AEG-1 participates in high glucose-induced activation of Rho kinase and epithelial-mesenchymal transition in proximal tubular epithelial cells

Wen-Ning Li， Jia-Li Wei， Ming Wu， Wei Wu， Yun Huang， Mao-Wei Xie， Hui Han

1Department of Nephrology， Hebei Medical University， Shijiazhuang 050011， Hebei， China

2Department of Nephrology， Hainan General Hospital， Haikou， Hainan 570311， China

3Hainan Health Department， Haikou， Hainan 570311， China

AEG-1 participates in high glucose-induced activation of Rho kinase and epithelial-mesenchymal transition in proximal tubular epithelial cells

Wen-Ning Li1，2#， Jia-Li Wei2#*， Ming Wu3， Wei Wu2， Yun Huang2， Mao-Wei Xie2， Hui Han2

1Department of Nephrology， Hebei Medical University， Shijiazhuang 050011， Hebei， China

2Department of Nephrology， Hainan General Hospital， Haikou， Hainan 570311， China

3Hainan Health Department， Haikou， Hainan 570311， China

ARTICLE INFO

Article history：

in revised form 20 October 2015

Accepted 3 November 2015

Available online 20 December 2015

Astrocyte elevated gene-1

Epithelial-mesenchymal transition

High glucose

Renal tubulointerstitial fibrosis

Rho kinase

Objective： To prove whether astrocyte elevated gene-1 （AEG-1） plays a role in high glucosestimulated Rho kinase activation and epithelial-mesenchymal transition （EMT） in human renal tubular epithelial （HK-2） cells. Methods： The protein levels of AEG-1， alpha-smooth muscle actin， E-cadherin and MYPT1 were determined by Western blot. Results： AEG-1 protein level was upregulated in HK-2 cells stimulated with high glucose. AEG-1 siRNA downregulated Rho kinase protein expression and blocked high glucose-induced EMT. Conclusions： Our results show that AEG-1 acts a key role in high glucoseinduced activation of Rho kinase and EMT in HK-2 cells.

1. Introduction

Tubulointerstitial fibrosis is a progressive pathomechanism in most chronic renal disorders. Excessive generation of extracellular matrix from the activated interstitial myofibroblasts takes part in the pathoganism of kidney interstitial fibrosis. Accumulating studies indicate that these myofibroblasts might come from tubular epithelial cells through epithelial- to- mesenchymal transition

（EMT）［1,2］. EMT is a process of losing polygonal shape and obtaining the myofibroblast phenotype［3］ and is thought to participate in the process of renal tubulointerstitial fibrosis［1,2］.

Astrocyte elevated gene-1 （AEG-1） was first discovered in primary human fetal astrocytes induced by HIV-1 or tumor necrosis factor- α［4］. Recent studies confirmed that AEG-1 shows an abnormally high expression in many malignant tumors［5,6］and plays a major role in cellular transformation， apoptosis inhibition， invasion， metastasis， angiogenesis and resistance to chemotherapeutic agents via activation of various signaling pathways［7-12］. Our previous report demonstrated that AEG-1 takes part in EMT process induced by TGF-beta1［13］.

Rho kinase， members of the Ras superfamily， are major effectors of small GTPase RhoA. Rho kinase act an crucial role in organization of actin cytoskeleton， cellular contraction， adhesion，migration and proliferation［14,15］. According to the previous reports，Rho kinase also take part in the process of EMT， while the exact mechanism remains to be elucidated. Our present study is to find out whether AEG-1 participates in high glucose-induced Rho kinase upregulation and EMT in HK-2 cells.

2. Materials and methods

2.1. Cell culture

Human proximal tubular （HK-2） cell was bought from cell bank of Xiangya Central Experiment Laboratory and prepared as described previously［13］. The protein expressions of a-smooth muscle actin（SMA）， E-cadherin and AEG-1 were detected by Western blot after incubation with 5.5 mM， 25 mM D-glucose or 5.5 mM D-glucose combined with 19.5 mM mannitol served as hyperosmosis for 48 h. In addition， HK-2 cells were incubated with high glucose for 48 h with or without AEG-1 siRNA transfection （Shanghai R&S Biotechnology， Co.， Ltd）.

2.2. RNA interference

AEG-1 （MTDH） siRNA oligonucleotide were synthesised by Shanghai R&S Biotechnology， Co. Ltd. No. of Gene Bank：NM_178812.3， F： 50-GACACUGGAGAUGCU AAUAUU- 30， R：50-UAUUAGCAUCUCCAGUGUCUU-30. Lipofectamine 2000 reagent （Invitrogen， Grand Island， NY， USA） was used to introduce siRNA into cells. Cells were stimulated with 5.5 mM， 25 mM D-glucose or 5.5 mM D-glucose combined with 19.5 mM mannitol for 48 h after transfected into HK-2 cells for five hours.

2.3. Western blot

Protein was prepared as described previously［13］ and detected with antibodies against AEG-1 （Abcam）， E-cadherin （BD Biotechnology），a-SMA （Sigma）， MYPT1 （Santa Cruz Biotechnology） and beta-actin（Sigma）.

2.4. Statistical test

Data were shown as means ± SD. Statistical test was conducted by ANOVA， and significance was defined as P ＜ 0.05.

3. Results

3.1. AEG-1 involved in high glucose-stimulated EMT in HK-2 cells

High glucose upregulated the protein levels of AEG-1 and a-SMA，but downregulated E-cadherin protein expression when compared with the control. AEG-1 siRNA potently reversed high glucoseinduced EMT （Figure 1）.

3.2. AEG-1 participation in activation of Rho kinase stimulated by high glucose.

High glucose significantly increased MYPT1 protein expression in HK-2 cells， which could be markedly reversed by AEG-1 siRNA（Figure 2）.

4. Discussion

Our results hold the hypothesis that AEG-1 acts a major effect in high glucose-stimulated EMT； while EMT， epithelial cell phenotypic changes include losing their polygonal morphology， expressing alpha-SMA and vimentin， and the inhibition of E-cadherin［16］. At present， our findings showed that high glucose upregulated AEG-1and a-SMA protein levels， but downregulated the protein levels of E-cadherin when compared with the control. Knockdown of AEG-1 potently reversed high glucose-induced EMT， suggesting that AEG-1 is helpful to high glucose-stimulated EMT.

Based on the previous reports， Rho kinase is involved in cytoskeletal arrangement［15］， EMT［13］ and in renal injury such as renal fibrosis in unilateral ureteral obstructive kidney disease［17］，and ischemic acute renal failure［18］. Nagatoya K et al［19］ reported that Y-27632， a specific ROCK inhibitor， blocks interstitial fibrosis in mouse kidneys with unilateral ureteral obstruction. In subtotally nephrectomized spontaneously hypertensive rats， fasudil might be a treatment optimism for renal injury in part through improving tubulointerstitial inflammation［20］. Meanwhile， Rho- kinase also regulates angiotensin II-stimulated kidney leision［21］. In this study，we also found out that AEG-1 depletion inhibited high glucoseinduced Rho kinase activation， indicating that AEG-1 is involved in high glucose-mediated upregulationof Rho kinase in proximal tubular epithelial cells.

Our results first show that tubular EMT-induced by high glucose is closely associated with Rho kinase signal pathway and AEG-1 activation. From these observations， we aim to recognize its role in AEG-1 activation stimulated by high glucose and AEG-1 may be a strategic target for therapeutic development against high glucoseinduced EMT. Moreover， AEG-1 depletion does not completely block EMT process， indicating that other signal events might be involved.

In conclusion， we display that AEG-1 involves in Rho kinase activation and EMT stimulated by high glucose in HK-2 cells.

Conflicts of interest statement

The authors have no financial conflicts of interest.

［1］ Burns WC， Kantharidis P， Thomas MC. The role of tubular epithelialmesenchymal transition in progressive kidney disease. Cells Tissues Organs 2007； 185（1-3）： 222-231.

［2］ Picard N， Baum O， Vogetseder A， Kaissling B， Le Hir M. Origin of renal myofibroblasts in the model of unilateral ureter obstruction in the rat. Histochem Cell Biol 2008； 130（1）： 141-155.

［3］ Essawy M， Soylemezoglu O， Muchaneta-Kubara EC， Shortland J，Brown CB， el Nahas AM. Myofibroblasts and the progression of diabetic nephropathy. Nephrol Dial Transplant 1997； 12（1）： 43-50.

［4］ Emdad L， Sarkar D， Su ZZ， Lee SG， Kang DC， Bruce JN，et al. Astrocyte elevated gene-1： recent insights into a novel gene involved in tumor progression， metastasis and neurodegeneration. Pharmacol Ther 2007；114（2）： 155-170.

［5］ Sarkar D， Emdad L， Lee SG， Yoo BK， Su ZZ， Fisher PB. Astrocyte elevated gene-1： far more than just a gene regulated in astrocytes. Cancer Res 2009； 69（22）： 8529-8535.

［6］ Yu C， Chen K， Zheng H， Guo X， Jia W， Li M， et al. Overexpression of astrocyte elevated gene-1 （AEG-1） is associated with esophageal squamous cell carcinoma （ESCC） progression and pathogenesis. Carcinogenesis 2009； 30（5）： 894-901.

［7］ Emdad L， Lee SG， Su ZZ， Jeon HY， Boukerche H， Sarkar D， et al. Astrocyte elevated gene-1 （AEG-1） functions as an oncogene and regulates angiogenesis. Proc Natl Acad Sci U S A 2009； 106（50）： 21300-21305.

［8］ Gnosa S， Shen YM， Wang CJ， Zhang H， Stratmann J， Arbman G， et al. Expression of AEG-1 mRNA and protein in colorectal cancer patients and colon cancer cell lines. J Transl Med 2012； 10（1）： 109.

［9］ Jiang T， Zhu A， Zhu Y， Piao D. Clinical implications of AEG-1 in liver metastasis of colorectal cancer. Med Oncol 2012； 29（4）： 2858-2863.

［10］ Ke ZF， Mao X， Zeng C， He S， Li S， Wang LT. AEG-1 expression characteristics in human non-small cell lung cancer and its relationship with apoptosis. Med Oncol 2013； 30（1）： 383.

［11］ Li C， Liu J， Lu R， Yu G， Wang X， Zhao Y， et al. AEG -1 overexpression：a novel indicator for peritoneal dissemination and lymph node metastasis in epithelial ovarian cancers. Int J Gynecol Cancer 2011； 21（4）： 602-608.

［12］ Wei J， Li Z， Ma C， Zhan F， Wu W， Han H， et al. Rho kinase pathway is likely responsible for the profibrotic actions of aldosterone in renal epithelial cells via inducing epithelial-mesenchymal transition and extracellular matrix excretion. Cell Biol Int 2013； 37（7）： 725-730.

［13］ Wei J， Li Z， Chen W， Ma C， Zhan F， Wu W. AEG-1 participates in TGF-beta1- induced EMT through p38 MAPK activation. Cell Biol Int 2013；37（9）： 1016-1021.

［14］ Nobes CD， Hall A. Rho， rac， and cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers，lamellipodia， and filopodia. Cell 1995； 81（1）： 53-62.

［15］ Haudenschild DR， D'Lima DD， Lotz MK. Dynamic compression of chondrocytes induces a Rho kinase-dependent reorganization of the actin cytoskeleton. Biorheology 2008； 45（3-4）： 219-228.

［16］ Masszi A， Fan L， Rosivall L， McCulloch CA， Rotstein OD， Mucsi I， et al. Integrity of cell-cell contacts is a critical regulator of TGF-beta 1-induced epithelial-to-myofibroblast transition： role for beta-catenin. Am J Pathol 2004； 165（6）： 1955-1967.

［17］ Fu P， Liu F， Su S， Wang W， Huang XR， Entman ML， et al. Signaling mechanism of renal fibrosis in unilateral ureteral obstructive kidney disease in ROCK1 knockout mice. J Am Soc Nephrol 2006； 17（11）： 3105-3114.

［18］ Teraishi K， Kurata H， Nakajima A， Takaoka M， Matsumura Y. Preventive effect of Y-27632， a selective Rho-kinase inhibitor， on ischemia/ reperfusion-induced acute renal failure in rats. Eur J Pharmacol 2004；505（1-3）： 205-211.

［19］ Nagatoya K， Moriyama T， Kawada N， Takeji M， Oseto S， Murozono T，et al. Y-27632 prevents tubulointerstitial fibrosis in mouse kidneys with unilateral ureteral obstruction. Kidney Int 2002； 61（5）： 1684-1695.

［20］ Kanda T， Wakino S， Hayashi K， Homma K， Ozawa Y， Saruta T. Effect of fasudil on Rho-kinase and nephropathy in subtotally nephrectomized spontaneously hypertensive rats. Kidney Int 2003； 64（6）： 2009-2019.

［21］ Rupérez M， Sánchez-López E， Blanco-Colio LM， Esteban V， Rodríguez-Vita J， Plaza JJ， et al. The Rho-kinase pathway regulates angiotensin II-induced renal damage. Kidney Int 2005； 68（Suppl 99）： S39-S45.

15 September 2015

Jia-Li Wei， M.D.， Department of Nephrology， Hainan General Hospital， Haikou， Hainan 570311， China.

Tel： 86-898-68622452

E-mail： wjl525@163.com

Fax： 86-898-68622456

Foundation project： This work was supported by Natural Science Foundation of China （81560124）， Social Development in Hainan Province Science and Technology Projects （No. 2012SF04， 2013SF04 and 2015SF43）.

#： These authors contributed equally to this work.