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First published online July 23, 2007
doi: 10.1242/10.1242/jcs.003566

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PKC and cofilin activation affects peripheral actin reorganization and cell-cell contact in cells expressing integrin 5 but not its tailless mutant

¹ Cancer Research Institute, College of Medicine, Seoul National University, 28, Yeongeon-dong, Jongno-gu, Seoul 110-799, Korea
² Department of Tumor Biology, Seoul National University, 28, Yeongeon-dong, Jongno-gu, Seoul 110-799, Korea
³ Department of Molecular and Clinical Oncology, Seoul National University, 28, Yeongeon-dong, Jongno-gu, Seoul 110-799, Korea
⁴ Laboratory of Angiogenesis and Chemoprevention, CPMDRC, College of Oriental Medicine, Kyunghee University, 1 Hoegidong, Dongdaemugu, Seoul 131-701, Korea

^* Author for correspondence (e-mail: sungkim7{at}khu.ac.kr; jwl{at}snu.ac.kr)

Accepted 2 June 2007

	Summary

Top Summary Introduction Results Discussion Materials and Methods References

 
Integrin-mediated cell adhesion transduces signaling activities for actin reorganization, which is crucially involved in cellular function and architectural integrity. In this study, we explored the possibility of whether cell-cell contacts might be regulated via integrin-51-mediated actin reorganization. Ectopic expression of integrin 5 in integrin-5-null intestinal epithelial cells resulted in facilitated retraction, cell-cell contact loss, and wound healing depending on Src and PI3K (phosphoinositide 3-kinase) activities by a reagent that affects actin organization. However, cytoplasmic tailless integrin 5 (hereafter referred to as 5/1) expression caused no such effects but rather sustained peripheral actin fibers, regardless of Src and PI3K signaling activities. Furthermore, integrin 5 engagement with fibronectin phosphorylated Ser643 of PKC, upstream of FAK and Src and at a transmodulatory loop with PI3K/Akt. Pharmacological PKC inactivation, dominant-negative PKC adenovirus or inactive cofilin phosphatase (SSH1L mutant) retrovirus infection of 5-expressing cells sustained peripheral actin organization and blocked the actin reorganizing-mediated loss of cell-cell contacts. Meanwhile, wild-type PKC expression sensitized 5/1-expressing cells to the actin disruptor to induce cell scattering. Altogether, these observations indicate that integrin 5, but not 5/1, mediates PKC phosphorylation and cofilin dephosphorylation, which in turn modulate peripheral actin organization presumably leading to an efficient regulation of cell-cell contact and migration.

Key words: PKC, Cofilin, Actin organization, Cell contacts, Integrin

	Introduction

Top Summary Introduction Results Discussion Materials and Methods References

 
Epithelial monolayer integrity is crucial for the function and homeostasis of epithelia. The epithelial monolayer is maintained by cell-cell contacts between adjacent cells through homophilic interactions between adhesion molecules including E-cadherin and others, and cell adhesions via integrin engagements to ECM proteins within basement membranes (Thiery, 2003). Cell-cell contact (i.e. tight and adherens junctions) and cell adhesion sites (i.e. focal adhesions) are linked to intracellular actin filaments through diverse protein-protein interactions. Therefore, epithelial monolayer integrity and intracellular actin organization influence each other bi-directionally. That is, disruption of cell contacts and/or adhesions can cause alterations in actin organization, and conversely aberrant actin organization can cause changes in cell contacts and/or adhesions. Disruption of this monolayer integrity can cause not only the functional impairment of normal epithelium but also the dissemination of cancerous cells from a primary tumor cell body during the early stages of metastasis (Hirohashi and Kanai, 2003; Thiery, 2002). However, the mutual regulatory linkages between cell-cell contacts and cell adhesions are largely unknown, but mechanistic investigations on the regulatory linkages between cell-cell contacts and cell adhesions are being expanded (Boyer et al., 2000).

Integrins, a group of cell adhesion receptors, are composed of an and a subunit. The integrins participate in the activation of diverse intracellular signaling molecules and in the reorganization of actin filaments (Brakebusch and Fassler, 2003; Carragher et al., 2003; Juliano et al., 2004). This participation is accomplished through direct signaling via integrin-mediated engagements of cells to ECM proteins, and by indirect signaling in collaboration with other membrane receptors (e.g. growth factor receptors, G-protein-coupled receptors or cytokine receptors) (Bhowmick et al., 2001; Eliceiri, 2001; Lee et al., 2004; Short et al., 2000; Yamada and Even-Ram, 2002). Interaction between integrins and ECMs at focal adhesions causes the clustering of integrins and the recruitment of signaling molecules and actin filaments to integrin cytoplasmic tails, and leads to the activation of integrin-mediated intracellular signal transduction (Hynes, 2002). Diverse intracellular signaling molecules, including focal adhesion kinase (FAK), Src family kinase (SFK), Erk, Akt/protein kinase B (PKB) and Rho GTPase family members including RhoA, Rac1 and CDC42 can be activated by integrin-mediated cell adhesion (Gilcrease, 2007; Juliano, 2002). Rho GTPase-mediated reorganization of actin filaments can involve their downstream effector molecules, which include LIMK, cofilin, MLCK and ROCK that affect actin polymerization and myosin light chain (MLC) phosphorylation-mediated intracellular contractility (Schmitz et al., 2000). Cofilin is known to sever actin filaments when it is dephosphorylated (Galkin et al., 2003). Cofilin Ser3 is dephosphorylated by Slingshot phosphatase (SSH) or chronophin (Huang et al., 2006; Ohta et al., 2003). These important molecules have been shown to regulate morphological changes via actin reorganization in diverse cell types.

Meanwhile, cell-cell contacts in epithelium involve homophilic interactions between E-cadherin at adherence junctions, claudin or occludin at tight junctions, or desmocollin or desmoglein at desmosomes (Thiery, 2003). These adhesion molecules recruit adaptors or signaling molecules at their cytoplasmic tails, so that the protein complexes are formed for their connections to actin or intermediate filaments (Weis and Nelson, 2006). Cell-cell contact loss can be caused by dramatic actin reorganization even without E-cadherin suppression or downregulation, however, the mechanistic aspects of the process remain largely unknown. Integrins are known to reorganize actin filaments, as explained above, but the contribution made by integrin-ECM engagement to the regulation of cell-cell contacts is not well understood. It was previously shown that Fer tyrosine kinase and Rab1 are involved in cross-talk between cell-cell contacts and focal adhesions (Balzac et al., 2005; Retta et al., 2006).

In this study, we investigated the significance of integrin-signaling-mediated actin organization in regulation of cell-cell contacts. Normal rat intestinal epithelial cells ectopically expressing wild-type (WT) or tailless (i.e. lacking for the cytoplasmic tail of the C-terminal 27 amino acids, including GFFKR residues) integrin 5 were used to define the integrin signaling. We observed that 5-expressing cells dynamically showed loss of cell-cell contacts, when they were treated with an actin-affecting reagent, whereas 5/1-expressing cells sustained peripheral actin bundles, such that the reagent-mediated cell-cell contact loss was not observed. Furthermore, the inactivation of PKC or cofilin caused persistent peripheral actin organization, and thus blocked the cell-cell contact loss of 5-expressing cells. These observations indicate that a reagent-mediated actin-reorganization leads to loss of cell-cell contact, through integrin 5, PKC phosphorylation-dependent and cofilin dephosphorylation-dependent peripheral actin reorganization.

	Results

Top Summary Introduction Results Discussion Materials and Methods References

 
Actin-reorganization-mediated loss of cell-cell contacts in RIE1 cells expressing integrin 5 but not its tailless mutant
To study how integrin signaling may regulate cell-cell contacts, we utilized stable cell lines ectopically expressing wild-type (WT) or mutant integrin 5 (RIE1-5 or RIE1-5/1, respectively). The mutant integrin 5 (5/1) lacks the C-terminal 27 amino acids of the cytoplasmic tail, leaving one amino acid proximal to the transmembrane domain. RIE1-5 and RIE1-5/1 cells generally showed polygonal epithelial morphologies with cell-cell contacts (Fig. 1A). The comparable expressions of the intact or tailless 5 integrin subunit in these cells were confirmed by immunoblotting (using anti-integrin 5 antibody recognizing an extracellular region or the cytoplasmic tail; Fig. 1B). The surface expression of both these subunits was confirmed by FACS analysis (data not shown), as shown previously (Lee and Juliano, 2000). The stable cells also showed similar growth rates (i.e. doubling times of 18±2 hours). Since, (1) we failed to find any growth factors (including 100 ng/ml hepatocyte growth factor) or cytokines capable of causing RIE1-5 cells to scatter and (2) cell scattering involves cell-cell contact disruption, presumably via actin-reorganization-based morphological changes, we investigated if a reagent-mediated actin reorganization might cause cell-cell contact loss in cells with integrin 5 WT or its tailless mutant expression. A putative anti-tumorigenic reagent (Lee et al., 2007), 6-(1-oxobutyl)-5,8-dimethoxy-1,4-naphthoquinone (OXO; Fig. 1C), was used because we have found it effective as an actin-disrupting reagent (see below). When RIE1 cells were treated with OXO at 10 µM, cells grew well with no significant change in doubling time (data not shown). However, treatment of RIE1-5 cells with OXO (at 10 µM for 24 hours) caused loss of -catenin and ZO1 from cell-cell contact sites, although this did not occur in RIE1-5/1 cells (Fig. 1D,E). These observations indicate that OXO treatment caused a loss of cell-cell contacts in RIE1 cells with intact integrin 5, but not those with tailless integrin 5. Unfortunately, we were not able to immunostain for E-cadherin, despite using three different commercial anti-E-cadherin antibodies and diverse protocols including different fixation methods, probably because endogenous E-cadherin levels were not high enough to be sensitized by the antibodies during the immunofluorescent staining approaches. However, its expression was detected by immunoblotting and found to be unchanged by OXO (data not shown).

View larger version (89K): [in this window] [in a new window]   Fig. 1. Cell-cell contact loss of RIE1-5, but not RIE1-5/1, cells after OXO treatment. (A) Stable cell lines ectopically expressing integrin 5 (RIE1-5) or tailless 5 (RIE1-5/1) were maintained, as described in Materials and Methods. Images for subconfluent cells in normal culture media were taken using a microscope equipped with a digital camera. Both cell lines generally form colonies. (B) Whole cell lysates from subconfluent cells were prepared for immunoblotting using an antibody against an extracellular region (5exo) or the cytoplasmic tail (5cyto) of the human integrin 5 subunit, or -tubulin. Data shown represent at least three independent experiments. (C) Chemical structure of 6-(1-oxobutyl)-5,8-dimethoxy-1,4-naphthoquinone (OXO). (D,E) Cells were seeded onto 10% FBS-DMEM-H-coated glass coverslips. Once confluent monolayers had been formed by incubation in 5% CO2 at 37°C, cells were treated with either vehicle (DMSO) or OXO (10 µM) for 24 hours, prior to being immunostained for ZO1 (D) or -catenin (E). Data shown are representative of three different experiments.

We next examined whether OXO treatment induced -smooth muscle actin (SMA, a marker for mesenchymal cell types) in RIE1-5, but not in RIE1-5/1, cells. As was expected, SMA expression was enhanced only in RIE1-5 cells by OXO treatment (Fig. 2A), indicating that OXO-mediated EMT was integrin 5 dependent. The activities of signaling molecules in cells left untreated or treated with OXO were then examined to explore the molecular basis underlying OXO-mediated EMT in RIE1-5, but not in RIE1-5/1, cells. Whereas RIE1-5 cells showed enhanced activities of FAK, SFK and Akt/PKB after OXO treatment, RIE1-5/1 cells did not (Fig. 2B). However, Erk1/2 activity was increased by OXO in both cell lines, which is consistent with the comparable growth rates of these cell lines, indicating a certain specificity of integrin signaling impairment in RIE1-5/1 cells (Fig. 2B).

View larger version (38K): [in this window] [in a new window]   Fig. 2. Signal activity specifically impaired in RIE1-5/1 cells by OXO treatment. (A,B) Subconfluent (7080% confluent) cells in 60 mm culture dishes were treated with either DMSO or 10 µM OXO for 24 hours. Protein amounts in whole cell lysates were normalized before standard western blotting for the indicated molecules. Data shown are representative of three independent experiments.

Since SFK and phosphoinositide 3-kinase (PI3K)/Akt are known to be important for EMT (Avizienyte and Frame, 2005), and their phosphorylations were increased by OXO treatment that also caused loss of cell-cell contacts (Fig. 2B), we next performed pharmacological inhibitor studies to determine if SFK or PI3K/Akt activity was involved in OXO-mediated EMT in RIE1-5 cells. Cells on coverslips precoated with normal culture medium were pretreated with DMSO, LY294002 (a specific PI3K inhibitor which consequently causes Akt/PKB inhibition), PP2 (a specific SFK inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine), or PP3 (a negative control for PP2, 4-amino-7-phenylpyrazol[3,4-d]pyrimidine), 30 minutes before 10 µM OXO treatment for 24 hours. Cell-cell contacts were then analyzed by immunostaining of ZO1 at contact sites. OXO-mediated RIE1-5 cell-cell contact loss was abolished by the pharmacological inhibition of SFK or PI3K/Akt, but not by PP3 treatment, whereas RIE1-5/1 cell-cell contact was maintained regardless of the reagent treatments (Fig. 3).

View larger version (64K): [in this window] [in a new window]   Fig. 3. Inhibition of PI3K and SFK blocked OXO-mediated scattering of RIE1-5 cells, but did not affect cell-cell contacts between RIE1-5/1 cells. Cells were seeded onto 10% FBS-DMEM-H-coated glass coverslips. Once cells formed monolayers, they were not pretreated or pretreated with LY294002 (LY, 20 µM), PP2 (10 µM), or PP3 (10 µM), 30 minutes before DMSO or 10 µM OXO treatment. Cells were processed for immunostaining against ZO1, 24 hours later, as described in Materials and Methods. Data shown are representative of three independent experiments.

Persistent actin organization at RIE1-5/1 cell peripheries

View larger version (87K): [in this window] [in a new window]   Fig. 4. Differential cofilin phosphorylation and peripheral actin reorganization in RIE1-5 and RIE1-5/1 cells. (A) Cells were seeded onto 10% FBS-DMEM-H-coated glass coverslips. After cells had adhered and spread typically, they were treated with either DMSO or 30 µM OXO for 30 minutes. Cells were then processed for actin staining using phalloidin-conjugated with Rhodamine, as described in Materials and Methods. Note that 30 µM OXO treatment disrupted actin filament organization only in RIE1-5 cells. (B) Cells on normal culture medium-coated coverslips were incubated with 20 µM DCHF-PBS for 30 minutes, prior to washing and visualization of ROS-positive cells by fluorescent microscopy. (C,D) Cells (at 7080% confluence) in 60 mm culture dishes were not pretreated or pretreated with the indicated pharmacological inhibitors, as in Fig. 3, prior to treatment with DMSO or 10 µM OXO for 24 hours (C) or with DMSO (i.e. 0 µM of cytochalasin D) or cytochalasin D at the indicated concentrations for 1 hour (D). Whole cell lysates were then prepared and normalized protein amounts were used in standard western blotting for the indicated molecules. Note that RIE1-5/1 cells showed more sustained pS3cofilin levels than RIE1-5 cells did, even after treatment with cytochalasin D. (E) Cells on normal culture medium-coated coverslips were treated with DMSO or cytochalasin D (0.05 or 0.2 µM) for 1 hour, prior to actin staining as in A. (F) Cells were manipulated as described in the legend of Fig. 3, with the exception of staining for actin using phalloidin-conjugated with Rhodamine. Data shown are representative of three independent experiments.

PKC inactivation sustained peripheral actin organization and blocked OXO-mediated cell-cell contact loss in RIE1-5 cells
It is possible that persistent peripheral actin organization in RIE1-5/1 cells might protect cell-cell contacts from OXO treatment. We next tried to identify signaling molecules that supported peripheral actin organization by using pharmacological reagents. Among the reagents tested, we found that treatment with rottlerin, a specific PKC inhibitor, enhanced peripheral actin organization in RIE1-5 cells (Fig. 5A). Therefore, we pretreated RIE1-5 cells with rottlerin prior to OXO treatment, and then examined -catenin localization at cell-cell contact sites. Rottlerin pretreatment of RIE1-5 cells did maintain cell-cell contacts even after OXO treatment (Fig. 5B). To validate the role of PKC in the blockade of OXO-mediated cell-cell contact loss, RIE1-5 cells were infected with adenovirus for dominant negative (DN) K376A PKC (Hirai et al., 1994) for 8 hours. Infected cells were then treated with DMSO or OXO for 24 hours prior to immunofluorescent staining. ZO1 or -catenin immunostaining showed that the expression of DN PKC in RIE1-5 cells abolished their OXO-mediated losses at cell-cell contact sites (Fig. 5C), consistent with the finding of the rottlerin inhibitor study. Next, we conversely examined if activation of PKC in RIE1-5/1 cells facilitated cell-cell contact loss by OXO treatment. Infection of RIE1-5/1 cells with adenovirus for WT PKC [overexpression of which enhances pS⁶⁴³PKC (Lee et al., 2005)] caused cell-cell contact loss after OXO treatment (Fig. 5D). However, WT PKC overexpression alone (without OXO treatment) did not cause loss of contact between RIE1-5/1 cells, presumably indicating that OXO treatment involves the activations of other signaling pathways or molecules, such as cortactin, MLC and/or Fer (see Discussion) in addition to PKC. It has previously been reported that A375-SM melanoma cell adhesion via integrin 51 engagement to fibronectin requires activation of PKC, but not of PKC (Mostafavi-Pour et al., 2003). Therefore, we examined if PKC was also involved in the OXO-mediated loss of cell-cell contact between RIE1-5 cells. Unlike PKC, infection of RIE1-5 or RIE1-5/1 cells with DN or WT PKC adenovirus, respectively, did not affect OXO-mediated effects on ZO1 localization (Fig. 5C,D, bottom panels), indicating that PKC is not involved in the OXO-induced loss of contacts between RIE1-5 cells. Furthermore, when actin organization was examined, the infection of RIE1-5 cells with DN PKC adenovirus was found to sustain peripheral actin organization even after OXO treatment (Fig. 5E, left). However, in the case of RIE1-5/1 cells, infection with WT PKC adenovirus failed to sustain peripheral actin organization after OXO treatment (Fig. 5E, right). Meanwhile, the phosphorylation of PKC Ser643 but not of Thr505 (i.e. pS⁶⁴³PKC and pT⁵⁰⁵PKC, respectively) appeared to be correlated with OXO-mediated cell-cell contact loss in RIE1-5 cells. In other words, pS⁶⁴³PKC was enhanced or remained unchanged when RIE1-5 or RIE1-5/1 cells were treated with OXO, respectively, although its basal level was marginally higher in RIE1-5/1 cells (Fig. 5F, lanes 1, 2, 4, and 5). Furthermore, DN PKC expression in RIE1-5 cells reduced OXO-mediated pS⁶⁴³PKC level, whereas WT PKC expression in RIE1-5/1 cells increased, as was expected (Fig. 5F, lanes 2, 3, 5, and 6). In addition, OXO-enhanced SMA expression level in RIE1-5 cells was decreased by DN PKC pre-infection (Fig. 5F, bottom, lanes 1, 2 and 3), whereas SMA level, unchanged by OXO treatment of RIE1-5/1, was increased by WT PKC expression (Fig. 5F, bottom, lanes 4, 5 and 6). pS⁶⁴³PKC-dependent cell-cell contact loss by OXO treatment appeared to be specific for RIE1-5 cells, since the pre-infection of RIE1 cells overexpressing integrin 4 (RIE1-4) with DN PKC adenovirus did not block OXO-induced cell contact loss (Fig. 5G).

View larger version (81K): [in this window] [in a new window]   Fig. 5. OXO-mediated cell-cell contact loss of RIE1-5 cells involves pS643PKC-dependent dynamic peripheral actin reorganization. (A,B) RIE1-5 cells (at 7080% confluence) on 10% FBS-DMEM-H-coated coverslips were treated with DMSO or 10 µM rottlerin for 24 hours (A), or with DMSO, 10 µM OXO alone or OXO plus rottlerin (10 µM OXO + 10 µM rottlerin) for 24 hours (B). Cells were then stained for actin (A) or immunostained for -catenin (B), as explained in Materials and Methods. (C-E) Cells were infected with adenovirus for GFP (Ad-cont), dominant negative K376A PKC (Ad-DN PKC) or K368R PKC (Ad-DN PKC), or wild-type PKC (Ad-WT PKC) or PKC (Ad-WT PKC) for 8 hours. After infection, media were replaced with fresh culture media. DMSO or 10 µM OXO was then treated for 24 hours, prior to immunofluorescent staining against ZO1 (left panels) or -catenin (right panels; C,D), or stained for actin using phalloidin-conjugated Rhodamine (E). (F) Cells in 60 mm culture dishes were infected with adenovirus for GFP (Con), K376A DN PKC (DN), or WT PKC (WT), for 8 hours. After the viruses had been washed out, cells were treated with DMSO or 10 µM OXO for 24 hours, before preparation of whole cell lysates. An equal amount of proteins was subjected to standard western blotting for the indicated molecules. (G) RIE1-4 cells were manipulated as in (C) prior to ZO1 immunofluorescent staining. Data shown are representative of three independent experiments.

Peripheral actin organization via cofilin phosphorylation
The phosphorylation of cofilin Ser3 (i.e. pS³cofilin) was also affected by PKC activity in RIE1 cells, and changes in pS³cofilin were found to be correlated with patterns of peripheral actin organization, as shown in Fig. 5E. The overexpression of DN PKC in RIE1-5 cells abolished OXO-mediated decrease in pS³cofilin, but the overexpression of WT PKC, and thereby the enhancement of pS⁶⁴³PKC, in RIE1-5/1 cells (Lee et al., 2005) decreased pS³cofilin upon OXO treatment, although pS³cofilin level in OXO-treated RIE1-5/1 was higher than that in OXO-treated RIE1-5 cells (Fig. 6A). Basal pS³cofilin level of RIE1-5/1 was also higher than that of RIE1-5 cells. Cofilin Ser3 is dephosphorylated by Slingshot phosphatase (SSH) thus activating it for actin filament severing (Huang et al., 2006; Ohta et al., 2003), and pS³cofilin can be increased by LIMK, ROCK (Schmitz et al., 2000) or PTEN (phosphatase and tensin homologue deleted in chromosome 10) protein level or activity (Nishita et al., 2004). Interestingly, however, SSH1L mRNA and LIMK1, ROCK1 and PTEN protein levels were similar in RIE1-5 and RIE1-5/1 cells (data not shown). Furthermore, other stable RIE1-5/1 cell lines showed consistently and generally higher pS³cofilin levels than other stable RIE1-5 cell lines (data not shown). Being consistent with the observations in Fig. 4C-E, these findings indicate that impaired integrin 5 signaling may somehow lead to a well polymerized actin status, probably via activated LIMK1 (see below) or other molecules. Nonetheless, it is possible that a reduction in pS³cofilin by OXO treatment in RIE1-5 cells, as shown in Fig. 4C, may lead to dynamic retractile actin organization.

View larger version (86K): [in this window] [in a new window]   Fig. 6. Dynamic reorganization of peripheral actin filament and cell-cell contact loss of RIE1-5 cells depend on PKC-dependent cofilin dephosphorylation after OXO treatment. (A-C) Cells in 60 mm culture dishes were infected with adenovirus for GFP (Cont), K376A DN PKC (DN), or WT PKC (WT), for 8 hours (A). Cells were infected or transiently transfected with adenovirus for K368R DN or WT PKC (B) or K437R or WT PKC-HA (C), respectively. (D-I) Cells on 10% FBS-DMEM-H-coated glass coverslips (D,E) or in 60 mm culture dishes (F,G) were infected with retrovirus for control empty vector (Rt-cont), inactive hSSH1L-CS mutant (Rt-hSSH1L-CS mutant), or wild-type hSSH1L (Rt-hSSH1L WT) for 24 hours. RIE1-5 cells were transiently transfected with control (Mock), (HA)3-Akt WT (H), LIMK1, or ROCK1 (I) plasmids, 24 hours before OXO treatment. After 24 hours without or with OXO treatment, cell lysates were prepared. An equal amount of proteins was used in standard western blotting for the indicated molecules (A,B,C,F,G,H,I). Cells on coverslips were stained for actin (D,E) or immunostained for cofilin or pS3cofilin (J). Data shown are representative of three independent experiments.

In a manner consistent with no OXO-induced cell scattering via PKC, as shown in Fig. 5C, PKC activity regulation did not further affect OXO-mediated pS³cofilin (Fig. 6B). In addition, PKC K437R dominant mutant or WT overexpression in RIE1-5 or RIE1-5/1 cells, respectively, did not further alter OXO-mediated pS³cofilin levels, indicating that PKC is not involved in the OXO-mediated effects on RIE1 cells (Fig. 6C).

To determine if indeed peripheral actin organization was regulated by cofilin phosphorylation status, RIE1-5 cells were infected with retrovirus for control empty vector or an inactive human cofilin phosphatase CS mutant, hSSH1L-CS, treated with DMSO or 10 µM OXO, and then stained for actin. Whereas control retrovirus-infected RIE1-5 cells showed morphological retraction and peripheral actin disruption after OXO treatment, hSSH1L-CS retrovirus-infected cells showed persistent peripheral actin bundles and no retraction even after OXO treatment (Fig. 6D). However, RIE1-5/1 cells infected with hSSH1L WT retrovirus showed morphological retraction via loss of peripheral actin organization even without OXO treatment (Fig. 6E). As was expected, the expression of hSSH1L CS mutant or WT forms increased or decreased pS³cofilin levels of RIE1-5 or RIE1-5/1 cells, respectively, compared to the basal level of each cell line (Fig. 6F). The effects of hSSH1L CS mutant or WT on cell-cell contacts, as shown in Fig. 6D,E, were found to be correlated with changes in SMA levels (Fig. 6G, left or right, respectively). Therefore, the disruption of peripheral actin organization in RIE1-5 cells by OXO treatment appeared to involve dephosphorylated, and thus active, cofilin, during OXO-mediated cell-cell contact loss.

It was previously reported that PI3K activity mediates SSH1L activation and pS⁴⁷³Akt colocalizes with cofilin but not with pS³cofilin, during the insulin-induced membrane protrusion of mammary MCF7 cells (Nishita et al., 2004). Therefore, we examined whether Akt was correlated with OXO-mediated decrease in pS³cofilin level of RIE1-5 cells. Overexpression of Akt WT reduced the basal level of pS³cofilin, which was further downregulated by OXO treatment (Fig. 6H). This suggests that OXO-enhanced PI3K/Akt activity is linked to SSH1L activation for less pS³cofilin in RIE1-5 cells. In addition, transient transfection experiments to reveal the upstream effector(s) of cofilin showed that LIMK1, but not ROCK1, overexpression increased basal pS³cofilin in RIE1-5 cells, which was inhibited by OXO treatment (Fig. 6I). This indicates that LIMK1 is upstream of cofilin when RIE1-5 cells are treated with OXO. It was also previously reported that a complex formation between SSH1L and LIMK1 inactivates LIMK1 and thus potentiates the cofilin dephosphorylation (Soosairajah et al., 2005), suggesting that both LIMK1 and SSH1L are upstream of cofilin during the OXO-mediated (peripheral) actin reorganization in RIE1-5 cells. Meanwhile, OXO-mediated pS³cofilin of DN PKC-infected RIE1-5 cells was similar or slightly lower than that of WT PKC-infected RIE1-5/1 cells, as shown in Fig. 6A. The former cells blocked OXO-mediated contact loss, but the latter cells lost contacts after OXO treatment. Therefore, it is also likely that the spatial regulation of pS³cofilin is required for peripheral actin regulation and cell-cell contact maintenance in the presence of OXO. To test it, we examined if OXO treatment regulated the spatial localizations of cofilin and pS³cofilin. When RIE1-5 cells were treated with OXO, certain nonphosphorylated cofilin was located at cell peripheries, but pS³cofilin was not (Fig. 6J, left and middle). Meanwhile, OXO-treated RIE1-5/1 cells still showed pS³cofilin at cell peripheries, but pS³cofilin was no longer observed at peripheries when cells were pre-infected with WT PKC adenovirus (Fig. 6J, right). This indicates that the spatial localization of pS³cofilin is important in the PKC-dependent OXO effects on contact loss of RIE1-5 cells.

PKC mediates signaling from integrin 5 to focal adhesion molecules
The roles of PKC in adhesion-mediated signal transduction to FAK have been previously described (Vuori and Ruoslahti, 1993), although no involvement of PKC in adhesion-mediated FAK activation has been shown (Miranti et al., 1999). Therefore, we investigated how pS⁶⁴³PKC was linked with focal adhesion molecules (e.g. FAK and SFK) and PI3K/Akt during integrin 5-dependent cell-cell contact loss after OXO treatment. We examined whether extracellular liganding of integrin 5, via replating cells on fibronectin (Fn), could enhance pS⁶⁴³PKC. RIE1-5 replated on Fn showed increased pS⁶⁴³PKC, but not pT⁵⁰⁵PKC, compared to that of suspended cells. However, RIE1-5/1 cells did not show any significant increase in pS⁶⁴³PKC and pT⁵⁰⁵PKC (Fig. 7A). Intact integrin 5-dependent pS⁶⁴³PKC increase appeared to correlate with complex formation between Ser643-phoshorylated PKC and intact integrin 5, but not tailless 5, although this complex was observed at a low level (<5% of input) and reverse coimmunoprecipitation (i.e. 5 coimmunoprecipitated by anti-Ser643-phoshorylated PKC) failed in our protocols (Fig. 7B and data not shown). Thus, the integrin 5 cytoplasmic tail might be important for complex formation and thus for PKC Ser643 phosphorylation/activation after OXO treatment. Since no conclusive evidence indicates that PKC Ser643 phosphorylation is crucial for its kinase activity (Li et al., 1997; Stempka et al., 1999), we examined both pS⁶⁴³PKC and PKC activity without or with OXO. When immunoprecipitated PKC was incubated with PKC-depleted extracts of RIE1-5 cells in the absence or presence of OXO to determine whether OXO itself affected PKC phosphorylation at Ser643, the pS⁶⁴³PKC level of the immunoprecipitates was found to be increased by the extracts, but not further changed by OXO (Fig. 7C, upper). In addition, in vitro PKC assay using recombinant PKC and peptide substrate showed that OXO did not affect PKC enzyme activity (Fig. 7C, bottom), indicating that the OXO effects on PKC are indirect.

View larger version (52K): [in this window] [in a new window]   Fig. 7. PKC mediates signaling from integrin 5 to focal adhesion molecules. (A) Cells were trypsinized, collected, suspended into DMEM-H-1% BSA, and rotated (60 rpm) at 37°C for 45 minutes. Then half of cells was kept in suspension and the other half was replated onto fibronectin-coated (10 µg/ml) 60 mm dishes for 2 hours. After incubation, whole cell lysates were prepared for immunoblots for the indicated molecules. (B) Whole cell extracts from the cells without or with OXO treatment were prepared and immunoprecipitated with anti-integrin 5 (clone P1D6). The immunoprecipitates and lysates were immunoblotted for pS643PKC or PKC. (C) Whole cell extracts from RIE1-5 cells were immunoprecipitated with anti-PKC. The PKC immunoprecipitates were incubated in a reaction buffer (25 mM Tris, pH 7.5, 10 mM MgCl2, 50 µM ATP and 1 mM DTT) without or with the PKC-depleted extracts (Extracts*; 10 µg protein/reaction) in the absence or presence of 10 µM OXO for 30 minutes at 25°C with shaking. After incubation, SDS-PAGE sample buffer was added to stop the reaction before immunoblotting for pS643PKC and PKC (upper panels). For in vitro PKC kinase assay (lower panel), determination of phosphorylation of Ser/Thr in the substrate by recombinant PKC (recPKC) using HTScanTM PKC kinase assay kit was performed following the manufacturer's protocols. The primary antibody of the kit and proper secondary antibody were used for immunoblots. (D) RIE1-5 cells were infected with adenovirus (Ad-Virus) encoding for a control protein (i.e. GFP, Cont) or WT PKC for 12 hours. Twenty four hours after the infection, cells were maintained in suspension (Sus) or replated onto fibronectin-coated (10 µg/ml; Fn) 60 mm dishes for 2 hours prior to cell lysates preparation and standard western blotting for the indicated molecules. (E,F) Whole cell lysates prepared as in Fig. 4C,D were normalized and used for standard western blotting for the indicated molecules. (G) RIE-4 or parental RIE1 WT cells in normal culture were harvested for integrin 4 and -tubulin immunoblots (upper panel). RIE1-4 cells were infected with control (Ad-cont) or DN PKC (Ad-DN PKC) adenovirus and treated with OXO (10 µM for 24 hours) prior to harvesting and immunoblotting for the indicated molecules. Data shown are representative of at least three independent experiments.

Furthermore, when RIE1-5 cells were replated on Fn after PKC WT virus infection to increase pS⁶⁴³PKC, pY³⁹⁷FAK, pY⁴¹⁶Src and pS⁴⁷³Akt were further enhanced (Fig. 7D), indicating that PKC acts upstream of them. In addition, pS⁶⁴³PKC was not altered even when pY³⁹⁷FAK was inhibited by cytochalasin D (5 µM), indicating that PKC is not downstream of FAK in this system (Fig. 7E). Moreover, OXO-mediated pS⁶⁴³PKC in RIE1-5 cells was partially or insignificantly reduced by PI3K or SFK inhibition, respectively, indicating that PKC is not downstream of SFK (Fig. 7F). These data indicate that PKC mediates signaling from integrin 5 to SFK and FAK.

RIE1-4 cells showed a reduced pS³cofilin level after OXO treatment (Fig. 7G), being consistent with OXO-mediated loss of contacts between RIE1-4 cells (Fig. 5G, upper). Although basal pY³⁹⁷FAK of RIE1-4 cells was increased by DN PKC infection, pre-infection with DN PKC adenovirus did not block the OXO-mediated effects on pS³cofilin, pS⁶⁴³PKC, pY³⁹⁷FAK, pY⁴¹⁶Src and pS⁴⁷³Akt (Fig. 7G). These observations are consistent with no blocking of OXO-mediated cell-cell contact loss (Fig. 5G, lower). Therefore, the pS⁶⁴³PKC dependency of the OXO effects appeared to be specific for integrin 5-expressing cells, whereas RIE1-WT or RIE1-4 cells showed loss of cell-cell contacts after OXO treatment presumably in a PKC-independent manner (Fig. 5G and data not shown).

OXO-mediated wound healing dependent on intact integrin 5 subunit
The observed OXO-mediated loss or maintenance of cell-cell contacts depending on WT or mutant integrin 5 signaling, respectively, may differentially support cell migration properties, since cell-cell contacts may resist cell migration. To test this possibility, we examined the wound healing abilities of RIE1-5 and RIE1-5/1 cells under diverse experimental conditions. First, when we examined the effects of OXO treatment on wound healing, we observed that wound healing by RIE1-5 cells was slightly improved, whereas wound healing by RIE1-5/1 cells was retarded after OXO treatment, without any improvement even after a longer period of treatment (Fig. 8A). This OXO-mediated retardation might be due to the maintenance of cell-cell contacts as well as the inhibition of basal intracellular migration machinery by OXO treatment. To determine whether wound healing abilities depend on SFK and PI3K/Akt activities and actin filaments, cells were wounded and pretreated with inhibitors against SFK (PP2 and its negative control PP3), PI3K/Akt (LY294002) or MLCK (ML9), 10 minutes before 10 µM OXO treatment. Degrees of wound healing were then compared. Pharmacological inhibition of PI3K/Akt, SFK or MLCK blocked OXO-enhanced wound healing by RIE1-5 cells, but PP3 did not (Fig. 8B, upper). By contrast, the wound healing abilities of RIE1-5/1 cells was unchanged under the same experimental conditions (Fig. 8B, bottom). Next, we compared wound healing abilities when cells were infected with control- or PKC-adenovirus. OXO-enhanced wound healing by RIE1-5 cells was blocked by DN PKC expression. However, wound healing by RIE1-5/1 cells that was unchanged by OXO treatment, was facilitated by WT PKC preinfection (Fig. 8C). These effects were correlated with blockades of cell-cell contact loss by pharmacological inhibition or DN PKC expression in RIE1-5 cells, and with induction of cell-cell contact loss by WT PKC expression in RIE1-5/1 cells (Figs 3 and 5). Thus, it is likely that the different degrees of wound healing observed after OXO treatment might be attributed to the integrin 5- and PKC-dependent regulations of cofilin-mediated peripheral actin organization and cell-cell contact.

View larger version (137K): [in this window] [in a new window]   Fig. 8. Cell-cell contact loss from the effects of OXO on peripheral actin filaments is correlated with wound healing abilities that are dependent on integrin 5, PKC, SFK and Akt/PKB activities. (A) Confluent RIE1 cells seeded onto 60 mm culture dishes were wounded, washed twice with 10% FBS-DMEM-H, and treated with DMSO or 10 µM OXO, as described in Materials and Methods. Phase contrast images were taken 17 or 24 hours after wounding and treatment. (B) Confluent cells in 60 mm culture dishes were wounded, washed, and then pretreated with LY294002 (LY, 20 µM), PP2 (10 µM), PP3 (10 µM) or ML9 (20 µM) 10 minutes before 10 µM OXO treatment. Another set of cells was treated with DMSO alone in parallel. After incubation for 20 hours, phase contrast images were taken. Note that ML9 treatment caused a certain level of cytotoxicity. (C) Cells in 60 mm culture dishes were infected with adenovirus for GFP (Ad-cont), K376A DN PKC (Ad-DN PKC), or WT PKC (Ad-WT PKC) for 8 hours. After the viruses had been washed out, the cells were treated with DMSO or 10 µM OXO for 16 hours, and then images were taken of the wound area. Dotted lines indicate the starting lines for wound healing. Data shown are representative of three independent experiments.

	Discussion

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Not only integrin-mediated cell adhesions to ECM but also cell-cell contacts at tight and adherence junctions through claudin or occludin and E-cadherin, respectively, link extracellular cues to intracellular actin filaments (Thiery, 2003). Thus, a bidirectional communication between focal adhesions and cell-cell contacts can be possible through actin cytoskeletal reorganization and signal transduction. However, mechanistic investigations of these aspects at the molecular level have been limited. This study provides evidences that actin reorganization effected by a chemical reagent could regulate cell-cell contact and SMA expression via PKC and cofilin phosphorylation and/or activity-mediated regulation of peripheral actin organization, differentially depending on integrin 5 WT or tailless mutant. Evidence obtained during this study suggests that PKC mediates signaling from integrin 5 to focal adhesion molecules (e.g. FAK and SFK) and PI3K/Akt to dynamically regulate cofilin dephosphorylation-dependent peripheral actin reorganization, and consequently EMT and wound healing ability, but that signaling via mutant integrin 5, which lacks the whole cytoplasmic tail (i.e. 5/1) cannot cause pS⁶⁴³PKC and dynamic regulation of peripheral actin organization (Fig. 9). Intact integrin 5-dependent pS⁶⁴³PKC increase after OXO treatment appeared to occur via OXO-mediated complex formation between integrin 5 and pS⁶⁴³PKC. The complex formation might occur only in limited regions, such as focal adhesions around cell peripheries, and only a small amount of the complex was thus formed. It cannot be ruled out that the formation of the complex may occur indirectly via another mediator such as integrin-linked kinase (ILK), syndecan or growth factor receptor.

View larger version (20K): [in this window] [in a new window]   Fig. 9. Working model of cell-cell contact loss and wound healing through integrin 5-PKC signal transduction to regulate cofilin dephosphorylation and peripheral actin reorganization. When actin filaments were affected presumably by extracellular cues or reagent-mediated morphological changes, integrin 5-mediated PKC Ser643 phosphorylation may cause the activation of focal adhesion molecules including FAK and SFK. The PI3K/Akt pathway may be linked to PKC through a transmodulatory loop (refer to text). Activation of focal adhesion molecules and PI3K/Akt correlates with the dephosphorylation of cofilin Ser3 upon OXO treatment, presumably via the activation of cofilin phosphatase, SSH1L (Nishita et al., 2004) and via SSH1L activation-mediated inhibition of LIMK1 activity (Soosairajah et al., 2005). In particular, OXO treatment can also cause localization of nonphosphorylated cofilin at cell peripheries. These can result in severing of peripheral actin filaments via the dephosphorylated and thus activated cofilin. This dynamic peripheral actin reorganization may lead to cell-cell contact loss, and thereby enhance wound healing. However, specific signaling linkage(s) to dynamically regulate PKC, FAK, SFK, and PI3K/Akt activity, and actin reorganization, is impaired in cells expressing integrin 5 lacking its whole cytoplasmic tail (right side) which appears to be crucial for association with and signaling to PKC, although signaling for Erk1/2-mediated cell proliferation, probably via intact transmembrane and extracellular domains is still possible. Therefore, integrin-PKC-mediated dynamic regulation of cofilin activity and peripheral actin reorganization is responsible for cell-cell contact loss in response to stimuli that cause morphological changes.

Since we failed to find any growth factors (including hepatocyte growth factor up to 100 ng/ml) or cytokines capable of causing RIE1 cell scattering, we instead used OXO to stimulate cells, which has been shown to affect actin organization. OXO itself did not generate ROS in RIE1 cells, although naphthoquinone compounds, such as OXO, have been shown to cause cytoskeletal alteration through ROS generation (Bellomo et al., 1990; Mirabelli et al., 1989). In addition, OXO did not affect the kinase activity of recombinant PKC in vitro. Thus, the OXO effects may be indirect possibly through actin reorganization or disruption. Although OXO treatment might bypass certain physiological cue-mediated processes to effect actin reorganization, the observations made in this study suggest that cell-cell contact status can be regulated by integrin-ECM adhesion via PKC and cofilin phosphorylation/activity-mediated peripheral actin reorganization.

We observed that intact integrin 5 enhanced pS⁶⁴³PKC, focal adhesion molecules (FAK and SFK) and Akt phosphorylation, and reduced cofilin phosphorylation presumably via activation of a cofilin phosphatase SSH1L after OXO treatment. However, integrin 4 and tailless 5/1 could not enhance pS⁶⁴³PKC, presumably because no complex was formed between 5/1 or 4 and pS⁶⁴³PKC. A similar integrin-subtype-specific linkage to PKC has previously been reported; signaling of integrin 5 (but not of 4) required PKC activity, especially PKC (Mostafavi-Pour et al., 2003). However, in this current study, integrin 5-mediated signaling for actin reorganization via cofilin dephosphorylation required PKC, but not PKC or PKC, indicating specific linkages between integrin and PKC subtypes. Furthermore, it was previously reported that 4-phorbol 12-myristate 13-acetate (PMA)-mediated cofilin dephosphorylation in neutrophils is blocked by bisindolylmaleimide I (BIM1, an antagonist of PKC, I, II, , and ), but not by Gö6976 (an antagonist of PKC, I, II and µ) (Zhan et al., 2003). These previous and current studies suggest that PKC can regulate cofilin dephosphorylation. Consistent with the findings of this study, it was shown that PKC causes EMT of urinary bladder carcinoma cells, whereas PKC/ promotes cell-cell contacts (Koivunen et al., 2004). Therefore, cell-cell contact maintenance of RIE1-5/1 cells, even after OXO treatment, might be attributed to their specific inability to enhance pS⁶⁴³PKC enough for dynamic regulation of peripheral actin reorganization. However, even tailless integrin 5/1 supported Erk1/2-mediated cell proliferation. This might be possible, because the transmembrane and/or extracellular domains of 5 or tailless 5/1 integrins could still transduce signaling for Erk1/2 activation through association with caveolin-1 and recruitment of Fyn (Wary et al., 1998), which might be strengthened by unidentified mechanisms in the presence of OXO, although OXO did not directly activate Erk1/2 during an in vitro Erk assay (data not shown). Alternatively, it cannot be ruled out that another unidentified target of OXO might mediate Erk1/2 activity, independently of the cytoplasmic tail of integrin 5.

How did integrin-mediated pS⁶⁴³PKC and PKC activity regulate cofilin (de)phosphorylation? It is unclear at this time whether PKC activates SSH1L directly. PKC was previously shown to be correlated with cofilin dephosphorylation; activation of PKC in resting neutrophils by treatment of PMA, an activator of PKC, markedly dephosphorylated cofilin (Djafarzadeh and Niggli, 1997; Zhan et al., 2003). In stimulated human peripheral blood T lymphocytes, the activation of the PKC-Ras-PI3K cascade was correlated with cofilin dephosphorylation (Samstag and Nebl, 2005). Furthermore, it was previously shown that PI3K activity in MCF7 cells causes cofilin dephosphorylation via the activation of SSH1L, and pS⁴⁷³Akt colocalizes with nonphosphorylated cofilin at the cell periphery (Nishita et al., 2004). We observed enhanced pS⁶⁴³PKC and pS⁴⁷³Akt, peripheral localization of nonphosphorylated cofilin, and retractile (peripheral) actin organization after OXO treatment of RIE1-5 (but not of RIE1-5/1) cells, which were blocked by expression of inactive hSSH1L mutant. We also observed that the overexpression of Akt WT could reduce pS³cofilin. Therefore, OXO-mediated pS⁶⁴³PKC and pS⁴⁷³Akt may be important for hSSH1L activation and thereby cofilin dephosphorylation at RIE1-5 cell peripheries.

The OXO-mediated activations of FAK and SFK in RIE1-5 cells might also result in cofilin dephosphorylation via their signaling linkages to PI3K/Akt, depending on complex formation between intact integrin 5 and pS⁶⁴³PKC. SFK activity has been shown to activate the PI3K/Akt pathway via a direct protein interaction (Pleiman et al., 1994), phosphorylation of the regulatory p85 subunit of PI3K (Cuevas et al., 2001), and inactivation of PTEN (Lu et al., 2003). Furthermore, pY³⁹⁷FAK is known to recruit Src or PI3K (Chen et al., 1996; Eide et al., 1995). This current study also showed that LIMK1 was upstream of cofilin when RIE1-5 cells were treated with OXO. Complex formation between SSH1L and LIMK1 was shown to cause inactivation of LIMK1 and thus potentiate cofilin dephosphorylation (Soosairajah et al., 2005), suggesting that both LIMK1 and SSH1L are upstream of cofilin during OXO-mediated (peripheral) actin reorganization in RIE1-5 cells. Also, in this study, OXO treatment caused removal of pS³cofilin from the peripheries of RIE1-5 cells, but not of RIE1-5/1 cells. In addition, peripheral pS³cofilin and sustained peripheral actin bundles of RIE1-5/1 cells were removed by PKC WT overexpression. Thus, intact integrin 5-dependent pS⁶⁴³PKC and activation of PKC downstream molecules including FAK, SFK and Akt might regulate SSH1L activation and LIMK1 inhibition, to cause cofilin dephosphorylation around cell peripheries to facilitate peripheral actin severing, morphological retraction, and consequent cell scattering (Fig. 9).

The roles of the different PKC isotypes and their spatiotemporal relationships at cell-cell contact sites were previously reported (Collazos et al., 2006). PKC activity has also been shown to be involved in integrin-mediated signaling. In the present study on the PKC-dependent EMT of RIE1 cells, the linkage between intact integrin 5 and PKC was found to be specific; integrin 4 and tailless 5 did not transduce signals to PKC and integrin 5 did not transduce signals to PKC or PKC. We also observed that engagement of intact integrin 5, but not of tailless 5/1, to fibronectin efficiently increased pS⁶⁴³PKC, upstream of FAK, SFK and Akt. General PKC activation has been shown to precede integrin 51-mediated FAK phosphorylation and cell spreading on fibronectin, through indirect effects of PKC on FAK phosphorylation (Vuori and Ruoslahti, 1993). We also previously reported that PKC expression and Ser643 phosphorylation induced by treating gastric carcinoma cells with TGF1 led to the inductions and activations of integrins 2 and 3, which were required for FAK activity-dependent cellular spreading and metastasis potential (Lee et al., 2005). In addition, RhoA-mediated actin organization and focal adhesion of gastric carcinoma cells were enhanced by PMA in a PKC activity-dependent manner (Lee et al., 2006). HEK293 cell adhesion also caused integrin activation and PKC phosphorylation in a PTEN activity-dependent manner, whereas suspended cells did not (Parekh et al., 2000). These previous and present studies support the notion that integrin transduces signals to FAK through PKC mediation. Although phosphorylation of FAK and Src were found to be regulated by PKC in the present study, inhibition of PI3K/Akt slightly reduced pS⁶⁴³PKC levels in the presence of OXO, indicating that the PI3K/Akt pathway partly regulates PKC. Thus, the relationship between PI3K/Akt and PKC might form a transmodulatory loop, because PKC could also regulate FAK, SFK and PI3K/Akt (see above). However, the significance of PKC regulation by Akt is not well understood at this time. The overexpression of WT PKC and thus enhanced Ser643 phosphorylation (Lee et al., 2005) blocked peripheral actin reorganization and caused cell-cell contact loss only after OXO treatment. This observation indicates that OXO causes more than PKC activation. The phosphorylation of cortactin and/or myosin light chain (MLC) might be regulated by integrin 5-dependent and PKC-independent signaling in the presence of OXO treatment (data not shown). Cortactin was previously shown to be involved in peripheral actin organization for cell-cell contacts (Helwani et al., 2004). Previously, non-receptor Fer tyrosine kinase released from disrupted adherence junction plaques was found to affect actin filaments and modulate integrin affinity for ECMs at focal adhesions (Arregui et al., 2000; Greer, 2002). We also observed in our system that kinase-dead Fer expression in RIE1-5/1 cells blocked peripheral actin organization (data not shown), indicating that RIE1-5/1 cells may have somehow highly activated Fer for persistent peripheral actin organization. It may be likely that OXO also regulates cortactin and/or Fer activity. We are currently carrying out investigations to determine how cortactin or Fer is involved in integrin 5-mediated signal transduction to regulate peripheral actin reorganization.

	Materials and Methods

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Cells
The integrin-5-null normal rat intestinal epithelial (RIE1) cells ectopically and stably expressing human integrin 5 (RIE1-5) or cytoplasmic tailless 5 [RIE1-5/1; in which the cytoplasmic tail of the C-terminal 27 amino acids, including GFFKR residues, was deleted, leaving one amino acid (Lys) proximal to the transmembrane domain] were previously described (Lee and Juliano, 2000). Stable RIE1 cells overexpressing human integrin 4 (RIE1-4) was prepared by a transfection of pcDNA3.1-4 (a kind gift from M. H. Ginsberg, University of California, San Diego, CA, USA) to RIE1 parental cells with a little endogenous 4 expression. The stable cells were maintained in DMEM-H (Gibco-BRL) culture medium containing 10% (v/v) fetal bovine serum (FBS), 0.25 µg/ml gentamycin (Calbiochem) and 200 µg/ml G418 (A.G. Scientific Inc., San Diego, CA, USA) at 37°C and 5% CO₂. The RIE1-5 cells were similar to the parental RIE1 (WT) cells in terms of actin organization and cell-cell contact formation in normal culture condition. Upon treatment with 6-(1-oxobutyl)-5,8-dimethoxy-1,4-naphthoquinone (OXO), the parental RIE1 WT cells showed cell scattering, as did RIE1-5 cells, and OXO treatment enhanced cell scattering and wound healing ability (data not shown). Expression of integrin 1 in RIE1 WT, RIE1-4, RIE1-5 and RIE1-5/1 cells was similar (data not shown).

Immunofluorescence microscopy
Cells were replated on 10% FBS-DMEM-H-coated glass coverslips and incubated overnight at 37°C to achieve typical adhesion and spreading. RIE1-5, RIE1-5/1 or RIE1-4 cells were infected with adenovirus for GFP or WT or dominant negative (DN) PKC (Hirai et al., 1994), or PKC for 8 hours prior to replating. In some instances, cells were infected for 24 hours with a pLNCX retrovirus (a modified control retroviral vector) or pLNCX-hSSH1L WT- or CS mutant-myc-(His)_6, in which the catalytic Cys residue is replaced with Ser. A modified pLNCX was used to subclone an inactive human Slingshot (hSSH1L) CS mutant cDNA insert (a kind gift from Tadashi Uemura, Kyoto University, Kyoto, Japan) at HindIII (5') and XhoI (3') sites. Cells were treated with vehicle (DMSO), OXO (at 10 or 30 µM in DMSO), or cytochalasin D (0.05 or 0.2 µM) for the indicated periods. In certain cases, cells were pretreated with LY294002 (20 µM; LC Laboratories®, Woburn, MA, USA), PP2 (10 µM; A.G. Scientific Inc.), PP3 (10 µM; A.G. Scientific Inc.) or rottlerin (10 µM; Calbiochem), 30 minutes before OXO treatment. Cells were then fixed with 3.7% formaldehyde in PBS, permeabilized with 0.5% Triton X-100 in PBS at room temperature (RT) for 10 minutes, and washed three times with PBS. The cells were then incubated with primary antibody for 1 hour at RT and washed with PBS (three times 10 minutes). The primary antibodies for cofilin, pS³cofilin (Cell Signaling Technology, Beverly, MA, USA), -catenin (Santa Cruz Biotechnology) and ZO1 (Zymed, S. San Francisco, CA, USA) were used. Cells were then incubated with anti-rabbit IgG-conjugated TRITC or FITC (Chemicon) in a dark, humidified chamber for 1 hour at RT. For actin staining, cells were stained with phalloidin-conjugated Rhodamine (Molecular Probes) for 1 hour at RT, washed three times with PBS, and mounted with a mounting solution (DakoCytomation, Germany). Mounted samples were examined using fluorescence microscopy (BX51TR, Olympus, Japan).

Cell lysates preparation and western blots
Whole cell lysates from cells treated with DMSO or OXO at the indicated concentrations and times or from cells kept in suspension or replated on fibronectin (10 µg/ml; Chemicon) were prepared, as described previously (Lee et al., 2005). Before cells were treated with DMSO, OXO, or other pharmacological inhibitors, cells were manipulated for transfection or infection with the indicated plasmids or viruses, respectively, as above. PKC K437R dominant mutant was generated from WT (a kind gift from J.-W. Soh, Inha University, Korea) using a QuickChange site-directed mutagenesis kit (Stratagene) and its sequence was confirmed by direct sequence analysis. Protein amounts in lysates were normalized, and then used in standard western blots using phospho-Y³⁹⁷FAK, phospho-Y⁴¹⁶Src, PKC, Src (Santa Cruz Biotechnology), Akt/PKB, phospho-S⁴⁷³Akt/PKB, phospho-T⁵⁰⁵PKC, phospho-S⁶⁴³PKC, pan-PKC, Erk1/2, phospho-Erk1/2, cofilin, phospho-S³cofilin (Cell Signaling Technology), -smooth muscle actin (SMA; Sigma-Aldrich), FAK, -tubulin, integrin 5 (BD Transduction Laboratories, San Jose, CA, USA), and the cytoplasmic tail of integrin 5 (Chemicon, Temecula, CA, USA).

ROS generation analysis
For the 2',7'-dichlorodihydrofluorescein (DCHF) assay to examine reactive oxygen species (ROS) generation by OXO treatment, 20 µM DCHF-PBS was added to RIE1 cells on normal culture medium-coated coverslips or in 96-well plates in the absence or presence of OXO treatment for 30 minutes or 24 hours. After incubation, cells on coverslips were washed and examined for fluorescence using a BX51TR microscope (Olympus, Japan) or cells in 96-well plates were washed and fluorescence was quantified using a plate reader at 530 nm.

Coimmunoprecipitation between integrin 5 and PKC
Subconfluent RIE1-5 or RIE1-5/1 cells without or with OXO treatment (10 µM for 24 hours) were harvested for whole cell extracts (Lee et al., 2005). An equal amount of protein was immunoprecipitated with anti-5 mAb (clone P1D6; Chemicon) and the immunoprecipitates were immunoblotted for pS⁶⁴³PKC and PKC.

Analysis of OXO effects on PKC Ser643 phosphorylation or kinase activity
Whole cell extracts were obtained from RIE1-5 cells using a buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 50 mM NaF, 1 mM sodium pyrophosphate, 0.1% SDS and 0.1% Triton X-100) and immunoprecipitated with anti-PKC. The PKC immunoprecipitates were mixed with an equal amount of a reaction buffer (25 mM Tris, pH 7.5, 10 mM MgCl₂, 50 µM ATP and 1 mM DTT) without or with the PKC-depleted extracts (10 µg protein/reaction) in the absence or presence of 10 µM OXO for 30 minutes at 25°C with shaking. After incubation, SDS-PAGE sample buffer was added to stop the reaction before immunoblotting for pS⁶⁴³PKC and PKC. For the in vitro PKC kinase assay, HTScan^TM PKC kinase assay kit (Cell Signaling Technology) was used according to the manufacturer's protocols. For determination of phosphorylation of Ser and Thr in the substrate peptide, the primary antibody of the kit and proper secondary antibody were used for immunoblots with a 0.1 µm pore size nitrocellulose transfer membrane.

Wound healing assay
When cells in 60 mm culture dishes had formed a confluent monolayer, wounds were made by scraping through the cell monolayer with a pipette tip. Cells were then washed twice with DMEM-H containing 10% FBS. In certain cases, cells were treated with pharmacological inhibitors, as explained above, 10 minutes before OXO treatment. For PKC expression studies, cells were infected with adenovirus for either GFP, WT or DN PKC for 8 hours, prior to wounding and treatment with DMSO or 10 µM OXO. After incubation at 37°C for the indicated periods, images were taken of the area around wounds for each condition, using a microscope equipped with a digital camera (CKX41, Olympus, Japan).

	Acknowledgments

 
This work was supported by a Korean Ministry of Health and Welfare fund (B050007) and CPMDRC (R13-2007-019-00000-0) to S.-H.K. and a Korea Research Foundation Grant (KRF-2006-311-C00491) and a KOSEF International Cooperation Fund (FO1-2006-000-10018-0) to J.W.L.

	References

Top Summary Introduction Results Discussion Materials and Methods References

 

Arregui, C., Pathre, P., Lilien, J. and Balsamo, J. (2000). The nonreceptor tyrosine kinase fer mediates cross-talk between N-cadherin and 1-integrins. J. Cell Biol. 149, 1263-1274.[Abstract/Free Full Text]

Avizienyte, E. and Frame, M. C. (2005). Src and FAK signalling controls adhesion fate and the epithelial-to-mesenchymal transition. Curr. Opin. Cell Biol. 17, 542-547.[CrossRef][Medline]

Balzac, F., Avolio, M., Degani, S., Kaverina, I., Torti, M., Silengo, L., Small, J. V. and Retta, S. F. (2005). E-cadherin endocytosis regulates the activity of Rap1: a traffic light GTPase at the crossroads between cadherin and integrin function. J. Cell Sci. 118, 4765-4783.[Abstract/Free Full Text]

Bellomo, G., Mirabelli, F., Vairetti, M., Iosi, F. and Malorni, W. (1990). Cytoskeleton as a target in menadione-induced oxidative stress in cultured mammalian cells. I. Biochemical and immunocytochemical features. J. Cell. Physiol. 143, 118-128.[CrossRef][Medline]

Bhowmick, N. A., Zent, R., Ghiassi, M., McDonnell, M. and Moses, H. L. (2001). Integrin 1 signaling is necessary for transforming growth factor- activation of p38MAPK and epithelial plasticity. J. Biol. Chem. 276, 46707-46713.[Abstract/Free Full Text]

Boyer, B., Valles, A. M. and Edme, N. (2000). Induction and regulation of epithelial-mesenchymal transitions. Biochem. Pharmacol. 60, 1091-1099.[CrossRef][Medline]

Brakebusch, C. and Fassler, R. (2003). The integrin-actin connection, an eternal love affair. EMBO J. 22, 2324-2333.[CrossRef][Medline]

Carragher, N. O., Westhoff, M. A., Fincham, V. J., Schaller, M. D. and Frame, M. C. (2003). A novel role for FAK as a protease-targeting adaptor protein: regulation by p42 ERK and Src. Curr. Biol. 13, 1442-1450.[CrossRef][Medline]

Chen, H. C., Appeddu, P. A., Isoda, H. and Guan, J. L. (1996). Phosphorylation of tyrosine 397 in focal adhesion kinase is required for binding phosphatidylinositol 3-kinase. J. Biol. Chem. 271, 26329-26334.[Abstract/Free Full Text]

Collazos, A., Diouf, B., Guerineau, N. C., Quittau-Prevostel, C., Peter, M., Coudane, F., Hollande, F. and Joubert, D. (2006). A spatiotemporally coordinated cascade of protein kinase C activation controls isoform-selective translocation. Mol. Cell. Biol. 26, 2247-2261.[Abstract/Free Full Text]

Cuevas, B. D., Lu, Y., Mao, M., Zhang, J., LaPushin, R., Siminovitch, K. and Mills, G. B. (2001). Tyrosine phosphorylation of p85 relieves its inhibitory activity on phosphatidylinositol 3-kinase. J. Biol. Chem. 276, 27455-27461.[Abstract/Free Full Text]

Djafarzadeh, S. and Niggli, V. (1997). Signaling pathways involved in dephosphorylation and localization of the actin-binding protein cofilin in stimulated human neutrophils. Exp. Cell Res. 236, 427-435.[CrossRef][Medline]

Eide, B. L., Turck, C. W. and Escobedo, J. A. (1995). Identification of Tyr-397 as the primary site of tyrosine phosphorylation and pp60src association in the focal adhesion kinase, pp125FAK. Mol. Cell. Biol. 15, 2819-2827.[Abstract]

Eliceiri, B. P. (2001). Integrin and growth factor receptor crosstalk. Circ. Res. 89, 1104-1110.[Abstract/Free Full Text]

Galkin, V. E., Orlova, A., VanLoock, M. S., Shvetsov, A., Reisler, E. and Egelman, E. H. (2003). ADF/cofilin use an intrinsic mode of F-actin instability to disrupt actin filaments. J. Cell Biol. 163, 1057-1066.[Abstract/Free Full Text]

Gilcrease, M. Z. (2007). Integrin signaling in epithelial cells. Cancer Lett. 247, 1-25.[CrossRef][Medline]

Greer, P. (2002). Closing in on the biological functions of Fps/Fes and Fer. Nat. Rev. Mol. Cell Biol. 3, 278-289.[CrossRef][Medline]

Helwani, F. M., Kovacs, E. M., Paterson, A. D., Verma, S., Ali, R. G., Fanning, A. S., Weed, S. A. and Yap, A. S. (2004). Cortactin is necessary for E-cadherin-mediated contact formation and actin reorganization. J. Cell Biol. 164, 899-910.[Abstract/Free Full Text]

Hirai, S., Izumi, Y., Higa, K., Kaibuchi, K., Mizuno, K., Osada, S., Suzuki, K. and Ohno, S. (1994). Ras-dependent signal transduction is indispensable but not sufficient for the activation of AP1/Jun by PKC. EMBO J. 13, 2331-2340.[Medline]

Hirohashi, S. and Kanai, Y. (2003). Cell adhesion system and human cancer morphogenesis. Cancer Sci. 94, 575-581.[CrossRef][Medline]

Huang, T. Y., DerMardirossian, C. and Bokoch, G. M. (2006). Cofilin phosphatases and regulation of actin dynamics. Curr. Opin. Cell Biol. 18, 26-31.[CrossRef][Medline]

Hynes, R. O. (2002). Integrins: bidirectional, allosteric signaling machines. Cell 110, 673-687.[CrossRef][Medline]

Juliano, R. L. (2002). Signal transduction by cell adhesion receptors and the cytoskeleton: functions of integrins, cadherins, selectins, and immunoglobulin-superfamily members. Annu. Rev. Pharmacol. Toxicol. 42, 283-323.[CrossRef][Medline]

Juliano, R. L., Reddig, P., Alahari, S., Edin, M., Howe, A. and Aplin, A. (2004). Integrin regulation of cell signalling and motility. Biochem. Soc. Trans. 32, 443-446.[CrossRef][Medline]

Koivunen, J., Aaltonen, V., Koskela, S., Lehenkari, P., Laato, M. and Peltonen, J. (2004). Protein kinase C / inhibitor Gö6976 promotes formation of cell junctions and inhibits invasion of urinary bladder carcinoma cells. Cancer Res. 64, 5693-5701.[Abstract/Free Full Text]

Lee, H. J., Lee, H. J., Song, G. Y., Li, G., Lee, J. H., Lu, J. and Kim, S. H. (2007). 6-(1-Oxobutyl)-5,8-dimethoxy-1,4-naphthoquinone inhibits lewis lung cancer by antiangiogenesis and apoptosis. Int. J. Cancer 120, 2481-2490.[CrossRef][Medline]

Lee, J. W. and Juliano, R. L. (2000). 51 integrin protects intestinal epithelial cells from apoptosis through a phosphatidylinositol 3-kinase and protein kinase B-dependent pathway. Mol. Biol. Cell 11, 1973-1987.[Abstract/Free Full Text]

Lee, M. S., Ko, S. G., Kim, H. P., Kim, Y. B., Lee, S. Y., Kim, S. G., Jong, H. S., Kim, T. Y., Lee, J. W. and Bang, Y. J. (2004). Smad2 mediates Erk1/2 activation by TGF-1 in suspended, but not in adherent, gastric carcinoma cells. Int. J. Oncol. 24, 1229-1234.[Medline]

Lee, M.-S., Kim, T. Y., Kim, Y.-B., Lee, S.-Y., Ko, S.-G., Jong, H.-S., Kim, T.-Y., Bang, Y.-J. and Lee, J. W. (2005). The signaling network of transforming growth factor 1, protein kinase C, and integrin underlies the spreading and invasiveness of gastric carcinoma cells. Mol. Cell. Biol. 25, 6921-6936.[Abstract/Free Full Text]

Lee, M. S., Kim, Y. B., Lee, S. Y., Kim, J. G., Kim, S. H., Ye, S. K. and Lee, J. W. (2006). Integrin signaling and cell spreading mediated by phorbol 12-myristate 13-acetate treatment. J. Cell. Biochem. 99, 88-95.[CrossRef][Medline]

Li, W., Zhang, J., Bottaro, D. P. and Pierce, J. H. (1997). Identification of serine 643 of protein kinase C- as an important autophosphorylation site for its enzymatic activity. J. Biol. Chem. 272, 24550-24555.[Abstract/Free Full Text]

Lu, Y., Yu, Q., Liu, J. H., Zhang, J., Wang, H., Koul, D., McMurray, J. S., Fang, X., Yung, W. K., Siminovitch, K. A. et al. (2003). Src family protein-tyrosine kinases alter the function of PTEN to regulate phosphatidylinositol 3-kinase/AKT cascades. J. Biol. Chem. 278, 40057-40066.[Abstract/Free Full Text]

Mirabelli, F., Salis, A., Vairetti, M., Bellomo, G., Thor, H. and Orrenius, S. (1989). Cytoskeletal alterations in human platelets exposed to oxidative stress are mediated by oxidative and Ca²⁺-dependent mechanisms. Arch. Biochem. Biophys. 270, 478-488.[CrossRef][Medline]

Miranti, C. K., Ohno, S. and Brugge, J. S. (1999). Protein kinase C regulates integrin-induced activation of the extracellular regulated kinase pathway upstream of Shc. J. Biol. Chem. 274, 10571-10581.[Abstract/Free Full Text]

Mostafavi-Pour, Z., Askari, J. A., Parkinson, S. J., Parker, P. J., Ng, T. T. and Humphries, M. J. (2003). Integrin-specific signaling pathways controlling focal adhesion formation and cell migration. J. Cell Biol. 161, 155-167.[Abstract/Free Full Text]

Nishita, M., Wang, Y., Tomizawa, C., Suzuki, A., Niwa, R., Uemura, T. and Mizuno, K. (2004). Phosphoinositide 3-kinase-mediated activation of cofilin phosphatase Slingshot and its role for insulin-induced membrane protrusion. J. Biol. Chem. 279, 7193-7198.[Abstract/Free Full Text]

Ohta, Y., Kousaka, K., Nagata-Ohashi, K., Ohashi, K., Muramoto, A., Shima, Y., Niwa, R., Uemura, T. and Mizuno, K. (2003). Differential activities, subcellular distribution and tissue expression patterns of three members of Slingshot family phosphatases that dephosphorylate cofilin. Genes Cells 8, 811-824.[Abstract]

Parekh, D. B., Katso, R. M., Leslie, N. R., Downes, C. P., Procyk, K. J., Waterfield, M. D. and Parker, P. J. (2000). 1-integrin and PTEN control the phosphorylation of protein kinase C. Biochem. J. 352, 425-433.[CrossRef][Medline]

Pleiman, C. M., Hertz, W. M. and Cambier, J. C. (1994). Activation of phosphatidylinositol-3' kinase by Src-family kinase SH3 binding to the p85 subunit. Science 263, 1609-1612.[Abstract/Free Full Text]

Retta, S. F., Balzac, F. and Avolio, M. (2006). Rap1: a turnabout for the crosstalk between cadherins and integrins. Eur. J. Cell Biol. 85, 283-293.[CrossRef][Medline]

Samstag, Y. and Nebl, G. (2005). Ras initiates phosphatidyl-inositol-3-kinase (PI3K)/PKB mediated signalling pathways in untransformed human peripheral blood T lymphocytes. Adv. Enzyme Regul. 45, 52-62.[CrossRef][Medline]

Schmitz, A. A., Govek, E. E., Bottner, B. and Van Aelst, L. (2000). Rho GTPases: signaling, migration, and invasion. Exp. Cell Res. 261, 1-12.[CrossRef][Medline]

Short, S. M., Boyer, J. L. and Juliano, R. L. (2000). Integrins regulate the linkage between upstream and downstream events in G protein-coupled receptor signaling to mitogen-activated protein kinase. J. Biol. Chem. 275, 12970-12977.[Abstract/Free Full Text]

Soosairajah, J., Maiti, S., Wiggan, O., Sarmiere, P., Moussi, N., Sarcevic, B., Sampath, R., Bamburg, J. R. and Bernard, O. (2005). Interplay between components of a novel LIM kinase-slingshot phosphatase complex regulates cofilin. EMBO J. 24, 473-486.[CrossRef][Medline]

Stempka, L., Schnolzer, M., Radke, S., Rincke, G., Marks, F. and Gschwendt, M. (1999). Requirements of protein kinase c for catalytic function. Role of glutamic acid 500 and autophosphorylation on serine 643. J. Biol. Chem. 274, 8886-8892.[Abstract/Free Full Text]

Thiery, J. P. (2002). Epithelial-mesenchymal transitions in tumour progression. Nat. Rev. Cancer 2, 442-454.[CrossRef][Medline]

Thiery, J. P. (2003). Epithelial-mesenchymal transitions in development and pathologies. Curr. Opin. Cell Biol. 15, 740-746.[CrossRef][Medline]

Vuori, K. and Ruoslahti, E. (1993). Activation of protein kinase C precedes 51 integrin-mediated cell spreading on fibronectin. J. Biol. Chem. 268, 21459-21462.[Abstract/Free Full Text]

Wary, K. K., Mariotti, A., Zurzolo, C. and Giancotti, F. G. (1998). A requirement for caveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth. Cell 94, 625-634.[CrossRef][Medline]

Weis, W. I. and Nelson, W. J. (2006). Re-solving the cadherin-catenin-actin conundrum. J. Biol. Chem. 281, 35593-35597.[Free Full Text]

Yamada, K. M. and Even-Ram, S. (2002). Integrin regulation of growth factor receptors. Nat. Cell Biol. 4, E75-E76.[CrossRef][Medline]

Zhan, Q., Bamburg, J. R. and Badwey, J. (2003). Products of phosphoinositide specific phospholipase C can trigger dephosphorylation of cofilin in chemoattractant stimulated neutrophils. Cell Motil. Cytoskeleton 54, 1-15.[CrossRef][Medline]

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