Induction of Na⁺/K⁺/2Cl⁻ cotransporter expression mediates chronic potentiation of intestinal epithelial Cl⁻ secretion by EGF

Abstract

Alterations in EGF receptor (EGFR) signaling occur in intestinal disorders associated with dysregulated epithelial transport. In the present study, we investigated a role for the EGFR in the chronic regulation of intestinal epithelial secretory function. Epithelial Cl− secretion was measured as changes in short-circuit current ( Isc) across voltage-clamped monolayers of T84 cells in Ussing chambers. Acute treatment of T84 cells with EGF (100 ng/ml, 15 min) chronically enhanced Isc responses to a broad range of secretagogues. This effect was apparent within 3 h, maximal by 6 h, and sustained for 24 h after treatment with EGF. The Na+/K+/2Cl− cotransporter (NKCC1) inhibitor bumetanide (100 μM) abolished the effect of EGF, indicating increased responses are due to potentiated Cl− secretion. Neither basal nor agonist-stimulated levels of intracellular Ca2+ or PKA activity were altered by EGF, implying that the effects of the growth factor are not due to chronic alterations in levels of second messengers. EGF increased the expression of NKCC1 with a time course similar to that of its effects on Cl− secretion. This effect of EGF was maximal after 6 h, at which time NKCC1 expression in EGF-treated cells was 199.9 ± 21.9% of that in control cells ( n = 21, P < 0.005). EGF-induced NKCC1 expression was abolished by actinomycin D, and RT-PCR analysis demonstrated EGF increased expression of NKCC1 mRNA. These data increase our understanding of mechanisms regulating intestinal fluid and electrolyte transport and reveal a novel role for the EGFR in the chronic regulation of epithelial secretory capacity through upregulation of NKCC1 expression.

The Na–K–Cl cotransporter in the brain edema of acute liver failure

“…We also found increased NKCC1 protein expression, which is known to also contribute to its increased activity [26,[61][62][63][64]. It is possible that increased protein expression may provide more substrate for its oxidation and nitration and subsequent phosphorylation of NKCC1.…”

Epidermal growth factor upregulates serotonin transporter in human intestinal epithelial cells via transcriptional mechanisms

“…EGF is a key regulator of a number of epithelial functions such as restitution, tight junction permeability, electrolyte transport, as well as nutrient and phosphate absorption (2,18,33,35,36,43,44,46,48). Also, EGFR-mediated signaling has been shown previously to be protective in cases of inflammation or infection by Clostridium difficile, indicating the emerging role of growth factors in treatment of intestinal disorders (14,29,41).…”

“…Previous studies have demonstrated that EGF influences intestinal epithelial transport processes by both rapid posttranslational events involving phosphorylation and/or transcriptional mechanisms via alteration in gene expression (2,18,33,35,36,43,44,46,48). Therefore, we first examined short-term effects of EGF on SERT function.…”

“…EGF mediates its effects via binding to its receptor, EGFR, expressed on the basolateral surface in human intestinal epithelial cells (7,30,31). EGF exhibits a wide range of physiological effects including stimulation of electrolyte and nutrient absorption and protective effects on intestinal mucosa in colitis such as the necrotizing colitis model and in human subjects (2,14,18,33,35,36,43,44,46,48). Our studies demonstrate for the first time that EGF upregulates SERT function and expression via EGFR and through mechanisms involving potential AP-1 cis-elements of the SERT promoters.…”

Activated PKCδ and PKCϵ Inhibit Epithelial Chloride Secretion Response to cAMP via Inducing Internalization of the Na+-K+-2Cl− Cotransporter NKCC1

“…The T84 colonic epithelial cell is a widely used and well characterized model for investigating the regulation of transepithelial chloride secretion (6,24,25). However, effective delivery of small interference RNA (siRNA) duplex and exogenous genes into T84 cell monolayers has proven to be a difficult technical challenge.…”