Lysophosphatidic acid 5 receptor induces activation of Na(+)/H(+) exchanger 3 via apical epidermal growth factor receptor in intestinal epithelial cells

Na(+) absorption is a vital process present in all living organisms. We have reported previously that lysophosphatidic acid (LPA) acutely stimulates Na(+) and fluid absorption in human intestinal epithelial cells and mouse intestine by stimulation of Na(+)/H(+) exchanger 3 (NHE3) via LPA(5) receptor. In the current study, we investigated the mechanism of NHE3 activation by LPA(5) in Caco-2bbe cells. LPA(5)-dependent activation of NHE3 was blocked by mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 and U0126, but not by phosphatidylinositol 3-kinase inhibitor LY294002 or phospholipase C-β inhibitor U73122. We found that LPA(5) transactivated the epidermal growth factor receptor (EGFR) and that inhibition of EGFR blocked LPA(5)-dependent activation of NHE3, suggesting an obligatory role of EGFR in the NHE3 regulation. Confocal immunofluorescence and surface biotinylation analyses showed that LPA(5) was located mostly in the apical membrane. EGFR, on the other hand, showed higher expression in the basolateral membrane. However, inhibition of apical EGFR, but not basolateral EGFR, abrogated LPA-induced regulation of MEK and NHE3, indicating that LPA(5) selectively activates apical EGFR. Furthermore, transactivation of EGFR independently activated the MEK-ERK pathway and proline-rich tyrosine kinase 2 (Pyk2). Similarly to MEK inhibition, knockdown of Pyk2 blocked activation of NHE3 by LPA. Furthermore, we showed that RhoA and Rho-associated kinase (ROCK) are involved in activation of Pyk2. Interestingly, LPA(5) did not directly activate RhoA but was required for transactivation of EGFR. Together, these results unveil a pivotal role of apical EGFR in NHE3 regulation by LPA and show that the RhoA-ROCK-Pyk2 and MEK-ERK pathways converge onto NHE3.