Transforming Growth Factor β1-induced Apoptosis in Podocytes via the Extracellular Signal-regulated Kinase-Mammalian Target of Rapamycin Complex 1-NADPH Oxidase 4 Axis

Ranjan Das,Shanhua Xu,Tuyet Thi Nguyen,Xianglan Quan,Seong-Kyung Choi,Soo-Jin Kim,Eun Young Lee,Seung-Kuy Cha,Kyu-Sang Park

doi:10.1074/jbc.m115.703116

Abstract

TGF-β is a pleiotropic cytokine that accumulates during kidney injuries, resulting in various renal diseases. We have reported previously that TGF-β1 induces the selective up-regulation of mitochondrial Nox4, playing critical roles in podocyte apoptosis. Here we investigated the regulatory mechanism of Nox4 up-regulation by mTORC1 activation on TGF-β1-induced apoptosis in immortalized podocytes. TGF-β1 treatment markedly increased the phosphorylation of mammalian target of rapamycin (mTOR) and its downstream targets p70S6K and 4EBP1. Blocking TGF-β receptor I with SB431542 completely blunted the phosphorylation of mTOR, p70S6K, and 4EBP1. Transient adenoviral overexpression of mTOR-WT and constitutively active mTORΔ augmented TGF-β1-treated Nox4 expression, reactive oxygen species (ROS) generation, and apoptosis, whereas mTOR kinase-dead suppressed the above changes. In addition, knockdown of mTOR mimicked the effect of mTOR-KD. Inhibition of mTORC1 by low-dose rapamycin or knockdown of p70S6K protected podocytes through attenuation of Nox4 expression and subsequent oxidative stress-induced apoptosis by TGF-β1. Pharmacological inhibition of the MEK-ERK cascade, but not the PI3K-Akt-TSC2 pathway, abolished TGF-β1-induced mTOR activation. Inhibition of either ERK1/2 or mTORC1 did not reduce the TGF-β1-stimulated increase in Nox4 mRNA level but significantly inhibited total Nox4 expression, ROS generation, and apoptosis induced by TGF-β1. Moreover, double knockdown of Smad2 and 3 or only Smad4 completely suppressed TGF-β1-induced ERK1/2-mTORactivation. Our data suggest that TGF-β1 increases translation of Nox4 through the Smad-ERK1/2-mTORC1 axis, which is independent of transcriptional regulation. Activation of this pathway plays a crucial role in ROS generation and mitochondrial dysfunction, leading to podocyte apoptosis. Therefore, inhibition of the ERK1/2-mTORC1 pathway could be a potential therapeutic and preventive target in proteinuric and chronic kidney diseases.

Highlights

We report on TGF-␤1-stimulated NADPH oxidase 4 (Nox4) up-regulation through ERK1/2 and mammalian target of rapamycin (mTOR) signaling, which accelerates oxidative stress-induced mitochondrial dysfunction and apoptosis in immortalized podocytes
We have reported that TGF-␤1 selectively up-regulates the transcription of Nox4 mRNA by Smad2/3, resulting in an elevated mitochondrial Nox4 protein level, oxidative stress, mitochondrial dysfunction, and apoptosis in podocytes [31]
This study identifies a distinct molecular mechanism of translational regulation of Nox4 protein that is considered another crucial step in TGF-␤1

Summary

Introduction

ERK1/2 plays an important role in TGF-␤1-induced mTOR activation and translational regulation of Nox4 protein. Treatment with simTOR suppressed TGF-␤1-induced Nox4 protein up-regulation and completely recovered TGF-␤1-stimulated ROS generation in podocytes (Fig. 2, B and C). Sip70S6K significantly reduced the total p70S6K protein level and protected TGF-␤1-induced Nox4 expression, ROS generation, and apoptosis in podocytes (Fig. 3, H–K).

Results

Conclusion