H-Ras mediates the inhibitory effect of epidermal growth factor on the epithelial Na+ channel.

Il-Ha Lee,Sung-Hee Song,Anuwat Dinudom,David I Cook,Michael B Butterworth

doi:10.1371/journal.pone.0116938

Il-Ha Lee, Sung-Hee Song + Show 3 more

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https://doi.org/10.1371/journal.pone.0116938

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Journal: PloS one	Publication Date: Mar 16, 2015
Citations: 8	License type: CC BY 4.0

Affiliation: Robert Bosch (Australia), University of Sydney

Abstract

The present study investigates the role of small G-proteins of the Ras family in the epidermal growth factor (EGF)-activated cellular signalling pathway that downregulates activity of the epithelial Na+ channel (ENaC). We found that H-Ras is a key component of this EGF-activated cellular signalling mechanism in M1 mouse collecting duct cells. Expression of a constitutively active H-Ras mutant inhibited the amiloride-sensitive current. The H-Ras-mediated signalling pathway that inhibits activity of ENaC involves c-Raf, and that the inhibitory effect of H-Ras on ENaC is abolished by the MEK1/2 inhibitor, PD98059. The inhibitory effect of H-Ras is not mediated by Nedd4-2, a ubiquitin protein ligase that regulates the abundance of ENaC at the cell surface membrane, or by a negative effect of H-Ras on proteolytic activation of the channel. The inhibitory effects of EGF and H-Ras on ENaC, however, were not observed in cells in which expression of caveolin-1 (Cav-1) had been knocked down by siRNA. These findings suggest that the inhibitory effect of EGF on ENaC-dependent Na+ absorption is mediated via the H-Ras/c-Raf, MEK/ERK signalling pathway, and that Cav-1 is an essential component of this EGF-activated signalling mechanism. Taken together with reports that mice expressing a constitutive mutant of H-Ras develop renal cysts, our findings suggest that H-Ras may play a key role in the regulation of renal ion transport and renal development.

Highlights

Amiloride-sensitive epithelial Na+ channels (ENaC) are expressed in the epithelium lining the distal collecting tubules of the kidney, distal colon and lung
The present study demonstrates a role for H-Ras in the epidermal growth factor (EGF)-activated cellular signalling pathway that regulates transepithelial Na+ absorption via ENaC
The positive effects of the siRNA directed against H-Ras (Fig. 1C) and of the dominant negative H-RasS17N (Fig. 2A) on the amiloride-sensitive current in M1 cells expressing endogenous ENaC and in Fischer rat thyroid (FRT) cells expressing exogenous ENaC indicate that endogenous H-Ras controls basal activity of ENaC and, that H-Ras is part of an intrinsic regulatory mechanism that keeps transepithelial Na+ absorption via ENaC at an appropriate level

Summary

Introduction

Amiloride-sensitive epithelial Na+ channels (ENaC) are expressed in the epithelium lining the distal collecting tubules of the kidney, distal colon and lung. They play a central role in regulating Na+ homeostatasis, blood pressure, and are important for maintaining total body [1] and alveolar fluid volumes [2]. EGFRs undergo dimerization and phosphorylation of specific tyrosine kinase residues at the cytosolic C-terminal [9, 10] These phosphorylated residues on the EGF receptor (EGFR), in turn, serve as docking sites for cytosolic signalling molecules, including those involved in the MAP kinase, JAK/STAT, phosphoinositol-3-kinase, and protein kinase C pathways [10]. Given the prominent role of Ras GTPases in growth factor signalling, we investigated the role of other isoforms of Ras in this signalling pathway

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