Sodium/hydrogen exchanger 8 affects sheet migration of human intestinal epithelial cells (HT29MTX) by influencing the actin cytoskeletal rearrangement

The gastrointestinal Na⁺/H⁺ transporter 8 (NHE8) is downregulated in the mucosa of patients with ulcerative colitis, and its deletion in the murine intestine causes a "colitis-like" phenotype. Since ulcerative colitis is characterized by repeated mucosal injury, we investigated the role of NHE8 in intestinal wound repair, by accessing its effect on intracellular pH (pH_i) regulation, cell proliferation, and migration. NHE8 expression was downregulated via shRNA lentiviral transduction in HT29MTX cells. The selected clonal cell line (HT29/shNHE8) with ∼80% reduced NHE8 mRNA expression displayed an increased proliferative but reduced migratory rate compared with the mock-transduced cells (HT29/pLKO1). The wound front of the HT29/shNHE8 cells consisted of both migrating and nonmigrating cells, and pH_i measured in this segment displayed the following values: pH_i(HT29/pLKO.1) = 7.35, pH_i(HT29/shNHE8_migrating) = 7.27, and pH_i(HT29/shNHE8_nonmigrating) = 7.1. The migrating HT29/shNHE8 cells exhibited a significantly increased NHE activity compared with migrating mock-transfected and nonmigrating cells, which was abolished by pharmacological NHE3 inhibition. NHE3 localized to the wound front of HT29/shNHE8, but not to that of HT29/pLKO.1 cells. Cell flattening and lamellipodia development were observed at the wound front in HT29/pLKO.1 cells, whereas the HT29/shNHE8 cells formed tight actin bundles and retained their apical-basal architecture. RAC1 and Cofilin-1, required for the generation of actin-based membrane protrusions, were absent at the wound front of HT29/shNHE8 cells but were expressed in migrating HT29/pLKO.1 cells, where RAC1 partially colocalized with NHE8. The results show that NHE8 downregulation reduces the pH_i and leads to enhanced epithelial cell proliferation, but impairs migration likely due to altered actin polymerization.<b>NEW & NOTEWORTHY</b> The Na⁺/H⁺ exchanger 8 (NHE8) plays an important role in the regulation of intracellular pH_i, cell proliferation, and epithelial sheet migration in HT29MTX intestinal cells. NHE8 knockdown cells lack the ability to dynamically rearrange the actin cytoskeleton at the wound front, resulting in a reduced migration rate. These observations provide insights into the molecular mechanism of the downregulation of this transporter in human ulcerative colitis and its role in epithelial restitution.