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Membrane type 1-matrix metalloproteinase expression is regulated by E-cadherin through the suppression of mitogen-activated protein kinase cascade
Cancer Letters 157 (2000) 115±121
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Membrane type 1-matrix metalloproteinase expression is regulated by E-cadherin through the suppression of mitogen-activated protein kinase cascade Toshiaki Ara a,*, Yoshiaki Deyama b, Yoshitaka Yoshimura b, Fumihiro Higashino c, Masanobu Shindoh c, Akira Matsumoto b, Hiroshi Fukuda a b
a First Department of Oral Surgery, School of Dentistry, Hokkaido University, Kita 13 Nishi 7, kita-ku, Sapporo 060-8586, Japan Department of Dental Pharmacology, School of Dentistry, Hokkaido University, Kita 13 Nishi 7, kita-ku, Sapporo 060-8586, Japan c Department of Oral Pathology, School of Dentistry, Hokkaido University, Kita 13 Nishi 7, kita-ku, Sapporo 060-8586, Japan
Received 24 December 1999; received in revised form 14 February 2000; accepted 15 February 2000
Abstract To elucidate the role of E-cadherin in matrix metalloproteinases (MMPs) expression, we transfected to squamous carcinoma cells with E-cadherin cDNA. HN5 cells and mock-transfected HN5-neo cells expressed proMMP-2 and active MMP-2. Ecadherin-transfected HN5-EC cells produced comparable proMMP-2 but low active MMP-2; and membrane type 1-MMP (MT1-MMP) mRNA declined. Phosphorylated ERK, a marker of mitogen-activated protein (MAP) kinase cascade, also declined in HN5-EC cells. The addition of anti-E-cadherin antibody resulted in the disappearance of these alterations in HN5-EC cells. These results suggest that E-cadherin suppresses MAP kinase cascade and down-regulates MT1-MMP. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: E-cadherin; Membrane type 1-matrix metalloproteinase; Mitogen-activated protein kinase cascade; Squamous carcinoma cell line
1. Introduction The invasion ability of tumor cells is one of the most important factors in tumor malignancy, and it is thought to be correlated with multiple factors such as cell growth, adhesiveness (cell±cell and cell±substratum adhesion), proteolytic enzyme production and cell motility [1]. The cell±cell adhesion of epithelial cells is mediated by E-cadherin, and E-cadherin plays important roles in the maintenance of epithelial cell polarity, the organization of epithelial tissues and epithelial cell differentiation [2]. The * Corresponding author. E-mail address: [email protected] (T. Ara).
reduction and loss of E-cadherin [3±5] and E-cadherin gene mutation [6±8] have been reported in many carcinomas. Recently, it is reported that the expression of E-cadherin and matrix metalloproteinases (MMPs) is inversely correlated in vivo [9,10] and that cell±cell adhesion mediated by E-cadherin regulates the expression of MMPs in vitro [11±13]. There have been a few reports concerning E-cadherin and MMPs expression; however, their interacting mechanism has not been documented in detail. Therefore, we transfected E-cadherin cDNA into an E-cadherin-negative squamous cell carcinoma cell line, HN5, to examine the alterations of MMPs and further their mechanisms.
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00494-8
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T. Ara et al. / Cancer Letters 157 (2000) 115±121
2. Materials and methods 2.1. Reconstruction of E-cadherin expression vector and cell cultures For the construction of human E-cadherin expression vector, E-cadherin cDNA was ampli®ed by reverse transcription-polymerase chain reaction (RT-PCR) with the use of a primer pair (5 0 GAAGGCT-AGCCAGACTCCAGCCCGCTCand 5 0 -G-GGGATCGATCTCTCTCCAG-3 0 GAGTCCCCTAGTC-3 0 ) and was inserted into NheI-ClaI site of pBK-CMV expression vector (Stratagene, La Jolla, CA). HN5 cells derived from moderately differentiated tongue squamous cell carcinoma [14], were kindly gifted by Dr A.S. Jones (Department of Otolaryngology/Head and Neck Surgery, Royal Liverpool University Hospital). They were cultured in Dulbecco's modi®ed Eagle medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum at 378C in humidi®ed 5% CO2. Transfection was performed by the electroporation method as described previously [15]. These cells were incubated with or without 10 mg/ml anti-E-cadherin antibody (HECD-1, Takara Biochemicals, Kyoto, Japan) or 50 mM PD98059 (Biomol Research Laboratories, Inc., Plymouth Meeting, MA). 2.2. Reverse transcription-polymerase chain reaction Total RNA was isolated with the use of ISOGEN (Nippon Gene Co., Toyama, Japan) from the cells in con¯uent cultures and was reverse transcribed with the use of Superscript Preampli®cation System (Gibco BRL), and was subjected to PCR. The primers for MMP-2 were 5 0 -CTGAACACCTTCTATGGCTG-3 0 and 5 0 -GTTGATCATGATGTCTGCCT-3 0 , those for membrane type 1 matrix metalloproteinase (MT1MMP) were 5 0 -AGGTGATCATCATTGAGGTGG-3 0 and 5 0 -ACAGAGAGAAGCAAGGAGGC-3 0 , those for tissue inhibitor of matrix metalloproteinase (TIMP-2) were 5 0 -GGAAGTGGACTCTGGAAACGACATT-3 0 and 5 0 -CTCCGATGTCGAGAAACTCCTGCTTG-3 0 and those for glyceraldehyde 3-phosphate dehydrogenase (G3PDH) were 5 0 -CGGAGTCAACGGATTTGGTCGTAT-3 0 and 5 0 -AGCCCTTCTCCATGGTGGTGAAGAC-3 0 . The PCR products were electrophoresed
in 1% agarose gel and visualized with SYBR Green I (Takara Biochemicals). 2.3. Immunoprecipitation and Western blotting The cells in con¯uent cultures were collected and dissolved in lysis buffer (50 mM Tris±HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF and 10 ml/ml aprotinin) for 30 min at 48C. The protein concentration was measured with the use of BCA Protein Assay Reagent kit (Pierce Chemical Co., Rockford, IL). Twenty micrograms of protein was mixed with HECD-1 for 2 h at 48C and then incubated with 20 ml of protein G-agarose (Boehringer Mannheim, Indianapolis, IN) for 1 h. The beads were washed three times with lysis buffer, then suspended in 20 ml of SDS sample buffer and boiled for 5 min. The supernatants were subjected to Western blotting. For Western blotting, 20 mg of protein was electrophoresed in polyacrylamide gel under reducing conditions and transferred onto polyvinylidene di¯uoride membrane (Hybond-P, Amersham Pharmacia Biotech, Uppsala, Sweden). The membrane was incubated with HECD-1, anti-a-catenin antibody (Transduction Laboratories Inc., Lexington, KY), anti-bcatenin antibody (Transduction Laboratories), antiERK1 antibody (Transduction Laboratories) or antip-ERK antibody (Santa Cruz Biotechnology) as primary antibody and with Horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology). Protein bands were visualized with ECL kit (Amersham Pharmacia Biotech). 2.4. Zymography The cells in con¯uent cultures were incubated in serum-free DMEM for 24 h. The conditioned media were collected and concentrated about 30-fold by Centriprep-10 (Amicon, Beverly, MA). Ten micrograms of protein was subjected to zymography as described previously [16]. 2.5. Immuno¯uorescent staining The cells were grown on 8-well Lab-Tek chamber (Nalge Nunc International, Rochester, NY) and then ®xed with 3% paraformaldehyde in phosphate-buffered
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saline (PBS) for 30 min. After several washing with PBS, the ®xed cells were permeabilized with in PBS containing 0.1% Triton X-100 (PBST) for 30 min. They were then incubated with HECD-1 for 60 min, followed by incubation with ¯ourescein isothiocyanate (FITC)-conjugated anti-mouse IgG antibody (DAKO Copenhagen, Denmark) for 30 min. After several washes with PBST, the cells were incubated with rhodamine±phalloidin (Molecular Probe, Eugene, OR) for 30 min and examined with a Zeiss LSM410 laser scanning microscope (Carl Zeiss, Inc., Thornwood, NY). 2.6. Densitometoric analysis The data of RT-PCR and Western blotting were semi-quantitatively analyzed using NIH Image 1.62 software on Macintosh computer. 3. Results 3.1. E-cadherin and catenin expressions and their association
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among all cell lines (Fig. 1C). MMP-9 band was not detected in any of all cell lines (data not shown). 3.3. MT1-MMP expression of transfected cells We examined mRNA level of MT1-MMP and TIMP-2 in these cell lines with or without HECD-1, which inhibits cell±cell adhesion. HN5 and HN5-neo cells showed the band of MT1-MMP mRNA with or without HECD-1. In contrast, the band was weak in HN5-EC-1 and HN5-EC-2 cells without HECD-1 but was comparable with those of HN5 and HN5-neo cells with HECD-1 (Fig. 2A). In densitometoric analysis, the intense of MT1-MMP mRNA of HN5-EC-1 and HN5-EC-2 cells without HECD-1 was about 20 and 15% that of HN5 cells, respectively, and that of HN5EC-1 and HN5-EC-2 cells with HECD-1 was 70 and 80%, respectively (Fig. 2B). The intensity of TIMP-2 mRNA was not signi®cantly varied with or without HECD-1 (from 85 to 135% that of HN5 cells without HECD-1).
We examine whether HN5 cells and each transfectant express E-cadherin, a-catenin and b-catenin, which associate to E-cadherin and regulate cell±cell adhesion. HN5 and HN5-neo cells did not express Ecadherin but HN5-EC cells did. All cell lines expressed both a- and b-catenin, and these molecules were co-immunoprecipitated with E-cadherin in HN5-EC-1 cells (Fig. 1A) and HN5-EC-2 cells (data not shown). 3.2. MMP expression of E-cadherin-transfected cells To examine if the transfection with E-cadherin affects the level of MMPs, we carried out zymography (Fig. 1B). HN5 cells had a intense zelatinolytic bands at 66 kDa and a faint band at 62 kDa. From their molecular weight these bands were thought to be proMMP-2 and active MMP-2, respectively. There was no apparent difference in proMMP-2 level among all cell lines, and active MMP-2 level of HN5-neo cells was nearly equal to that of HN5 cells. However, active MMP-2 level of HN5-EC cells was signi®cantly low (about 40% in densitometoric analysis). MMP-2 mRNA level was nearly equal
Fig. 1. E-cadherin expression and the effects on MMP-2 level. (A) E-cadherin and catenin expression and their association. (B) Zymography of HN5 cells and each transfectant in con¯uent cultures. Arrows indicate the positions of proMMP-2 (66 kDa) and active MMP-2 (62 kDa). (C) RT-PCR of MMP-2.
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the cell lines with or without HECD-1 (Fig. 4A). pERK bands (44 and 42 kDa) were weak in HN5EC-1 and HN5-EC-2 cells compared with HN5 and HN5-neo cells without HECD-1. In contrast, pERK bands in HN5-EC-1 and HN5-EC-2 cells were more intense with HECD-1 than those without HECD-1 (Fig. 4B). In densitometoric analysis, the intense of pERK of HN5-EC-1 and HN5-EC-2 cells was about 27 and 44% without HECD-1, respectively, and about 60 and 66% with HECD-1 that of HN5 cells, respectively (Fig. 4C). 3.6. Distribution of actin cytoskeleton of transfected cells
Fig. 2. Effects of E-cadherin expression on MT1-MMP and TIMP-2 mRNA level. The cells in con¯uent cultures were incubated in the absence (2) or presence of HECD-1 (10 mg/ml) for 24 h. (A) mRNA expressions of MT1-MMP and TIMP-2. (B) Densitometoric analysis.
3.4. Mitogen-activated protein kinase cascade activity and MT1-MMP expression HN5 cells were incubated with control dimethyl sulfoxide (DMSO) or mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059, for 24 h. The MAP kinase (ERK) level was not altered with or without PD98059 (Fig. 3A), while phosphorylated ERK (pERK) level was declined by PD98059 (Fig. 3B), and MT1-MMP mRNA was also declined (Fig. 3C). In densitometoric analysis, the intense of pERK and MT1-MMP mRNA was decreased to about 50% (data not shown). 3.5. MAP kinase cascade activity of transfected cells Total amount of ERK1 was not altered in any of all
E-cadherin was not obvious in HN5 cells (Fig. 5a) and actin ®laments distributed throughout the membrane and cytoplasm (Fig. 5c). Similar ®ndings of E-cadherin and actin ®laments were observed in HN5-neo cells (data not shown). In contrast, HN5EC cells showed intense expression of E-cadherin on the cell membrane (Fig. 5b) and actin ®laments were accumulated mostly on the cell membrane in the same distribution as that of E-cadherin (Fig. 5d) and that was con®rmed by the image that merged E-cadherin and actin (Fig. 5f). Similar ®ndings were observed in HN5-EC-2 cells (data not shown). 4. Discussion In this report, we showed that active MMP-2 was declined by E-cadherin transfection. Since the
Fig. 3. Effects of PD98059 on the phosphorylation of ERK and the expression of MT1-MMP mRNA. HN5 cells in con¯uent cultures were incubated with DMSO (2) or 50 mM PD98059 (PD) for 24 h. Western blotting was performed with the use of anti-ERK1 antibody (A) and anti-p-ERK antibody (B). (C) RT-PCR of MT1-MMP.
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that down-regulate MT1-MMP mRNA, some possible explanations are considered. One possibility is the signal pathway through MAP kinase cascade. It has been reported that E1AF, an ets oncogene family transcription factor, activates the expression of MT1-MMP [19] and that ets family transcription factors is activated through MAP kinase cascade [20]. Indeed, hepatocyte growth factor, which activates the MAP kinase cascade, enhances MT1MMP mRNA in MDCK cells [21]. Since MAP kinase (ERK), a downstream factor in MAP kinase cascade, is phosphorylated and activated by MAP kinase kinase, we evaluated MAP kinase cascade activity with pERK in this report. The results that PD98059 decreased both pERK and MT1-MMP suggest that MT1-MMP is regulated through MAP kinase cascade. Moreover, pERK was decreased by E-cadherin-transfection and was partially recovered by the inhibition of cell±cell adhesion. These results suggest that E-cadherin expression suppresses MAP kinase cascade and further down-regulates MT1MMP. The existence of some other pathways is Fig. 4. ERK expression and its phosphorylation level in the absence or presence of HECD-1. The cells in con¯uent cultures were incubated in the absence (2) or presence of HECD-1 (10 mg/ml) for 24 h. Western blotting was performed using anti-ERK1 antibody (A) and anti-p-ERK antibody (B). (C) Densitometoric analysis of pERK.
levels of MMP-2 mRNA and pro MMP-2 were not altered, the decrease of active MMP-2 must have occurred at the posttranslational level. It is known that proMMP-2 is activated by MT1-MMP [17] and repressed by TIMP-2 [18] and we revealed that MT1-MMP mRNA declined signi®cantly but not TIMP-2. The ®nding that cadherin±catenin complex was formed in E-cadherin-transfected cells indicates the contribution of E-cadherin to cell±cell adhesion. Moreover, the inhibition of cell±cell adhesion of HN5-EC cells with anti-E-cadherin antibody recovered MT1-MMP mRNA expression to those of HN5 and HN5-neo cells, suggesting that not the overexpression of E-cadherin but cell±cell adhesion regulates MT1-MMP expression. Recently, it is reported that E-cadherin-positive cell line produced little MT1-MMP compared with E-cadherin-negative cell [9], supporting our results. For the mechanisms
Fig. 5. Immuno¯uorescent staining for E-cadherin and actin ®laments. (a,c,e) HN5 cells. (b,d,f) HN5-EC-1 cells. (a,b) E-cadherin staining. (c,d) Actin ®laments staining in corresponding ®elds. (e,f) Merged images of E-cadherin and actin. Bars, 50 mm.
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considered to be the reason for the inhibition of cell±cell adhesion not completely recovering the pERK level. Second, the organization of actin cytoskeleton has been reported to have a potent role for extracellular matrix degradation. When the cytoskeletal structure is disrupted by colchicine, cytochalasin or the anti-E-cadherin antibody, the expression of some proteases is up-regulated [22,23]. Moreover, ®broblasts, which adhere to extracellular matrix, express little MT1-MMP mRNA but the cells mechanically relaxed in collagen lattices lose actin bundles and express MT1-MMP mRNA [24]. In this report, we found that E-cadherin transfection altered distribution of actin ®laments as reported previously [25,26] and that active MMP-2 and MT1-MMP level declined. These ®ndings strongly suggest that the structure of actin cytoskeleton is closely correlated with MT1MMP expression. Although it is considered that Ecadherin-mediated cytoskeletal organization suppress MAP kinase cascade, this mechanism remain elucidated. In conclusion, we demonstrated that E-cadherin-transfection apparently led to downregulation of MT1-MMP mRNA and that this alteration might be mediated possibly through the suppression of MAP kinase cascade and/or cytoskeletal organization. Although the mechanisms of signal pathways after E-cadherin expression are not fully understood yet, it is suggested that Ecadherin expression and MT1-MMP level are not necessarily independent events and that E-cadherin is one of the important factors that affect MT1MMP expression level. Acknowledgements We thank Drs A.S. Jones and N. Andrew (Department of Otolaryngology/Head and Neck Surgery, Royal Liverpool University Hospital) for providing HN5 cells. References [1] E. Kawahara, Y. Okada, I. Nakanishi, K. Iwata, S. Kojima, S. Kumagai, E. Yamamoto, The expression of invasive behavior of differentiated squamous carcinoma cell line evaluated by an in vitro invasion model, Jpn. J. Cancer Res. 84 (1993) 409±418.
[2] M. Takeichi, Cadherin cell adhesion receptors as a morphogenetic regulator, Science 251 (1991) 1451±1455. [3] J.H. Schipper, U.H. Frixen, J. Behrens, A. Unger, K. Jahnke, W. Birchmeier, E-cadherin expression in squamous cell carcinomas of head and neck: inverse correlation with tumor dedifferentiation and lymph node metastasis, Cancer Res. 51 (1991) 6328±6337. [4] Y. Shimoyama, S. Hirohashi, S. Hirano, M. Noguchi, Y. Shimosato, M. Takeichi, O. Abe, Cadherin cell-adhesion molecules in human epithelial tissues and carcinomas, Cancer Res. 49 (1989) 2128±2133. [5] R. Moll, M. Mitze, U.H. Frixen, W. Birchmeier, Differential loss of E-cadherin expression in in®ltrating ductal and lobular breast carcinomas, Am. J. Pathol. 143 (1993) 1731±1742. [6] K.F. Becker, M.J. Atkinson, U. Reich, H.H. Huang, H. Nekarda, J.R. Siewert, H. Ho¯er, Exon skipping in the Ecadherin gene transcript in metastatic human gastric carcinomas, Hum. Mol. Genet. 2 (1993) 803±804. [7] T. Oda, Y. Kanai, T. Oyama, K. Yoshiura, Y. Shimoyama, W. Birchmeier, T. Sugimura, S. Hirohashi, E-cadherin gene mutations in human gastric carcinoma cell lines, Proc. Natl. Acad. Sci. USA 91 (1994) 1858±1862. [8] Y. Kanai, T. Oda, H. Tsuda, A. Ochiai, S. Hirohashi, Point mutation of the E-cadherin gene in invasive lobular carcinoma of the breast, Jpn. J. Cancer Res. 85 (1994) 1035±1039. [9] M. Polette, C. Gilles, S. de Bentzmann, D. Gruenert, J.M. Tournier, P. Birembaut, Association of ®broblastoid features with the invasive phenotype in human bronchial cancer cell lines, Clin. Exp. Metastasis 16 (1998) 105±112. [10] Y. Kitadai, L.M. Ellis, S.L. Tucker, G.F. Greene, C.D. Bucana, K.R. Cleary, Y. Takahashi, E. Tahara, I.J. Fidler, Multiparametric in situ mRNA hybridization analysis to predict disease recurrence in patients with colon carcinoma, Am. J. Pathol. 149 (1996) 1541±1551. [11] M. Miyaki, K. Tanaka, R. Kikuchi-Yanoshita, M. Muraoka, M. Konishi, M. Takeichi, Increased cell±substratum adhesion, and decreased gelatinase secretion and cell growth, induced by E-cadherin transfection of human colon carcinoma cells, Oncogene 11 (1995) 2547±2552. [12] A.H. Borchers, L.A. Sanders, G.T. Bowden, Regulation of matrilysin expression in cells of squamous cell carcinoma by E-cadherin-mediated cell±cell contact, J. Cancer Res. Clin. Oncol. 123 (1997) 13±20. [13] A. Llorens, I. Rodrigo, L. Lopez-Barcons, M. Gonzalez-Garrigues, E. Lozano, A. Vinyals, M. Quintanilla, A. Cano, A. Fabra, Down-regulation of E-cadherin in mouse skin carcinoma cells enhances a migratory and invasive phenotype linked to matrix metalloproteinase-9 gelatinase expression, Lab. Invest. 78 (1998) 1131±1142. [14] D.M. Easty, G.C. Easty, R.L. Carter, P. Monaghan, L.J. Butler, Ten human carcinoma cell lines derived from squamous carcinomas of the head and neck, Br. J. Cancer 43 (1981) 772±785. [15] J. Sambrook, E.F. Fritsch, T. Maniatis, Expression of cloned genes in cultured mammalian cells, in: I. Nina (Ed.), Molecular Cloning: a Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, New York, 1989, pp. 16.54±16.55.
T. Ara et al. / Cancer Letters 157 (2000) 115±121 [16] C. Heussen, E.B. Dowdle, Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates, Anal. Biochem. 102 (1980) 196±202. [17] H. Sato, M. Seiki, Membrane-type matrix metalloproteinases (MT-MMPs) in tumor metastasis, J. Biochem. 119 (1996) 209±215. [18] H. Kolkenbrock, D. Orgel, A. Hecker-Kia, W. Noack, N. Ulbrich, The complex between a tissue inhibitor of metalloproteinases (TIMP-2) and 72-kDa progelatinase is a metalloproteinase inhibitor, Eur. J. Biochem. 198 (1991) 775±781. [19] H. Habelhah, F. Okada, M. Kobayashi, K. Nakai, S. Choi, J. Hamada, T. Moriuchi, M. Kaya, K. Yoshida, K. Fujinaga, M. Hosokawa, Increased E1AF expression in mouse ®brosarcoma promotes metastasis through induction of MT1-MMP expression, Oncogene 18 (1999) 1771±1776. [20] B. Rabault, M.F. Roussel, C.T. Quang, J. Ghysdael, Phosphorylation of Ets1 regulates the complementation of a CSF-1 receptor impaired in mitogenesis, Oncogene 13 (1996) 877± 881. [21] Y. Kadono, K. Shibahara, M. Namiki, Y. Watanabe, M. Seiki, H. Sato, Membrane type 1-matrix metalloproteinase is
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involved in the formation of hepatocyte growth factor/scatter factor-induced branching tubules in madin-darby canine kidney epithelial cells, Biochem. Biophys. Res. Commun. 251 (1998) 681±687. U.H. Frixen, Y. Nagamine, Stimulation of urokinase-type plasminogen activator expression by blockage of Ecadherin-dependent cell±cell adhesion, Cancer Res. 53 (1993) 3618±3623. F.M. Botteri, K. Ballmer-Hofer, B. Rajput, Y. Nagamine, Disruption of cytoskeletal structures results in the induction of the urokinase-type plasminogen activator gene expression, J. Biol. Chem. 265 (1990) 13327±13334. J.J. Tomasek, N.L. Halliday, D.L. Updike, J.S. Ahern-Moore, T.K. Vu, R.W. Liu, E.W. Howard, Gelatinase A activation is regulated by the organization of the polymerized actin cytoskeleton, J. Biol. Chem. 272 (1997) 7482±7487. D.F. Aghib, P.D. McCrea, The E-cadherin complex contains the src substrate p120, Exp. Cell Res. 218 (1995) 359±369. Y. Doki, H. Shiozaki, H. Tahara, M. Inoue, H. Oka, K. Iihara, T. Kadowaki, M. Takeichi, T. Mori, Correlation between Ecadherin expression and invasiveness in vitro in a human esophageal cancer cell line, Cancer Res. 53 (1993) 3421±3426.
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Membrane type 1-matrix metalloproteinase expression is regulated by E-cadherin through the suppression of mitogen-activated protein kinase cascade Toshiaki Ara a,*, Yoshiaki Deyama b, Yoshitaka Yoshimura b, Fumihiro Higashino c, Masanobu Shindoh c, Akira Matsumoto b, Hiroshi Fukuda a b
a First Department of Oral Surgery, School of Dentistry, Hokkaido University, Kita 13 Nishi 7, kita-ku, Sapporo 060-8586, Japan Department of Dental Pharmacology, School of Dentistry, Hokkaido University, Kita 13 Nishi 7, kita-ku, Sapporo 060-8586, Japan c Department of Oral Pathology, School of Dentistry, Hokkaido University, Kita 13 Nishi 7, kita-ku, Sapporo 060-8586, Japan
Received 24 December 1999; received in revised form 14 February 2000; accepted 15 February 2000
Abstract To elucidate the role of E-cadherin in matrix metalloproteinases (MMPs) expression, we transfected to squamous carcinoma cells with E-cadherin cDNA. HN5 cells and mock-transfected HN5-neo cells expressed proMMP-2 and active MMP-2. Ecadherin-transfected HN5-EC cells produced comparable proMMP-2 but low active MMP-2; and membrane type 1-MMP (MT1-MMP) mRNA declined. Phosphorylated ERK, a marker of mitogen-activated protein (MAP) kinase cascade, also declined in HN5-EC cells. The addition of anti-E-cadherin antibody resulted in the disappearance of these alterations in HN5-EC cells. These results suggest that E-cadherin suppresses MAP kinase cascade and down-regulates MT1-MMP. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: E-cadherin; Membrane type 1-matrix metalloproteinase; Mitogen-activated protein kinase cascade; Squamous carcinoma cell line
1. Introduction The invasion ability of tumor cells is one of the most important factors in tumor malignancy, and it is thought to be correlated with multiple factors such as cell growth, adhesiveness (cell±cell and cell±substratum adhesion), proteolytic enzyme production and cell motility [1]. The cell±cell adhesion of epithelial cells is mediated by E-cadherin, and E-cadherin plays important roles in the maintenance of epithelial cell polarity, the organization of epithelial tissues and epithelial cell differentiation [2]. The * Corresponding author. E-mail address: [email protected] (T. Ara).
reduction and loss of E-cadherin [3±5] and E-cadherin gene mutation [6±8] have been reported in many carcinomas. Recently, it is reported that the expression of E-cadherin and matrix metalloproteinases (MMPs) is inversely correlated in vivo [9,10] and that cell±cell adhesion mediated by E-cadherin regulates the expression of MMPs in vitro [11±13]. There have been a few reports concerning E-cadherin and MMPs expression; however, their interacting mechanism has not been documented in detail. Therefore, we transfected E-cadherin cDNA into an E-cadherin-negative squamous cell carcinoma cell line, HN5, to examine the alterations of MMPs and further their mechanisms.
0304-3835/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(00)00494-8
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2. Materials and methods 2.1. Reconstruction of E-cadherin expression vector and cell cultures For the construction of human E-cadherin expression vector, E-cadherin cDNA was ampli®ed by reverse transcription-polymerase chain reaction (RT-PCR) with the use of a primer pair (5 0 GAAGGCT-AGCCAGACTCCAGCCCGCTCand 5 0 -G-GGGATCGATCTCTCTCCAG-3 0 GAGTCCCCTAGTC-3 0 ) and was inserted into NheI-ClaI site of pBK-CMV expression vector (Stratagene, La Jolla, CA). HN5 cells derived from moderately differentiated tongue squamous cell carcinoma [14], were kindly gifted by Dr A.S. Jones (Department of Otolaryngology/Head and Neck Surgery, Royal Liverpool University Hospital). They were cultured in Dulbecco's modi®ed Eagle medium (DMEM; Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum at 378C in humidi®ed 5% CO2. Transfection was performed by the electroporation method as described previously [15]. These cells were incubated with or without 10 mg/ml anti-E-cadherin antibody (HECD-1, Takara Biochemicals, Kyoto, Japan) or 50 mM PD98059 (Biomol Research Laboratories, Inc., Plymouth Meeting, MA). 2.2. Reverse transcription-polymerase chain reaction Total RNA was isolated with the use of ISOGEN (Nippon Gene Co., Toyama, Japan) from the cells in con¯uent cultures and was reverse transcribed with the use of Superscript Preampli®cation System (Gibco BRL), and was subjected to PCR. The primers for MMP-2 were 5 0 -CTGAACACCTTCTATGGCTG-3 0 and 5 0 -GTTGATCATGATGTCTGCCT-3 0 , those for membrane type 1 matrix metalloproteinase (MT1MMP) were 5 0 -AGGTGATCATCATTGAGGTGG-3 0 and 5 0 -ACAGAGAGAAGCAAGGAGGC-3 0 , those for tissue inhibitor of matrix metalloproteinase (TIMP-2) were 5 0 -GGAAGTGGACTCTGGAAACGACATT-3 0 and 5 0 -CTCCGATGTCGAGAAACTCCTGCTTG-3 0 and those for glyceraldehyde 3-phosphate dehydrogenase (G3PDH) were 5 0 -CGGAGTCAACGGATTTGGTCGTAT-3 0 and 5 0 -AGCCCTTCTCCATGGTGGTGAAGAC-3 0 . The PCR products were electrophoresed
in 1% agarose gel and visualized with SYBR Green I (Takara Biochemicals). 2.3. Immunoprecipitation and Western blotting The cells in con¯uent cultures were collected and dissolved in lysis buffer (50 mM Tris±HCl (pH 7.4), 1% NP-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM sodium orthovanadate, 1 mM NaF, 1 mM PMSF and 10 ml/ml aprotinin) for 30 min at 48C. The protein concentration was measured with the use of BCA Protein Assay Reagent kit (Pierce Chemical Co., Rockford, IL). Twenty micrograms of protein was mixed with HECD-1 for 2 h at 48C and then incubated with 20 ml of protein G-agarose (Boehringer Mannheim, Indianapolis, IN) for 1 h. The beads were washed three times with lysis buffer, then suspended in 20 ml of SDS sample buffer and boiled for 5 min. The supernatants were subjected to Western blotting. For Western blotting, 20 mg of protein was electrophoresed in polyacrylamide gel under reducing conditions and transferred onto polyvinylidene di¯uoride membrane (Hybond-P, Amersham Pharmacia Biotech, Uppsala, Sweden). The membrane was incubated with HECD-1, anti-a-catenin antibody (Transduction Laboratories Inc., Lexington, KY), anti-bcatenin antibody (Transduction Laboratories), antiERK1 antibody (Transduction Laboratories) or antip-ERK antibody (Santa Cruz Biotechnology) as primary antibody and with Horseradish peroxidaseconjugated secondary antibody (Santa Cruz Biotechnology). Protein bands were visualized with ECL kit (Amersham Pharmacia Biotech). 2.4. Zymography The cells in con¯uent cultures were incubated in serum-free DMEM for 24 h. The conditioned media were collected and concentrated about 30-fold by Centriprep-10 (Amicon, Beverly, MA). Ten micrograms of protein was subjected to zymography as described previously [16]. 2.5. Immuno¯uorescent staining The cells were grown on 8-well Lab-Tek chamber (Nalge Nunc International, Rochester, NY) and then ®xed with 3% paraformaldehyde in phosphate-buffered
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saline (PBS) for 30 min. After several washing with PBS, the ®xed cells were permeabilized with in PBS containing 0.1% Triton X-100 (PBST) for 30 min. They were then incubated with HECD-1 for 60 min, followed by incubation with ¯ourescein isothiocyanate (FITC)-conjugated anti-mouse IgG antibody (DAKO Copenhagen, Denmark) for 30 min. After several washes with PBST, the cells were incubated with rhodamine±phalloidin (Molecular Probe, Eugene, OR) for 30 min and examined with a Zeiss LSM410 laser scanning microscope (Carl Zeiss, Inc., Thornwood, NY). 2.6. Densitometoric analysis The data of RT-PCR and Western blotting were semi-quantitatively analyzed using NIH Image 1.62 software on Macintosh computer. 3. Results 3.1. E-cadherin and catenin expressions and their association
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among all cell lines (Fig. 1C). MMP-9 band was not detected in any of all cell lines (data not shown). 3.3. MT1-MMP expression of transfected cells We examined mRNA level of MT1-MMP and TIMP-2 in these cell lines with or without HECD-1, which inhibits cell±cell adhesion. HN5 and HN5-neo cells showed the band of MT1-MMP mRNA with or without HECD-1. In contrast, the band was weak in HN5-EC-1 and HN5-EC-2 cells without HECD-1 but was comparable with those of HN5 and HN5-neo cells with HECD-1 (Fig. 2A). In densitometoric analysis, the intense of MT1-MMP mRNA of HN5-EC-1 and HN5-EC-2 cells without HECD-1 was about 20 and 15% that of HN5 cells, respectively, and that of HN5EC-1 and HN5-EC-2 cells with HECD-1 was 70 and 80%, respectively (Fig. 2B). The intensity of TIMP-2 mRNA was not signi®cantly varied with or without HECD-1 (from 85 to 135% that of HN5 cells without HECD-1).
We examine whether HN5 cells and each transfectant express E-cadherin, a-catenin and b-catenin, which associate to E-cadherin and regulate cell±cell adhesion. HN5 and HN5-neo cells did not express Ecadherin but HN5-EC cells did. All cell lines expressed both a- and b-catenin, and these molecules were co-immunoprecipitated with E-cadherin in HN5-EC-1 cells (Fig. 1A) and HN5-EC-2 cells (data not shown). 3.2. MMP expression of E-cadherin-transfected cells To examine if the transfection with E-cadherin affects the level of MMPs, we carried out zymography (Fig. 1B). HN5 cells had a intense zelatinolytic bands at 66 kDa and a faint band at 62 kDa. From their molecular weight these bands were thought to be proMMP-2 and active MMP-2, respectively. There was no apparent difference in proMMP-2 level among all cell lines, and active MMP-2 level of HN5-neo cells was nearly equal to that of HN5 cells. However, active MMP-2 level of HN5-EC cells was signi®cantly low (about 40% in densitometoric analysis). MMP-2 mRNA level was nearly equal
Fig. 1. E-cadherin expression and the effects on MMP-2 level. (A) E-cadherin and catenin expression and their association. (B) Zymography of HN5 cells and each transfectant in con¯uent cultures. Arrows indicate the positions of proMMP-2 (66 kDa) and active MMP-2 (62 kDa). (C) RT-PCR of MMP-2.
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the cell lines with or without HECD-1 (Fig. 4A). pERK bands (44 and 42 kDa) were weak in HN5EC-1 and HN5-EC-2 cells compared with HN5 and HN5-neo cells without HECD-1. In contrast, pERK bands in HN5-EC-1 and HN5-EC-2 cells were more intense with HECD-1 than those without HECD-1 (Fig. 4B). In densitometoric analysis, the intense of pERK of HN5-EC-1 and HN5-EC-2 cells was about 27 and 44% without HECD-1, respectively, and about 60 and 66% with HECD-1 that of HN5 cells, respectively (Fig. 4C). 3.6. Distribution of actin cytoskeleton of transfected cells
Fig. 2. Effects of E-cadherin expression on MT1-MMP and TIMP-2 mRNA level. The cells in con¯uent cultures were incubated in the absence (2) or presence of HECD-1 (10 mg/ml) for 24 h. (A) mRNA expressions of MT1-MMP and TIMP-2. (B) Densitometoric analysis.
3.4. Mitogen-activated protein kinase cascade activity and MT1-MMP expression HN5 cells were incubated with control dimethyl sulfoxide (DMSO) or mitogen-activated protein (MAP) kinase kinase inhibitor, PD98059, for 24 h. The MAP kinase (ERK) level was not altered with or without PD98059 (Fig. 3A), while phosphorylated ERK (pERK) level was declined by PD98059 (Fig. 3B), and MT1-MMP mRNA was also declined (Fig. 3C). In densitometoric analysis, the intense of pERK and MT1-MMP mRNA was decreased to about 50% (data not shown). 3.5. MAP kinase cascade activity of transfected cells Total amount of ERK1 was not altered in any of all
E-cadherin was not obvious in HN5 cells (Fig. 5a) and actin ®laments distributed throughout the membrane and cytoplasm (Fig. 5c). Similar ®ndings of E-cadherin and actin ®laments were observed in HN5-neo cells (data not shown). In contrast, HN5EC cells showed intense expression of E-cadherin on the cell membrane (Fig. 5b) and actin ®laments were accumulated mostly on the cell membrane in the same distribution as that of E-cadherin (Fig. 5d) and that was con®rmed by the image that merged E-cadherin and actin (Fig. 5f). Similar ®ndings were observed in HN5-EC-2 cells (data not shown). 4. Discussion In this report, we showed that active MMP-2 was declined by E-cadherin transfection. Since the
Fig. 3. Effects of PD98059 on the phosphorylation of ERK and the expression of MT1-MMP mRNA. HN5 cells in con¯uent cultures were incubated with DMSO (2) or 50 mM PD98059 (PD) for 24 h. Western blotting was performed with the use of anti-ERK1 antibody (A) and anti-p-ERK antibody (B). (C) RT-PCR of MT1-MMP.
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that down-regulate MT1-MMP mRNA, some possible explanations are considered. One possibility is the signal pathway through MAP kinase cascade. It has been reported that E1AF, an ets oncogene family transcription factor, activates the expression of MT1-MMP [19] and that ets family transcription factors is activated through MAP kinase cascade [20]. Indeed, hepatocyte growth factor, which activates the MAP kinase cascade, enhances MT1MMP mRNA in MDCK cells [21]. Since MAP kinase (ERK), a downstream factor in MAP kinase cascade, is phosphorylated and activated by MAP kinase kinase, we evaluated MAP kinase cascade activity with pERK in this report. The results that PD98059 decreased both pERK and MT1-MMP suggest that MT1-MMP is regulated through MAP kinase cascade. Moreover, pERK was decreased by E-cadherin-transfection and was partially recovered by the inhibition of cell±cell adhesion. These results suggest that E-cadherin expression suppresses MAP kinase cascade and further down-regulates MT1MMP. The existence of some other pathways is Fig. 4. ERK expression and its phosphorylation level in the absence or presence of HECD-1. The cells in con¯uent cultures were incubated in the absence (2) or presence of HECD-1 (10 mg/ml) for 24 h. Western blotting was performed using anti-ERK1 antibody (A) and anti-p-ERK antibody (B). (C) Densitometoric analysis of pERK.
levels of MMP-2 mRNA and pro MMP-2 were not altered, the decrease of active MMP-2 must have occurred at the posttranslational level. It is known that proMMP-2 is activated by MT1-MMP [17] and repressed by TIMP-2 [18] and we revealed that MT1-MMP mRNA declined signi®cantly but not TIMP-2. The ®nding that cadherin±catenin complex was formed in E-cadherin-transfected cells indicates the contribution of E-cadherin to cell±cell adhesion. Moreover, the inhibition of cell±cell adhesion of HN5-EC cells with anti-E-cadherin antibody recovered MT1-MMP mRNA expression to those of HN5 and HN5-neo cells, suggesting that not the overexpression of E-cadherin but cell±cell adhesion regulates MT1-MMP expression. Recently, it is reported that E-cadherin-positive cell line produced little MT1-MMP compared with E-cadherin-negative cell [9], supporting our results. For the mechanisms
Fig. 5. Immuno¯uorescent staining for E-cadherin and actin ®laments. (a,c,e) HN5 cells. (b,d,f) HN5-EC-1 cells. (a,b) E-cadherin staining. (c,d) Actin ®laments staining in corresponding ®elds. (e,f) Merged images of E-cadherin and actin. Bars, 50 mm.
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considered to be the reason for the inhibition of cell±cell adhesion not completely recovering the pERK level. Second, the organization of actin cytoskeleton has been reported to have a potent role for extracellular matrix degradation. When the cytoskeletal structure is disrupted by colchicine, cytochalasin or the anti-E-cadherin antibody, the expression of some proteases is up-regulated [22,23]. Moreover, ®broblasts, which adhere to extracellular matrix, express little MT1-MMP mRNA but the cells mechanically relaxed in collagen lattices lose actin bundles and express MT1-MMP mRNA [24]. In this report, we found that E-cadherin transfection altered distribution of actin ®laments as reported previously [25,26] and that active MMP-2 and MT1-MMP level declined. These ®ndings strongly suggest that the structure of actin cytoskeleton is closely correlated with MT1MMP expression. Although it is considered that Ecadherin-mediated cytoskeletal organization suppress MAP kinase cascade, this mechanism remain elucidated. In conclusion, we demonstrated that E-cadherin-transfection apparently led to downregulation of MT1-MMP mRNA and that this alteration might be mediated possibly through the suppression of MAP kinase cascade and/or cytoskeletal organization. Although the mechanisms of signal pathways after E-cadherin expression are not fully understood yet, it is suggested that Ecadherin expression and MT1-MMP level are not necessarily independent events and that E-cadherin is one of the important factors that affect MT1MMP expression level. Acknowledgements We thank Drs A.S. Jones and N. Andrew (Department of Otolaryngology/Head and Neck Surgery, Royal Liverpool University Hospital) for providing HN5 cells. References [1] E. Kawahara, Y. Okada, I. Nakanishi, K. Iwata, S. Kojima, S. Kumagai, E. Yamamoto, The expression of invasive behavior of differentiated squamous carcinoma cell line evaluated by an in vitro invasion model, Jpn. J. Cancer Res. 84 (1993) 409±418.
[2] M. Takeichi, Cadherin cell adhesion receptors as a morphogenetic regulator, Science 251 (1991) 1451±1455. [3] J.H. Schipper, U.H. Frixen, J. Behrens, A. Unger, K. Jahnke, W. Birchmeier, E-cadherin expression in squamous cell carcinomas of head and neck: inverse correlation with tumor dedifferentiation and lymph node metastasis, Cancer Res. 51 (1991) 6328±6337. [4] Y. Shimoyama, S. Hirohashi, S. Hirano, M. Noguchi, Y. Shimosato, M. Takeichi, O. Abe, Cadherin cell-adhesion molecules in human epithelial tissues and carcinomas, Cancer Res. 49 (1989) 2128±2133. [5] R. Moll, M. Mitze, U.H. Frixen, W. Birchmeier, Differential loss of E-cadherin expression in in®ltrating ductal and lobular breast carcinomas, Am. J. Pathol. 143 (1993) 1731±1742. [6] K.F. Becker, M.J. Atkinson, U. Reich, H.H. Huang, H. Nekarda, J.R. Siewert, H. Ho¯er, Exon skipping in the Ecadherin gene transcript in metastatic human gastric carcinomas, Hum. Mol. Genet. 2 (1993) 803±804. [7] T. Oda, Y. Kanai, T. Oyama, K. Yoshiura, Y. Shimoyama, W. Birchmeier, T. Sugimura, S. Hirohashi, E-cadherin gene mutations in human gastric carcinoma cell lines, Proc. Natl. Acad. Sci. USA 91 (1994) 1858±1862. [8] Y. Kanai, T. Oda, H. Tsuda, A. Ochiai, S. Hirohashi, Point mutation of the E-cadherin gene in invasive lobular carcinoma of the breast, Jpn. J. Cancer Res. 85 (1994) 1035±1039. [9] M. Polette, C. Gilles, S. de Bentzmann, D. Gruenert, J.M. Tournier, P. Birembaut, Association of ®broblastoid features with the invasive phenotype in human bronchial cancer cell lines, Clin. Exp. Metastasis 16 (1998) 105±112. [10] Y. Kitadai, L.M. Ellis, S.L. Tucker, G.F. Greene, C.D. Bucana, K.R. Cleary, Y. Takahashi, E. Tahara, I.J. Fidler, Multiparametric in situ mRNA hybridization analysis to predict disease recurrence in patients with colon carcinoma, Am. J. Pathol. 149 (1996) 1541±1551. [11] M. Miyaki, K. Tanaka, R. Kikuchi-Yanoshita, M. Muraoka, M. Konishi, M. Takeichi, Increased cell±substratum adhesion, and decreased gelatinase secretion and cell growth, induced by E-cadherin transfection of human colon carcinoma cells, Oncogene 11 (1995) 2547±2552. [12] A.H. Borchers, L.A. Sanders, G.T. Bowden, Regulation of matrilysin expression in cells of squamous cell carcinoma by E-cadherin-mediated cell±cell contact, J. Cancer Res. Clin. Oncol. 123 (1997) 13±20. [13] A. Llorens, I. Rodrigo, L. Lopez-Barcons, M. Gonzalez-Garrigues, E. Lozano, A. Vinyals, M. Quintanilla, A. Cano, A. Fabra, Down-regulation of E-cadherin in mouse skin carcinoma cells enhances a migratory and invasive phenotype linked to matrix metalloproteinase-9 gelatinase expression, Lab. Invest. 78 (1998) 1131±1142. [14] D.M. Easty, G.C. Easty, R.L. Carter, P. Monaghan, L.J. Butler, Ten human carcinoma cell lines derived from squamous carcinomas of the head and neck, Br. J. Cancer 43 (1981) 772±785. [15] J. Sambrook, E.F. Fritsch, T. Maniatis, Expression of cloned genes in cultured mammalian cells, in: I. Nina (Ed.), Molecular Cloning: a Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, New York, 1989, pp. 16.54±16.55.
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