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Genes to Cells (2007) 12, 787-796. doi:10.1111/j.1365-2443.2007.01092.x
© 2007 Blackwell Publishing or its licensors
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T-cadherin enhances cell–matrix adhesiveness by regulating ß1 integrin trafficking in cutaneous squamous carcinoma cells

 Yohei Mukoyama1,2,*, Atsushi Utani1, Seiya Matsui1,2, Shuxia Zhou3, Yoshiki Miyachi1 and Norihisa Matsuyoshi1

1 Department of Dermatology, Graduate School of Medicine, Kyoto University, 54 kawahara-cho, Shogo-in, Sakyo-ku, Kyoto 606-8317, Japan
2 Drug Discovery Research, Research Laboratories, Kyoto R&D Center, Maruho Co., Ltd., 92 Awata-cho, Chudoji, Shimogyo-ku, Kyoto 600-8815, Japan
3 Life Sciences, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
T-cadherin is a glycosyl-phosphatidylinositol (GPI) anchored cadherin molecule. We previously reported that T-cadherin is normally expressed on the basal keratinocytes of the epidermis and is down-regulated in cutaneous squamous cell carcinoma (SCC). We found that expression of T-cadherin in cutaneous squamous carcinoma cells regulated level of surface ß1 integrin, which functioned as extracellular matrix (ECM) receptor. Involvement of T-cadherin in ß1 integrin trafficking was studied using three different stable cell lines with cytomegalovirus (CMV)-driven over-expression, tetracycline (Tet)-inducible expression and RNAi-mediated suppressed expression of T-cadherin. Pulse–chase analysis using a cholesterol-depleting reagent and a tyrosine kinase inhibitor showed that ß1 integrin mainly internalized via caveolae. Over-expression of T-cadherin suppressed the internalization of both ß1 integrin and cholera toxin (CTX), a marker of caveolae-mediated endocytosis. By Western blot analysis of tyrosine-kinase target molecules, we demonstrated a reduced level of EGF receptor (EGFR)-phosphorylation in T-cadherin over-expressing cells. In addition, studies using EGF and EGFR specific inhibitors revealed that EGFR activation stimulated ß1 integrin internalization. Taking these results together, T-cadherin may modulate cell–matrix adhesion in basal keratinocytes as well as invasive potency in SCC by regulating surface level of ß1 integrin.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
Loss of cadherin expression is frequently observed in various carcinomas and it may mediate metastasis or invasion of carcinoma cells through detachment from adjacent cells. T-cadherin (CDH 13, H-cadherin) is a unique member of the cadherin superfamily that lacks the transmembrane and cytoplasmic domains, and is instead linked to the plasma membrane via a glycosyl-phosphatidylinositol (GPI) anchor (Ranscht & Dours-Zimmermann 1991). Since cytoplasmic domain of classical cadherin plays a crucial role in its homophilic binding (Nagafuchi & Takeichi 1988; Stappert & Kemler 1994; Yap et al. 1998), the lack of this domain in T-cadherin strongly suggests the existence of other functions. We previously demonstrated that T-cadherin was normally expressed on the basal keratinocytes of the epidermis and was down-regulated in cutaneous squamous cell carcinoma (SCC) (Takeuchi et al. 2002; Zhou et al. 2002). Furthermore, T-cadherin did not alter cell–cell adhesion of squamous carcinoma cells, but inhibited cell proliferation (Mukoyama et al. 2005).

Like other GPI-anchored proteins, T-cadherin has been shown to distribute in lipid rafts/caveolae (Philippova et al. 1998). Lipid rafts are cholesterol and sphingolipid-rich subdomains of the plasma membrane, containing GPI-anchored proteins and signal transduction proteins such as Src-family kinases (Brown & London 2000; Maxfield 2002). The size and composition of lipid rafts can be varied in response to intra- or extracellular stimuli, and consequently favor specific protein–protein interactions, resulting in the activation of signaling cascade (Simons & Toomre 2000). Caveolae are related domains of lipids raft and mediate clathrin-independent endocytosis (Anderson 1998; Nabi & Le 2003). Recent studies have shown that ß1 integrin can be internalized via caveolae (Marjomaki et al. 2002; Upla et al. 2004). On the epidermis, basal keratinocytes expressed ß1 integrin, and these cells utilize integrin heterodimer to adhere to underlying basement membrane that is rich in extracellular matrix (ECM) proteins including collagens, fibronectin and laminins (Peltonen et al. 1989; Carter et al. 1990; Watt 2002). By these facts, we speculate that GPI-anchored T-cadherin may affect the function of the ß1 integrin.

Using cutaneous squamous carcinoma cells, we demonstrated that T-cadherin changed the surface expression of ß1 integrin through reducing its internalization. Because EGF receptor (EGFR) is known to concentrate in caveolae (Smart et al. 1995; Mineo et al. 1999), and EGF-stimulation modified cell–matrix adhesion or migration (Bill et al. 2004; Shirk & Kuver 2005), we also analyzed the involvement of EGFR signaling in the interactions of T-cadherin and ß1 integrin.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
T-cadherin increases cell-surface ß1 integrin expression

To determine whether T-cadherin affect the expression level of ß1 integrin, we used a human cutaneous squamous carcinoma cell line HSC-1 (Kondo & Aso 1981) and its transfectants with different levels of T-cadherin expression. HSC-1 TCAD cells (over-expression of T-cadherin driven by the CMV promoter) and HSC-1 RNAI cells (knockdown of T-cadherin via plasmid-based RNA interference) were stable transfectants of HSC-1 cells that were selected and picked up from single cell colonies. Immunofluorescence staining showed that HSC-1 RNAI cells exhibited weak signals of ß1 integrin as compared to the HSC-1 cells under unpermeabilized conditions (Fig. 1a). By contrast, the immunofluorescence of ß1 integrin at cell–cell contacts of HSC-1 TCAD cells was more intense than that of the HSC-1 cells (Fig. 1a). Similar results were obtained using other clones (data not shown).


Figure 1
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  		Figure 1  T-cadherin increases cell-surface ß1 integrin without changes in the total amount. (a) Confluent cultures of HSC-1, HSC-1 RNAI or HSC-1 TCAD were fixed with PLP under unpermeabilized conditions. Cells were double-stained with mAb for T-cadherin (red) and ß1 integrin (green). The signals were visualized by confocal microscopy under the same setting to compare the fluorescence intensity. Scale bar: 100 µm. (b) Cell-surface proteins or total cellular proteins of HSC-1 cells (lanes 1 and 4), HSC-1 RNAI cells (lanes 2 and 5) and HSC-1 TCAD cells (lanes 3 and 6) were collected as described under Experimental procedures. The collected lysates were subjected to Western blot analysis for ß1 integrin and T-cadherin. E-cadherin was used as a loading control. The signal intensity of ß1 integrin was determined by densitometry (lower panel).

 
We next performed Western blot analysis. To assess the cell-surface proteins, we used the surface biotinylation method. As shown in Fig. 1b, left panel, HSC-1 RNAI exhibited reduced expression of surface biotinylated ß1 integrin, and conversely, HSC-1 TCAD exhibited increased expression. The differences of the levels of surface ß1 integrin expression among the three cell lines were in accord with that of immunofluorescence staining. Meanwhile, two major bands of ß1 integrin were detected in total cellular protein extracts (Fig. 1b, right panel). The lower band was considered to be a precursor of ß1 integrin that is incompletely in glycosylated form, and the upper band was considered to be a mature protein. In contrast to surface ß1 integrin, the levels of total cellular ß1 integrin in the three cell lines were not parallel to that of T-cadherin; that is, the three cell lines expressed similar levels of total cellular ß1 integrin (Fig. 1b, right panel).

T-cadherin enhances cell–matrix adhesiveness

We next examined whether T-cadherin-mediated up-regulation of surface ß1 integrin could affect cell–matrix adhesion. To explore this, we compared adhesiveness to ECM of HSC-1, HSC-1 RNAI and HSC-1 TCAD cells. In all cell lines, few cells adhered to BSA, used as a negative control (Fig. 2a). Augmented adhesion was observed on poly-D-lysine, a positively charged protein, and on ECM including collagen type I, collagen type IV, fibronectin and laminin (Fig. 2a). Although there were no significant differences in adhesiveness to poly-D-lysine among the cell lines, adhesion to all of the tested types of ECM was significantly enhanced by the over-expression of T-cadherin (Fig. 2a). Additionally, adhesion to collagen type I was significantly reduced by the knockdown of T-cadherin (Fig. 2a).


Figure 2
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  		Figure 2  T-cadherin enhances ß1 integrin-mediated cell–ECM adhesiveness. (a) HSC-1, HSC-1 RNAI and HSC-1 TCAD cells were suspended in DMEM and allowed to adhere to wells that had been pre-coated with bovine serum albumin (BSA), poly-D-lysine (PDL), collagen I (CL I), collagen IV (CL IV), fibronectin (FN) or laminin (LN) at 37 °C for 30 min. Numbers of adhered cells were examined by a WST-1 method. Results are the means ± SD of three independent wells. **P < 0.01, *P < 0.05 versus HSC-1 by the Kruskal–Wallis rank test with Scheffe's multiple comparison test. (b) Cells were pre-incubated with or without blocking mAb for ß1 integrin M13 (67 µg/mL) for 15 min at 4 °C, and then allowed to adhere to the wells. Results are the means ± SD of three independent wells. **P < 0.01 versus absence of M13 by Student's t test.

 
To examine the specificity of ß1 integrin in the cell–ECM adhesion, we used blocking antibody for ß1 integrin, M13 (Fig. 2b). The adhesion of HSC-1 cells or HSC-1 TCAD cells to collagen type I was completely blocked by M13. Furthermore, M13 blocked the adhesion of HSC-1 TCAD cells to collagen type IV, fibronectin and laminin. These results indicated that the cell adhesions to all of tested ECM were dependent on ß1 integrin. Taken these results together, we concluded that T-cadherin enhanced cell–matrix adhesiveness by increasing functional ß1 integrin expression.

T-cadherin reduces ß1 integrin trafficking

The results that over-expression of T-cadherin increased surface ß1 integrin expression without changing the total amount are consistent with the hypothesis that T-cadherin is involved in ß1 integrin trafficking. To evaluate this, we performed pulse–chase analysis. In starved HSC-1 cells, ß1 integrin began to be internalized from the regions of cell–cell contacts during the initial 15 min of chase and was sequentially transported to the inside of the cells in a chase-time-dependent manner (Fig. 3a,b). Labeled ß1 integrin at the regions of cell–cell contacts largely disappeared by 60 min. In contrast to HSC-1 cells, HSC-1 TCAD cells showed no striking internalization of ß1 integrin at 15 min, and labeled ß1 integrin at the regions of cell–cell contacts partly remained at 60 min. These results indicated that over-expression of T-cadherin delayed the internalization of ß1 integrin, which consequently may induce accumulation of ß1 integrin at the cell-surface. We could not perform pulse–chase analysis using HSC-1 RNAI cells, because the expression levels of ß1 integrin at cell–cell contacts of this cell line were not adequate even before a start of chasing.


Figure 3
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  		Figure 3  T-cadherin suppresses internalization of ß1 integrin. (a, b) HSC-1 cells and HSC-1 TCAD cells were pulse-labeled with mAb for ß1 integrin and then chased for the time indicated in serum-free medium. After fixation and permeabilization, the signal was visualized by confocal microscopy under the same setting in each cell lines. Scale bar: 25 µm. (b) ß1 integrin located at cell–cell contact or inside of the cell were selected, respectively, and the immunofluorescence intensities of the both regions were quantitatively measured by their histogram. Results represent means ± SD of five separated cells. (c, d) HSC-1 Trex TCAD cells were incubated in the presence or absence of tetracycline (Tet) for 24 h. (c) The cells were lysed and equal amounts of cell lysates were subjected to Western blot analysis for T-cadherin. (d) Using the Tet-treated or untreated cells, pulse–chase analysis for ß1 integrin was performed for 60 min and the intensity of the signals at the regions of cell–cell contacts was measured. Results are described as percentage of initial intensity (at 0 min) and represent the means ± SD of five separated cells. *P < 0.05, **P < 0.01 versus Tet-untreated cells by the Kruskal–Wallis rank test with Scheffe's multiple comparison test.

 
To analyze the relationship between the expression level of T-cadherin and the internalization of ß1 integrin, we used tetracycline (Tet)-inducible T-cadherin-expressing cell line, HSC-1 Trex TCAD. As shown in Fig. 3c, HSC-1 Trex TCAD cells expressed low levels of T-cadherin in the absence of Tet, and the expression of T-cadherin was up-regulated by addition of Tet in a dose-dependent manner. Using this cell line, we performed the pulse–chase analysis in the presence or absence of Tet. After 60 min of chase, ß1 integrin at the regions of cell–cell contacts was dose-dependently remained by Tet (Fig. 3d). These results show that reduction of ß1 integrin internalization is dependent on T-cadherin expression levels and even small increase of T-cadherin expression achieved by 10 ng/mL of Tet is sufficient to suppress ß1 integrin internalization.

T-cadherin specifically suppresses caveolae-mediated endocytosis

To evaluate the pathway of the ß1 integrin internalization, pulse–chase analysis was performed in the presence of various inhibitors. Cholesterol-depleting reagent methyl-ß-cyclodextrin (MBCD) and tyrosine kinases inhibitor genistein, but not clathrin-dependent endocytosis inhibitor chlorpromazine, suppressed the internalization of ß1 integrin at the regions of cell–cell contacts (Fig. 4a). These sensitivities to the inhibitors were in agreement with the features of caveolae-mediated endocytosis (Parton et al. 1994). To elucidate whether T-cadherin affected the caveolae-mediated endocytosis, we used FITC-labeled cholera toxin (CTX), which is a marker of caveolae-mediated endocytosis (Montesano et al. 1982; Parton et al. 1994; Wolf et al. 1998). CTX was rapidly internalized into HSC-1 cells at 30 min (Fig. 4b), and this translocation was mostly prevented by MBCD or genistein, but not by chlorpromazine (Fig. 4c). We also confirmed that endocytosis of CTX was suppressed by over-expression of T-cadherin (Fig. 4d). These results suggest that T-cadherin suppressed caveolae-mediated endocytosis and that was associated with suppression of ß1 integrin internalization. Since GPI-anchored proteins including T-cadherin distribute in lipid rafts/caveolae (Philippova et al. 1998), it is possible that over-expression of other GPI-anchored proteins also suppress caveolae-mediated endocytosis. To examine this possibility, a GPI-anchored heparan sulfate proteoglycan glypican-1-expressing HSC-1 cell line, HSC-1 GP1 was obtained. The expression of glypican-1 on HSC-1 GP1 cells was confirmed by immunofluorescence staining with mAb for heparan sulfate (Fig. 4e). HSC-1 GP1 cells did not delay the internalization of ß1 integrin as compared to the parental HSC-1 cells (Fig. 4f). We also confirmed that endocytosis of CTX was not suppressed by over-expression of glypican-1 (Fig. 4f). These results support the specificity of T-cadherin in involvement of ß1 integrin trafficking and caveolae-mediated endocytosis.


Figure 4
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  		Figure 4  T-cadherin specifically suppresses caveolae-mediated endocytosis. (a, c) HSC-1 cells were not treated or treated with methyl-ß-cyclodextrin (MBCD, 10 mM), genistein (GEN, 10 µg/mL) or chlorpromazine (CPM, 28 µM) for 30 min. Then pulse–chase analysis for ß1 integrin with 60 min chase (a) or CTX with 30 min chase (c) was performed. The intensity of signals at the regions of cell–cell contacts was measured under the same setting. Results are the means ± SD of five separated cells. **P < 0.01 versus non-treated-HSC-1 after chasing by the Kruskal–Wallis rank test with Scheffe's multiple comparison test. (b) HSC-1 cells were pulse-labeled with FITC-CTX and then chased in serum-free medium for 30 min. The signal was visualized by confocal microscopy. Scale bar: 25 µm. (d) Pulse–chase analysis for CTX was performed with HSC-1 cells and HSC-1 TCAD cells. (e) Glypican-1-over-expressing cells (HSC-1 GP1) and the parental HSC-1 cells were stained with mAb for heparan sulfate and the signals were visualized by confocal microscopy under same setting. Scale bar: 25 µm. (f) Using HSC-1 cells, HSC-1 TCAD cells and HSC-1 GP1 cells, pulse–chase analysis was performed. After chase of 60 min for ß1 integrin or 30 min for CTX the intensity of the signals at the regions of cell–cell contact was measured. Results are described as percentage of initial intensity (at 0 min) and represent the means ± SD of five separated cells. **P < 0.01 versus HSC-1 cells by the Kruskal–Wallis rank test with Scheffe's multiple comparison test.

 
T-cadherin reduces tyrosine-phosphorylation of EGFR

Since inhibition of tyrosine kinase activity is known to suppress caveolae-mediated endocytosis (Parton et al. 1994), we examined the effect of T-cadherin on phosphotyrosine levels. As shown in Fig. 5a, genistein-treated HSC-1 cells exhibited a low level of a tyrosine-phosphorylated 160-kDa protein compared to non-treated HSC-1 cells (arrow). Interestingly, HSC-1 TCAD cells exhibited a marked reduction of phosphotyrosine content of this protein. Based on its molecular weight, we speculated that this protein might be an EGFR. To confirm this, we used anti-EGFR- and anti-phospho EGFR (at Tyr845)-specific polyclonal antibodies. Predictably, EGFR was detected at the same position (Fig. 5b; EGFR). Genistein-treatment reduced the phosphotyrosine content of EGFR on HSC-1 cells without changing the expression level of EGFR (Fig. 5b; P-EGFR, EGFR). HSC-1 TCAD cells showed a marked reduction of tyrosine-phosphorylation of EGFR (Fig. 5b; P-EGFR). Additionally, the expression level of EGFR on HSC-1 TCAD cells was slightly lower than that of HSC-1 cells (Fig. 5b; EGFR). These results indicated that T-cadherin adversely affected the activation of EGFR by reducing its expression and increasing tyrosine-phosphorylation. In addition, the levels of caveolin-1, a main component and rate-limiting molecule for caveolae-formation, were equal among the cell lines (Fig. 5b; caveolin-1).


Figure 5
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  		Figure 5  T-cadherin reduces tyrosine-phosphorylation of EGFR. (a) Equal amounts of cellular proteins from non-treated HSC-1 cells (–) and HSC-1 TCAD cells, or genistein-treated HSC-1 cells (GEN) were subjected to Western blot analysis for phosphotyrosine (P-tyr). (b) Western blot analysis was performed for detection of EGFR, phospho-EGFR (P-EGFR) and caveolin-1. To determine the relative intensities of EGFR and phospho-EGFR, densitometry was performed (lower panel).

 
EGFR signaling is involved in ß1 integrin trafficking

To explore whether the T-cadherin-mediated increase of surface ß1 integrin expression is abrogated by activation of EGFR signaling, initially we tested the tyrosine-phosphorylation of EGFR on HSC-1 TCAD cells by EGF-stimulation. The addition of EGF induced the tyrosine-phosphorylation of EGFR up to sevenfold within 2 min, and the level of phospho-EGFR reached a peak at 15 min (Fig. 6a; P-EGFR). During this period, the expression level of EGFR decreased in a time-dependent manner (Fig. 6a; EGFR). This reduction of EGFR might be caused by ligand-dependent degradation. Since EGFR on HSC-1 TCAD cells retained responsiveness to EGF, we assessed the effect of EGF on surface ß1 integrin expression. EGF-treatment decreased the expression of ß1 integrin at the regions of cell–cell contacts of HSC-1 TCAD cells (Fig. 6b), although the total cellular content of ß1 integrin was unchanged during this treatment period (Fig. 6a; ß1 integrin). We next examined the involvement of EGFR signaling in ß1 integrin trafficking. In HSC-1 cells, internalization of ß1 integrin at the regions of cell–cell contacts was partly suppressed by the addition of EGFR kinase inhibitor, PD168393 or AG1478, indicating that the EGFR signaling is involved in internalization of ß1 integrin (Fig. 6c). The above results taken together indicates that the reduced internalization of ß1 integrin by T-cadherin over-expression is, at least in part, attributed to the reduction of tyrosine-phosphorylation of EGFR.


Figure 6
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  		Figure 6  EGF abrogates T-cadherin-mediated accumulation of ß1 integrin on the cell-surface. (a) HSC-1 TCAD cells were not treated or treated with EGF (100 ng/mL) for the indicated time periods. Equal amounts of cellular protein were subjected to Western blot analysis for EGFR, phospho-EGFR and ß1 integrin. Densitometry was performed to determine the relative intensities of EGFR and phospho-EGFR (right panel). (b) HSC-1 TCAD cells were not treated or treated with EGF (100 ng/mL) for 30 min. After fixation under unpermeabilized conditions, cells were stained with mAb for ß1 integrin. Signals were visualized by confocal microscopy. Scale bar: 100 µm. (c) HSC-1 cells were not treated or treated with PD168393 (PD, 3 µM) or AG1478 (AG, 3 µM) for 30 min. Then cells were pulse-labeled with mAb for ß1 integrin and then chased for 60 min. The signal was visualized by confocal microscopy under the same setting, and the intensity of signals at the regions of cell–cell contacts was measured. Results are the means ± SD of five separated cells. **P < 0.01 versus non-treated-HSC-1 at 60 min by the Kruskal–Wallis rank test with Scheffe's multiple comparison test.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In the present study, we showed novel molecular interactions between T-cadherin and ß1 integrin. T-cadherin increased surface ß1 integrin expression and consequently enhanced cell–matrix adhesiveness. This is the first report that a member of cadherin family, T-cadherin, controls cell–matrix adhesion via surface expression of an integrin.

T-cadherin suppressed internalization of ß1 integrin and CTX. CTX is a ligand for ganglioside GM1 and is a marker of caveolae-mediated endocytosis. Although it is taken up through clathrin in some cells (Shogomori & Futerman 2001; Torgersen et al. 2001), endocytosis of CTX on HSC-1 cells is suppressed by cholesterol depletion (MBCD-treatment), indicating that its endocytosis is through caveolae as seen in the other cells (Montesano et al. 1982; Wolf et al. 1998). Similarly, the internalization of ß1 integrin was suppressed by cholesterol depletion, indicating the involvement of caveolae in ß1 integrin trafficking. This result is in accordance with the previous studies that showed ß1 integrin internalized via caveolae (Marjomaki et al. 2002; Upla et al. 2004; Sharma et al. 2005). Taken together, these results suggest that T-cadherin plays a role in the regulation of surface ß1 integrin expression by suppressing caveolae-mediated endocytosis, and accumulation of surface ß1 integrin on T-cadherin over-expressing cells is, at least in part, caused by this mechanism. Furthermore, we showed that reduction of ß1 integrin internalization is dependent on T-cadherin expression levels and even small increase of T-cadherin expression is sufficient to suppress ß1 integrin internalization. These results suggest a possible physiological relevance of T-cadherin in ß1 integrin trafficking. In addition, the facts that over-expression of glypican-1, another GPI-anchored protein, did not affect internalization of ß1 integrin, as well as that of CTX, indicate that the reduction of ß1 integrin trafficking and caveolae-mediated endocytosis is specific to T-cadherin. Over-expression of T-cadherin enhanced ß1 integrin-mediated cell adhesiveness to collagen type I, collagen type IV, fibronectin and laminin. On the other hand, the cells with knockdown of T-cadherin, which expressed half levels of surface ß1 integrin as compared to the parental cells, did not exhibit clear reduction of the adhesiveness in this adhesion assay. The sensitivity of the adhesion assay using pre-coated-plates may not be high enough to detect a loss of adhesion activity due to a decreased level of surface ß1 integrin. If amounts of the pre-coated substrates such as collagens, fibronectin and laminin were reduced to a proper level, weaker adhesion activity of the cells with less ß1 integrins by knockdown of T-cadherin may be demonstrated.

We also found that T-cadherin over-expressing cells exhibited a marked reduction of phosphotyrosine content of EGFR. Similar to cholesterol depletion, inhibition of tyrosine kinase suppressed the internalization of both ß1 integrin and CTX. It has been reported that EGF-stimulation induces caveolae-mediated endocytosis (Lu et al. 2003), and also modifies cell–matrix adhesion and migration (Bill et al. 2004; Shirk & Kuver 2005). Interestingly, EGF-stimulation to T-cadherin over-expressing cells transiently enhanced the levels of phospho-EGFR and concurrently decreased the expression of ß1 integrin on the cell-surface. We further confirmed that specific inhibitors of EGFR kinase suppressed the internalization of ß1 integrin. These data suggest that reduced phosphorylation of EGFR by T-cadherin is connected with increased expression of surface ß1 integrin.

Although the mechanisms by which T-cadherin affects the phosphorylation of EGFR remain unclear, one possibility is that T-cadherin regulates Src-family kinases and it interacts with EGFR. The lipid rafts are postulated to be involved in GPI-anchored protein signaling via Src-family kinase. For instance, antibody-mediated cross-linking of GPI-anchored proteins induces a rapid activation of Src-family kinases and a transient increase in the tyrosine-phosphorylation of several substrates (Kasahara & Sanai 2000). EGFR is one of the substrates of Src-family kinases, and Tyr845 have been identified as Src-dependent phosphorylation site (Biscardi et al. 1999). Here we showed that over-expression of T-cadherin resulted in reduced phosphorylation of Tyr845 on EGFR. Therefore, it is speculated that distribution of T-cadherin at cell–cell contacts reduce mobility of lipid raft on plasma membrane, and subsequently inhibits activating of Src-family kinases. Further studies are needed to elucidate the precise mechanisms.

In normal epidermis, T-cadherin is localized at the basal layer and is down-regulated in cutaneous SCC (Takeuchi et al. 2002; Zhou et al. 2002). The present results, combined with our previous results showing T-cadherin as a negative regulator of proliferation of cutaneous squamous carcinoma cells (Mukoyama et al. 2005), suggest crucial roles of T-cadherin in epidermal tumorigenesis. Down-regulation of T-cadherin may affect integrin function and EGFR activation as well as regulation of cell cycle, leading to a breakdown of epidermal homeostasis and consequently induce transformation of keratinocytes with changes in cellular behavior such as uncontrolled proliferation, aggressive invasion and distant metastasis.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
Cell lines

Human cutaneous squamous carcinoma cell line HSC-1 was used in this study (Kondo & Aso 1981). The methods of establishment of HSC-1 TCAD cell line, HSC-1 RNAI cell line and HSC-1 Trex TCAD cell line, and their corresponding constructs (T-cadherin-pIRES neo2 plasmid, T-cadherin-siRNA-pSilencer 3.1-H1 hygro plasmid, and T-cadherin-pcDNA5/TO and pcDNA6/TR, respectively) were described previously (Mukoyama et al. 2005). Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum and antibiotics (streptomycin and penicillin).

Plasmid and transfection

Details of the procedures to construct human glypican-1 expression vector containing cytomegalovirus promoter, IRES and a puromycin-resistant gene were described previously (Utani et al. 2001). HSC-1 cells were transfected with glypican-1 expression vector using lipofectamine 2000 reagent (Invitrogen) and selected with 0.4 µg/mL puromycin (Sigma). Then, HSC-1 GP1 cell lines were picked up from single cell colonies.

Antibodies and reagents

Details of the procedures used to prepare rat anti-T-cadherin monoclonal antibody (TCD-1) were described previously (Mukoyama et al. 2005). Other antibodies used for detection were mouse anti-human ß1 integrin monoclonal antibody (JB1A; CHEMICON International), mouse anti-human E-cadherin monoclonal antibody (HECD-1) (Shimoyama et al. 1989), mouse anti-phosphotyrosine monoclonal antibody (PY20; BD Bio science), rabbit anti-human epidermal growth factor receptor (EGFR) polyclonal antibody (Cell Signaling Technology), rabbit anti-human phospho-EGFR polyclonal antibody (Cell Signaling Technology), rabbit anti-human caveolin-1 polyclonal antibody (N-20; Santa Cruz Biotechnology), FITC-conjugated anti-heparan sulfate monoclonal antibody (10E4; Seikagaku Kogyo) and mouse anti-ß-actin monoclonal antibody (ab6276; abcam). HRP-conjugated goat anti-rat IgG (Dako Cytomation), HRP-conjugated goat anti-mouse IgG (Dako Cytomation), HRP-conjugated goat anti-rabbit IgG (Cell Signaling Technology), Alexa Fluor 546-conjugated goat anti-rat IgG (Molecular Probe) and Alexa Fluor 488-conjugated goat anti-mouse IgG (Molecular Probe) were used as secondary antibodies. Anti-ß1 integrin blocking antibody, M13, was a gift from K. M. Yamada, NIDCR, NIH, Bethesda, MD. Chlorpromazine, MBCD, genistein and FITC-labeled CTX were purchased from Sigma Chemical. PD168393 and AG1478 were obtained from Calbiochem. Recombinant human EGF was obtained from Techne Corp.

Immunofluorescence staining

The cells cultured on chamber slides were fixed with PLP (10 mM sodium periodate, 75 mM lysine and 2.14 mg/mL paraformaldehyde) for 10 min at room temperature. After blocking with 5% skim milk in PBS for 30 min at room temperature, the slides were incubated with a mixture of primary antibodies and subsequently incubated with a mixture of secondary antibodies. Images were visualized by confocal laser scanning microscopy using a Zeiss LSM 510 system (Carl Zeiss Microscope).

Western blot analysis

To collect total cellular proteins, cells were lysed with RIPA buffer (Santa Cruz Biotechnology) containing inhibitor mixture: phenylmethylsulfonyl fluoride, aprotinin, leupeptin, pepstatin and sodium vanadate. Protein concentrations were measured with the Bio-Rad DC protein assay (BIO-RAD). Equal amounts of protein were boiled with 2-mercaptoethanol and separated by SDS-PAGE, followed by transfer to a nitrocellulose membrane sheet. After incubation with primary antibody and HRP-conjugated secondary antibody, signals were detected using the ECL system (Amersham Pharmacia Biotech).

Recovery of cell-surface protein

Monolayer cultures of cells were washed with ice-cold PBS and then incubated with 0.5 mg/mL of sulfo-NHS-SS-biotin, which is a thiol-cleavable amine-reactive biotinylation reagent (PIERCE), for 30 min at room temperature. After washing with PBS to remove non-reacted biotinylation reagent, the cells were lysed in RIPA buffer. Equal amounts of cellular protein were incubated with avidin-beads in PBS. Unbiotinylated proteins were washed off by centrifugation. The samples were analyzed by Western blot analysis.

Adhesion assays

Cells were harvested with 0.01% trypsin in the presence of 2 mM CaCl2 and 0.5 mM MgSO4, and seeded into plates (1 x 104 cells/cm2) pre-coated with ECM including poly-D-lysine, collagen type I, collagen type IV, fibronectin or laminin (BD biosciences). BSA-coated plates were used as a negative control. After incubation for 30 min, the wells were washed with PBS to remove non-adherent cells. Adherent cells were counted using a WST-1, a tetrazolium salt, according to the procedure provided by the manufacturer (Takara Bio).

Pulse–chase analysis

Cultured cells on slide chambers were incubated with anti-ß1 integrin JB1A for 5 min at 37 °C, and then the excess antibody was washed off with medium at 4 °C. The pulse-labeled cells were chased by incubation in serum-free medium for various periods of time at 37 °C, fixed with 4% paraformaldehyde, then permeabilized with 0.4% Triton X-100. After blocking with 5% skim milk, the slides were incubated with Alexa Fluor 488-conjugated secondary antibody and visualized by confocal microscopy. The immunofluorescence intensities of ß1 integrin at the regions of cell–cell contacts or inside of the cells were measured using the histogram function of the Zeiss LSM 510 system. Both the regions were selected using the area button under the histogram function.

Statistical analysis

Statistical analysis was performed using Student's t test and the Kruskal–Wallis rank test with Scheffe's multiple comparison test. Statistical significance was defined as a value of P < 0.05.


   Acknowledgements
 
We are grateful to Professor S. Kondo for kindly providing us with the human cutaneous squamous carcinoma cell line, HSC-1. We also would like to thank Dr S. Hirohashi and Professor M. Takeichi for the gifts of the mouse anti-human E-cadherin antibody, HECD-1.


   Footnotes
 
Communicated by: Shigeo Hayashi

* Correspondence: E-mail: mukoyama_bux{at}mii.maruho.co.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Accepted: 19 March 2007




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