The electroneutral Na+-HCO3- cotransporter NBCn1 (SLC4A7) modulates colonic enterocyte pHi, proliferation, and migration

Abstract

NBCn1 (SLC4A7) is one of the two major Na⁺-HCO₃^- cotransporters in the human colonic epithelium, expressed predominantly in the highly proliferating colonocytes at the cryptal base. Increased NBCn1 expression levels are reported in tumors, including colorectal cancer. The study explores its importance for maintenance of the intracellular pH (pH_i), as well as the proliferative, adhesive, and migratory behavior of the self-differentiating Caco2BBe colonic tumor cell line. In the self-differentiating Caco2BBe cells, NBCn1 mRNA was highly expressed from the proliferative stage until full differentiation. The downregulation of NBCn1 expression by RNA interference affected proliferation and differentiation and decreased intracellular pH (pH_i) of the cells in correlation with the degree of knockdown. In addition, a disturbed cell adhesion and reduced migratory speed were associated with NBCn1 knockdown. Murine colonic Nbcn1^-/- enteroids also displayed reduced proliferative activity. In the migrating Caco2BBe cells, NBCn1 was found at the leading edge and in colocalization with the focal adhesion markers vinculin and paxillin, which suggests that NBCn1 is involved in the establishment of cell-matrix adhesion. Our data highlight the physiological significance of NBCn1 in modulating epithelial pH homeostasis and cell-matrix interactions in the proliferative region of the colonic epithelium and unravel the molecular mechanism behind pathological overexpression of this transporter in human colorectal cancers.NEW & NOTEWORTHY The transporter NBCn1 plays a central role in maintaining homeostasis within Caco2BBe colonic epithelial cells through its regulation of intracellular pH, matrix adhesion, migration, and proliferation. These observations yield valuable insights into the molecular mechanism of the aberrant upregulation of this transporter in human colorectal cancers.

Keywords: acid/base balance; cell migration; colon cancer; pHi regulation; sodium bicarbonate cotransporter.

Graphical abstract

Figure 1.

Expression of NBCn1 in human tissue and Caco2BBe cells. A: gene expression of NBCn1 is upregulated in human colon cancer tissue compared with the healthy colon tissue. The Log₂ mRNA expression data are provided by GENT2 database (29) available at http://gent2.appex.kr/gent2/ and are illustrated in Tukey box plot. B: expression of NBCn1 (Slc4A7) mRNA at different stages of Caco2BBe culture is relatively stable over the time of the culture. C: SLC26A3 encodes for an apical Cl^–/HCO₃^– exchanger and is a differentiation marker of Caco2BBe cells. SLC26A3 mRNA expression is enhanced after the confluent monolayer starts to differentiate. Means ± SE, ANOVA. ns, not significant; preconfl., preconfluence cultures with 50% cell density. **P < 0.01, ***P < 0.001.

Figure 2.

Generation of NBCn1 knockdown Caco2BBe cells. Caco2BBe cells were transduced using lentiviral particles to generate NBCn1 knockdown (KD) cells, selected with hygromycin B and then harvested for Western and qPCR analysis. The knockdown efficiency was analyzed by quantifying NBCn1 mRNA by RT-qPCR, using RPLP0 as a control gene (A) as well as by Western blot, using β-actin as loading control (B and C). For further analysis, we selected two clones, namely KD#2 clone with 97% mRNA and 90% protein and KD#4 with 66% mRNA and 57% protein downregulation in comparison with the control, which was transduced with universal scrambled shRNA. Means ± SE, ANOVA, *P < 0.05, ***P < 0.001.

Figure 3.

NBCn1-KD affects expression of SLC26A3. Representative immunoblot image showing expression of SLC26A3 protein in control and NBCn1-KD clones of Caco2BBe. Control and NBCn1-KD#4 cultures were harvested at 1 wk post-confluence and NBCn1-KD#2 cultures were harvested 1 wk after the initial 14 days of antibiotic selection (A). β-Actin was used as a loading control. Relative SLC26A3 signals were quantified and illustrated in ratio to control after normalization to the β-actin signal (B). NBCn1-KD substantially reduces protein expression of SLC26A3. Means ± SE, ANOVA. ns, not significant. ***P < 0.001.

Figure 4.

NBCn1 downregulation reduces steady-state pH_i in Caco2BBe cells. A: steady-state intracellular pH was measured by single chamber perfusion fluorometry for NBCn1-KD#2 cells and control cells which were grown and transduced on cover slips, directly after 2 wk of selection with hygromycin. B: steady-state intracellular pH was measured by double perfusion fluorometry for NBCn1-KD#4 cells, control cells, and wild-type (WT) nontransduced Caco2BBe culture. For these samples, the cultures were established in flasks, then seeded on transwell inserts and assayed in subconfluent state. The same culture conditions and times were used for WT and KD cells. Both NBCn1-KD clones have a significantly lowered steady-state pH_i compared with their paired controls. The control cultures grown in the presence of hygromycin have a lower steady-state pH_i than the nontransduced WT cells. Means ± SE, Student’s t test or ANOVA. *P < 0.05, **P < 0.01.

Figure 5.

NBCn1 downregulation affects cell proliferation. A: NBCn1-KD#2 and KD#4 cells were seeded in multiple wells of culture plates at 9000 cells per cm², and maintained for up to 18 days. Cell proliferation was determined by detaching and counting cells from selected culture wells. Control cultures reached confluency at day 11. B: short-term proliferation of Caco2BBe control and KD#4 cells was assessed by the crystal violet method 18 h after seeding in a 96-well plate. The results are given as ratio of the control samples. C: cell-matrix adhesion of NBCn1- KD#2 and KD#4 cells was determined separately in comparison with control cells. Per each well 100,000 cells were seeded in 96-well plates and incubated for 2 h. The ratio of the adherent cells was determined by crystal violet assay after removing nonadherent cells. Means ± SE, ANOVA. **P < 0.01, ***P < 0.001.

Figure 6.

BrdU proliferation assay of Nbcn1^+/+ and Nbcn1^–/– colonoids. A: RT-qPCR of Nbcn1 variants in the mouse colonoid cultures. MEAD-Nbcn1 (the expression of which is controlled by P1 promoter) is the primary variant of Nbcn1 in Nbcn1^+/+ mice colonoid cultures. Expression of MEAD-Nbcn1 is significantly abolished in colonoid cultures from Nbcn1^–/– mice. MERF-Nbcn1 (regulated by the P2 promoter), exhibits basal expression levels with no notable distinction between Nbcn1^+/+ and Nbcn1^–/– cultures; two-way ANOVA. B: mid-colon organoids from Nbcn1^+/+ and Nbcn1^–/– mice in day 2 culture were labeled with BrdU for 12 h and the signal intensity for incorporated BrdU was measured in resuspended cells. Organoids at passage 5 from three mice pairs; Student’s t test, *P < 0.05. C: representative light microscopy images of Nbcn1^+/+ and Nbcn1^–/– organoids which were used for BrdU labeling. The cultures are visually similar. Also, when the diameter of all organoids on the field of vision was assessed for all days and all organoid preparations, the average diameter of Nbcn1^+/+ and Nbcn1^–/– organoids was not significantly different (data not shown). Scale bar = 200 µm.

Figure 7.

NBCn1 knockdown reduces Caco2BBe cell migration. A and B: wound scratch assay was used to determine the migratory activity of NBCn1 knockdown Caco2BBe cells and control cells. Representative images showing the extent of wound healing over 12-h periods from control and NBCn1-KD#4 cells. The dashed line indicates the initial scratch area at the beginning of the assay. Scale bar = 100 µm. All images are shown at the same magnification. C: the area that the cells covered was named wound gap closure and was calculated as a percentage of the initial wound area that was covered by the cells after 12 h. D: NBCn1 mRNA knockdown efficiency in the migration experiments. The experiment showed significantly reduced migratory activity in KD#4 cells. Student’s t test, *P < 0.05, ***P < 0.001.

Figure 8.

NBCn1 localizes to the leading edge of the Caco2BBe cells. Localization of NBCn1 and two protein markers for focal adhesions, namely vinculin and paxillin, was investigated in subconfleunt Caco2BBe wild-type cells. At the plasma membrane, NBCn1 was mainly found at the leading edge, where it in part colocalized with focal adhesion markers paxillin or vinculin. Colocalization analysis and visualization (bottom) were performed in Fiji using the “Colocalization Finder” plugin. DAPI, nuclei; Phalloidin, F-actin.