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Genes to Cells (2009) 14, 703-716. doi:10.1111/j.1365-2443.2009.01303.x
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Molecular mechanisms regulating dissociation of cell–cell junction of epithelial cells by oxidative stress

 Junko Inumaru1,2{dagger}, Osamu Nagano1,3{dagger}, Eri Takahashi1,2, Takatsugu Ishimoto1, Satoshi Nakamura4, Yoshimi Suzuki4, Shin-ichiro Niwa4, Kazuo Umezawa5, Hidenobu Tanihara2 and Hideyuki Saya1,3

1 Division of Gene Regulation, Institute for Advanced Medical Research, Keio University School of Medicine, Tokyo 160-8582, Japan
2 Department of Ophthalmology and Visual Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan
3 CREST, Japan Science and Technology Agency, Tokyo 102-0075, Japan
4 Link Genomics, Inc., Tokyo 103-0024, Japan
5 Faculty of Science and Technology, Keio University, Yokohama 223-0061, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
Oxidative stress is regarded as a causative factor in aging and various degenerative diseases. Here, we show the mechanism by which oxidative stress induces disruption of cell–cell junctions using retinal pigment epithelial cells. We demonstrated that reactive oxygen species (ROS)-mediated activation of Src kinase increases the tyrosine phosphorylation state of p120-catenin and rapidly triggers translocation of p120-catenin and internalization of N-cadherin from the cell–cell adhesion sites to an early endosomal compartment. Endosomal accumulation of p120-catenin resulted in stress fiber formation and cell–cell dissociation through the activation of Rho/Rho kinase pathway. However, these cytoskeletal remodeling and cell–cell dissociation induced by oxidative stress were transient, due to the activation of nuclear factor-{kappa}B (NF-{kappa}B) and the expression of manganese superoxide dismutase (Mn-SOD). Using the NF-{kappa}B specific inhibitor DHMEQ, we found that NF-{kappa}B is part of a negative feedback loop to control intracellular ROS levels. Finally, we demonstrated that H2O2 treatment alone does not induce the epithelial mesenchymal transition (EMT) in retinal pigment epithelial cells, which can be induced by TNF-{alpha} treatment. These findings suggest that oxidative stress is a crucial factor to induce the cell–cell dissociation, an initial step of EMT, but does not provide sufficient signals to establish and to maintain the EMT.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Epithelial mesenchymal transition (EMT) has been recently recognized not only as a physiological mechanism for development and tissue remodeling, but also as a pathological mechanism in the progression of various diseases including inflammation, fibrosis and cancer (Thiery 2002). During EMT, cell–cell adhesion molecules are inactivated and in some cases destroyed, while cell–matrix adhesion increases, which leads to promotion of cell motility and migration. Fibrogenesis during wound healing or following organ inflammation is dependent on the proliferation and migration of new fibroblastic cells generated by EMT (Venkov et al. 2007). For instance, proliferative vitreoretinopathy (PVR), which is one of the major complications of rhegmatogenous retinal detachment surgery, is characterized by the formation of scar-like fibrous tissue containing myofibroblasts derived from transdifferentiated retinal pigment epithelial cells (Saika et al. 2004). This developmental source of PVR is closely related to disruption of cell–cell adhesion by EMT. Thus, the maintenance of epithelial integrity is considered to be important for the suppression of various fibrotic diseases.

Cadherin is a major component of the adherens junction (AJ) and provides cell–cell adhesion through Ca2+-dependent, homophilic binding between cadherin molecules on adjacent epithelial cells. The cytoplasmic domain of classical cadherin is highly conserved between different subtypes, including E-, N- and P-cadherin and binds directly to several cytoplasmic proteins including β-catenin and p120-catenin. Biochemical modification of those catenins is known to regulate cadherin function. Recent studies have demonstrated that cadherin mediated cell–cell adhesion is controlled by Src-dependent tyrosine phosphorylation of cadherin associated components. In particular, p120-catenin, which was first identified as a Src substrate, has emerged as an important regulatory component to stabilize cadherins at the cell membrane by modulating cadherin membrane trafficking and degradation (Anastasiadis & Reynolds 2001; Chen et al. 2003; Halbleib & Nelson 2006; Xiao et al. 2007). Furthermore, the phosphorylation of tyrosine residues in p120-N-terminal regulatory domain was shown to induce dissociation of cadherin-mediated cell–cell adhesion (Aono et al. 1999; Ozawa & Ohkubo 2001). However, the physiological and pathological conditions that promote p120-catenin tyrosine phosphorylation and the molecular link between the phosphorylation and cell–cell dissociation are not fully understood.

Oxidative stress is widely regarded as a potential causative factor in aging and diverse degenerative diseases. It has been also reported that oxidative stress induces cell–cell dissociation in various epithelial cell types (Nigam et al. 1998). Among them, the retinal pigment epithelium is particularly susceptible to oxidative stress because of its high consumption of oxygen, its high proportion of polyunsaturated fatty acids and its exposure to visible light (Beatty et al. 2000). Increased oxidative stress disrupts retinal pigment epithelial cell–cell junction and barrier integrity, which may be associated with the pathogenesis of age-related macular degeneration (AMD). These evidences suggest a causative relationship between oxidative stress and disruption of cell–cell junctions, which is the early event of EMT.

Here, we have identified the mechanism by which oxidative stress induces the disruption of cell–cell junctions using normal retinal pigment epithelial cells. Reactive oxygen species (ROS)-mediated activation of Src kinase increases tyrosine phosphorylation of p120-catenin and rapidly triggers translocation of p120-catenin with cadherin to the endosomal compartment from the sites of cell–cell adhesion. Furthermore, endosomal accumulation of p120-catenin in response to oxidative stress results in the activation of Rho/Rho kinase pathway, leading to dissociation of cell–cell contact and cytoskeletal remodeling. Through those analyses, we have attempted to answer the important question whether or not the dissociation of cell–cell contact by oxidative stress is sufficient for induction and maintenance of EMT.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
H2O2 treatment induces dissociation of cell–cell attachment in ARPE-19 cells

We first investigated the effect of oxidative stress on epithelial cell morphology and structures. ARPE-19, which are normal retinal pigment epithelial cells, were treated with 200 µM H2O2 (Fig. 1a) and morphological changes were evaluated by immunostaining of actin and {alpha}-tubulin. Cell–cell attachments were disrupted and increased stress fiber formation was observed as early as 1 h post-exposure to 200 µM H2O2. However, cell–cell adhesions were gradually recovered thereafter and were completely restored to the pretreatment status by 24 h. Furthermore, stress fiber formation was also disappeared at 24 h after H2O2 treatment (Fig. 1a). These results suggest that H2O2 treatment triggers short-term cell–cell dissociation and cytoskeletal remodeling in ARPE-19 cells. A similar temporal sequence of morphological changes induced by H2O2 treatment has previously been reported in other cell lines (Nigam et al. 1998).


Figure 1
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  		Figure 1  H2O2 treatment induces transient cell–cell dissociation and N-cadherin translocation in ARPE-19 cells. (a) Disruption of cell–cell attachment by H2O2 treatment. ARPE-19 cells were treated with 200 µM H2O2 for the indicated times. Actin filaments were stained with Alexa 488-Phalloidin (upper panels) and anti-{alpha}-tubulin was used to clearly visualize the cell–cell attachment (lower panels). Arrows (black) indicate actin stress fiber formation. Arrowheads (white) indicate the areas of cell–cell dissociation. (b) ARPE-19 cells express primarily N-cadherin, not E-cadherin. Immunoblot analysis of whole cell lysates extracted from untreated ARPE-19 and MCF7 cells by using antibodies against N-cadherin, E-cadherin and β-actin. (c) Translocation of N-cadherin from plasma membrane to a cytoplasmic compartment in response to H2O2 treatment. ARPE-19 cells were cultured in serum free medium in the presence or absence of 1 mM N-acetylcistein (NAC) overnight and then stimulated with 200 µM H2O2. Cells were stained with anti-N-cadherin. Arrows indicate N-cadherin which accumulates to form a focal patch in cytoplasm of each H2O2-treated cells (1 h treatment). NAC pre-treatment effectively blocked the cytoplasmic translocation of N-cadherin from plasma membrane. (d) N-cadherin expression levels were not altered by H2O2 treatment. Whole cell lysates from untreated ARPE-19 cell (lane 1) and H2O2-treated cells (lanes 2–4) were immunoblotted with anti-N-cadherin, and with anti-β-actin as a loading control.

 
For most epithelial cells, E-cadherin plays a role in assembly of cell–cell junctions and generation of an epithelial phenotype. N-cadherin is usually absent from most epithelial cells and, when cells are transformed, the expression of N-cadherin impairs rather than supports junction formation, inducing a motile, mesenchymal phenotype (Islam et al. 1996; Hazan et al. 2004). In contrast, retinal pigment epithelial cells, which originate from neural ectoderm express N-cadherin, rather than E-cadherin, as their normal dominant cadherin and form stable, zonula adherens-type junctions using N-cadherin (McKay et al. 1997; Kaida et al. 2000). In ARPE-19 cells, we confirmed that N-cadherin is the major cadherin component of cell–cell junctions (Fig. 1b). We thus examined the effect of H2O2 on expression and localization of N-cadherin in ARPE-19 cells (Fig. 1c). Immunocytochemical analysis revealed that N-cadherin localization to the cell border decreased and a fraction of N-cadherin accumulated to form a focal patch in the cytoplasm by 1 h after initiation of H2O2 treatment. Subsequently, most N-cadherin returned to the plasma membrane by 6 h after initiation of H2O2 treatment (Fig. 1c, upper panels). Western blot analysis, however, showed that overall levels of N-cadherin in ARPE-19 cells were little affected by H2O2 treatment (Fig. 1d). The transient cytoplasmic translocation of N-cadherin and cell–cell dissociation by H2O2 treatment were completely blocked by the pretreatment of cells with an antioxidant N-acetylcystein (NAC) (Fig. 1c, lower panels). These findings indicate that oxidative stress induces N-cadherin internalization from the plasma membrane, leading to transient dissociation of cell–cell attachments.

Inhibition of protein tyrosine phosphatases by H2O2 treatment induces translocation of p120-catenin and N-cadherin to the cytoplasm

Tyrosine phosphorylation of cadherin complexes has major effects on the stability of AJs. Inhibition of protein tyrosine phosphatases (PTPs) was previously reported to increase tyrosine phosphorylation of cadherin complexes through Src kinase activation and disruption of cell–cell adhesion sites (Sallee et al. 2006). Furthermore, given that ROS are potent inhibitors of PTPs (Meng et al. 2002), oxidative stress may induce cell–cell contact dissociation by inhibition of PTPs and activation of Src kinase. In fact, treatment with sodium orthovanadate (SOV), a PTP inhibitor, induced translocation of N-cadherin to cytoplasm to form the focal patches and dissociation of cell–cell contacts in ARPE-19 cells within 1 h (Fig. 2a), similar to H2O2-treatment of ARPE-19 cells.


Figure 2
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  		Figure 2  Translocation of N-cadherin and p120-catenin to the endosomal compartment by oxidative stress or inhibition of protein tyrosine phosphatases (PTPs). (a) ARPE-19 cells were treated with or without 50 µM sodium orthovanadate (SOV), a PTP inhibitor, for 1 h and stained with anti-N-cadherin or anti-p120-catenin antibody. Arrows indicate the cytoplasmic focal accumulation of N-cadherin or p120-catenin. Arrowheads indicate the areas of cell–cell dissociation. (b) Cytoplasmic translocation of p120-catenin by H2O2 treatment. ARPE-19 cells were cultured in serum free medium in the presence or absence of 1 mM NAC overnight and then stimulated with 200 µM H2O2 for the indicated times. Cells were stained with anti-p120-catenin. Arrows indicate p120-catenin accumulation in the cytoplasm compartment. The p120-catenin internalization was effectively blocked by NAC treatment. (c) Expression levels of p120-catenin were not altered in H2O2-treated cells. Whole cell lysates from untreated ARPE-19 cell and cells treated with H2O2 for 1 h, 6 h and 24 h were immunoblotted with anti-p120-catenin, and with anti-β-actin as a loading control. (d) Subcellular localization of p120-catenin in H2O2-treated cells. ARPE-19 cells treated with 200 µM H2O2 were subjected to double staining with antibodies against p120-catenin and markers for the Golgi apparatus (TR ceramide), lysosomes (Lysotracker) or the early endosome (anti-EEA-1). Arrow indicates co-localization of p120-catenin with EEA-1. (e) Subcellular localization of N-cadherin in H2O2-treated cells. Cells were treated with H2O2 for 1 h and then stained with antibodies against N-cadherin and EEA-1. Arrow indicates co-localization of N-cadherin with EEA-1.

 
p120-catenin interacts with the intracellular domain of cadherin and plays a role in lateral clustering of cadherins and regulation of cadherin stability(Daniel 2007). In addition, phosphorylation of p120-catenin by Src kinase triggers the dissociation of cadherin complexes (Alema & Salvatore 2007). Therefore, we examined the effect of SOV treatment on localization of p120-catenin. Although p120-catenin is localized to cell–cell junctions in untreated ARPE-19 cells, SOV treatment induced translocation of p120-catenin to cytoplasm to form the focal patches within 1 h (Fig. 2a). These results indicate that the inhibition of PTP triggers N-cadherin and p120-catenin translocation from cell–cell adhesion site to the cytoplasmic focal patch. Furthermore, H2O2-mediated ROS generation induced similar translocation of p120-catenin to cytoplasm within 1 h (Fig. 2b). Similar to the dynamics of N-cadherin localization, p120-catenin returned to the plasma membrane by 6 h after initiation of the treatments. Western blotting analysis revealed that p120-catenin protein levels were also not significantly changed by the H2O2 treatments (Fig. 2c). These results suggest that the ROS-mediated PTP inactivation triggers transient cytoplasmic translocation of N-cadherin and p120-catenin without affecting their protein stabilities.

To clarify the subcellular localization of the p120-catenin and N-cadherin after 1 h of H2O2 treatment, we used compartment specific immunofluorescent markers for the endosome (EEA-1), Golgi (TR ceramide®) and lysosome (Lysotracker®). The result showed that p120-catenin co-localized with EEA-1, but not with TR ceramide or Lysotracker in H2O2-treated cells (Fig. 2d). Moreover, N-cadherin also co-localized with EEA-1 positive compartments (Fig. 2e). These findings suggest that both p120-catenin and N-cadherin transiently translocate to the EEA-1 positive endosomal compartments after exposure to oxidative stress.

Activation of Src kinase by oxidative stress-induced inactivation of PTPs leads to translocation of p120-catenin and N-cadherin

It has been reported that Src-mediated tyrosine phosphorylation perturbes the plasma membrane cadherin complex formation (Matsuyoshi et al. 1992). Furthermore, p120-catenin was described first as a prominent Src and receptor tyrosine kinase (RTK) substrate (Reynolds et al. 1989). We next investigated the role of Src activation in translocation of p120-catenin and N-cadherin in H2O2-treated ARPE-19 cells. The internalization of N-cadherin and p120-catenin induced by H2O2-treatment was completely blocked by depletion of Src expression by siRNA (Fig. 3a). We then investigated the role of Src kinase on the p120-catenin tyrosine phosphorylation in response to oxidative stress. Lysates from cells treated with H2O2 or SOV for 1 h were immunoprecipitated with anti-p120-catenin antibody and immunoblotted with anti-Src and anti-pTyr antibodies (Fig. 3b). In H2O2-treated cells, p120-catenin was associated with Src kinase and the tyrosine phosphorylation of p120-catenin was significantly increased. The same phenomenon was also observed when cells were treated with SOV. Interestingly, p120-catenin isoform 3, which is known as epithelial isoform (Montonen et al. 2001), was predominantly tyrosine-phosphorylated by the treatment of H2O2 or SOV (Fig. 3b). These results indicate that the inactivation of PTPs by oxidative stress leads to the tyrosine-phosphorylation of epithelial isoform of p120-catenin by Src kinase and subsequent translocation of p120-catenin and N-cadherin to the endosome, resulting in the dissociation of cell–cell junctions.


Figure 3
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  		Figure 3  Role of Src kinase in cytoplasmic translocation of p120-catenin and N-cadherin in H2O2-treated cells. (a) Depletion of Src kinase inhibits H2O2-induced internalization of N-cadherin and p120-catenin translocaion. ARPE-19 cells transfected with Src siRNA for 4 days were treated with or without H2O2 for 1 h in a serum-free medium and then stained with antibodies against N-cadherin and p120-catenin. (b) Interaction of p120-catenin and Src kinase in H2O2-treated cells. Whole cell lysates prepared from ARPE-19 cells treated with 50 µM SOV or 200 µM H2O2 for 1 h were immunoblotted with anti-Src and anti-p120-catenin (left panels). Lysates were immunoprecipitated with anti-p120-catenin and the immunoprecipitates were probed with antibodies against Src, p120-catenin and phosphorylated tyrosine (p-Tyr). The lower molecular weight form of p120-catenin (isoform 3) is detected by anti-p-Tyr.

 
Negative feedback suppression of ROS level through NF-{kappa}B activation

Oxidative stress-induced translocation of p120-catenin is transient and both p120-catenin and N-cadherin return from endosome to the cell membrane within 6 h after initiation of H2O2 treatment (Figs 1c, 2b). We hypothesized that recovery of cell–cell junctions, which is mediated by recovery of the N-cadherin complex formation at plasma membrane, is caused by the reduction of intracellular ROS level. To address this question, we examined the localization of N-cadherin and p120-catenin at 6 h after repeated treatments (multiple treat) with H2O2 at 0, 1 and 3 h to maintain higher ROS levels. N-cadherin and p120-catenin remained in the cytoplasm at 6 h after the initial treatment (Fig. 4a). There was no change in the expression level of N-cadherin and p120-catenin proteins in cells challenged with repeated H2O2 treatments (Fig. 4b). This finding suggests that there is a negative-feedback mechanism suppressing the signals induced by oxidative stress in ARPE-19 cells.


Figure 4
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  		Figure 4  NF-{kappa}B-mediated suppression of oxidative stress elicits a rapid recovery of cell–cell junctions in H2O2-treated cells. (a) ARPE-19 cells were treated with 100 µM H2O2 three times (at 0, 1 and 3 h). At 6 h after the first treatment, cells were stained with antibodies against N-cadherin and p120-catenin. Arrows indicate N-cadherin or p120-catenin retained in endosomal compartments. (b) Expression levels of p120-catenin and N-cadherin in cells shown in (A) were evaluated by immunoblot analysis. (c) NF-{kappa}B phosphorylation in ARPE-19 cells treated with 200 µM H2O2 for the indicted times was detected by an anti-phosphorylated p65 antibody. Lysate from cells treated with TNF-{alpha} (100 ng/mL) for 1 h (lane 2) was used as a positive control. (d) Effect of Src depletion on phosphorylation of NF-{kappa}B. ARPE-19 cells were transfected with either control (GL-2) or Src siRNA. After 72 h, cells were incubated in serum free medium for 24 h and then stimulated with 200 µM H2O2 for the indicated times. Cells were harvested and immunoblotted with antibodies against the indicated proteins (left panels). The intensities of the bands were measured with MacBAS2000 and the phosphorylated form/total NF-{kappa}B ratios were calculated (right panel). (e) Delayed induction of Mn-SOD expression by H2O2 treatment. RT-PCR analysis of Mn-SOD mRNA expression in untreated cells (lane 1), cells treated with H2O2 for the indicated times (lanes 2–4), cells pretreated with DHMEQ and then H2O2 (lanes 5–7) and TNF-{alpha} treated cells, as a positive control (lane 8). (f) Quantitative RT-PCR analysis of Mn-SOD expression in untreated cells, cells treated with H2O2 for 6 h, cells pretreated with DHMEQ and then H2O2 and TNF-{alpha} treated cells, as a positive control. *P < 0.05. (g) Inhibition of NF-{kappa}B nuclear translocation interferes with the return of p120-catenin to the plasma membrane in H2O2-treated cells. Cells were incubated overnight in the absence (upper panels) or presence (lower panels) of 100 ng/mL DHMEQ in serum free medium and then incubated with 200 µM H2O2. Cells were fixed at the indicated times and examined for p120-catenin localization. Arrows indicate p120-catenin retained in cytoplasm of DHMEQ-treated cells at 6 h after H2O2 treatment.

 
NF-{kappa}B is a major signal transducer of oxidative stress-induced signals and has been recently reported to be regulated by p120-catenin (Perez-Moreno et al. 2006; Reynolds 2007). NF-{kappa}B also acts as a suppressor of intracellular ROS formation in response to tumor necrosis factor-{alpha} (TNF-{alpha}) by increasing transcription of manganese superoxide dismutase (Mn-SOD) (Shen & Pervaiz 2006). Therefore, we investigated the status of NF-{kappa}B in H2O2-treated ARPE-19 cells. H2O2 treatment, similar to TNF-{alpha} treatment, rapidly induced phosphorylation of NF-{kappa}B from as early as 15 min and the phosphorylation was maintained up to 6 h after treatment (Fig. 4c). This oxidative stress-induced NF-{kappa}B phophorylation was significantly suppressed by siRNA depletion of Src (Fig. 4d), suggesting that the Src/p120-catenin signaling is involved in the NF-{kappa}B phosphorylation. We also found that expression of Mn-SOD is induced by the H2O2 treatment (Fig. 4e,f). The exogenous oxidative stress might be blocked by phosphorylation and, thus, activation of NF-{kappa}B and subsequent induction of Mn-SOD in the cells. Pretreatment with the specific NF-{kappa}B inhibitor DHMEQ, which inhibits the nuclear translocation of NF-{kappa}B (Ariga et al. 2002), interfered with the return of p120-catenin from endosome to the cell–cell junction by 6 h after H2O2 treatment (Fig. 4g). These data suggest a model in which cells can rapidly reduce the intracellular ROS level and recover from oxidative stress-induced dissociation of cell–cell junction through activation of NF-{kappa}B by p120-catenin/Src complex within the endosomal compartments.

H2O2 treatment-induced Rho/Rho kinase activation leads to cytoskeletal remodeling, but not NF-{kappa}B activation

It has been reported that p120-catenin localized at the plasma membrane suppresses the activity of Rho by forming a complex with p190RhoGAP (Wildenberg et al. 2006). Furthermore, phosphorylation of p120-catenin by Src family kinases regulates its interaction with Rho (Castaño et al. 2007). We first examined the involvement of p120-catenin and Rho in the H2O2 induced cytoskeletal remodeling. To examine the role of p120-catenin in the cellular morphology and cytoskeleton of ARPE-19 cells, we depleted p120-catenin expression by siRNA. p120-catenin siRNA triggered the morphological change from epithelial to mesenchymal phenotypes and reduced cell–cell contact (Fig. 5A). In addition, the formation of stress fiber was enhanced in p120-catenin depleted cells [Fig. 5B(b)]. This morphological change and stress fiber formation of p120-catenin depleted cells were suppressed by treatment with the Rho kinase inhibitor Y27632 [Fig. 5B(c)]. This result suggests that the presence of p120-catenin constitutively suppresses Rho/Rho kinase pathway in ARPE-19 cells. Furthermore, the stress fiber formation induced by H2O2 treatment was suppressed by Y27632 treatment [Fig. 5B(d,e)]. We also found that Mn-SOD siRNA also triggered the enhanced stress fiber formation, similar to p120-catenin depleted cells (Fig. 5C). These findings suggest that increased ROS level followed by the translocation of p120-catenin to endosome activates Rho/Rho kinase pathway, leading to the cytoskeletal remodeling.


Figure 5
Figure 5
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  		Figure 5  Oxidative stress liberates Rho/Rho kinase pathway from p120-catenin-mediated suppression, leading to the stress fiber formation but not NF-{kappa}B activation. (A) Role of p120-catenin in the maintenance of epithelial integrity in ARPE-19 cells. ARPE-19 cells were transfected with control (GL2) or p120-catenin siRNA and cultured for 4 days. Expression of p120-catenin in those cells was examined by immunoblot analysis (left panels). Cellular morphology was examined by phase-contrast microscopy (right panels). Note that p120-catenin siRNA induced dissociation of cell–cell contact. (B) ARPE-19 cells trasfected with control siRNA (a) and p120-catenin siRNA (b) for 4 days were stained with antibody against p120-catenin and rhodamine-phalloidin (F-actin). p120-catenin siRNA transfected cells were treated with 10 µM Y27632 for 1 h before staining (c). ARPE-19 cells were treated with 200 µM H2O2 for 1 h in serum free medium in the presence (e) or absence (d) of Y27632 and stained with anti-p120-catenin antibody and rhodamine-phalloidin (F-actin). Arrows indicate actin stress fiber formation. (C) Depletion of p120-catenin or Mn-SOD enhances stress fiber formation in ARPE-19 cells. ARPE-19 cells were transfected with control (GLs), p120-catenin or Mn-SOD siRNA and cultured for 4 days. The cells were fixed and stained with p120-catenin or rhodamine-phalloidin. Arrows indicate the enhanced stress fiber formation. Note that p120-catenin staining positive cells (siRNA untransfected cells) represent normal actin stress fiber (arrowheads). Enhanced stress fiber formation and diffuse cytoplasmic staining of p120-catenin were observed in Mn-SOD siRNA transfected cells. (D) Rho activity assays. ARPE-19 cells were treated with 200 µM H2O2 for the indicated times. Total protein extracts from each cell were incubated with GST-Rhotekin RBD (Rho binding domain) bound to glutathione-coupled agarose beads to selectively pull down the GTP-bound active form of Rho. The input cell lysates and the beads-bound proteins were then subjected to immunoblot analysis with antibodies to RhoA (left panels). The intensities of the bands were measured with MacBAS2000, and the active form/total RhoA ratios were calculated (right panel). (E) The NF-{kappa}B phosphorylation induced by H2O2 treatment was not inhibited by the Rho kinase inhibitor Y27632. ARPE-19 cells were incubated with (lanes 5–8) or without (lanes 1–4) 10 µM Y27632 for 1 h. The cells were then treated with 200 µM H2O2 for the indicated times. Cell lysates were subjected to the immunoblot analysis with indicated antibodies (left panels). The intensities of the bands were measured with MacBAS2000, and the phosphorylated form/total NF-{kappa}B ratios were calculated (right panel).

 
We then examined whether H2O2 treatment increase the active form of Rho. In Rho activity assay, H2O2 treatment displayed an increase in the amount of GTP-bound (active) Rho, while total Rho protein levels were unchanged (Fig. 5D). These results suggest that H2O2 treatment triggers cytoskeletal reorganization through the Rho activation. Recent studies revealed that NF-{kappa}B can be phosphorylated by Rho-kinase in p120-catenin deficient keratinocytes (Perez-Moreno et al. 2006). Therefore, we hypothesized that the p120-catenin/Rho/Rho-kinase pathway is not only involved in the H2O2-induced cytoskeletal remodeling but also in the H2O2-induced activation of NF-{kappa}B signaling pathway. However, contrary to our expectation, the NF-{kappa}B phosphorylation induced by H2O2 treatment was not inhibited by the Rho kinase inhibitor Y27632 (Fig. 5E), suggesting that NF-{kappa}B activation by H2O2 treatment is not driven by activation of Rho/Rho-kinase pathway.

H2O2 treatment alone is not sufficient for EMT induction

The EMT process is initiated by cell–cell dissociation, which is preceded by internalization of cadherins and progressive disappearance of cadherins from cell–cell contact areas (Boyer et al. 1989). To investigate whether oxidative stress-induced cell–cell dissociation is sufficient for induction of EMT, we examined morphological changes and EMT markers in H2O2-treated ARPE-19 cells. To maintain cell–cell dissociation, cells were repeatedly treated with H2O2 (multiple treatments). We have recently shown that TNF-{alpha} induces morphological changes typical of EMT in ARPE-19 cell with an increased expression of ECM components, including fibronectin and hyaluronic acid, and down-regulation of an epithelial marker, keratin-18 (Takahashi et al., manuscript in preparation). Furthermore, TNF-{alpha} induced translocation of N-cadherin to the cytoplasm and fibroblast-like morphological change. These changes led to the down-regulation of epithelial integrity in ARPE-19 cells and consequently induced the formation of characteristic cell aggregation (Fig. 6a). However, H2O2-treated cells did not display such morphological and molecular changes characteristic of EMT (Fig. 6b,c). These results suggest that oxidative stress is required for cell–cell dissociation, but that it is not sufficient to cause and/or maintain EMT status in ARPE-19 cells.


Figure 6
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  		Figure 6  Oxidative stress alone is not sufficient for induction of EMT in ARPE-19 cells. (a) TNF-{alpha} induces multicellular aggregate formation in ARPE-19 cells. ARPE-19 cells were cultured with or without 100 ng/mL TNF-{alpha} for 3 days. The cells were fixed and immunostained with anti-N-cadherin antibody and Hoechst 33342 (nuclear staining). (b) EMT marker expression in TNF-{alpha} and H2O2 treated cells. Confluent monolayer of ARPE-19 cells were incubated in medium containing 100 ng/mL TNF-{alpha} or 100 µM H2O2. The protein levels of keratin18, fibronectin, β-catenin and β-actin were determined by immunoblot analysis. (c) Morphological changes of cells treated with 100 ng/mL TNF-{alpha} and 200 µM H2O2 for 3 days were observed by phase-contrast microscopy. Arrows indicate characteristic cell aggregation found when EMT is induced in ARPE-19 cells by TNF-{alpha} treatment. The cell aggregation was not identified in H2O2 treated cells.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we have shown that oxidative stress induces the collapse of cell–cell junctions in retinal pigment epithelial cells followed by rapid recovery of cell–cell contacts and we have identified the underlying mechanisms of this process. We found that oxidative stress-induced cell–cell dissociation is caused primarily by endocytosis of N-cadherin mediated by Src-dependent phosphorylation and internalization of p120-catenin.

It was previously reported that integrity of cell–cell contact is disrupted by TGF-β-induced EMT in renal proximal tubular cells (Masszi et al. 2004). Their study showed that the disruption of cell–cell contact activates β-catenin signaling and Rho/Rho kinase pathway, leading to the activation of {alpha}-smooth muscle actin (SMA) promoter to promote EMT (Masszi et al. 2004, Fan et al. 2007). In our present study, we found that H2O2 treatment induces the translocation of p120-catenin to endosome, which leads to the loss of epithelial integrity, activates Rho/Rho kinase pathway and results in enhanced stress fiber formation, which are similar steps to the initial phase of EMT. However, H2O2-mediated activation of Rho/Rho kinase pathway is transient and failed to induce or maintain the mesenchymal phenotypes. In fact, we found that H2O2-induced Src activation triggers the activation of NF-{kappa}B, leading to Mn-SOD expression which reduces oxidative stress. Thus, we demonstrate that oxidative stress-induced cell–cell dissociation might be required for the initial step of EMT, but is not sufficient for stable induction of EMT.

p120-catenin is an important regulatory component of the cadherin adhesive complex. A core function of p120-catenin in epithelial cells is to stabilize cadherins at the cell membrane by modulating cadherin membrane trafficking and degradation (Xiao et al. 2007). Our observations showed that H2O2 treatment rapidly induces internalization of p120-catenin and N-cadherin as early as 1 h after the treatment, which leads to dissociation of cell–cell junctions and cytoskeletal remodeling via Rho activation. Furthermore, we demonstrate here that p120-catenin and N-cadherin are consistently coprecipiated in response to H2O2 treatment. Thus, they may translocate together from plasma membrane to endosomal compartment.

The stability of cell–cell adhesions is regulated by protein tyrosine phosphorylation of cell adhesion molecules and their associated components, with high levels of phosphorylation promoting disassembly of junctions (Sallee et al. 2006). p120-catenin was initially identified as a major substrate of Src kinase, and Src-medicated phosphorylation of p120-catenin is a candidate mechanism for regulation of E-cadherin trafficking and reduction in cell–cell adhesion in cancer cells in which Src levels are elevated (Avizienyte et al. 2002). Our findings show that H2O2 treatment promotes interaction of p120-catenin with Src kinase and results in tyrosine phosphorylation of the epithelial isoform (isoform 3) of p120-catenin. In addition, depletion of Src by siRNA completely blocked the internalization of both p120-catenin and N-cadherin and inhibited H2O2-induced cell–cell dissociation. Therefore, the phosphorlation of p120-catenin isoform 3 through the interaction with Src at cell–cell junction may be a key mechanism for the cell–cell dissociation caused by oxidative stress.

p120-catenin was previously found to interact with a receptor-like protein tyrosine phosphatase (RPTP), which negatively regulates the Src-mediated phosphorylation of p120-catenin(Mariner et al. 2001). Furthermore, oxidative stress was shown to induce a conformational change in the D2 domain of RPTP through oxidation of the active-site cystein residue, leading to inhibition of RPTP activity (Blanchetot et al. 2002; Meng et al. 2002; Ostman et al. 2006). Importantly, we demonstrated that inhibition of PTP activity by SOV induces translocation of p120-catenin and N-cadherin to endosome and the cell–cell dissociation, showing a phenotype similar to that observed in H2O2-treated cells. Taken together, these results suggest that oxidative stress-induced cell–cell dissociation is caused by inactivation of RPTP, leading to Src-mediated phosphorylation and translocation of p120-catenin.

The oxidative stress-induced translocation of p120-catenin and N-cadherin is transient and the cell–cell junction is rapidly recovered. Given that multiple treatments with H2O2 sustained the endosomal localization of p120-catenin and the cell–cell dissociation, it is likely that oxidative stress was rapidly reduced after the initial treatment. Furthermore, NF-{kappa}B was activated by H2O2 and the translocation of p120-catenin to endosome, suggesting that NF-{kappa}B participates in the intracellular redox reaction. We found that H2O2 treatment induces transcription of an NF-{kappa}B-controlled gene encoding the antioxidant enzyme Mn-SOD, lowering the ROS levels. Long-term high ROS levels may be detrimental and lead to cell death or an acceleration in aging (Finkel & Holbrook 2000). Therefore, this negative feedback mechanism may act as a host defense mechanism to lower ROS levels. In addition, we speculate that the fine-tuning of ROS levels by this mechanism regulates cell–cell interactions and consequently affects tissue remodeling.

Recent observations have suggested a functional link between p120-catenin and NF-{kappa}B activation. p120-catenin forms a complex with p190RhoGAP to inhibit Rho activity at cell junctions (Perez-Moreno et al. 2006; Wildenberg et al. 2006) and, depletion of p120-catenin thereby induces phosphorylation of NF-{kappa}B by the Rho-kinase. Based on these findings, we examined whether oxidative stress-induced internalization of p120-catenin leads to NF-{kappa}B phosphorylation through the Rho/Rho-kinase pathway. Depletion of Src inhibited the internalization of p120-catenin and suppressed the phosphorylation of NF-{kappa}B by H2O2. However, a Rho-kinase inhibitor, which inhibits H2O2-induced cytoskeletal remodeling, did not suppress H2O2-induced NF-{kappa}B phosphorylation. Therefore, other unknown pathways or mechanisms may link p120-catenin translocation and NF-{kappa}B activation.

EMT processes are initiated by endocytosis and degradation of cadherin, resulting in cell–cell dissociation (Palacios et al. 2005; Lee et al. 2006). Given that oxidative stress induces the disruption of the cell junctions, we examined the possibility that oxidative stress is associated with EMT induction. A previous study demonstrated that stromelysin-1/matrix metalloproteinase-3 (MMP-3) induced EMT through expression of Rac1b which causes an increase in ROS levels (Radisky et al. 2005), implicating ROS in EMT. In the present study, although H2O2 treatment disrupted the cell–cell junctions, it did not induce events typically found in EMT, such as upregulation of fibronectin and down-regulation of keratin-18, which were observed when ARPE-19 cells were treated with TNF-{alpha}. Therefore, oxidative stress alone is not sufficient for induction of EMT and redox regulation through NF-{kappa}B activation is critical for suppression of EMT. Furthermore, retinal cells may have a high oxidation-reduction capability in order to prevent the disruption of the epithelium because the retina is constitutively exposed to oxidative stress. In the future, the differences in the effects of oxidative stress on retinal pigment epithelial cells and cells in the other organs should be investigated.

In summary, we have demonstrated that an oxidative stress induces the dissociation of cell–cell junctions following the translocation of p120-catenin to endosome and that its translocation is associated with the reduction in intracellular oxidative stress mediated NF-{kappa}B (Fig. 7). This negative feedback mechanism is crucial for maintenance of epithelial structure under highly oxidative conditions. Thus, disruption of epithelial integrity due to injury or aging may be the result of insufficient reduction of intracellular oxidants.


Figure 7
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  		Figure 7  Proposed model of the negative-feedback regulation of ROS-induced cell–cell dissociation. In unstimulated retinal pigment epithelial cells, p120-catenin forms a complex with N-cadherin and stabilizes cell–cell contacts by Rac-mediated signals. Oxidative stress inactivates PTPs and induces Src-mediated phosphorylation of p120-catenin, leading to the translocation of both p120-catenin and N-cadherin to the endosomal compartment and the displacement of p190RhoGAP from plasma membrane. Thus, p120-catenin translocation results in the activation of Rho/Rho kinase pathway, leading to the cytoskeletal remodeling and other signaling pathway. However, Src-mediated activation of NF-{kappa}B induces transcriptional expression of antioxidant Mn-SOD, which reduces intracellular ROS level and restores the epithelial integrity.

 

   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cells and cell culture

All experiments were performed using ARPE-19, a human diploid retinal pigment epithelial cell line. The cells were grown in Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham (Sigma) with 10% bovine serum at 37 °C in an atmosphere containing 5% CO2. TNF-{alpha} (BD Bioscience Pharminogen; 100 ng/mL),  Sodium orthovanadate (SOV) (Sigma; 50 µM), N-acetylcysteine (NAC) (Sigma, 1 mM), NF-{kappa}B inhibitor [Dehydroxymethylepoxyquinomicin (DHMEQ); 100 ng/mL], Rho kinase (ROCK) inhibitor (Calbiochem Y27632, 10 µM) were used. DHMEQ was synthesized by us from 2,5-dihidroxyaniline as described previously (Suzuki et al. 2004).

Antibodies

Monoclonal antibodies against N-cadherin, p120 catenin and fibronectin were purchased from BD Transduction Laboratories. Polyclonal antibodies against β-catenin and NF-{kappa}B were obtained from Santa Cruz Biotechnology. A monoclonal antibody specific for the phosphorylated form of NF-{kappa}B was purchased from Cell signaling Technology. A monoclonal antibody against keratin18 and a polyclonal antibody against EEA-1 were purchased from abcam. A monoclonal antibody against β-actin was obtained from Sigma. Horseradish peroxidase conjugated secondary antibodies were purchased from Amersham Biosciences. Rhodamin-phalloidin was purchased from Molecular Probes, Inc. A fluorescein conjugated secondary antibody was obtained from BIOSOURCE. A Cy5TM conjugated rabbit IgG antibody was obtained from GE Healthcare. BODIPY® TR ceramide and Lysotracker were purchased from Molecular Probes.

RNA interference

A human SRC RNAi duplex was purchased from Invitrogen. p120-catenin and Mn-SOD siRNA were obtained from Japan Bioservice. A double-stranded RNA targeting luciferase (GL-2) was used as a control. Transfection was performed using Oligofectamine (Invitrogen), according to the manufacturer's protocol.

Western blot analysis

Cultured cells were directly lysed with SDS sample buffer (2% SDS, 10% glycerol, 0.1 M dithiothreitol, 120 mM Tris-HCL, pH 6.8, 0.02% bromophenol blue) and boiled for 5 min. Samples containing equal amounts of cell lysate were electrophoresed on a SDS-polyacrylamide gel and transferred to nitrocellulose filters with a constant current of 140 mA for 90 min. The filters were blocked in PBS containing 5% skim milk for 20 min at room temperature and then incubated with primary antibodies diluted in PBS containing 0.03% Tween20 overnight at 4 °C. The filters were washed and then incubated for 40 min with the appropriate secondary antibodies diluted in PBS containing 0.03% Tween20, and specific proteins were detected using an enhanced chemiluminescence system (Amersham Pharmacia Biotech).

Immunofluorescence microscopy analysis

ARPE-19 cells grown on 35-mm culture dishes were fixed with 4% paraformaldehyde for 15 min followed by 0.2% Triton-X100 in PBS for 5 min. After washing with PBS, the cells were incubated in primary antibodies diluted PBS containing 0.3% bovine serum albumin (BSA) overnight at 4 °C, washed three times and incubated with the secondary antibodies in PBS containing 0.3% BSA for 60 min at room temperature. In some experiments, cells were stained with PI during the secondary antibody incubation. After being washed with PBS, samples were mounted in 80% glycerol and visualized using a confocal microscope (Fluoview, Olympus, Tokyo, Japan) equipped with an argon gas laser and appropriate filters sets to allow simultaneous recording of fluorescein. Fluorescence micrographs were recorded using PLAPO 60x objectives and were sampled at a resolution of 1024 x 1024 pixels and 8-bit color.

Quantification of Mn-SOD mRNA levels by RT-PCR

To evaluate Mn-SOD mRNA expression after H2O2 treatment, we performed RT-PCR using the following primers: 5'-CTT TCA GTT ACA TTC TCC CAG TTG-3' (Mn-SOD-S) and 5'-GAC ACT TAC AAA TTG CTG CTT GTC C-3' (Mn-SOD-AS). First strand cDNA, which served as the PCR template, was synthesized from 1 µg of total RNA purified using an RNeasy minikit (Qiagen). The reverse tyranscription (RT) reaction was performed using an oligo(dT) primer and Superscript II reverse transcriptase (Invitrogen). PCR was performed with 1 µL of RT reaction, 1.25 units of rTaq DNA polymerase (Takara), 2 mM MgCl2 and 0.8 mM dNTP mixture in a final volume of 50 µL. PCR conditions were a 5-min initial denaturation at 94 °C followed by 25 cycles at 94 °C for 30 s, 57 °C for 30 s and 72 °C for 30 s. PCR products were resolved by electrophoresis in a 1.5% agarose gel and visualized by staining with ethidium bromide.

Quantitative real time PCR was also performed using a Thermal Cycler Dice Real Time system (TaKaRa) and the following primer sets:

Mn-SOD, Forward: 5'-CAAAGGGAGATGTTACAGCC-3', Reverse: 5'-TTAGGGCTGAGGTTTGTCCA-3'; GAPDH, Forward: 5'-TGAAGGTCGGAGTCAACGATTTGGT-3', Reverse: 5'-GAAGATGGTGATGGGATTTC-3'. The PCR conditions were as follows: 95 °C for 2 min, 40 cycles of 95 °C for 30 s and 60 °C for 30 s, followed by dissociation curve analysis to confirm specificity. Data are presented as means ± SD of triplicates.

Rho activity assay

ARPE-19 cells were lysed in Mg2+ containing lysis buffer [25 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 10% glycerol, 10 mM Mgcl2, 1 mM EDTA, 10 mM NaF, 2 mM Na3VO4, protease inhibitor (Sigma)] and GTP-bound form of Rho was affinity precipitated with Rhotekin-RBD (Rho binding domain) beads (Upstate). Affinity precipitated Rho was quantified from cell lysates by Western blot analysis with an antibody against Rho (Upstate).


   Acknowledgements
 
We thank all members of the Saya lab for valuable suggestions and discussion. This work was supported by a grant for Cancer Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (to H.S.) and a research grant from National Institute of Biomedical Innovation, Japan (to H.S.).


   Footnotes
 
Communicated by: Kozo Kaibuchi

{dagger}Both authors contributed equally to this work. Back

* Correspondence: hsaya{at}a5.keio.jp


   References
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 Introduction
 Results
 Discussion
 Experimental procedures
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Accepted: 8 March 2009




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