Disruption of the cell-cell junction with concomitant changes in the expression of junctional proteins is a hallmark of cancer cell invasion and metastasis. The role of adherent junction proteins has been studied extensively in cancer, but the roles of tight junction (TJ) proteins are less well understood. Claudins are recently identified members of the tetraspanin family of proteins, which are integral to the structure and function of TJs. Recent studies show changes in expression/cellular localization of claudins during tumorigenesis; however, a causal relationship between claudin expression/localization and cancer has not been established. Here, we report an increased expression of claudin-1 in human primary colon carcinoma and metastasis and in cell lines derived from primary and metastatic tumors. We also report frequent nuclear localization of claudin-1 in these samples. Genetic manipulations of claudin-1 expression in colon cancer cell lines induced changes in cellular phenotype, with structural and functional changes in markers of epithelial-mesenchymal transition. Furthermore, we demonstrate that changes in claudin-1 expression have significant effects on growth of xenografted tumors and metastasis in athymic mice. We further provide data suggesting that the regulation of E-cadherin expression and beta-catenin/Tcf signaling is a possible mechanism underlying claudin-1-dependent changes.

Background: LPA induces HIF-1␣ expression, but the underlying mechanism remains unknown. Results: LPA induced HIF-1␣ by decreasing p53 expression, and additionally, KLF5 transactivated HIF-1␣ expression. Conclusion: KLF5 and p53 reciprocally regulate HIF-1␣ expression. Significance: This study provides a new dimension to understanding how LPA promotes colon cancer.

cell line represents absorptive polarized intestinal epithelial cells that express multiple forms of Na ϩ /H ϩ exchanger (NHE) in their plasma membranes. Caco-2 cells express the major apical NHE isoform NHE3, but low NHE3 expression together with inefficient transfection often hamper intended studies. In this study, we examined whether SK-CO15 cells could be used to study NHE3 regulation. SK-CO15 cells grown on Transwell inserts developed polarized epithelial cells with microvilli. The transfection efficiency of SK-CO15 cells was markedly higher compared with Caco-2 cells, an advantage in gene transfer and knockout. SK-CO15 cells expressed NHE1, NHE2, and NHE3. NHE3 expression was significantly greater in these cells than Caco-2, and NHE3 comprised more than half of total NHE activity. Apical expression of NHE3 in SK-CO15 cells was confirmed by confocal immunofluorescence and surface biotinylation. NHE regulatory factors NHERF1 and NHERF2, which are important for regulation of NHE3 activity, were expressed in these cells. Stimulatory response of NHE3 in SK-CO15 cells was assessed by dexamethasone and lysophosphatidic acid (LPA). Treatment with dexamethasone for 24 -48 h increased NHE3 expression and activity. Similarly to Caco-2 cells, SK-CO15 cells lacked the expression of the LPA receptor LPA 5, but exogenous expression of LPA5 resulted in acute stimulation of NHE3. Forskolin acutely inhibited NHE3 activity in SK-CO15 cells, further attesting the validity of these cells. We conclude that SK-CO15 cells with the amenity for transfection and high endogenous NHE3 expression are a new and better cell model for NHE3 regulatory investigation than widely used Caco-2 cells.intestine; epithelia POLARIZED INTESTINAL EPITHELIAL cells form an interface separating the internal from external environments and maintain homeostasis between intestinal lumen and the body interior. The plasma membranes of polarized epithelial cells are divided into apical and basolateral domains with asymmetric distribution of cytoplasmic organelles by vectorial sorting mechanisms (27). Na ϩ /H ϩ exchange is a major route of Na ϩ absorption in the small intestine and colon (9). Intestinal epithelial cells express multiple forms of Na ϩ /H ϩ exchangers (NHEs; Slc9a) among which the type 3, NHE3 (Slc9a3), is the primary brush-border NHE. The functions and mechanisms of NHE3 regulation by hormones and growth factors have been investigated since its cloning in the early 1990s using several cell model systems. Among these, PS120 Chinese hamster lung fibroblasts and AP1 Chinese hamster ovarian cells provide an ideal cell system for reductionist approach to characterize NHEs (26, 28). However, the nonepithelial origins of PS120 and AP1 cells often led to question the physiological validity of the findings. Originated from human intestines, T84 and HT29 human colon carcinoma epithelial cell lines are not suitable, as these cells represent secretory crypt epithelial cells (5, 6). The Caco-2 human adenocarcinoma cell line, on the other hand, express severa...

Background: Regulation of NHE3 by ubiquitination has not been reported. Results: Human NHE3, but not non-primates NHE3s, is ubiquitinated by Nedd4-2 and undergoes internalization at an increased rate. Conclusion: Human NHE3 is uniquely regulated by ubiquitination. Significance: This study provides a new mechanism of regulating NHE3 in human that may be relevant to diseases associated with increased Na ϩ and fluid absorption.

The distribution of two proteins in Naegleria gruberi, N-␥TRP (Naegleria ␥-tubulin-related protein) and N-PRP (Naegleria pericentrin-related protein), was examined during the de novo formation of basal bodies and flagella that occurs during the differentiation of N. gruberi.