* Department of Pathology, College of Medicine, University of Arizona, Tucson, Arizona; and
Department of Medical Pharmacology and Toxicology, College of Medicine, Texas A&M University System Health Science Center, College Station, Texas
Received November 30, 2000; accepted February 20, 2001
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ABSTRACT |
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Key Words: cadherin; catenin; oxidative stress; precision-cut liver slices.
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INTRODUCTION |
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A common link between several liver diseases is the disruption of normal intracellular redox status, commonly referred to as oxidative stress (reviewed in Kaplowitz and Tsukamoto, 1996). Oxidative stress has been associated with hepatic bacterial and viral infections (Larrea et al., 1998; Sipowicz et al., 1997
), diabetes (Traverso et al., 1999
), xenobiotic-induced hepatotoxicity (Unemura et al., 1999
), chronic ethanol consumption (Fataccioli et al., 1999
), and hepatocellular carcinoma (Factor et al., 1998
). Findings from animal models are supported by data from human patients demonstrating that the oxidized form of plasma ubiquinone is significantly increased and plasma ascorbate levels decreased in chronic active hepatitis, liver cirrhosis, and hepatocellular carcinoma (Yamamoto et al., 1998
). Although oxidative stress has been extensively linked to hepatocyte death (Rosser and Gores, 1995
), little is known about the potential impact of non-lethal oxidative stress on hepatocyte function. Disruption of cellcell adhesion, however, is associated with the accumulation of inflammatory cells, an event associated with several hepatopathologies associated with oxidative stress (Kaplanski et al., 1997
). Thus, intercellular adhesion may be disrupted by oxidative stress in the liver. This suggestion is supported by the finding that elevated circulating levels of adhesion molecules are associated with hepatitis, cirrhosis, and cancer (Katayama et al., 1994
; Lucka et al., 1998
).
Previous work has shown that chemically induced oxidative stress disrupts protein interactions of the ß-catenin with both E-cadherin and -catenin (Parrish et al., 1999
). However, the impact of oxidative stress on the N-cadherin and
-catenin complexes has not been investigated. In hepatocellular carcinomas, a significant loss of E-cadherin, but not N-cadherin, is seen (Kozyraki et al., 1996
), suggesting that distinct complexes may have fundamental differences in respect to complex stability. The current studies were designed to examine the impact of non-cytolethal oxidative stress on the expression and protein interactions of each of the cadherin/catenin complexes present in the liver.
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MATERIALS AND METHODS |
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Precision-cut mouse liver slices.
Male CD-1 mice (2025 g) were purchased from Charles River (Wilmington, MA). Following a 1-week acclimation period, the livers were excised and chilled in Krebs-bicarbonate buffer. Cylindrical cores (8-mm diameter) were cut and slices (275 µm thick) were produced using the Brendel/Vitron tissue slicer. The slices were incubated on mesh screen rollers with DMEM/F12 medium for 1 h at 37°C. At this time, slices were challenged with diamide (DA; 25 or 250 µM) or tert-butylhydroperoxide (tBHP; 5- or 50 µM) for 6 h. These concentrations are not associated with cytolethality as assessed by biochemical evaluation (Parrish et al., 1999). All experiments were performed within 3 weeks following the arrival of the mice.
Western blot/immunoprecipitation.
Slices were homogenized in 500 µl of lysis buffer (4% SDS) and 20 µg of protein separated on an 8% polyacrylamide gel. Proteins were transferred to nitrocellulose membranes and blocked overnight with a 5% non-fat milk solution. Monoclonal antibodies (Transduction Laboratories) against N-cadherin, -catenin, and p120 (all 1:5000) were added for 2 h at room temperature. Membranes were washed and incubated with a secondary antibody conjugated to horseradish peroxidase (1:12500) prior to washing and detection via ECL.
For immunoprecipitation studies, slices were homogenized in immunoprecipitation buffer (1% Triton X-100, 150 mM NaCl2, 10 mM Tris, pH 7.4, 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium orthovanadate, 0.2 mM PMSF, 0.5% NP-40) and 300 µg of protein incubated for 1 h with 3 µg of antibody (ß- and -catenin, polyclonal, Santa Cruz; p120 monoclonal, Transduction Laboratories). GammaBind plus Sepharose® (20 µl) was added and the samples incubated overnight. The immunoprecipitates were collected by centrifugation and processed for Western-blot analysis.
Confocal microscopy.
Following chemical challenges, slices were snap-frozen in isopentane pre-chilled in an LN2 bath. Thin (5 µm) frozen sections were placed on slides. The slides were fixed for 10 min in 20°C acetone followed by air drying. Primary antibodies were applied for 30 min, followed by 3 5-min PBS washes, before application of secondary antibodies for 30 min. After secondary antibody binding, specimens were then washed again 3 x 5 min in PBS, rinsed briefly in water and then fixed in ethanol for 5 min, air-dried, and mounted.
Confocal laser scanning-immunofluorescence microscopy was done on a Zeiss LSM 410 UV (Carl Zeiss). For double-label fluorescence, an argon/krypton ion laser operating at 488 nm and 568 nm was used together with a long-pass filter 590 for visualization of Cy 3 fluorescence and a band-pass filter 515540 for visualization of FITC and Cy 2 fluorescence, respectively. RGB images were taken in high-resolution mode using 1,024 x 1,024 image points (pixels) and 8-s scan time. Noise levels were reduced by line averaging of the scans.
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RESULTS |
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DISCUSSION |
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The finding that oxidative stress disrupts the E-cadherin/ß-catenin complex is supported by several studies suggesting that oxidative stress disrupts either cell-cell adhesion or, specifically, the cadherin/catenin complex. Hydrogen peroxide induces tyrosine phosphorylation of a number of proteins similar in molecular weight to cadherins and catenins, and disrupts the barrier function of several intestinal cell lines (Rao et al., 1997). In addition, the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of oxidative stress (Hu and Cotgreave, 1995
), causes internalization of E-cadherin from the plasma membrane to the cytosol without decreasing the total amount of protein (Jansen et al., 1996
). Exposure of endothelial cell monolayers to hydrogen peroxide is also associated with internalization of cadherins (Kevil et al., 1998
). Thus, oxidative stress may be a common mechanism of disruption of the cadherin/catenin complex for a number of chemical or physiological stimuli.
Oxidative stress is an important epigenetic mechanism of hepatocarcinogenesis in a number of studies (reviewed in Klaunig et al., 1998). Disruption of gap junctional intercellular communication (GJIC) is a sensitive target of oxidative stress (Sai et al., 1998, reviewed in Trosko and Ruch et al., 1998). This is of interest given the relationship between the cadherin/catenin complex and GJIC. The cadherin/catenin complex is requisite for the formation of gap junctions, and intercellular communication (Jongen et al., 1991). This interpretation is supported by the finding that antibodies to cadherin inhibit gap junction formation (Frenzel and Johnson, 1996
) and that disruption of cadherin expression or complex assembly correlates with loss of GJIC (Jansen et al., 1996
). Thus, the effect of chemically-induced disruption of the E-cadherin/ß-catenin complex in hepatocytes on GJIC is an exciting area for future studies of epigenetic mechanisms of hepatocarcinogenesis.
The demonstration of both susceptible and non-susceptible complexes within hepatocyte populations offers exciting possibilities in the development/progression of hepatocellular carcinomas. In human hepatocellular carcinoma, a striking down-regulation of E-cadherin, but not N-cadherin has been reported (Kozyraki et al., 1996). This is supported by more recent data suggesting the decreased expression of E-cadherin, but not N-cadherin, precedes down-regulation of
- and
-catenin expression in human hepatocellular carcinomas (Nuruki et al., 1998
). The findings that E-cadherin appears to be more sensitive to disruption is consistent with our results, which demonstrate that oxidative stress selectively disrupts the E-cadherin/ß-catenin complex in hepatocytes.
In summary, we have demonstrated a selective disruption of E-cadherin/ß-catenin complexes in precision-cut mouse liver slices. Although the molecular mechanism underlying the sensitivity of the E-cadherin/ß-catenin complex to chemically induced oxidative stress remains undefined, this pattern of disruption parallels that seen during the development of human hepatocellular carcinomas. These changes were observed in an integrated in vitro system that allows for examination of the influence of organ architecture and cellular heterogeneity on the cell adhesion complex. Given the increased awareness of the influence of xenobiotics on cell adhesion, these results provide the basis for further investigation of the role of oxidative stress on the cadherin/catenin complex.
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ACKNOWLEDGMENTS |
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NOTES |
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REFERENCES |
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---|
Boterberg, T., Vennekens, K. M., Thienpont, M., Mareel, M. M., and Bracke, M. E. (2000). Internalization of the E-cadherin/catenin complex and scattering of human mammary carcinoma cells MCF-7/AZ after treatment with conditioned medium from human skin squamous carcinoma cells COLO 16. Cell. Adhes. Commun. 7, 299310.[ISI][Medline]
Brancolini, C., Lazarevic, D., Rodriguez, J., and Schneider. C. (1997). Dismantling cellcell contacts during apoptosis is coupled to a caspase-dependent proteolytic cleavage of beta-catenin. J. Cell. Biol. 139, 759771.
Bush, K. T., Tsukamoto, T., and Nigam, S. K. (2000). Selective degradation of E-cadherin and dissolution of E-cadherin-catenin complexes in epithelial ischemia. Am. J. Physiol. Renal Physiol. 278, F847852.
Butz, S., and Larue, L. (1995). Expression of catenins during mouse embryonic development and in adult tissues. Cell Adhes. Commun. 3, 337352.[ISI][Medline]
Factor, V. M., Kiss, A., Woitach, J. T., Wirth, P. J., and Thorgeirsson, S. S. (1998). Disruption of redox homeostasis in the transforming growth factor-alpha/c-myc transgenic mouse model of accelerated hepatocarcinogenesis. J. Biol. Chem. 273, 1584615853.
Fataccioli, V., Andraud, E., Gentil, M., French, S. W., and Rouach, H. (1999). Effects of chronic ethanol administration on rat liver proteasome activities: Relationship with oxidative stress. Hepatology 29, 1420.[ISI][Medline]
Frenzel, E. M., and Johnson, R. G. (1996). Gap junction formation between cultured embryonic lens cells is inhibited by an antibody to N-cadherin. Dev. Biol. 179, 116.[ISI][Medline]
Gallin, W.J., Prediger, E. A., Edelman, G. M., and Cunningham, B. A. (1985). Isolation of a cDNA clone for the liver cell adhesion molecule (L-CAM). Proc. Natl. Acad. Sci. U.S.A. 82, 28092813.[Abstract]
Hu, J., and Cotgreave, I. A. (1995). Glutathione depletion potentiates 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced inhibition of gap junctional intercellular communication in WB-F344 rat liver epithelial cells: Relationship to intracellular oxidative stress. Chem. Biol. Interact. 95, 291307.[ISI][Medline]
Ihara, A., Koizumi, H., Hashizume, R., and Uchikoshi, T. (1996). Expression of epithelial cadherin and alpha- and beta-catenins in nontumoral livers and hepatocellular carcinomas. Hepatology 23, 14411447.[ISI][Medline]
Jansen, L. A., Mesnil, M., and Jongen, W. M. (1996). Inhibition of gap junctional intercellular communication and delocalization of the cell adhesion molecule E-cadherin by tumor promoters. Carcinogenesis 17, 15271531.[Abstract]
Jongen, W. M., Fitzgerald, D. J., Asamoto, M., Piccoli, C., Slaga, T. J., Gros, D., Takeichi, M., and Yamasaki, H. (1991). Regulation of connexin 43-mediated gap junction intercellular communication by Ca2+ in mouse epidermal cells is controlled by E-cadherin. J. Cell Biol. 114, 545555.[Abstract]
Kaplanski, G., Farnarier, C., Payan, M. J., Bongrand, P., and Durand, J. M. (1997). Increased levels of soluble adhesion molecules in the serum of patients with hepatitis C. Correlation with cytokine concentrations and liver inflammation and fibrosis. Dig. Dis. Sci. 42, 22772284.[ISI][Medline]
Kaplowitz, N., and Tsukamoto, H. (1996). Oxidative stress and liver disease. Prog. Liver Dis. 14, 131159.[Medline]
Katayama, M., Hirai, S., Kamihagi, K., Nakagawa, K., Yasumoto, M., and Kato, I. (1994). Soluble E-cadherin fragments increased in circulation of cancer patients. Br. J. Cancer 69, 580585.[ISI][Medline]
Kemler, R. (1992). Classical cadherins. Semin. Cell Biol. 3, 149155.[Medline]
Kevil, C. G., Ohno, N., Gute, D. C., Okayama, N., Robinson, S. A., Chaney, E., and Alexander, J. S. (1998). Role of cadherin internalization in hydrogen peroxide-mediated endothelial permeability. Free Radic. Biol. Med. 24, 10151022.[ISI][Medline]
Klaunig, J. E., Xu, Y., Isenberg, J. S., Bachowski, S., Kolaja, K. L., Jiang, J., Stevenson, D. E., and Walborg, E. F., Jr. (1998). The role of oxidative stress in chemical carcinogenesis. Environ. Health Perspect. 106(Suppl. 1), 289195.[ISI][Medline]
Kozyraki, R., Scoazec, J. Y., Flejou, J. F., D'Errico, A., Bedossa, P., Terris, B., Fiorentino, M., Bringuier, A. F., Grigoni, W., and Feldmann, G. (1996). Expression of cadherins and -catenin in primary epithelial tumors of the liver. Gastroenterology 110, 11371149.[ISI][Medline]
Kreft, B., Berndorff, D., Bottinger, A., Finnemann, S., Wedlich, D., Hortsch, M., Tauber, R., and Gebner, R. (1997). LI-cadherin-mediated cellcell adhesion does not require cytoplasmic interactions. J. Cell Biol. 136, 11091121.
Larrea, E., Beloqui, O., Munoz-Navas, M. A., Civeira, M. P., and Prieto, J. (1998). Superoxide dismutase in patients with chronic hepatitis C virus infection. Free Radic. Biol. Med. 24, 12351241.[ISI][Medline]
Linnemann, D., Gaardsvoll, H., Dalseg, A. M., Zhernosekov, D., Lundgren, T., Edvardsen, K., and Bock, E. (1994). Characterization of N-cadherin messenger RNA and polypeptide expression in rat. Int. J. Dev. Neuroscience 12, 441450.[ISI][Medline]
Lucka, L., Sel, S., Danker, K., Horstkorte, R., and Reutter, W. (1998). Carcinoembryonic antigen-related cell adhesion molecule C-CAM is greatly increased in serum and urine of rats with liver diseases. FEBS Lett. 438, 3740.[ISI][Medline]
Mandel, L. J., Doctor, R. B., and Bacallao, R. (1994). ATP depletion: A novel method to study junctional properties in epithelial tissues: II. Internalization of Na+,K+-ATPase and E-cadherin. J. Cell Sci. 107, 3313324.
Nathke, I. S., Hinck, L., Swedlow, J. R., Papkoff, J., and Nelson, W. J. (1994). Defining interactions and distributions of cadherin and catenin complexes in polarized epithelial cells. J. Cell. Biol. 125, 13411352.[Abstract]
Nollet, F., Kools, P., and van Roy, F. (2000). Phylogenetic analysis of the cadherin superfamily allows identification of six major subfamilies besides several solitary members. J. Mol. Biol. 299, 551572.[ISI][Medline]
Nuruki, K., Toyoyama, H., Ueno, S., Hamanoue, M., Tanabe, G., Aikou, T., and Ozawa, M. (1998). E-cadherin but not N-cadherin expression is correlated with the intracellular distribution of catenins in human hepatocellular carcinomas. Oncol. Rep. 5, 11091114.[ISI][Medline]
Parrish, A. R., Catania, J. M., Orozco, J., and Gandolfi, A. J. (1999). Chemically induced oxidative stress disrupts the E-cadherin/catenin cell adhesion complex. Toxicol. Sci. 51: 8086.[Abstract]
Rao, R. K., Baker, R. D., Baker, S. S., Gupta, A., and Holycross, M. (1997). Oxidant-induced disruption of intestinal epithelial barrier function: Role of protein tyrosine phosphorylation. Am. J. Physiol. 273, G812823.
Reynolds, A. B., Daniel, J., McCrea, P. D., Wheelock, M. J., Wu, J., and Zhang, Z. (1994). Identification of a new catenin: The tyrosine kinase substrate p120cas associates with E-cadherin complexes. Mol. Cell Biol. 14, 83338342.[Abstract]
Ringwald, M., Schuh, R., Vestweber, D., Eistetter, H., Lottspeich, F., Engel, J., Dolz, R., Jahnig, F., Epplen, J., and Mayer, S. (1987). The structure of cell adhesion molecule uvomorulin. Insights into the molecular mechanism of Ca2+-dependent cell adhesion. EMBO J. 6, 36473653.[Abstract]
Rosser, B. G., and Gores, G. J. (1995). Liver-cell necrosis: Cellular mechanisms and clinical implications. Gastroenterology 108, 252275.[ISI][Medline]
Sai, K., Upham, B. L., Kang, K. S., Hasegawa, R., Inoue, T., and Trosko, J. E. (1998). Inhibitory effect of pentachlorophenol on gap junctional intercellular communication in rat liver epithelial cells in vitro. Cancer Lett. 130, 917.[ISI][Medline]
Schmieser, K., Hammond, E. M., Roberts, S., and Grand, R. J. (1998). Specific cleavage of gamma catenin by caspases during apoptosis. FEBS Lett. 433, 5157.[ISI][Medline]
Sipowicz, M. A., Chomarat, P., Diwan, B. A., Anver, M. A., Awasthi, Y. C., Ward, J. M., Ride, J. M., Kasprzak, K. S., Wild, C. P., and Anderson, L. M. (1997) Increased oxidative DNA damage and hepatocyte overexpression of cytochrome P450 isoforms in hepatocytes of mice infected with Helicobacter hepaticus. Am. J. Pathol. 151, 933941.
Traverso, N., Menini, S., Odetti, P., Pronzato, M. A., Cottalasso, D., and Marinari, U. M. (1999). Lipoperoxidation in hepatic subcellular compartments of diabetic rats. Free Radic. Biol. Med. 26, 538547.[ISI][Medline]
Trosko, J. E., and Ruch, R. J. (1998). Cell-cell communication in carcinogenesis. Front. Biosci. 15, D208236.
Tsukita, S., Tsukita, S., Nagafuchi, A., and Yonemura, S. (1992). Molecular linkage between cadherins and actin filaments in cell-cell adherens junction. Curr. Opin. Cell Biol. 4, 834839.[Medline]
Unemura, T., Kai, S., Hasegawa, R., Sai, K., Kurokawa, Y., and Williams, G. M. (1999). Pentachlorophenol (PCP) produces liver oxidative stress and promotes but does not initiate hepatocarcinogenesis in B6C3F1 mice. Carcinogenesis 20, 11151120.
Wanner, R. B., Glowacki, F., Kolde, G., and Wittig, B. (1999). The loss of desmosomes after retinoic acid treatment results in an apparent inhibition of HaCaT keratinocyte differentiation. Arch. Dermatol. Res. 291, 346353.[ISI][Medline]
Wu, J.-C., Gregory, C. W., and DePhilip, R. M. (1993). P-cadherin and E-cadherin are co-expressed in MDCK cells. Bioc. Biophys. Res. Commun. 195, 13291335.
Wu, Q., and Maniatis, T. (1999). A striking organization of a large family of human neural cadherin-like cell adhesion genes. Cell 97, 779790.[ISI][Medline]
Yamamoto, Y., Yamashita, S., Fujisawa, A., Kokura, S., and Yoshikawa, T. (1998). Oxidative stress in patients with hepatitis, cirrhosis, and hepatoma evaluated by plasma antioxidants. Biochem. Biophys. Res. Commun. 247, 166170.[ISI][Medline]