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 (pHi) 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 pHi measured in this segment displayed the following values: pHi(HT29/pLKO.1) = 7.35, pHi(HT29/shNHE8migrating) = 7.27, and pHi(HT29/shNHE8nonmigrating) = 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 pHi and leads to enhanced epithelial cell proliferation, but impairs migration likely due to altered actin polymerization.NEW & NOTEWORTHY The Na+/H+ exchanger 8 (NHE8) plays an important role in the regulation of intracellular pHi, 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.