Suppression of Par-4 Protects Human Renal Proximal Tubule Cells from Apoptosis Induced by Oxidative Stress

Background: Oxidative stress is an important inducer of cell apoptosis and plays a key role in the development of renal inflammation. The prostate apoptosis response factor-4 (Par-4) gene was originally identified in prostate cells undergoing apoptosis. Subsequently, Par-4 was found to possess potent pro-apoptotic activity in various cellular systems. However, it remains unclear whether Par-4 is involved in oxidant injury of renal tubular epithelial cells. Aims: To determine the role of Par-4 in renal proximal tubular cell apoptosis induced by oxidative stress. Methods: Par-4 gene expression was silenced by small interfering RNA. Renal proximal tubular cells were then exposed to hydrogen peroxide and the effect of Par-4 silencing on apoptosis and expression of phosphorylated Akt and vascular endothelial growth factor was determined. Results: Hydrogen peroxide induced apoptosis and increased Par-4 expression in human renal proximal tubular epithelial cells. Par-4 silencing significantly protected renal proximal tubular cells from apoptosis via activating the PI3K/Akt signaling pathway as Akt phosphorylation was enhanced. Par-4 silencing also ameliorated Received: June 30, 2009 Accepted: June 22, 2010 Published online: September 1, 2010 Chang-Ying Xing Division of Nephrology, Department of Internal Medicine The First Affiliated Hospital of Nanjing Medical University Guangzhou Road 300, Nanjing 210029, Jiangsu (China) Tel. +86 139 5165 3266, Fax +86 258 629 1273, E-Mail cyxing_nanjing @ 163.com © 2010 S. Karger AG, Basel 1660–2129/11/1173–0053$38.00/0 Accessible online at: www.karger.com/nee D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /1 9/ 20 17 1 2: 27 :4 9 P M Sun /Lu /Zhou /Xing Nephron Exp Nephrol 2011;117:e53–e61 e54 Prostate apoptosis response factor-4 (Par-4) was initially identified in human prostate cell lines. Subsequently, Par-4 was found to possess potent pro-apoptotic activity in various cellular systems in response to numerous stimuli. The Par-4 gene maps to chromosome 12q21, a region frequently deleted in malignant tumors, and encodes a 38-kDa protein. Par-4 contains a leucine zipper domain in the carboxyterminal region that interacts with a variety of proteins, including the atypical protein kinases (aPKCs), PKC and PKCl/I. Par-4 protein localizes predominantly to the nucleus but is also present in the cytoplasm. However, nuclear entry of Par-4 was essential for its pro-apoptotic activity [13–17] . Recently, Lee et al. [18] demonstrated that the ectopic expression of Par-4 sensitized human renal cancer cells to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). However, it remains unclear whether Par4 is involved in the apoptosis of renal tubular epithelial cells induced by oxidant injury. The serine/threonine kinase Akt is a critical component of the intracellular signaling pathway that supports cell survival and inhibits apoptosis [19–21] . The nonphosphorylated form of AKT is virtually inactive, and phosphorylation at Ser-473 generates active pAKT [19– 22] . Vascular endothelial growth factor (VEGF) is a critical mediator of angiogenesis [20, 21] and plays a role in the pathogenesis of diabetic retinopathy, nephropathy and vascular disease [23, 24] . VEGF is both an autocrine and paracrine factor and VEGF expression is increased in the kidneys of patients with type 1 [25] and type 2 diabetes [26] . In this context, increased VEGF expression is involved in glomerular and tubular hypertrophy, proteinuria and glomerular hyperfiltration [23, 24] . Activation of the PI3K/Akt pathway increases transcriptional activation of the VEGF promoter via the transcription factor Sp1 [27] . However, little is known about the effect of oxidative stress on the regulation of VEGF expression in human renal tubular epithelial cells. In the present study, we provide evidence that the silencing of Par-4 gene expression promotes PI3K/Akt signal transduction and protects renal proximal tubular cells from apoptosis induced by oxidative stress. Materials and Methods Chemicals and Reagents Fetal bovine serum and Dulbecco’s modified Eagle’s medium were obtained from GibcoBRL (Gaithersburg, Md., USA). Ribonuclease (RNase) and propidium iodide (PI) were purchased from Sigma Chemical (St. Louis, Mo., USA). Caspase-3 assay kits and the caspase-3 inhibitor were purchased from Calbiochem (Cambridge, Mass., USA). Antibodies against Par-4, Akt, phosphorylated Akt (Ser-473) and VEGF were purchased from Santa Cruz Biotechnology (Santa Cruz, Calif., USA). Lipofectamine was purchased from Invitrogen Life Technologies Inc. (Carlsbad, Calif., USA). Cell Culture Commercially available human proximal tubular epithelial cells prepared from human kidney biopsies were purchased from Cambrex Biosciences (East Rutherford, N.J., USA). Renal proximal tubular epithelial cells were grown in a renal epithelial growth medium as recommended by the manufacturer. HK-2 cells, a human renal proximal tubular epithelial cell line, were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 g/ml streptomycin. Subconfluent cells were washed once and made quiescent by incubation in serumand supplement-free medium for 24 or 48 h prior to experiments. Preparation of Nuclear Extracts and Whole Cell Extracts Cells were washed twice with ice-cold phosphate-buffered saline and collected after centrifugation at 300 g for 5 min. Cells were resuspended in ice-cold buffer containing 10 m M Hepes, pH 7.8, 10 m M KCl, 1.5 m M MgCl 2 , 1 m M dithiothreitol, 0.1 m M EDTA, 1 m M phenylmethylsulfonyl fluoride, 10  g/ml aprotinin, and 2  g/ml pepstatin and kept on ice for 15 min. Cells were lysed in 0.1% Nonidet P-40 and vortexed for 10s, and the nuclear pellet was recovered after centrifugation at 13,000 g for 10 s at 4 ° C. The nuclear pellet was resuspended in ice-cold buffer containing 20 m M Hepes, pH 7.8, 0.4 M NaCl, 1 m M EDTA, 1.5 m M MgCl 2 , 1 m M dithiothreitol, 20% glycerol, 1 m M phenylmethylsulfonyl fluoride, 10  g/ml aprotinin, and 2  g/ml pepstatin and incubated on ice for 20 min with shaking. Nuclear extracts were retained after centrifugation at 14,000 rpm for 5 min at 4 ° C, and the supernatant was separated into aliquots and stored at –80 ° C. Protein concentration was determined by the Bradford method (Bio-Rad). Whole cell extracts were prepared by sonication in Laemmli sample buffer (10% glycerol, 5% -mercaptoethanol, 2.3% SDS, 62.5 m M Tris-HCl (pH 6.8), and 0.1% bromophenol blue). Western Blot Analysis Fifty micrograms of protein from either nuclear or whole cell extracts were loaded on precast SDS/Tris/glycine gels (Bio-Rad). After electrophoresis, proteins were transferred to nitrocellulose membranes for subsequent blotting with the appropriate primary antibody. Membranes were then incubated with the appropriate secondary antibody linked to horseradish peroxidase for 1 h at room temperature. After washes in TBST (25 m M Tris, pH 8.0, 125 m M NaCl and 0.1% Tween-20), the blot was incubated in detection reagent (ECL Advance Western blotting detection kit) and exposed to a Hyperfilm ECL film (Pierce). Tubulin and nuclear protein Histone H1 served as loading controls. Small Interfering RNA-Based (siRNA) Experiments A siRNA strategy was employed to silence Par-4 expression in renal proximal tubular cells. The experiments were performed as described previously [17] . Par-4 and scrambled control siRNAs were generated using the procedure of siSTRIKE TM U6 Hairpin Cloning Systems (Promega). The Par-4 siRNA had the following D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /1 9/ 20 17 1 2: 27 :4 9 P M Suppression of Par-4 Protects Human RPTCs from Apoptosis Nephron Exp Nephrol 2011;117:e53–e61 e55 sense strand sequences: 5 -ACCGTCACAGCCGTTTGAATATATT TCAAGAGAATATATTCAAA CGGCTGTGACTTTTTC3 . Sense and antisense strands were annealed and ligated into the linearized psiSTRIKE Vector following the manufacturer’s directions. Sequence analysis of randomly picked transformed clones was used to confirm the sequence integrity of the Par-4 shRNA plasmids. Cells were transfected with siRNA or the indicated constructs using Lipofectamine 2000 (Invitrogen) in Opti-MEM I for 24 h, and then the medium was changed back to growth medium for additional incubation. Green fluorescent protein phMGFP vector was co-transfected to determine transfection efficiency by flow cytometry. Forty-eight hours after the transfection, total RNA was prepared using TRI Reagent according to the manufacturer’s instructions and used to perform real-time quantitative PCR analysis. The level of target RNA suppression in transfected cells was determined by normalizing for transfection efficiency. Flow Cytometric Analysis of Apoptosis Renal proximal tubular cells were grown to the exponential phase, seeded at a density of 2 ! 10 6 cells per 60-mm dish, and transfected with the indicated concentrations of Par-4 siRNA plasmids. Cells were then exposed to hydrogen peroxide (H 2 O 2 ) or control medium for various periods of time. The cells were collected by trypsinization and washed in PBS. After incubation with 5 l of Annexin-V FITC and 10 l of PI (50  g/ml) at room temperature for 15 min in the dark, cells were analyzed by flow cytometry using a FACS Calibar. The percentage of cells undergoing apoptosis was calculated and the result presented relative to the levels of apoptosis in controls. Measurement of Caspase-3 Activity Caspase-3 activity was measured by a caspase-3 fluorometric protease assay kit (MBL) following the manufacturer’s instructions. The fluorogenic synthetic peptide DEVD-7-amino-4-trifluoromethylcoumarin (AFC) was used as a substrate for caspase-3 and the fluorescence of the released AFC was measured with an excitation wavelength of 360 nm and an emission wavelength of 530 nm. Measurement of VEGF Production in the Culture Supernatants The VEGF levels of cell culture supernatant were determined using ELISA kits (R&D Systems, Minneapolis, Minn., USA) according to the manufacturer’s instructions. Briefly, 200 l of cell culture supernatants, controls or standards were added to wells previously coated with human monoclonal anti-VEGF antibody. After 2 h of incubation, wells were washed and incubated with an enzyme-linked polyclonal anti-VEGF antibody. Following another wash, a substrate solution was added to wells with color development being proportional to the amount of bound VEGF. The plate was read on a Dynex plate reader with an absorbance of 450 nm. Results were calculated from the standard curve generated from several known VEGF concentrations. Statistical Analysis When appropriate, data were expressed as mean 8 SE. Data were analyzed by factorial ANOVA and Fisher’s least significant difference test when appropriate. Statistical significance was accepted at p ! 0.05. Results H 2 O 2 Induced Apoptosis and Increased Par-4 Protein Expression in Human Renal Proximal Tubular Epithelial Cells We employed H 2 O 2 to initiate intracellular oxidative stress, as H 2 O 2 is involved in renal disease and is readily permeable to the plasma membrane [28] . A treatment dose of 0.75 m M H 2 O 2 was chosen according to preliminary studies (data not shown) and cell apoptosis was monitored over a period of up to 24 h after H 2 O 2 treatment. Obvious morphological changes were observed in renal proximal tubular cells within 2 h with cell shrinkage (the morphological hallmark of apoptosis) developing afterwards. Flow cytometric analysis using FITC-labeled annexin V to bind phosphatidylserine was employed to assess the level of apoptosis whilst cell viability was assessed by staining with fluorescent PI that binds DNA. Treatment with 0.75 m M H 2 O 2 induced significant time-dependent apoptosis of renal proximal tubular cells starting 6 h after H 2 O 2 treatment ( fig. 1 ). Western blotting was performed to determine the involvement of Par-4 in the regulation of H 2 O 2 -induced apoptosis in renal proximal tubule cells. As the pro-apoptotic function of Par-4 has been attributed to its nuclear translocation, Western blotting was performed on nuclear extracts. H 2 O 2 induced a time-dependent increase in Par-4 expression in renal proximal tubular cells compared to control with similar results obtained in HK-2 cells ( fig. 2 a). Since the activation of caspase-3 has been implicated as a common downstream effector of diverse apoptotic pathways [29] , we examined caspase-3 activation in renal proximal tubule cells exposed to H 2 O 2 . Treatment with H 2 O 2 significantly increased caspase-3 activation in both human primary renal proximal tubular epithelial cells and HK-2 cells ( fig. 2 b). These data indicate that H 2 O 2 induced apoptosis in human renal proximal tubular epithelial cells and that this was associated with increased Par-4 protein expression. Par-4 Suppression Protects Renal Proximal Tubular Cells from H 2 O 2 -Induced Apoptosis via Activation of the PI3K/Akt Signaling Pathway To investigate whether Par-4 suppression may protect renal proximal tubular cells from oxidative stress-induced apoptosis, we performed siRNA experiments. Renal proximal tubular cells were transfected with either Par-4-specific siRNA, control scrambled siRNA, or no D ow nl oa de d by : 54 .7 0. 40 .1 1 11 /1 9/ 20 17 1 2: 27 :4 9 P M Sun /Lu /Zhou /Xing Nephron Exp Nephrol 2011;117:e53–e61 e56 siRNA (mock transfection). Par-4 mRNA and protein expression was assessed by real-time quantitative PCR analysis and Western blotting respectively. Par-4-specific siRNA effectively reduced Par-4 mRNA and protein expression ( fig. 3 a). Control and Par-4-specific siRNA transfected renal proximal tubular cells were then exposed to 0.75 m M H 2 O 2 for 24 h. Flow cytometric analysis indicated that the suppression of Par-4 expression significantly attenuated H 2 O 2 -induced apoptosis ( fig. 3 b). Because it has been demonstrated that Par-4 deficiency leads to activation of the Akt pathway in vivo and in several cellular systems [18, 26, 27] , we assessed the effects of Par-4 silencing on the PI3K/Akt signaling pathway in renal proximal tubular cells exposed to H 2 O 2 . Primary renal proximal tubular cells and HK-2 cells were treated with 0.75 m M H 2 O 2 for between 5 min and 24 h at which point lysates were analyzed by Western blotting. H 2 O 2 treatment increased Akt phosphorylation (Ser-473) that peaked at 10 min and declined to basal levels within 3 h ( fig. 4 a). We then sought to determine whether the antiapoptotic effect of Par-4 suppression was related to sustained activation of the PI3K/AKT signaling pathway. Renal proximal tubular cells were transfected with Par4-specific siRNA followed by treatment with the PI3K inhibitor LY294002 (20  M ) or medium alone for 1 h. Then, cells were exposed to 0.75 m M H 2 O 2 for 10 min and Akt phosphorylation (Ser-473) detected with Western blotting. Transfection of cells with Par-4-specific siRNA resulted in increased Akt phosphorylation in H 2 O 2 -treated renal proximal tubular cells ( fig. 4 b). However, LY294002 0 0 h 20 40 60 80 100 A po pt ot ic c el ls (% )