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

Abstract

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.

Human intestinal epithelial cell line SK-CO15 is a new model system to study Na+/H+exchanger 3

“…2B). We showed previously that LPA 5 phosphorylates Erk1/2 via transactivation of the epidermal growth factor receptor (40). Consistent with the robust expression of HA-LPA 5 , LPA markedly activated Erk1/2 in SK-CO15 cells.…”

“…SK-CO15, Caco-2, and Caco-2bbe cells were maintained in DMEM supplemented with 10% FBS, penicillin (50 mU/ml), streptomycin (50 g/ml), 1 mM sodium pyruvate, 15 mM HEPES, and 1ϫ nonessential amino acids. Caco-2bbe stably expressing NHE3 was previously described (20,40). When necessary, cells were transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA).…”

“…Expression of lysophosphatidic acid receptor type 5 (LPA5) in Caco-2bbe cells was attained by using lentivirus harboring hemagglutinin (HA)-tagged LPA 5, as previously reported (20,40).…”

Genetic markers of bevacizumab-induced hypertension

“…Another SNP involving an A to G mutation at position 61 of the 5 0 untranslated region of EGF (rs4444903), which results in higher epidermal growth factor levels in GG-genotype carriers [50], was also associated with bevacizumab-induced hypertension in our study. This observation is noteworthy as the epidermal growth factor receptor (EGFR) may contribute to the development of hypertension by regulating vascular tone and renal sodium handling [51]. Synthetic EGFR inhibitors also reduce blood pressure in some experimental models of hypertension and have been suggested as a novel target for antihypertensive therapy [52].…”

Calmodulin Kinase II Constitutively Binds, Phosphorylates, and Inhibits Brush Border Na+/H+ Exchanger 3 (NHE3) by a NHERF2 Protein-dependent Process

“…EGF stimulates NHE3 via basolateral membrane EGF receptors (36) and via apical EGF receptor transactivation (37). Thus, Caco-2BBe/HA-NHE3 cells had NHE3 activity determined in the presence/absence of 30 M KN-62, and the effects compared with a maximally effective concentration of EGF added both apically and basolaterally with determination of whether both stimulatory processes were additive.…”