-cadherin-based AJs during liver development. Results Morphological changes of fetal hepatocytes upon induction of differentiation As we demonstrated previously, stimulation of fetal hepatocytes with 10 ng/ml of OSM in the presence of 1 × 10−7 M Dex promotes their differentiation as evidenced by the induction of differentiation marker genes [tyrosine aminotransferase (TAT) and glucose-6-phosphatase (G6Pase)] for the neonatal liver and by the up-regulation of glycogenic activity (Kamiya et al., 1999; Ito et al., 2000). Besides these functional parameters of liver development, we also noted that OSM in combination with Dex induced marked morphological changes as compared with Dex alone (Figure 1A). In the presence of OSM and Dex (OSM/Dex), the cytosol was highly granulated and the nucleus was clear and round. Moreover, cell–cell contact became clearer in the OSM/Dex-stimulated cells, raising the interesting possibility that cell adhesion is strengthened by OSM and Dex. To test this possibility, we analyzed the ultrastructure of contact sites of cells treated with Dex or OSM/Dex (Figure 1B and C). When cells were cultured with Dex alone, no obvious adhesion structure was found between adjacent cells, even though a junctional zone was visible (Figure 1B). In contrast, an electron-dense structure, which resembles the morphology of AJs, was observed along with the apical part of lateral membranes in the cells cultured with OSM/Dex (Figure 1C), and desmosome-like structures were also observed in the junctional zone (Figure 1C, inset). In addition, a number of microvilli were found on the apical membranes (Figure 1C, lower panel) and there were glycogen and lipid granules in the cytoplasm (Figure 1C). These observations suggest that fetal hepatocytes treated with OSM/Dex develop a well-polarized epithelial morphology similar to that in mature hepatocytes and that OSM in combination with Dex modulates formation of cell adhesion(s) during the differentiation. Figure 1.Morphological changes induced by OSM. Fetal hepatocytes derived from murine liver at E14.5 were cultured for 6 days with Dex or OSM/Dex. (A) Phase contrast images of cultured hepatocytes. OSM/Dex-stimulated cells exhibited tight cell–cell contact, a dense cytoplasm and a clear and round nucleus. (B and C) Ultrastructure of fetal hepatocytes. Cells cultured with Dex (B) or OSM/Dex (C) were processed for electron microscopy. Upper panels: junction areas between adjacent cells. Arrows indicate junctional zones between adjacent cells. Markers shown are Lg, lipid granule; Gg, glycogen granule; If, intermediate filament. The insert in (C) shows desmosome-like structures that are indicated by arrows. Lower panels: apical membrane domain. Mv, microvilli. Download figure Download PowerPoint OSM and Dex regulate AJ formation in a distinct manner In order to elucidate the molecular basis for regulation of cell adhesion by these external signals, we examined the subcellular localization and expression of cell adhesion molecules by immunofluorescence and biochemical analyses. In the absence of OSM and Dex, E-cadherin was expressed weakly and distributed diffusely (Figure 2A). When cells were incubated with Dex alone, expression of E-cadherin was markedly increased, although the pattern of subcellular localization was not significantly altered. Interestingly, stimulation with both OSM and Dex dramatically changed the distribution of E-cadherin, inducing its localization at cell–cell junctions (Figure 2A). Moreover, a view of the x–z axis revealed that E-cadherin was concentrated in the apical parts of lateral membranes in the presence of OSM and Dex (Figure 2B, arrowhead). Such localization of E-cadherin at junctional zones in lateral membranes was also observed in cells treated with OSM alone, although the protein level of E-cadherin was very low (Figure 2A). These results suggest that Dex up-regulates the protein expression of E-cadherin, while OSM modulates its subcellular localization. In addition, immunostaining of β-catenin revealed that the protein was regulated similarly by Dex and OSM (Figure 2C and data not shown). As in the case of immunostaining, western blot analysis of total protein extracts demonstrated that Dex increased protein levels of E-cadherin and catenins (Figure 3A). Importantly, however, OSM did not change the protein levels regardless of whether the cells were stimulated with Dex or not (Figure 3A), indicating that OSM specifically mobilizes these proteins to cell–cell junctions without affecting their protein levels. Figure 2.OSM induces formation of adherens junctions, but not tight junctions. Fetal hepatocytes were cultured for 6 days without OSM/Dex (No factor) or with OSM, Dex or OSM/Dex. The localization of cell adhesion molecules was examined by immunofluorescence analysis. (A and B) Subcellular distribution of E-cadherin in cultured hepatocytes. Cells were fixed and stained with anti-E-cadherin antibody. (A) x–y views; (B) x–z views of the cells. Bars and arrows indicate the bottom and top of the cells, respectively. E-cadherin was highly concentrated at the apical parts of lateral membrane (arrowheads). (C and D) Intracellular localization of β-catenin and ZO-1. Cells were fixed and stained with antibodies against β-catenin (C) or ZO-1 (D). (E) Co-localization of E-cadherin and the actin cytoskeleton. Cells were stained with anti-E-cadherin antibody (green, left panels) and rhodamine–phalloidin (red, middle panels). Each image was overlaid (yellow, right panels). Upper panels: Dex-treated cells. Lower panels: OSM/Dex-treated cells. Scale bars: 10 μm. Download figure Download PowerPoint Figure 3.Expression of AJ components and subcellular localization of E-cadherin. Fetal hepatocytes were cultured for 6 days with stimulation as indicated in the figure. (A) Total protein levels of AJ components in fetal hepatocytes. Cell extracts from each culture were analyzed by western blotting using antibodies against E-cadherin, and α-, β- and γ-catenins. The location of each protein is indicated by an arrow. (B) Detergent solubility assay in cultured hepatocytes. Cell extracts were fractionated into soluble (S) and insoluble (I) fractions as described in Materials and methods. E-cadherin protein levels in each fraction were determined by western blotting using anti-E-cadherin antibody. The abundance of E-cadherin in each fraction was estimated by quantitative analysis using the NIH image program, and the ratios of [S] or [I] in [S + I] at each culture condition are shown. Download figure Download PowerPoint Since it was known that the formation of the tight AJ complex reduces the solubility of E-cadherin in a mild cell lysis condition (Angres et al., 1996; Potempa and Ridley, 1998), we next examined the solubility of E-cadherin in different culture conditions (Figure 3B). In the culture conditions of no factor and OSM alone, nearly 100% of E-cadherin was present in the soluble fraction obtained by 0.5% NP-40 extraction and no E-cadherin was found in the insoluble fraction. Stimulation of the cells with Dex alone resulted in an increase of total E-cadherin and the accumulation of E-cadherin in the insoluble fraction (17%), and OSM/Dex further increased the percentage of insoluble E-cadherin up to 32%. Consistent with the results of solubility assays, a small portion of the actin cytoskeleton stained by phalloidin was co-localized with E-cadherin even in Dex-stimulated cells. Importantly, OSM/Dex strongly induced co-localization of actin fibers with E-cadherin at cell–cell junctions (Figure 2E), suggesting that a tight AJ complex was formed at the junctional zones on stimulation with OSM/Dex. Since all these characteristics are a hallmark of the AJ structure, our results suggest that OSM and Dex promote formation of the AJ complex, at least partly, through the regulation of the subcellular localization and protein expression of AJ components, respectively. Finally, we analyzed the expression and localization of the ZO-1 protein, a key component of tight junctions (Stevenson et al., 1986), in different culture conditions (Figure 2D). As with the components of AJ, the protein level of ZO-1 was also up-regulated by Dex. However, ZO-1 was localized at cell junction sites even in the absence of OSM and Dex, and neither expression levels nor the distribution of ZO-1 was affected by OSM (Figure 2D). These results suggest that the formation of tight junctions is independent of OSM signaling and that in the presence of Dex alone hepatocytes are connected to each other by cell adhesion structures including tight junctions but not E-cadherin-based AJs. Ras is essential for OSM-induced E-cadherin localization at cell–cell junctions OSM activates STAT3 and Ras, two major signaling components of IL-6 family cytokines, in various cell types including fetal hepatocytes (Fukada et al., 1996; Taga and Kishimoto, 1997; Ito et al., 2000). To investigate the OSM signaling pathway that regulates AJ formation, we expressed mutants of STAT3 and Ras in fetal hepatocytes and examined their effects on OSM-induced E-cadherin localization. Since ΔSTAT3 is a dominant-negative mutant of STAT3 that lacks the C-terminal transactivation domain, western blotting with antibody against the N-terminal region of STAT3 verified the expression of ΔSTAT3 as a protein smaller than the endogenous STAT3 in fetal hepatocytes (Figure 4I). Expression of ΔSTAT3, however, had no effect on E-cadherin localization at cell–cell junctions in cells cultured with OSM/Dex (Figure 4C). In contrast, expression of RasN17, a dominant-negative form of Ras, completely blocked AJ formation in response to OSM, i.e. E-cadherin was distributed throughout the cell membrane even in the presence of OSM and Dex (Figure 4D and I). Likewise, the OSM-induced β-catenin localization was inhibited by RasN17, but not by ΔSTAT3 (data not shown). These results indicate that Ras is vital for OSM-triggered E-cadherin/β-catenin localization at cell–cell junctions in fetal hepatocytes. Figure 4.Effects of dominant-negative forms of signal molecules on OSM-triggered E-cadherin localization at cell–cell junctions. Fetal hepatocytes were infected on day 0 with the pMIG retroviral vector carrying no signaling molecule (A, B and E), ΔSTAT3 (C), RasN17 (D), RhoN19 (tagged with myc) (F), RacN17 (G) or Cdc42N17 (tagged with Flag) (H), and cultured for 6 days with Dex (A) or OSM/Dex (B–H). Viral infection was verified by green fluorescence of GFP (left panels). The localization of E-cadherin in each culture was examined by immunofluorescence analysis using anti-E-cadherin antibody (middle panels). Merged images are shown in the right hand panels. Scale bars: 10 μm. (I) Ectopic expression of dominant-negative molecules in fetal hepatocytes. Cell extracts from each culture were analyzed by western blotting using specific antibodies. ΔSTAT3 is detected as a smaller band than the endogenous STAT3. Expression of RasN17 and RacN17 was verified by augmented expression of Ras and Rac1, respectively (asterisk; non-specific band). Expression of RhoN19 (tagged with myc) and Cdc42N17 (tagged with Flag) was determined by immunoblotting using antibodies against myc and Flag, respectively. Because expression levels of endogenous STAT3 were comparable in each sample, STAT3 was used as an internal control. (J) Inhibitory effect of dominant-negative forms of the Rho family proteins on c-fos transcription in NIH-3T3 cells. To confirm whether retrovirus vectors carrying dominant-negative forms of the Rho family are functional, NIH-3T3 cells infected with these retrovirus vectors were transiently transfected with a reporter plasmid linked to the c-fos promoter and pRL plasmid and cultured with or without 2% serum for 24 h. Luciferase activity was determined using the Dual Luciferase Reporter Assay System. Download figure Download PowerPoint The Rho family proteins, Rho, Rac and Cdc42, are known to be necessary for regulation of the E-cadherin-mediated AJs in MDCK cells and keratinocytes, which constitutively express E-cadherin (Braga et al., 1997; Kuroda et al., 1997; Takaishi et al., 1997). To examine whether the Rho family proteins are required for OSM-triggered localization of E-cadherin, we constructed pMIG retrovirus vectors carrying dominant-negative forms of Rho, Rac and Cdc42 (RhoN19, RacN17 and Cdc42N17). We first examined their function in NIH-3T3 cells, in which c-fos transcription by serum stimulation is known to be regulated by the Rho family proteins (Alberts et al., 1998; Kim and Kim, 1998; Wang et al., 1998). Expression of these mutants suppressed c-fos induction, indicating that these mutants function as a dominant-negative molecule (Figure 4J). Although expression of these mutants in fetal hepatocytes was verified by western blotting (Figure 4I), none of these mutants had any effect on the OSM-induced E-cadherin localization (Figure 4E–H). These data suggest that the Rho family proteins are not actively involved in the OSM signaling for AJ formation in fetal hepatocytes. These results indicate that Ras is a major OSM-mediated intracellular signal for AJ formation. A novel role for K-Ras in OSM-triggered E-cadherin localization The classical Ras family consists of H-Ras, N-Ras and K-Ras. It was shown that knockout (KO) mice lacking both H-Ras and N-Ras develop normally and are fertile (Umanoff et al., 1995), whereas K-Ras KO mice die at around E15 and exhibit abnormality in various organs including the liver (Johnson et al., 1997; Koera et al., 1997). Moreover, K-Ras-deficient embryonic stem (ES) cells failed to contribute to the liver in chimeric mice generated from blastocysts injected with the ES cells (Johnson et al., 1997). These results indicate an indispensable role for K-Ras during liver development and led us to consider the possibility that K-Ras is involved specifically in OSM-mediated signaling in fetal hepatocytes. To test this possibility, we took advantage of KO mice lacking the K-Ras gene, and first examined the expression of α-fetoprotein (AFP) mRNA, a marker of immature hepatocytes (Shiojiri et al., 1991). K-Ras−/− liver at the E14.5 stage expressed AFP abundantly and the levels were comparable with wild-type and K-Ras+/− livers in the littermates (Figure 5A). These results suggest that there are cells with the characteristics of fetal hepatocytes at E14.5 in K-Ras−/− mice and that liver development before this stage may not be compromised. In the K-Ras−/− hepatocytes isolated from an individual liver at E14.5, stimulation with OSM/Dex induced mRNA expression of differentiation markers (TAT and G6Pase), and the levels of their expression were again comparable with those in wild-type cells (Figure 5B), indicating that K-Ras−/− fetal hepatocytes have a potential in response to OSM. Interestingly, however, K-Ras−/− fetal hepatocytes failed to mobilize E-cadherin to cell–cell junctions in response to OSM, while the protein level of E-cadherin was not altered in these cells (Figure 5C). In contrast, OSM did stimulate E-cadherin localization in H-Ras−/− KO cells (data not shown). Furthermore, no obvious perturbation of E-cadherin localization was found even in H-Ras−/− N-Ras−/− double KO cells (Figure 5C). To eliminate the possibility that these observations were unexpected consequences of gene disruption, we introduced the normal Ras alleles into E14.5 K-Ras−/− cells (Figure 6). E-cadherin distribution was diffuse even in K-Ras−/− hepatocytes treated with OSM/Dex (Figures 5C and 6A). In contrast, E-cadherin was clearly localized at cell–cell junctions in the cells transduced with the K-Ras allele, as shown by the arrows in Figure 6B. These data indicate that introduction of the K-Ras allele into K-Ras−/− fetal hepatocytes restored the normal distribution of E-cadherin in response to OSM. Moreover, expression of H-Ras only partially restored the response to OSM, and N-Ras had no effect at all (Figure 6C and D). These results indicate that K-Ras acts as a specific downstream mediator of OSM signaling in the regulation of E-cadherin localization in fetal hepatocytes. Figure 5.A critical role for K-Ras in liver development. (A) Expression of the early hepatic marker gene, α-fetoprotein (AFP). A 10 μg aliquot of total RNA extracted from wild-type, K-Ras+/− or K-Ras−/− fetal liver at E14.5 was loaded in each lane (rRNA; loading control). mRNA expression of AFP was determined by northe