An acidic extracellular pH induces Src kinase-dependent loss of β-catenin from the adherens junction☆
Introduction
Interstitial acidification occurs in inflammatory sites, in peritoneal fluids during laparatomy, and in the tumor microenvironment [1], [2]. The pHe in a variety of solid tumors ranges from 5.8 to 7.4 [3], [4], [5]. Mechanisms reported to be responsible for this acidity include the production of lactic acid and ATP hydrolysis in hypoxic tumors [3]. In addition, effects of CO2 pneumoperitoneum on tumor growth have been reported. CO2 pneumoperitoneum stimulates the growth of malignant colonic cells and subcutaneous tumors [6]. In addition to a decrease in partial oxygen pressure in the abdominal wall, dramatic decreases in extra- and intra-cellular pH values are observed during CO2 insufflation in vitro [7]. Severe peritoneal acidosis down to a pH of 6.59–6.74 has been observed after prolonged CO2 pneumoperitoneum and may help tumor metastasis to port sites [8]. Thus, alterations in the peritoneal pH (6.59–6.74) may be responsible for providing an environment favorable to tumor cell implantation during laparoscopy [8]. Extracellular acidification is often associated with tumor progression [9], [10], [11], but the mechanism is unknown.
The AJ is composed of transmembrane cadherin molecules and the associated proteins, β-catenin and α-catenin [12]. At the plasma membrane adjacent to the AJ, β-catenin binds directly to the cytoplasmic region of E-cadherin and α-catenin, the latter of which is linked to the actin-based cytoskeleton [13], [14]. Disruption of AJs precedes mesenchymal transition and enhances invasion and metastasis in several types of cancer cells [15]. In many invasive carcinomas, expression of E-cadherin and β-catenin is either reduced or absent [16], [17], [18]. Moreover, in epithelial cells, β-catenin plays a role in both cell adhesion at the AJ and in Wnt signal transduction in the cytoplasm/nucleus [19]. Retention of β-catenin in the E-cadherin/catenin complex at the AJ is regulated by tyrosine phosphorylation of β-catenin [20]. Three Src family kinases, Fer, Fyn, and c-Src, are involved in phosphorylation of β-catenin at different tyrosine residues [21]. For example, transformation of MDCK cells with a temperature-sensitive v-Src mutant results in increased tyrosine phosphorylation of E-cadherin and β-catenin, a rapid loosening of cell–cell contact, and promotion of invasiveness [22]. Treatment with the protein tyrosine phosphatase inhibitor, pervanadate, increases tyrosine phosphorylation of β-catenin and leads to its redistribution from the cell–cell junction to the cytoplasm or the nucleus [23], [24].
In order to gain a further insight into the effect of an acidic microenvironment on cell adhesion, we cultured HepG2 cells, a human hepatocellular carcinoma cell line, in vitro at an acidic pHe and examined whether this disrupted the integrity of the AJ. The distribution and changes in the phosphorylation levels of β-catenin were assessed by immunofluorescence microscopy, immunoprecipitation, and Western blot analyses. Possible signaling pathways accounting for the phosphorylation of β-catenin were analyzed. We provide evidence that an acidic pHe causes c-Src-dependent tyrosine phosphorylation of β-catenin and decreases the binding of E-cadherin to β-catenin, which leads to destabilization of the AJ and increased cell migration and invasion.
Section snippets
Cell culture
The human hepatocellular carcinoma cell line HepG2 (ATCC, HB8065), purchased from the American Type Culture Collection (Rockville, MD), was grown in growth medium consisting of Dulbecco’s modified Eagle medium containing non-essential amino acids, sodium pyruvate, Earle’s balanced salt solution, 100 IU/ml of penicillin, 100 μg/ml of streptomycin, and 10% fetal calf serum (all from Gibco BRL, Grand Island, NY) in a 5% CO2 humidified atmosphere at 37 °C. The cells were plated at 2 × 105 cells/ml in 35
Culturing at acidic pHe disrupts compact colony formation of HepG2 cells
We first examined the effect of an acidic culture medium on cell proliferation and cell death. Cell viability, assessed by the MTT assay, showed that incubating the cells in acidic culture media (pH 6.6) was not cytotoxic for HepG2 cells (Fig. 1A). Propidium iodide vital staining and DAPI (4′,6-diamidino-2-phenylindole dilactate) nuclear staining showed very few apoptotic or necrotic cells after 24 h incubation at pH 6.6, the percentages being comparable to those in the pH 7.4 group (data not
Discussion
In this study, we examined the effect of an acidic pHe on the AJ and the migratory capability of HepG2 cells. After incubation in pH 6.6 medium, HepG2 cells acquired mesenchymal characteristics, including a loss of β-catenin from the cell junction and spreading and migratory phenotypes. At this pH, rapid phosphorylation of c-Src was observed. Activation of c-Src kinase was shown to be responsible for the tyrosine phosphorylation of β-catenin, and this reduced the binding to E-cadherin, as
Acknowledgements
Grant sponsors: Far Eastern Memorial Hospital, Taiwan and the National Science Council, Republic of China (grant NSC 95-2314-B-002-192). We thank Dr. Thomas Barkas for his critical reading and correction of this manuscript.
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Grant sponsors: Far Eastern Memorial Hospital, Taiwan and the National Science Council (Grant NSC 96-2320-B-002-005).