Knockdown of sodium-calcium exchanger 1 induces epithelial-to-mesenchymal transition in kidney epithelial cells

Abstract

Mesenchymal-to-epithelial transition (MET) and epithelial-to-mesenchymal transition (EMT) are important processes in kidney development. Failure to undergo MET during development leads to the initiation of Wilms tumor, whereas EMT contributes to the development of renal cell carcinomas (RCC). The role of calcium regulators in governing these processes is becoming evident. We demonstrated earlier that Na⁺/Ca²⁺ exchanger 1 (NCX1), a major calcium exporter in renal epithelial cells, regulates epithelial cell motility. Here, we show for the first time that NCX1 mRNA and protein expression was down-regulated in Wilms tumor and RCC. Knockdown of NCX1 in Madin-Darby canine kidney cells induced fibroblastic morphology, increased intercellular junctional distance, and induced paracellular permeability, loss of apico-basal polarity in 3D cultures, and anchorage-independent growth, accompanied by expression of mesenchymal markers. We also provide evidence that NCX1 interacts with and anchors E-cadherin to the cell surface independent of NCX1 ion transport activity. Consistent with destabilization of E-cadherin, NCX1 knockdown cells showed an increase in β-catenin nuclear localization, enhanced transcriptional activity, and up-regulation of downstream targets of the β-catenin signaling pathway. Taken together, knockdown of NCX1 in Madin-Darby canine kidney cells alters epithelial morphology and characteristics by destabilization of E-cadherin and induction of β-catenin signaling.

Keywords: MDCK; Wilms tumor; beta-catenin (B-catenin); cadherin-1 (CDH1) (epithelial cadherin) (E-cadherin); epithelial cell; epithelial-mesenchymal transition (EMT); kidney; renal cell carcinoma; sodium-calcium exchanger.

Figure 1.

NCX1 mRNA and protein is down-regulated in renal cancers. A, analysis of microarray data GSE11151 comparing NCX1 transcript levels across different types of RCC: clear cell RCC (n = 26), papillary RCC (n = 19), chromophobe RCC (n = 4), and Wilms tumor (n = 4). For calculation of p values, RCC subtypes were compared with adult normal kidney (n = 3), whereas Wilms tumor was compared with fetal normal kidney (n = 2). **, p < 0.005; ***, p < 0.001. B and C, immunoblots showing NCX1 protein level in matched normal (N) and tumor (T) tissues of Wilms tumor and RCC. Arrows, 120-kDa full-length NCX1 protein. The bands at 160 kDa represent unreduced NCX1 protein. β-Actin was used as a loading control. D, box and whisker plot showing the densitometric quantitation of NCX1 protein normalized to β-actin protein. Asterisks indicate that the reduction in NCX1 protein is statistically significant for both Wilms tumor and RCC (**, p < 0.005). E, bar graphs showing mean NCX1 mRNA levels in Wilms tumor and RCC normalized to β-tubulin mRNA. Error bars, S.E. ***, p < 0.001; ****, p < 0.0001.

Figure 2.

NCX1 knockdown changes epithelial characteristics in MDCK cells. A, representative immunoblots showing the levels of total and cell surface NCX1 and PMCA in MDCK and NCX1-KD cells. β-Actin was used as a loading control. B, representative phase-contrast images of MDCK and NCX1-KD cells in culture for 24 h showing cell morphology. Note the change in morphology in NCX1-KD cells. Scale bar, 100 μm. C, representative immunofluorescence images of cells stained with anti-β-catenin antibody. Such images were used for determination of cell length. Red lines, cell length quantitated in D. Scale bar, 20 μm. D, the graph shows average cell length of MDCK, MDCK-Scr, and NCX1-KD cells in μm from three independent experiments with at least 20 cells/experiment. Error bars, S.E. ****, p < 0.0001; ns, not significant.

Figure 3.

NCX1-KD cells form non-polarized cysts in 3D Matrigel and exhibit-enhanced rate of proliferation. A, representative confocal images of MDCK and NCX1-KD cysts in 3D Matrigel^TM cultures stained for β-catenin (green), F-actin (red), and nuclei (blue). Majority of MDCK cysts had a single hollow lumen with distinct apical and basal polarity, whereas NCX1-KD cells failed to form polarized cysts. Some NCX1-KD cysts had multiple lumens (bottom panel). Scale bar, 10 μm. B, bar graph comparing the mean number of polarized or non-polarized cysts in MDCK and NCX1-KD cells. Error bars, S.E. from three independent experiments (n = 100 cysts/experiment). **, p < 0.005; ***, p < 0.001. C, graph comparing MDCK and NCX1-KD cyst dimension from three independent experiments (n = 20 each). ***, p < 0.001. D, the bar graph denotes the rate of cell proliferation in MDCK and NCX1-KD cells in the BrdU assay. Error bars, S.E. from three independent experiments in triplicate. *, p < 0.05.

Figure 4.

NCX1 knockdown reduces tightness of epithelial cell contact. A, graph shows normalized TER measurements from a representative run using ECIS system. Similar results were obtained in three independent experiments performed in triplicate. B, graph showing normalized TER measurements captured during a calcium switch from low to high calcium. The arrow indicates the time when cells were switched to high calcium. A representative graph from three independent experiments done in duplicate is shown. C, the fluorescence intensity corresponding to the amount of TAMRA in the bottom chamber provides a measure of the rate of paracellular permeability in MDCK and NCX1-KD cells. Error bars, S.E. (p = 0.0272). D, representative transmission electron micrographs showing junctions between adjacent cells in MDCK or NCX1-KD cells. Arrows, intersection of apical and lateral membrane between two cells in contact; asterisks, desmosomes in the lateral membrane. Note that NCX1-KD cells have wide space between adjacent cells. The bottom panels show magnified views of cell–cell contact points near the apical surface. Bars, 200 nm.

Figure 5.

NCX1 interacts with E-cadherin and regulates its membrane expression independent of NCX1 ion transport activity. A, immunoblot representing total and membrane E-cadherin expression along with total β-catenin levels. β-Actin was used as a loading control. B, representative immunoblots of exocytosis (quenched with or without NHS-acetate) and endocytosis (reduced or non-reduced) of E-cadherin in MDCK and NCX1-KD cells. C, immunoblots of E-cadherin and β-actin in soluble (S) and pellet (P) fractions in MDCK and NCX1-KD cells following detergent extraction. D, co-immunoprecipitation assay showing E-cadherin, β-catenin, and NCX1 protein in IgG, anti-NCX1, and anti-E-cadherin immunoprecipitates (IP). E-cadherin and β-catenin antibodies were simultaneously added for the detection of both proteins on the same blot. E, immunofluorescence images showing co-localization of NCX1 and E-cadherin in MDCK cells. Bar, 10 μm. F, lysates of MDCK cells treated with DMSO or KB-R7943 were immunoprecipitated using anti-E-cadherin antibody and immunoblotted for NCX1 and E-cadherin. Note that E-cadherin co-immunoprecipitated with NCX1 in NCX1-inhibited cells. G, representative immunoblots showing E-cadherin and β-actin in detergent-soluble (S) and pellet (P) fractions of MDCK monolayers treated with DMSO (control) or KB-R7943.

Figure 6.

Activation of β-catenin signaling pathway in NCX1-KD cells. A, immunostaining of MDCK and NCX1-KD cells with β-catenin is shown (green). Nuclei were stained with TO-PRO3 (blue). Arrows highlight nuclear localization of β-catenin in NCX1-KD cells. Scale bar, 10 μm. B, the transcriptional activity of β-catenin in MDCK and NCX1-KD cells was determined by a Dual-Luciferase assay. The graph represents mean -fold change in β-catenin transcriptional activity calculated from three independent experiments in triplicate. *, p = 0.0225. C, the graph shows transcript levels of cyclin D1 and Axin2 normalized to GAPDH from three independent experiments in triplicate (*, p < 0.05). D, representative images showing the NCX1-KD cysts treated with vehicle or XAV-939 for 96 h and stained with phalloidin and β-catenin. Scale bar, 25 μm.

Figure 7.

NCX1 regulates the expression of epithelial and mesenchymal markers. A, representative immunoblots of NCX1, α-smooth muscle actin (α-sma), and N-cadherin in MDCK and NCX1-KD cells. β-Actin was used as a loading control. B, representative confocal images showing immunofluorescence staining for fibronectin and phospho-NF-κB in MDCK and NCX1-KD cells. Scale bar, 25 μm. C, representative confocal images showing immunofluorescence staining for fibronectin and phospho-NF-κB in NCX1-KD cells treated with vehicle or XAV-939. Scale bar, 10 μm.

Figure 8.

NCX1 knockdown cells exhibit anchorage-independent growth. A, representative images showing MDCK and NCX1-KD colonies in soft agar. The bottom panels show phase-contrast images of the colonies. Scale bar, 30 μm. B, graph showing the average number of colonies in soft agar from two independent experiments in triplicate. Error bars, S.E. ****, p < 0.0001.

Figure 9.

Schematic representation of the effect of NCX1 knockdown in renal epithelial cells. NCX1 complexes with E-cadherin on the membrane in renal epithelial cells and the reduction of NCX1 in NCX1-KD cells cause a decrease in E-cadherin membrane expression and stability. This might be the reason for the increase in intercellular membrane distance and formation of non-polarized cysts, as shown in previous studies (79, 80). Furthermore, this also leads to an increase in β-catenin transcriptional activity that is associated with increase in proliferation (28), anchorage-independent growth (60), and increase in EMT markers (59). This model illustrates the mechanism by which the loss of NCX1 initiates morphological and functional characteristics of cells undergoing EMT, thereby leading to cancer progression.