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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 (pHi), 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 (pHi) 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. [ABSTRACT FROM AUTHOR]

Cation and anion transport in the colonocyte apical membrane is highly spatially organized along the cryptal axis. Because of lack of experimental accessibility, information about the functionality of ion transporters in the colonocyte apical membrane in the lower part of the crypt is scarce. The aim of this study was to establish an in vitro model of the colonic lower crypt compartment, which expresses the transit amplifying/progenitor (TA/PE) cells, with accessibility of the apical membrane for functional study of lower crypt-expressed Na+/H+ exchangers (NHEs). Colonic crypts and myofibroblasts were isolated from human transverse colonic biopsies, expanded as three-dimensional (3D) colonoids and myofibroblast monolayers, and characterized. Filter-grown colonic myofibroblast–colonic epithelial cell (CM-CE) cocultures (myofibroblasts on the bottom of the transwell and colonocytes on the filter) were established. The expression pattern for ion transport/junctional/stem cell markers of the CM-CE monolayers was compared with that of nondifferentiated (EM) and differentiated (DM) colonoid monolayers. Fluorometric pHi measurements were performed to characterize apical NHEs. CM-CE cocultures displayed a rapid increase in transepithelial electrical resistance (TEER), paralleled by downregulation of claudin-2. They maintained proliferative activity and an expression pattern resembling TA/PE cells. The CM-CE monolayers displayed high apical Na+/H+ exchange activity, mediated to >80% by NHE2. Human colonoid–myofibroblast cocultures allow the study of ion transporters that are expressed in the apical membrane of the nondifferentiated colonocytes of the cryptal neck region. The NHE2 isoform is the predominant apical Na+/H+ exchanger in this epithelial compartment. [ABSTRACT FROM AUTHOR]

Intestinal NaCl, HCO3-, and fluid absorption are strongly dependent on apical Na+/H+ exchange. The intestine expresses three presumably apical sodium-hydrogen exchanger (NHE) isoforms: NHE2, NHE3, and NHE8. We addressed the role of NHE8 [solute carrier 9A8 (SLC9A8)] and its interplay with NHE2 (SLC9A2) in luminal proton extrusion during acute and chronic enterocyte acidosis and studied the differential effects of NHE8 and NHE2 on enterocyte proliferation. In contrast to NHE3, which was upregulated in differentiated versus undifferentiated colonoids, the expression of NHE2 and NHE8 remained constant during differentiation of colonoids and Caco2Bbe cells. Heterogeneously expressed Flag-tagged rat (r)Nhe8 and human (h)NHE8 translocated to the apical membrane of Caco2Bbe cells. rNhe8 and hNHE8, when expressed in NHE-deficient PS120 fibroblasts showed higher sensitivity to HOE642 compared to NHE2. Lentiviral shRNA knockdown of endogenous NHE2 in Caco2Bbe cells (C2Bbe/shNHE2) resulted in a decreased steady-state intracellular pH (pHi), an increased NHE8 mRNA expression, and augmented NHE8-mediated apical NHE activity. Lentiviral shRNA knockdown of endogenous NHE8 in Caco2Bbe cells (C2Bbe/shNHE8) resulted in a decreased steady-state pHi as well, accompanied by decreased NHE2 mRNA expression and activity, which together contributed to reduced apical NHE activity in the NHE8-knockdown cells. Chronic acidosis increased NHE8 but not NHE2 mRNA expression. Alterations in NHE2 and NHE8 expression/activity affected proliferation, with C2Bbe/shNHE2 cells having lower and C2Bbe/shNHE8 having higher proliferative capacity, accompanied by amplified ERK1/2 signaling pathway and increased EGFR expression in the latter cell line. Thus, both Na+/H+ exchangers have distinct functions during cellular homeostasis by triggering different signaling pathways to regulate cellular proliferation and pHi control. [ABSTRACT FROM AUTHOR]

NBCn1 (SLC4A7) is one of the two major Na + -HCO 3 - 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.

Loss of function mutations in the SLC26A3 gene cause chloride-losing diarrhea in mice and humans. Although systemic adaptive changes have been documented in these patients and in the corresponding knockout mice, how colonic enterocytes adapt to loss of this highly expressed and highly regulated luminal membrane anion exchanger remains unclear. To address this question, SLC26A3 was deleted in the self-differentiating Caco2BBe colonic cell line by the CRISPR/Cas9 technique. We selected a clone with loss of SLC26A3 protein expression and morphological features indistinguishable from those of the native cell line. Neither growth curves nor development of transepithelial electrical resistance (TEER) differed between wild-type (WT) and SLC26A3 knockout (KO) cells. Real-time qPCR and Western analysis in SLC26A3-KO cells revealed an increase in AE2 expression without significant change in NHE3 expression or localization. Steady-state pH i and apical and basolateral Cl - /HCO 3 - exchange activities were assessed fluorometrically in a dual perfusion chamber with independent perfusion of luminal and serosal baths. Apical Cl - /HCO 3 - exchange rates were strongly reduced in SLC26A3-KO cells, accompanied by a surface pH more acidic than that of WT cells. Steady-state pH i was not significantly different from that of WT cells, but basolateral Cl - /HCO 3 - exchange rates were higher in SLC26A3-KO than in WT cells. The data show that CRISPR/Cas9-mediated SLC26A3 deletion strongly reduced apical Cl - /HCO 3 - exchange rate and apical surface pH, but sustained a normal steady-state pH i due to increased expression and function of basolateral AE2. The low apical surface pH resulted in functional inhibition of NHE-mediated fluid absorption despite normal expression of NHE3 polypeptide. NEW & NOTEWORTHY SLC26A3 gene mutations cause chloride-losing diarrhea. To understand how colonic enterocytes adapt, SLC26A3 was deleted in Caco2BBe cells using CRISPR/Cas9. In comparison to the wild-type cells, SLC26A3 knockout cells showed similar growth and transepithelial resistance but substantially reduced apical Cl - /HCO 3 - exchange rates, and an acidic surface pH. Steady-state intracellular pH was comparable between the WT and KO cells due to increased basolateral AE2 expression and function.

Intestinal NaCl, HCO 3 - , and fluid absorption are strongly dependent on apical Na + /H + exchange. The intestine expresses three presumably apical sodium-hydrogen exchanger (NHE) isoforms: NHE2, NHE3, and NHE8. We addressed the role of NHE8 [solute carrier 9A8 (SLC9A8)] and its interplay with NHE2 (SLC9A2) in luminal proton extrusion during acute and chronic enterocyte acidosis and studied the differential effects of NHE8 and NHE2 on enterocyte proliferation. In contrast to NHE3, which was upregulated in differentiated versus undifferentiated colonoids, the expression of NHE2 and NHE8 remained constant during differentiation of colonoids and Caco2Bbe cells. Heterogeneously expressed Flag-tagged rat (r)Nhe8 and human (h)NHE8 translocated to the apical membrane of Caco2Bbe cells. rNhe8 and hNHE8, when expressed in NHE-deficient PS120 fibroblasts showed higher sensitivity to HOE642 compared to NHE2. Lentiviral shRNA knockdown of endogenous NHE2 in Caco2Bbe cells (C2Bbe/shNHE2) resulted in a decreased steady-state intracellular pH (pH i ), an increased NHE8 mRNA expression, and augmented NHE8-mediated apical NHE activity. Lentiviral shRNA knockdown of endogenous NHE8 in Caco2Bbe cells (C2Bbe/shNHE8) resulted in a decreased steady-state pH i as well, accompanied by decreased NHE2 mRNA expression and activity, which together contributed to reduced apical NHE activity in the NHE8-knockdown cells. Chronic acidosis increased NHE8 but not NHE2 mRNA expression. Alterations in NHE2 and NHE8 expression/activity affected proliferation, with C2Bbe/shNHE2 cells having lower and C2Bbe/shNHE8 having higher proliferative capacity, accompanied by amplified ERK1/2 signaling pathway and increased EGFR expression in the latter cell line. Thus, both Na + /H + exchangers have distinct functions during cellular homeostasis by triggering different signaling pathways to regulate cellular proliferation and pH i control.