Human intestinal epithelial cell line SK-CO15 is a new model system to study Na⁺/H⁺exchanger 3

Abstract

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...

Restoration of Na+/H+ exchanger NHE3-containing macrocomplexes ameliorates diabetes-associated fluid loss

“…Caco-2bbe and SK-CO15 cells were obtained from C. Parkos at Emory University, Atlanta, Georgia, USA, and E. Rodriguez-Boulan at Weill Medical College, Cornell University, Ithaca, New York, USA, respectively. Caco-2bbe/NHE3V and SK-CO15 cells were cultured as previously described (32). Cells were grown on Transwell filters (Corning) for 5 to 7 days after confluence to ensure differentiation prior to all assays.…”

“…Lysophosphatidic acid (LPA) is a growth factor-like lipid molecule that mediates multiple biological effects through 6 LPA receptors (31). We have shown previously that LPA activates NHE3 via the LPA5 receptor in Caco2bbe cells and mouse ileum (11,32). The activation of NHE3 by LPA is independent of PI3K and is regulated by the ERK-RSK2 cascade (33), suggesting that LPA and insulin might regulate NHE3 through different pathways.…”

Disrupted apical exocytosis of cargo vesicles causes enteropathy in FHL5 patients with Munc18-2 mutations

“…In MVID, disturbed interaction of Slp4a/ Stx3 in apical fusion processes was previously shown to impair the trafficking and localization of apical transmembrane transporters in enterocytes (26), e.g., sodium-hydrogen exchanger 3 (NHE3) and glucose transporter 5 (GLUT5), which are relevant for enterocyte physiology (35)(36)(37). Therefore, we further analyzed the localization of these proteins in Munc18-KO cells.…”

PRMT5 C-terminal Phosphorylation Modulates a 14-3-3/PDZ Interaction Switch

“…To test this hypothesis, we chose to work in SK-CO15 cells, which are human intestinal epithelial cells that express high levels of NHERF2 (39). First, we performed cell fractionation studies to determine whether endogenous PRMT5 is localized to the membrane.…”