Role of ERK 1 / 2-mTORC 1-Nox 4 in TGF-β 1-induced podocyte apoptosis 1 Transforming Growth Factor-β 1-induced Apoptosis in Podocytes via Extracellular-signal-regulated Kinase-Mammalian Target of Rapamycin Complex 1-NADPH Oxidase 4 Axis

Transforming Growth Factor-β (TGF-β) is a pleiotropic cytokine which accumulates during kidney injuries resulting in various renal diseases. We have previously reported that TGF-β1 induces selective upregulation of mitochondrial Nox4 playing critical roles in podocyte apoptosis. Here, we investigated the regulatory mechanism of Nox4 upregulation by mTORC1 activation on TGF-β1-induced apoptosis in immortalized podocytes. TGF-β1 treatment markedly increased phosphorylation of mTOR and its downstream target p70S6K and 4EBP1. Blocking TGF-β receptor-I by SB431542 completely blunted phosphorylation of mTOR, p70S6K and 4EBP1. Transient adenoviral over-expression of mTORWT and constitutively active mTORΔ augmented TGF-β1-treated Nox4 expression, ROS generation and apoptosis, while mTOR-KD suppressed above changes. In addition, knock-down of mTOR by simTOR mimicked the effect of mTOR-KD. Inhibition of mTORC1 by low dose of rapamycin or sip70S6K protected podocytes through attenuation of Nox4 expression and subsequent oxidative stress-induced apoptosis by TGF-β1. Pharmacological inhibition of MEK-ERK cascade, but not PI3K-Akt-TSC2 pathway, abolished TGFβ1-induced mTOR activation. Inhibition of neither ERK1/2 nor mTORC1 reduced the TGF-β1stimulated increase of Nox4 mRNA level, however, 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-mTOR activation. Our data suggest that TGF-β1 increases translation of Nox4 through Smad-ERK1/2mTORC1 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 ERK1/2-mTORC1 pathway could be a potential therapeutic and preventive target against proteinuric and chronic kidney diseases. -----------------------------------------------------------Podocyte damage is one of the key determinants of majority of diabetic and nondiabetic glomerular diseases leading to chronic kidney diseases (CKD) and end-stage renal disease (ESRD) (1). Leakage of plasma proteins http://www.jbc.org/cgi/doi/10.1074/jbc.M115.703116 The latest version is at JBC Papers in Press. Published on November 12, 2015 as Manuscript M115.703116 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. at U N IV O F N E B R A SK A L incoln on D ecem er 7, 2015 hp://w w w .jb.org/ D ow nladed from Role of ERK1/2-mTOR-Nox4 in TGF-β1-induced podocyte apoptosis 2 in urine indicates onset of renal diseases which are associated with podocyte injury (2-4). Recent evidences show that mammalian target of rapamycin (mTOR) signaling activation is an important mediator of diabetic nephropathy in mice and human (5,6). Increased mTOR activity have been reported in different glomerular diseases and mTORC1 inhibition by rapamycin and everolimus shows beneficial effects against diabetic nephropathy, focal segmental glomerulosclerosis (FSGS), minimal change disease, membranous nephropathy etc (7-10). But contrasting evidences also indicate that mTORC1 inhibition by immunosuppressive drug rapamycin leads proteinuria, podocyte apoptosis and develops primary FSGS in renal transplant recipients (11). Therefore, it might be important to investigate detail molecular mechanisms to answer this puzzle and identify therapeutic targets. mTOR is an atypical serine/threonine kinase belonging to the member of PIKK family of protein kinase and conserved in yeast to human (12,13). It forms two functionally different multiprotein complexes – mTOR complex 1 (mTORC1), sensitive to rapamycin and mTOR complex 2 (mTORC2), rapamycin insensitive (14). Functional mTORC1 consists of mTOR, mLST8 and raptor, while mTORC2 includes mTOR, mLST8, mSIN1 and rictor. These signaling pathways regulate major physiological events including regulation of ribosome biogenesis and protein translation (15), lipid homeostasis (16) and mitochondrial biogenesis (17), however is also implicated in various disease progression such as type 2 diabetes and obesity (14). mTORC1 activation results in phosphorylation of ribosomal protein S6 via p70S6K and eukaryotic translation initiation factor 4E binding protein (4EBP) which potentiates initiation of protein translation (18,19). However, mTORC2 regulates cell survival through phosphorylation of Akt (Ser473) and cytoskeleton organization through phosphorylation of PKCα (20,21). Tuberous sclerosis 1 (TSC1)/hamartin forms heterodimer with tuberous sclerosis 2 (TSC2)/tuberin to negatively regulate mTORC1 activity. Many growth factors including insulin, IGF, transforming growth factor-β (TGF-β) and nutrients such as amino acids exert their cellular inputs through modulation of mTOR complexes (22,23). Akt, ERK1/2 and AMPK are well known intermediate signaling molecules which transmit extracellular signal to mTOR (15,24). TGF-β is a multi-functional cytokine involved in many cellular processes including migration, growth inhibition, invasion, extracellular matrix remodeling, epithelial mesenchymal transition, and fibrosis (25). Increased activity of TGF-β plays critical roles in progression of renal diseases in animal model and human renal diseases where podocyte loss is highly evident (26,27). Furthermore, accumulation of TGF-β and its pathogenic roles in primary FSGS, progressive glomerulosclerosis and diabetic nephropathy have been reported (26,28). In addition, adenovirusmediated TGF-β1 gene transfer to kidney glomeruli leads to proteinuria in rats (29). It is recently reported that podocyte specific overexpression of TGF-β induces segmental glomerulosclerosis with dysfunction in endothelial cells and podocyte depletion (30). We also previously published that TGF-β1 induces podocyte apoptosis through transcriptional upregulation of NADPH oxidase 4 (Nox4) by activation of Smad pathway (31). There is also evidence that high glucose-mediated inactivation of AMPK activates mTOR in a TSC2 dependent manner, which accounts for Nox4-mediated podocyte depletion in diabetic mice (32). But the possibility of mTOR activation by TGF-β1 in podocyte apoptosis has not been addressed so far. Moreover, molecular mechanism of Nox4 regulation by TGF-β1-mTOR axis has not been explored so far in detail. In this study we observed that Nox4 is upregulated by TGF-β1 via rapamycin sensitive mTORC1 activation as Nox4-induced oxidative stress and mitochondrial dysfunctions are ameliorated with rapamycin treatment or silencing of mTOR or its downstream target p70S6K. ERK1/2 plays an important role in TGF-β1induced mTOR activation and translational regulation of Nox4 protein. Finally, Smad signaling regulates TGF-β1-mediated ERK1/2 phosphorylation in podocytes. Therefore, we suggest that inhibition of ERK1/2-mTOR pathway at U N IV O F N E B R A SK A L incoln on D ecem er 7, 2015 hp://w w w .jb.org/ D ow nladed from Role of ERK1/2-mTOR-Nox4 in TGF-β1-induced podocyte apoptosis 3 could provide an effective protection against podocyte depletion in glomerular kidney diseases. EXPERIMENTAL PROCEDURES Cell culture and drugs Immortalized mouse podocytes were obtained from the laboratory of Prof. Peter Mundel at Harvard Medical School, USA and was cultured as described previously (31). Cells were grown on collagen coated 100mm dishes using low glucose Dulbecco's Modified Eagles Medium supplemented with 5% fetal bovine serum (FBS) and 100U/ml penicillin, and 100μg/ml streptomycin. Cell proliferation was achieved at 33°C in the presence of 20U/ml mouse recombinant interferon-γ and for differentiation these cells were thermo-shifted to 37°C in the absence of interferon-γ for 10~14 days. Before experiments, cells were changed with media containing 0.2% FBS for 24 hr. Chemicals were purchased as following SB431542 (Catalog #S4317) and rapamycin (Catalog #R8781) from Sigma (St. Louis, MO, USA), U0126 (Catalog #9903) and PD184352 (Catalog #12147) from Cell Signaling Technology (Danvers, MA, USA) and cycloheximide (Catalog #14126) from Cayman Chemical (Ann Arbor, MI, USA). Quantitative Real-time PCR To isolate total RNA, podocytes were grown on 100mm dishes. Cells were washed twice with PBS before trypsinization. RNA was isolated according to the RNeasy kit (Catalog #74134, Qiagen GmbH, Hilden, Germany). cDNA was synthesized from 0.5-1μg of RNA with a reverse transcription kit (Applied Bioscience, Foster City, CA, USA) using oligo-dT in a reaction volume of 20-40μl according to the company instruction. cDNA was subjected to real-time PCR to evaluate mRNA expression of Nox4 with the help of a sequence-specific primer (forward5’CCACAGACCTGGATTTGGAT-3’, reverse5’TGGTGACAGGTTTGTTGCTC-3’). β-actin (forward-5’-AAGAGCTATGAGCTGCCTGA-3’, reverse-5’-CACAGGATTCCATACCCAAG-3’) primer was considered as a reference control. Experiments were performed in triplicate in a real time PCR machine (7900HT, Applied Bioscience) using SYBR Green PCR Master Mix (Catalog #204143, Qiagen GmbH). Data analysis was performed according to ΔΔCT method (33). Western blot For isolation of total protein podocytes were differentiated on 100mm dishes or on 6-well plates. The cells were carefully washed twice with ice cold PBS and lysed with cold RIPA buffer (Thermo Scientific, Rockford, IL, USA) containing protease and phosphatase inhibitor cocktail (Thermo Scientific, Catalog #88665 and Catalog #88667). The crude lysates were centrifuged in a table-top centrifuge at 13,200 rpm for 20 min at 4°C and clear supernatants were carefully transferred to a new eppendorf tube. BCA protein assay kit (Thermo scientific) was used to determine concentration of total protein. Equal amount of proteins were loaded to 6%~10% SDS PAGE and transferred to polyvinylidenedifluoride (PVDF) membrane (Millipore Corporation, Billerica, MA). The membrane was blocked with either with 6% skim milk or 5% BSA followed by primary antibody incubati