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The Department of Molecular Biology and Biochemistry, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita, Osaka 565-0871, Japan

	Abstract

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In epithelial cells, tight junctions (TJs) and adherens junctions (AJs) form junctional complexes. At AJs, cadherins and nectins are the major cell-cell adhesion molecules. Nectins first form cell–cell adhesions and then recruit cadherins to the nectin-based cell–cell adhesion sites to form AJs in coordination with the activation of integrin _vß₃, followed by the formation of TJs. We previously demonstrated that when MDCK cells precultured at a low Ca²⁺ concentration were treated with the protein kinase C (PKC) activator 12-O-tetradecanoyl-phorbol-13-acetate (TPA), incomplete AJs and a TJ-like structure were achieved. However, it remains unknown how PKC is activated and how it regulates the formation of cell–cell junctions. When MDCK cells precultured at a low Ca²⁺ concentration were treated with TPA, incomplete AJs were formed without the activation of integrin _vß₃. Treatment of cells with TPA also enhanced the phosphorylation of FAK, which transmits the outside-in signal of integrin and plays a role in the nectin-induced formation of AJs. In addition, inhibition of PKC suppressed the formation of AJs. These results indicate that the activation of PKC functions downstream of integrin _vß₃ and upstream of FAK, and is important for the nectin-induced formation of AJs.

	Introduction

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The formation of cell–cell junctions is indispensable for normal tissue organization. Cell–cell junctions in epithelial cells are comprised of several specialized junctional apparatuses: mainly, tight junctions (TJs) and adherens junctions (AJs). TJs are localized at the most apical side of cell–cell junctions. Their two major functions are to act as a barrier, preventing the passage of soluble molecules through the gaps between cells, and as a fence, keeping the cell surface proteins and lipids in the basolateral region separate from those in the apical region (Tsukita et al. 1999; Tsukita & Furuse 2002). On the other hand, AJs play a role in mechanically connecting adjacent cells to resist strong contractile forces and to maintain tissue structures (Gumbiner 1996). It was illustrated that the establishment of AJs is also a prerequisite for the consequent formation of TJs (Gumbiner et al. 1988; Watabe-Uchida et al. 1998). However, several studies have shown that, under some conditions, the formation of TJs is not completely dependent on the formation and maintenance of AJs (Balda et al. 1993; Fukuhara et al. 2002b; Okamoto et al. 2005; Yamada et al. 2006; Capaldo & Macara 2007). Thus, the requirement of AJs for the formation of TJs is now controversial.

Cadherins and nectins are the two major types of cell–cell adhesion molecule (CAM) present at AJs. Cadherins are key Ca²⁺-dependent, single transmembrane CAMs constituting a family with over 80 members that are expressed in various kinds of cells (Takeichi 1991; Yagi & Takeichi 2000). Among the variety of cadherin family members, E-cadherin, which is expressed in epithelial cells, has been most extensively studied in the context of physical cell–cell adhesion and its trans-interaction-induced intracellular signaling in both physiological and pathological states. E-Cadherin binds directly to ß-catenin, which in turn interacts with -catenin, resulting in a linkage between E-cadherin and actin filaments (F-actin) that reinforces the cell–cell junctional connection. On the other hand, nectins are a group of recently discovered, Ca²⁺-independent immunoglobulin-like CAMs consisting of four members: nectin-1, nectin-2, nectin-3 and nectin-4 (Takai & Nakanishi 2003). Nectins are also associated with F-actin through afadin, a nectin- and F-actin-interacting molecule, and they regulate the actin cytoskeleton to form AJs.

Our recent studies using MDCK cells as a model epithelial cell line revealed that nectins play key roles in the initial step of AJ formation and the subsequent formation of TJs. In the process of forming AJs, the trans-interaction of nectins first occurs at the initial cell–cell contact sites and then induces the activation of Rap1, Cdc42 and Rac small G proteins (Ogita & Takai 2006). Cdc42 and Rac reorganize the actin cytoskeleton, which recruits the cadherin–catenin complex to the nectin-based cell–cell adhesion sites. At this time, the cell–cell adhesion activity of E-cadherin is weak, but activated Rap1, which interacts with afadin, enhances the adhesion activity of E-cadherin through the binding of p120^ctn, eventually leading to the formation of AJs by the trans-interaction of cadherin (Sato et al. 2006). In addition, nectins are also essential for the subsequent assembly of TJ components, including claudin, occludin and junctional adhesion molecule (JAM), at the apical side of AJs, to form TJs (Takai et al. 2003; Takai & Nakanishi 2003; Nakanishi & Takai 2004).

There is also cross-talk between cell–cell junctions and cell–matrix junctions (Pignatelli 1998; Siu & Cheng 2004). It has been shown that cell–cell junctions are positively or negatively regulated by integrin-mediated cell–matrix junctions (Monier-Gavelle & Duband 1997; Schreider et al. 2002). We recently found that integrin _vß₃ is physically associated with nectin-3 and is involved in nectin-induced signaling and the formation of AJs (Sakamoto et al. 2006). During the formation of AJs in NIH3T3 cells, the high-affinity form of integrin _vß₃ co-localizes with nectin-3 at the primordial cell–cell contact sites, and these two CAMs cooperatively induce the activation of c-Src, which is a key molecule for the formation of AJs. After the establishment of AJs, the high-affinity form of integrin _vß₃ is converted to the low-affinity form, which continues to co-localize with nectin-3. This supports the importance of cross-talk between cell–matrix and cell–cell adhesions in the formation of AJs.

It has been shown that the pharmacological activation of protein kinase C (PKC) by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in MDCK cells cultured at a low Ca²⁺ concentration induces the formation of incomplete AJs and a TJ-like structure (Balda et al. 1993; van Hengel et al. 1997; Asakura et al. 1999; Cowell & Garrod 1999; Fukuhara et al. 2002b; Okamoto et al. 2005). At incomplete AJs, non-trans-interacting, but not trans-interacting, E-cadherin and its associating catenins are recruited to the nectin-based cell–cell adhesion sites; thus, incomplete AJs exhibit weaker cell–cell adhesion than mature AJs that include trans-interacting E-cadherin (Okamoto et al. 2005). The existence of incomplete AJs was certified in our recent study showing that GFP-E-cadherin, which does not trans-interact under conditions of a low Ca²⁺ concentration but associates instead with - and ß-catenins and p120^ctn, was recruited to nectin-based cell–cell adhesion sites when MDCK cells precultured at a low Ca²⁺ concentration were treated with TPA (Okamoto et al. 2005). In addition, the rat monoclonal antibody (mAb) against E-cadherin (ECCD2), which recognizes trans-interacting E-cadherin, did not detect the signal for E-cadherin at the TPA-induced cell–cell adhesion sites of MDCK cells, but the mouse mAb (610182), which recognizes the cytoplasmic region of E-cadherin, could demonstrate the concentration of E-cadherin there. These results reinforce the recruitment of non-trans-interacting E-cadherin to the cell–cell adhesion sites in the presence of TPA at a low Ca²⁺ concentration. This also indicates that the trans-interaction of E-cadherin is not necessary for the formation of a TJ-like structure by the action of TPA. On the other hand, several studies have reported that integrins, such as integrin _IIbß₃ and integrin _vß₃, regulate the activation of PKC through its outside-in signaling (Buensuceso et al. 2005; Rucci et al. 2005), and that activation of PKC by phorbol esters induces activation of FAK (Lewis et al. 1996). Based on these observations, we examined in this study the role and mode of action of PKC in the formation of AJs.

	Results

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Transient activation of integrin _vß₃ during the formation of AJs in MDCK cells

We previously showed in a wound healing assay using NIH3T3 cells that the high-affinity form of integrin _vß₃ co-localizes with nectin-3 at the primordial cell–cell adhesion sites, and that this high-affinity form of integrin _vß₃ becomes converted into the low-affinity form after the establishment of AJs (Sakamoto et al. 2006). First, we examined whether this phenomenon is also observed in the formation of AJs in MDCK cells, using a Ca²⁺ switch assay. When MDCK cells were precultured at a low Ca²⁺ concentration (2 µM), the immunofluorescence signals for AJ and TJ constituents, except for afadin, were not observed at the spot-like cell–cell contact sites as described (Asakura et al. 1999; Fukuhara et al. 2002b; Katata et al. 2003). Because the signals for nectins were not detected at the cell–cell adhesion sites in MDCK cells probably due to the sensitivity of the Abs against canine nectins (Fukuhara et al. 2002a), we stained MDCK cells with an anti-afadin Ab instead of the anti-nectin Abs. Under these culture conditions, the signal for integrin _vß₃ as determined by the mAb LM609, which recognizes both the high- and low-affinity forms of integrin _vß₃, was broadly distributed along the cell periphery (Fig. 1A). By contrast, the signal for talin, which links integrin to the actin cytoskeleton and activates integrin through inside-out signaling (Tadokoro et al. 2003), was not detected anywhere, suggesting that integrin _vß₃ is the low-affinity form under these conditions.

View larger version (45K): [in this window] [in a new window]   Figure 1  Involvement of integrin vß3 activation in the formation of AJs. (A) Distribution of integrin vß3, but not talin, along the cell periphery in MDCK cells precultured in a low Ca2+ medium. MDCK cells were precultured at 2 µM Ca2+ for 3 h and then immunostained with the mAb LM609 against integrin vß3 or an anti-talin mAb, as well as an anti-afadin pAb. (B) Concentration of AJ components as well as integrin vß3 and talin at AJs in MDCK cells re-cultured at a normal Ca2+ concentration. MDCK cells were precultured at 2 µM Ca2+ for 3 h and re-cultured at 2 mM Ca2+ for 1 h. Cells were immunostained with the indicated Abs. (C) Disappearance of talin, but not integrin vß3, after the establishment of AJs in MDCK cells. MDCK cells were precultured at 2 µM Ca2+ for 3 h and re-cultured at 2 mM Ca2+ for 6 h. Cells were immunostained with the indicated Abs. Scale bars, 10 µm. The results are representative of three independent experiments.

Formation of incomplete AJs and a TJ-like structure by activation of PKC without activation of integrin _vß₃

It has been shown that activation of PKC induces the formation of incomplete AJs and a TJ-like structure in MDCK cells (Okamoto et al. 2005). To further explore the relationship between PKC and integrin in this process, MDCK cells were precultured in a low Ca²⁺ medium and treated with the PKC activator TPA, keeping the Ca²⁺ concentration low. At 1 h after treatment with TPA, the immunofluorescence signals for afadin and -catenin were concentrated at cell–cell adhesion sites as observed in MDCK cells re-cultured at a normal Ca²⁺ concentration, whereas the signal for E-cadherin determined by the mAb ECCD2 was not detected anywhere (Fig. 2 and data not shown). The signal for occludin, a TJ marker, was also concentrated at cell–cell adhesion sites as previously described (Okamoto et al. 2005) (data not shown). Although the signal for integrin _vß₃ was concentrated at cell–cell adhesion sites as observed in cells that had been re-cultured at a normal Ca²⁺ concentration, the signal for talin was hardly visible (Fig. 2), suggesting that integrin _vß₃ at these cell–cell adhesion sites is of the low-affinity form. These results provide the important information that activation of PKC is required to enable cells to form incomplete AJs and a TJ-like structure without the activation of integrin, and that PKC functions downstream of integrin during the formation of cell–cell junctions.

View larger version (38K): [in this window] [in a new window]   Figure 2  TPA-induced formation of incomplete AJs without integrin vß3 activation. MDCK cells precultured at 2 µM Ca2+ for 3 h were treated with 100 nM TPA for 1 h. Cells were then immunostained with the indicated Abs. Scale bars, 10 µm. The results are representative of three independent experiments.

We previously showed that the activation of FAK and c-Src tyrosine kinases play essential roles in the nectin-induced formation of AJs and that both nectin-induced signaling and integrin-induced, FAK-mediated signaling converge at the step of activation of c-Src during the formation of AJs (Fukuhara et al. 2004; Fukuyama et al. 2005; Kawakatsu et al. 2005; Sakamoto et al. 2006). Therefore, we examined whether FAK and c-Src are necessary for the formation of incomplete AJs initiated by the activation of PKC. To verify the involvement of FAK during the PKC-mediated formation of cell–cell adhesion, we generated FRNK-MDCK cells, which are MDCK cells that stably express HA-tagged FRNK. FRNK lacks the catalytic domain of FAK and functions as a negative regulator of this kinase (Parsons 2003). We confirmed that the formation of AJs induced by re-culturing cells at a normal Ca²⁺ concentration was impaired in FRNK-MDCK cells compared with wild-type MDCK cells (Fig. 3A). When wild-type MDCK and FRNK-MDCK cells were precultured in a low Ca²⁺ medium and treated with TPA, we observed the formation of fewer incomplete AJs in FRNK-MDCK cells than in wild-type MDCK cells (Fig. 3B). To exclude the clonal effects of these FRNK-MDCK cells, we produced several lines of FRNK-MDCK cells and confirmed that the similar results were obtained from at least three different lines of FRNK-MDCK cells (data not shown). Moreover, re-culturing MDCK cells at a normal Ca²⁺ concentration or treating the cells with TPA actually increased the amount of FAK phosphorylation (Fig. 3C). These results indicate that the activation of FAK by PKC is necessary for the PKC-induced formation of incomplete AJs.

View larger version (42K): [in this window] [in a new window]   Figure 3  Involvement of FAK in the formation of AJs. (A) Requirement of FAK activation in the formation of AJs in MDCK cells re-cultured at a normal Ca2+ concentration. Wild-type MDCK cells and MDCK cells stably transfected with HA-FRNK were precultured at 2 µM Ca2+ for 3 h and re-cultured at 2 mM Ca2+ for 1 h. Cells were immunostained with an anti--catenin pAb. (B) Requirement of FAK activation in the formation of incomplete AJs in MDCK cells treated with TPA at a low Ca2+ concentration. Wild-type MDCK cells and MDCK cells stably transfected with HA-FRNK were precultured at 2 µM Ca2+ for 3 h and further treated with 100 nM TPA for 1 h. Cells were immunostained with an anti--catenin pAb. (C) Activation of FAK in MDCK cells re-cultured at a normal Ca2+ concentration or treated with TPA while cultured in a low Ca2+ medium. MDCK cells precultured at 2 µM Ca2+ for 3 h (Low) were re-cultured at 2 mM Ca2+ (Normal Ca2+) or treated with 100 nM TPA (TPA) for the indicated periods of time. Cell lysates were immunoblotted with anti-phospho-specific FAK (pY397) and FAK mAbs. Scale bars in (A) and (B), 10 µm. The results are representative of three independent experiments.

View larger version (54K): [in this window] [in a new window]   Figure 4  Involvement of c-Src in the formation of AJs. (A) Requirement of the activation of c-Src in MDCK cells re-cultured at a normal Ca2+ concentration. MDCK cells precultured at 2 µM Ca2+ for 3 h were re-cultured at 2 mM Ca2+ in the presence of 10 µM PP2 or PP3 for 1 h. Cells were immunostained with an anti--catenin pAb. (B) Requirement of c-Src activation in MDCK cells treated with TPA at a low Ca2+ concentration. MDCK cells precultured at 2 µM Ca2+ for 3 h were treated with 100 nM TPA in the presence of 10 µM PP2 or PP3 for 1 h. Cells were immunostained with an anti--catenin pAb. (C) Rapid formation of AJs in c-Src-CA-MDCK cells. MDCK cells and c-Src-CA-MDCK cells were precultured at 2 µM Ca2+ for 3 h and treated with TPA for the indicated periods of time. Cells were immunostained with an anti--catenin pAb (red) and an anti-afadin mAb (green). Scale bars, 10 µm. The results are representative of three independent experiments.

Activation of PKC by integrin _vß₃ during the formation of AJs

We then sought to explore which isoform of PKC is activated and involved in the formation of AJs. Because TPA activates classical and novel PKC isoforms, we screened MDCK cells for the expression of different PKC isoforms and found abundant PKC expression in these cells (data not shown). Thereafter, we focused on the role of PKC in the formation of AJs. We examined whether this isoform is activated by integrin _vß₃ during the formation of cell–cell adhesion. Because PKC translocates from the cytoplasm to the plasma membrane to exert its kinase activity (Shirai & Saito 2002), the translocation of PKC was monitored during the formation of cell–cell adhesion. When MDCK cells were precultured in a low Ca²⁺ medium, a weak immunofluorescence signal for PKC was broadly distributed throughout the cytoplasm (Fig. 5A). Then, by re-culturing cells at a normal Ca²⁺ concentration, PKC moved to the plasma membrane, especially to cell–cell adhesion sites (Fig. 5A). This translocation was also observed following the treatment of cells with TPA. In addition, in the presence of the integrin _vß₃ inhibitor echistatin, the translocation of PKC to the plasma membrane induced by re-culturing cells at a normal Ca²⁺ concentration was suppressed, and the formation of AJs during the Ca²⁺ switch assay was inhibited (Fig. 5B). By contrast, the formation of cell–cell junctions in MDCK cells precultured in a low Ca²⁺ medium and treated with TPA was not impaired, even in the presence of echistatin. Furthermore, we found that a PKC inhibitor calphostin C blocked the translocation of PKC and, thus, suppressed the formation of cell–cell junctions in the Ca²⁺ switch assay (Fig. 5C). These results indicate that activation of PKC is induced by the activation of integrin _vß₃ and is necessary for the formation of AJs.

View larger version (89K): [in this window] [in a new window]   Figure 5  Integrin-induced activation of PKC and its involvement in the formation of AJs. (A) Activation of PKC in MDCK cells re-cultured at a normal Ca2+ concentration or treated with TPA at a low Ca2+ concentration. MDCK cells precultured at 2 µM Ca2+ for 3 h (Low Ca2+) were re-cultured at 2 mM Ca2+ (Normal Ca2+) or treated with 100 nM TPA (TPA) for 1 h. Cells in each condition were then immunostained with an anti-PKC pAb. (B) Inactivation of PKC by inhibition of integrin vß3 in MDCK cells re-cultured at a normal Ca2+ concentration. MDCK cells precultured at 2 µM Ca2+ for 3 h were re-cultured at 2 mM Ca2+ in the presence or absence of 370 nM echistatin for 1 h. Cells were immunostained with an anti-PKC pAb and an anti-afadin mAb. (C) Suppression of the formation of AJs by inhibition of PKC activation. MDCK cells precultured at 2 µM Ca2+ for 3 h were re-cultured at 2 mM Ca2+ in the presence or absence of 500 nM calphostin C for 1 h. Cells were immunostained with an anti-PKC pAb and an anti-afadin mAb. Scale bars, 10 µm. The results are representative of three independent experiments.

	Discussion

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We previously showed that when MDCK cells cultured in a low Ca²⁺ medium were treated with TPA, non-trans-interacting E-cadherin and its binding molecules, including - and ß-catenins and p120^ctn, are assembled at nectin-based cell–cell adhesion sites and thus, incomplete AJs are formed (Okamoto et al. 2005). In this assay, TJ constituents such as claudin, occludin and JAM are also recruited to cell–cell adhesion sites to form a TJ-like structure. Based on this result, we further examined in this study how TPA functions in the signaling cascade described above to achieve the formation of incomplete AJs. At a low Ca²⁺ concentration, the activation of integrin _vß₃ is inhibited; however, the addition of TPA induces the formation of incomplete AJs and a TJ-like structure without the activation of integrin _vß₃, indicating that PKC acts downstream of integrin _vß₃. Consistent with this, the treatment of cells with TPA in the presence of echistatin, an integrin _vß₃ inhibitor, also induces the formation of incomplete AJs in MDCK cells cultured in a low Ca²⁺ medium, although echistatin suppresses the formation of AJs in MDCK cells re-cultured at a normal Ca²⁺ concentration as previously described (Sakamoto et al. 2006). The inhibition of the integrin signal by echistatin results in the change in the cell shape and the attenuation of cell–matrix adhesion, such as focal adhesion. However, even in the presence of echistatin, the recruitment of the nectin-afadin complex to the cell–cell contact sites is not altered. Thus, it is likely that the cell attachment between the adjacent cells is maintained in the process of the formation of cell–cell junctions independently of the inhibition of integrin and that this inhibition is simply involved in the signal cross-talk, especially the nectin-induced signaling.

To precisely determine the position where PKC functions in the signaling cascade leading to formation of AJs, MDCK cells preincubated in a medium containing PP2 or MDCK cells stably expressing HA-FRNK (FRNK-MDCK cells) were treated with TPA during culture in a low Ca²⁺ medium. We found that the formation of incomplete AJs is impaired in MDCK cells preincubated with PP2 as well as in FRNK-MDCK cells. Furthermore, the treatment of MDCK cells with TPA increases the amount of FAK phosphorylation, an effect that is also observed in MDCK cells re-cultured at a normal Ca²⁺ concentration. Taken together, we conclude that PKC functions downstream of integrin _vß₃ and upstream of FAK in the formation of AJs (Fig. 6).

View larger version (22K): [in this window] [in a new window]   Figure 6  A schematic model for the involvement of PKC in the nectin- and integrin vß3-induced recruitment of E-cadherin in the formation of AJs. (A) Signaling pathways involved in the formation of AJs in MDCK cells precultured at a low Ca2+ concentration and re-cultured at a normal Ca2+ concentration. Small red circles in this figure indicate Ca2+ bound to E-cadherin and integrin vß3. (B) Signaling pathways involved in the formation of AJs in MDCK cells precultured at a low Ca2+ concentration and treated with TPA. Details are described in the Discussion section.

We assume that PKC, one of the isoforms in classical PKCs, is mainly involved in the Ca²⁺- and TPA-induced formation of AJs and incomplete AJs, respectively, because PKC is abundantly expressed in MDCK cells and is activated during the formation of AJs as seen by its translocation from the cytoplasm to the plasma membrane where cell–cell junctions occur. However, we cannot absolutely exclude the possibility that other PKC isoforms also participate in the formation of AJs. Among the three (classical, novel and atypical) classes of the PKC family, only classical PKCs are activated by both Ca²⁺ and phorbol esters. Novel PKCs are sensitive to phorbol esters but not Ca²⁺, and atypical PKCs are insensitive to both Ca²⁺ and phorbol esters. Thus, it would be reasonable to assume that activation of the PKC isoform is essential for the formation of AJs in MDCK cells re-cultured at a normal Ca²⁺ concentration and in the cells treated with TPA during culture in a low Ca²⁺ medium.

c-Src is a key molecule in the signaling pathway involved in the nectin-induced and integrin-dependent formation of AJs and TJs. Thus, we investigated the importance of c-Src activation in the formation of AJs. For this purpose, we generated c-Src-CA-MDCK cells, which stably express a constitutively active mutant of c-Src. When cultured in a low Ca²⁺ medium, these cells, unfortunately, did not form cell–cell junctions. However, these cells formed AJs after re-culturing at a normal Ca²⁺ concentration, or incomplete AJs after treatment of cells with TPA, more rapidly than wild-type MDCK cells formed these structures. These results indicate that the activation of c-Src is necessary, but not sufficient, for the formation of AJs. It also suggests that activated PKC induced by TPA has two roles: one is activating the FAK-c-Src signaling that leads to the activation of Cdc42 and Rac to recruit cadherin–catenin complexes to nectin-based cell–cell adhesion sites as shown previously (Fukuhara et al. 2003; Sakamoto et al. 2006; Sato et al. 2006) and in this study, and the other is facilitating the recruitment of the cadherin–catenin complex to nectin-based cell–cell adhesion sites in cooperation with unidentified molecules, as part of a signaling pathway not yet uncovered (Fig. 6). It is also unclear whether FAK has other unknown functions, as PKC does, and this will be difficult to determine because there is currently no useful constitutively active mutant of FAK.

Cadherins require Ca²⁺ to enhance their cell–cell adhesion activity and to induce their trans-interaction (Takeichi 1991). Thus, it is considered that the formation of trans-interacting cadherin-based AJs in the Ca²⁺ switch assay is mainly due to a Ca²⁺-dependent up-regulation of the adhesion activity of cadherins. Consistent with this, TPA-induced activation of PKC, which functions downstream of integrin _vß₃, at a low Ca²⁺ concentration enables E-cadherin and its binding catenins to assemble at nectin-based cell–cell adhesion sites in MDCK cells, but fails to increase the cell–cell adhesion activity of E-cadherin because of a lack of Ca²⁺, resulting in the formation of incomplete AJs with non-trans-interacting E-cadherin (Okamoto et al. 2005). However, given that like cadherins, integrins also require Ca²⁺ to become active and to form cell–matrix junctions (Leitinger et al. 2000), both cadherins and integrins are likely to play critical roles in the formation of AJs in a cooperative manner in the presence of Ca²⁺. This is supported by the finding that inhibition of integrin _vß₃ by echistatin apparently suppresses the formation of AJs in MDCK cells re-cultured in a normal Ca²⁺ medium.

In conclusion, we show for the first time the role and mode of action of PKC in the formation of AJs; namely, that it functions downstream of integrin and upstream of FAK. The activation of PKC is sufficient for the formation of cell–cell junctions even in the absence of integrin activation. To reveal the mechanism of AJ formation and the role of the molecules involved in this process will be quite important for the proper understanding of cellular functions in both physiological and pathological states. Thus, the findings of this study may provide new insight into the fields of medical science and cell biology.

	Experimental procedures

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Expression vectors

The mammalian expression vector for HA-tagged FRNK (COOH-terminal 396 amino acids of FAK; pCX4pur-HA-FRNK) was kindly provided by Dr T. Akagi (Osaka Bioscience Institute, Suita, Japan). The expression vector for a constitutively active form of c-Src (pUSE-Src(Y529F); c-Src-CA), in which the Tyr residue at position 529 is replaced with Phe, was purchased from Upstate.

Abs and chemicals

The mouse anti-afadin mAb and the rabbit anti-afadin polyclonal Ab (pAb) were prepared as described (Sakisaka et al. 1999). The rat anti-occludin mAb (MOC37) was purchased from Sanko Junyaku. Rabbit anti--catenin and rabbit anti-PKC pAbs were purchased from Sigma and Santa-Cruz, respectively. The mouse mAbs listed below were also purchased from commercial sources: anti-integrin _vß₃ (LM609) (Chemicon); anti-talin (8D4) (Sigma); anti-FAK (clone 77) (Pharmingen); and anti-phospho-FAK (Tyr³⁹⁷) (clone 18) (Pharmingen) Abs. Horseradish peroxidase-conjugated and fluorophore-labeled secondary Abs were obtained from Chemicon and Molecular Probes, respectively. TPA and echistatin were purchased from Sigma, and PP2, PP3 and calphostin C were from Calbiochem-Novabiochem.

Cell culture and transfection

MDCK cells kindly supplied by Dr W. Birchmeier (Max Delbrueck Center for Molecular Medicine, Berlin, Germany) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum. MDCK cells stably expressing FRNK (FRNK-MDCK cells) were generated by transfection of the pCX4pur-HA-FRNK vector into MDCK cells and selection by 5 µg/mL puromycin. Similarly, MDCK cells stably expressing c-Src-CA (c-Src-CA-MDCK) were produced by transfection of the pUSE-Src(Y529F) vector into MDCK cells and selection by 500 µg/mL of G418. For DNA transfection, a Nucleofection system (Amaxa) was used according to the manufacturer's instruction.

Ca²⁺ switch assay and immunofluorescence microscopy

Ca²⁺ switch experiments using MDCK cells were performed as previously described (Fukuhara et al. 2002b; Okamoto et al. 2005). In brief, cells (1 x 10⁵) were seeded on 18-mm glass coverslips in 12-well culture dishes. At 48 h after seeding, the culture medium was replaced with DMEM (2 mM Ca²⁺) without serum for 1 h. Cells were then precultured in DMEM with 5 mM EGTA (2 µM Ca²⁺) for 3 h. After this cultivation, cells were re-cultured in DMEM (2 mM Ca²⁺) for 1 h or treated with 100 nM TPA keeping the Ca²⁺ concentration in the culture medium low (2 µM Ca²⁺) for 1 h. For treatment with inhibitors, Calphostin C was added to the medium at 1 h before the end of the low Ca²⁺ precultivation, while the others were added at the end of the low Ca²⁺ precultivation. After these procedures, cells were fixed with a mixture of 50% acetone and 50% methanol on ice for 1 min or with 3.7% formaldehyde at room temperature for 15 min, and then permeabilized with 0.2% Triton X-100 at room temperature for 15 min. After blocking with 1% bovine serum albumin for 1 h, samples were incubated with the indicated primary Abs for 1 h and the fluorophore-labeled secondary Abs for 30 min, and then observed using an LSM 510 META confocal laser scanning microscope (Carl Zeiss).

Western blotting

Samples for Western blotting were prepared as follows: cells were washed twice with ice-cold PBS, lyzed in Laemmli buffer (Laemmli 1970) containing 1 mM Na₃VO₄, 10 mM NaF and a phosphatase inhibitor cocktail (Sigma), sonicated 3 times for 10 s with 20 s cooling periods, and boiled for 5 min. The protein concentrations of the samples were determined using an RC DC protein assay kit (Bio-Rad) with BSA as a reference protein. Samples were separated by SDS-PAGE, followed by Western blotting.

	Acknowledgements

 
We thank Drs T. Akagi and W. Birchmeier for providing us with the expression vectors for FRNK and MDCK cells, respectively. This work was supported by grants-in-aid for Scientific Research and for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (2006).

	Footnotes

 
Communicated by: Eisuke Nishida

^* Correspondence: E-mail: ytakai{at}molbio.med.osaka-u.ac.jp

	References

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Received: 30 January 2007
Accepted: 15 February 2007

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