Differential MAPK Pathways Utilized for HGF- and EGF-dependent Renal Epithelial Morphogenesis

Abstract

Cells derived from the inner medullary collecting duct undergo in vitro branching tubulogenesis to both the c-met receptor ligand hepatocyte growth factor (HGF) as well as epidermal growth factor (EGF) receptor ligands. In contrast, many other cultured renal epithelial cells respond in this manner only to HGF, suggesting that these two receptors may use independent signaling pathways during morphogenesis. We have therefore compared the signaling pathways for mIMCD-3 cell morphogenesis in response to EGF and HGF. Inhibition of the p42/44 mitogen-activated protein kinase (MAPK) pathway with the mitogen-activated protein kinase kinase (MKK1) inhibitor PD98059 (50 M) markedly inhibits HGF-induced cell migration with only partial inhibition of EGF-induced cell motility. Similarly, HGF-dependent, but not EGF-dependent, branching morphogenesis was more greatly inhibited by the MKK1 inhibitor. Examination of EGF-stimulated cells demonstrated that extracellular-regulated kinase 5 (ERK5) was activated in response to EGF but not HGF, and that activation of ERK5 was only 60% inhibited by 50 M PD98059. In contrast, the MKK inhibitor U0126 markedly inhibited both ERK1/2 and ERK5 activation and completely prevented HGF-and EGF-dependent migration and branching process formation. Expression of dominant negative ERK5 (dn-BMK1) likewise inhibited EGF-dependent branching process formation, but did not affect HGF-dependent branching process formation. Our results indicate that activation of the ERK1/ERK2 signaling pathway is critical for HGF-induced cell motility/morphogenesis in mIMCD-3 cells, whereas ERK5 appears to be required for EGF-dependent morphogenesis.In renal epithelial cells, cell morphogenesis is an important process in embryonic development and repair of injured tubules. Two cell types commonly utilized to examine renal epithelial morphogenesis are murine mIMCD-3 collecting duct cells and canine MDCK tubular cells. In mIMCD-3 cells, both the c-met receptor ligand hepatocyte growth factor (HGF) 1 and the epidermal growth factor receptor (EGFR) ligands, transforming growth factor-␣ and epidermal growth factor (EGF), are capable of mediating branching morphogenesis (1, 2). In contrast, despite the presence of the EGFR in both cell types, only HGF is capable of mediating branching morphogenesis in MDCK cells, suggesting that these two receptors initiate morphogenesis utilizing similar, but not identical, signaling pathways. Upon activation, both the c-met receptor and the EGFR are tyrosine-phosphorylated and subsequently interact with GRB-2 and GAB1 (GRB-2-associated binding protein) (3, 4). These adapter molecules allow the activated receptor to initiate signaling through phosphoinositide 3-kinase (PI3K), phospholipase C-␥ (PLC␥), SHPTP and mitogen-activated protein kinase (MAPK) (3, 5-10). We have found that activation of the PI3K results in an increase in receptor-mediated activation of PLC␥ and that these signaling pathways then mediate PKC activation as an important component of the cell migratory response (11,12). H...

ERK Negatively Regulates the Epidermal Growth Factor-mediated Interaction of Gab1 and the Phosphatidylinositol 3-Kinase

“…Our recent determination that HGF-stimulated epithelial cell morphogenesis requires ERK1/2 activation provides a potential physiologic role for ERK-regulated PI3K activation (18). Interestingly, in this same study, we found that EGF, but not HGF, activates ERK5 in addition to ERK1/2.…”

“…U0126 has been shown to inhibit MEK1, MEK 2 (20), and MEK5 (18) at concentrations less than 50 M, but it does not inhibit MEK3, MEK4, MEK6, MEK7, protein kinase C␣, protein kinase A, PDK1, or other tested serine/threonine kinases (20). U0126 was used rather than PD98059, because we have found that EGF-mediated ERK5 activation, which is critical for EGF-mediated mIMCD-3 cell morphogenesis, is fully inhibited by U0126 but only partially inhibited by PD98059 at concentrations Յ100 M (18). Following EGF stimulation, cells were lysed in 800 l of ice-cold radioimmune precipitation lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2 g/ml aprotinin, 2 g/ml leupeptin, 2 g/ml pepstatin A, 10 mM NaF, 1 mM phenylmethylsulfonyl fluoride, and 1 mM Na 3 VO 4 .…”

“…1A, upper panel). To examine the role of ERK in regulating this association, cells were pretreated with 10 M U0126, a concentration of the MEK inhibitor that prevents both ERK1/2 and ERK5 activation (18). Inhibition of ERK activation resulted in a marked increase in the EGF-stimulated association of p85 with Gab1.…”

Expression of Neutrophil Gelatinase-associated Lipocalin Regulates Epithelial Morphogenesis in Vitro

“…Ngal Stimulates Transient ERK Activation-We have demonstrated previously that cell migration and branching morphogenesis stimulated by HGF and EGF require the activation of the MAPK pathway (5,26). The ability of Ngal to activate MAPK was therefore examined in cells stimulated with Ngal as compared with HGF.…”

Cell Confluence Regulates Hepatocyte Growth Factor-Stimulated Cell Morphogenesis in a β-Catenin-Dependent Manner

“…Akt activation is downregulated in confluent cells. Previous studies in our laboratory and others have shown that HGFstimulated cell migration requires activation of several intracellular signaling pathways, including the PI 3-K and MAPK pathways (9,15,19). HGF-stimulated activation of the downstream proteins Akt (for the PI 3-K pathway) and ERK (for the MAPK pathway) was therefore examined under nonconfluent and confluent conditions using activation state-specific antibodies.…”