BRIEF REPORT |
Localization of Alkaline Phosphatase and Proteins Related to Intercellular Junctions in Rat Hepatoma Cell Line McA-RH 7777
Department of Anatomy, School of Allied Health Sciences, Kitasato University, Sagamihara, Kanagawa, Japan
Correspondence to: Kohsuke Chida, Dept. of Anatomy, School of Allied Health Sciences, Kitasato University, Sagamihara, Kanagawa 228-8555, Japan. E-mail: chida{at}ahs.kitasato-u.ac.jp
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(J Histochem Cytochem 52:979983, 2004)
Key Words: alkaline phosphatase connexin 32 E-cadherin ZO-1 rat hepatoma McA-RH 7777 immunohistochemistry
-GLUTAMYLTRANSPEPTIDASE (GGT) and alkaline phosphatase (ALP) are localized in the plasma membrane in normal adult rat hepatocytes, and display increased concentrations in rat neoplastic nodules and hepatoma (Karasaki 1975
; Suzuki et al. 1987
). GGT and ALP in the McA-RH 7777 rat hepatoma cell line are reportedly restricted to cytoplasm around the nucleus (Chida 2000
). During the SG2-phases of the cell cycle in cells synchronously cultured in excess thymidine and hydroxyurea, the majority of McA-RH 7777 cells assume a flat form, making many cell-to-cell contacts, and both enzymes are localized at the borders between adjacent cells and cytoplasm around the nucleus (Chida 2000
). This indicates that contacts between adjacent McA-RH 7777 cells promote translocation of GGT or ALP from cytoplasm to the plasma membrane. However, the mechanisms promoting translocation of GGT and ALP after contact between adjacent McA-RH 7777 cells have not been fully elucidated.
Dexamethasone and dimethylsulfoxide in cultured fetal rat hepatocytes reportedly promote the differentiation of hepatocytes and translocation of GGT and ALP from cytoplasm to the plasma membrane (Chida 1998). This indicates that translocation of GGT and ALP in hepatocytes is related to development of hepatocyte polarity. Polarity of the plasma membrane in epithelial cells develops as a result of intercellular junctional complexes between adjacent cells (Wodarz 2002
). In normal adult rat hepatocytes, ALP is predominantly localized in the bile canalicular domain of the plasma membrane and is considered to hydrolyze phosphate compounds as phospholipids excreted into the bile canalicular lumen. Moreover, the formation of intercellular junctions between adjacent hepatocytes divides the plasma membrane to two domains, apical and basolateral, and produces bile canaliculi as apical spaces. The formation of intercellular junctions between adjacent rat hepatocytes is therefore expected to be closely related to localization of ALP and necessary for ALP function. The present study investigated localization of ALP and three proteins related to intercellular junctions, E-cadherin (adherens junctions), ZO-1 (tight junctions), and connexin-32 (gap junctions) in McA-RH 7777 cells to examine whether the formation of intercellular junctions is related to translocation of ALP from cytoplasm to the plasma membrane after contact between adjacent McA-RH 7777 cells.
McA-RH 7777 cells (American Type Culture Collection; Rockville, MD) derived from Morris rat hepatoma were cultured in -minimum essential medium containing 10% fetal bovine serum (
-MEM 13.1; Dainippon Phamaceutical, Osaka, Japan). McA-RH 7777 cells were seeded at a concentration of 2 x 104 cells/ml in eight-well collagen-coated BioCoat Culture Slides (Nippon Becton Dickinson; Tokyo, Japan) and cultured for 3 days. Other McA-RH 7777 cells were seeded at a concentration of 105 cells/ml in identical slides and synchronized at S- phase of the cell cycle by culturing in medium containing excess thymidine and hydroxyurea, as described previously (Chida 2000
). Synchronized cells were then cultured in
-MEM for 48 hr.
McA-RH 7777 cells were fixed in Zamboni solution (2% paraformaldehyde, 15% saturated picric acid in 0.1 M phosphate buffer, pH 7.4) for 10 min at RT. After washing with 0.01 M phosphate-buffered saline solution (PBSS, pH 7.2) containing 0.85% NaCl and 0.05% saponin, cells were immersed in 0.1% Triton X-100 solution dissolved in PBSS for 5 min at RT. Cells were then incubated for 1 hr at RT in anti-rat ALP anti-serum (Chida 1993a,b
) diluted 50-fold with PBSS, anti-human E-cadherin monoclonal antibodies (BD Bioscience Pharmingen; San Diego, CA) diluted 500-fold with PBSS, anti-mouse ZO-1 monoclonal antibodies (Chemicon International; Temecula, CA) diluted 1000-fold with PBSS, or anti-rat connexin 32 monoclonal antibodies (Zymed Laboratories; South San Francisco, CA) diluted 250-fold with PBSS. For control, normal rabbit serum or mouse IgG was used in place of antiserum or antibodies in the same solutions. Cells were washed with PBSS and reacted for 30 min at RT with fluorescein isothiocyanate (FITC)-labeled anti-rabbit or anti-mouse IgG antibodies (Medical and Biological Laboratories; Nagoya, Japan). Double staining for ALP and mannosidase II, a Golgi marker, was performed in some cells cultured at low concentration. For double staining, cells were incubated for 1 hr at RT in mixed solution containing anti-rat ALP antiserum diluted 50-fold with PBSS and anti-rat mannosidase II monoclonal antibody (Covance; Research Products, Richmond, CA) diluted 10,000-fold with PBSS. After washing with PBSS, cells were reacted for 30 min at RT with mixed solution containing rhodamine-labeled anti-rabbit and FITC-labeled anti-mouse IgG antibodies (Medical and Biological Laboratories). All cells were mounted in Fluoro-Guard Antifade Reagent (Bio-Rad; Hemel Hempstead, UK) and examined under confocal laser scanning microscopy (Radiance2100; Bio-Rad).
In McA-RH 7777 cells cultured at low concentration, ALP was restricted to cytoplasm around the nucleus (Figure 1A) . Moreover, double staining for ALP and mannosidase II indicated locations of ALP in cytoplasm basically consistent with those of mannosidase II (Figures 1C and 1D). During the SG2 stages of the cell cycle in synchronous McA-RH 7777 cells, ALP was localized at the borders between adjacent cells and in cytoplasm around the nucleus (Figure 1B).
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In mouse or rat livers, colchicine administration or bile duct ligation leads to morphological changes of the junctional complex of hepatocytes and to partial breakdown of the permeability barrier between the canalicular lumen and the intercellular space between adjacent hepatocytes (Metz et al. 1977; Rassat et al. 1982
). ALP in rat liver becomes localized not only in the bile canalicular membrane but also in the basolateral membrane by the above treatment (Araki et al. 1995
; Chida et al. 1995
). These facts indicate that translocalization of ALP in the plasma membrane of rat hepatocytes is closely related to the formation of intercellular junctions between adjacent hepatocytes. However, at present we cannot assert whether the formation of adherens junctions between adjacent McA-RH 7777 cells triggers translocation of ALP from Golgi complexes to the plasma membrane. Moreover, tyrosine phosphorylation of junctional proteins reportedly disrupts the junctional complex (Rao et al. 2002
). However, whether ALP functions as a phosphatase enzyme to protect intercellular junctions from such tyrosine phosphorylation of junctional proteins is unknown.
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Literature Cited |
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