Mo1809 Enteropathogenic E. coli Non-Lee Encoded Effectors NleH1 and NleH2 Attenuate Activation of Mitogen-Activated Protein Kinases

Abstract

BACKGROUND: Regulation of the brush border (BB) Na+/H+ exchanger, NHE3, accounts for most of the recognized digestion-related changes in neutral Na+ absorption, as well as most of the inhibition of Na+ absorption that occurs in diarrheal diseases. NHE3 regulation usually occurs by changes in the rates of endocytosis and/or exocytosis. NHE3 traffics between the plasma membrane and recycling endosomes under basal and regulated conditions. Constitutive NHE3 endocytosis occurs through a clathrin-dependent process. In intestinal epithelial cells, carbachol (CCH) elevation of intracellular Ca2+ ([Ca]i) decreases NHE3 activity and stimulates endocytosis as early as one minute; however, the mechanism involved in calcium-mediated endocytosis of NHE3 remains to be determined. Furthermore, a pool of BB NHE3 resides in lipid rafts which contributes to basal, but not cAMP-mediated, NHE3 trafficking, suggesting an alternative mechanism exists for NHE3 endocytosis. Recently, the Rho GTPase, Cdc42, was demonstrated to play an integral role in cholesterol-sensitive, clathrin-independent endocytosis. However, it has not been determined whether [Ca]imediated NHE3 endocytosis occurs through a clathrin-independent, lipid raft-dependent pathway involving Cdc42. PURPOSE: Therefore, the current study was designed to test the hypothesis that (1) inhibition of clathrin-mediated endocytosis (CME) prevents constitutive, but not CCH-mediated, endocytosis of NHE3, and (2) CCH-mediated endocytosis of NHE3 occurs through a lipid raft, activated Cdc42-dependent pathway that does not involve clathrin. METHODS: 12 day post-confluent WT, Rab5 and Cdc42 shRNA knockdown (KD) Caco-2/BBe cells grown on filters were transiently infected with a 3HA-NHE3 adenovirus construct and studied 48 hours later. NHE3 activity was measured by fluorimetry using the pH-sensitive dye, BCECF, in cells treated with vehicle or 10μM CCH to elevate [Ca]i as well as 10mM methyl-β-cyclodextrin (MβCD) to disrupt lipid rafts. CME was blocked by chlorpromazine (CPZ) or K+ depletion. Cdc42 activity was determined in total cell lysates obtained (with 0.1% Triton X-100) from cells treated with vehicle or CCH at various time points. RESULTS: Basal NHE3 activity, but not CCH-inhibition, was increased in WT cells treated with CPZ or K+ depletion as well as in Rab5 KD cells supporting that constitutive NHE3 endocytosis is regulated by CME. CCH rapidly stimulated Cdc42 activity as early as 1min. CCH-mediated inhibition of NHE3 activity was abolished in WT cells treated with MβCD as well as in Cdc42 KD cells. CONCLUSIONS: CCH-mediated inhibition of NHE3 activity is not dependent on clathrin or Rab5 and is regulated by lipid rafts and requires Cdc42. Elevation of [Ca]i may specifically stimulate a novel clathrin-independent endocytic process to regulate protein trafficking in intestinal epithelial cells.