Accepted Manuscript Title: Resveratrol increases anti-aging Klotho gene expression via the activating transcription factor 3/c-Jun complex-mediated signaling pathway Author: Shih-Che Hsu Shih-Ming Huang Ann Chen Chiao-Yin Sun Shih-Hua Lin Jin-Shuen Chen Shu-Ting Liu Yu-Juei Hsu PII: DOI: Reference:

S1357-2725(14)00200-3 http://dx.doi.org/doi:10.1016/j.biocel.2014.06.002 BC 4359

To appear in:

The International Journal of Biochemistry & Cell Biology

Received date: Revised date: Accepted date:

4-1-2014 19-4-2014 2-6-2014

Please cite this article as: Hsu, S.-C., Huang, S.-M., Chen, A., Sun, C.-Y., Lin, S.H., Chen, J.-S., Liu, S.-T., and Hsu, Y.-J.,Resveratrol increases anti-aging Klotho gene expression via the activating transcription factor 3/c-Jun complex-mediated signaling pathway, International Journal of Biochemistry and Cell Biology (2014), http://dx.doi.org/10.1016/j.biocel.2014.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Hsu et al.

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Highlights  Resveratrol up-regulated ATF3 and p-c-Jun expression in a dose-dependent manner.  ATF3 or c-Jun overexpression enhanced Klotho mRNA expression in the NRK-52E cells.  Dominant-negative ATF3 or c-Jun mutant abrogated the Klotho induction by resveratrol.  ATF3 may form a heterodimer with c-Jun onto the Klotho promoter.

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Resveratrol increases anti-aging Klotho gene expression via the activating transcription factor 3/c-Jun complex-mediated

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signaling pathway

Shih-Che Hsu1, Shih-Ming Huang1,2, Ann Chen1,4, Chiao-Yin Sun5,6, Shih-Hua

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Lin1,3, Jin-Shuen Chen1,3, Shu-Ting Liu2,and Yu-Juei Hsu1,3,*

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Graduate Institute of Medical Sciences, National Defense Medical Center,

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Taipei, Taiwan; 2Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan; 3Division of Nephrology, Department of Medicine, 4Department

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of Pathology, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan; 5Division of Nephrology, Chang Gung Memorial Hospital,

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Taiwan

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Keelung, Taiwan; 6School of Medicine, Chang Gung University, Taoyuan,

Running Title: Resveratrol increases renal Klotho gene expression

*

Corresponding author at:

Division of Nephrology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, No. 325, Sec. 2, Cheng-Kung Rd., Neihu 114, Taipei, Taiwan. TEL: +886-2-87927213; FAX: +886-2-87927134 E-MAIL: [email protected] (Yu-Juei Hsu)

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Abstract The Klotho gene functions as an aging suppressor gene. Evidence from animal models suggests that induction of Klotho expression may be a potential

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treatment for age-associated diseases. However, the molecular mechanism involved in regulating renal Klotho gene expression remains unclear. In this

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study, we determined that resveratrol, a natural polyphenol, induced renal

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Klotho expression both in vivo and in vitro. In the mouse kidney, resveratrol administration markedly increased both Klotho mRNA and protein expression.

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In resveratrol-treated NRK-52E cells, increased Klotho expression was accompanied by the upregulation and nuclear translocation of activating

enhanced

the

transcriptional

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transcription factor 3 (ATF3) and c-Jun. ATF3 or c-Jun overexpression activation

of

Klotho.

Conversely,

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resveratrol-induced Klotho expression was attenuated in the presence of

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dominant-negative ATF3 or c-Jun. Coimmunoprecipitation and a chromatin immunoprecipitation assay revealed that ATF3 physically interacted with c-Jun

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and that the ATF3/c-Jun complex directly bound to the Klotho promoter

through ATF3- and AP-1-binding elements. C-Jun cotransfection augmented the effects of ATF3 on Klotho transcription in vitro. Although Sirtuin 1 mRNA

expression was induced by resveratrol and involved in regulating Klotho mRNA expression, it was not the primary cause for the aforementioned ATF3/c-Jun pathway. In summary, resveratrol enhances the renal expression of the anti-aging Klotho gene, and the transcriptional factors ATF3 and c-Jun functionally interact and coordinately regulate the resveratrol-mediated transcriptional activation of Klotho.

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Keywords: Klotho; activating transcription factor 3 (ATF3); c-Jun; resveratrol;

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anti-aging

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1. Introduction The Klotho gene functions as an aging suppressor gene (Wang and Sun, 2009). Mice exhibiting genetic ablation of Klotho develop accelerated aging including

arteriosclerosis,

a

short

lifespan,

hyperphosphatemia,

skin

vascular

atrophy,

osteopenia,

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phenotypes,

calcification,

pulmonary

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emphysema, parkinsonian gait, and cognitive impairment (Kuro-o et al., 1997). The Klotho gene encodes a single-pass transmembrane polypeptide and is

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predominantly expressed in the distal convoluted tubule of the kidney (Wang

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and Sun, 2009). Klotho exists in membrane and soluble secreted forms. The membrane Klotho forms a complex with multiple fibroblast growth factor (FGF)

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receptors and functions as an obligatory cofactor for FGF23, a bone-derived phosphaturic hormone that causes the kidney to increase renal phosphate

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excretion (Urakawa et al., 2006). The secreted Klotho is cleaved from the

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membrane form by secretases expressed on the cell surface and is released into the circulation to act as an endocrine hormone (Kuro-o, 2012). Emerging

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evidence suggests that both the membrane and secreted Klotho forms are closely linked to accelerated aging. Klotho or FGF23 ablation in mice results in phosphate retention and a premature aging phenotype, which is ameliorated by

a

low-phosphate

diet.

This

suggests

a

potential

link

between

hyperphosphatemia and aging (Nakatani et al., 2009). In addition, secreted Klotho modulates several critical signaling pathways involved in regulating longevity, including insulin/insulin-like growth factor-1, Wnt, transforming growth factor (TGF)-β1, and oxidative stress (Utsugi et al., 2000). Renal Klotho expression is reduced in several rodent models of human diseases characterized by oxidative stress or accelerated aging, such as 5

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hypertension, diabetes mellitus, ischemia–reperfusion injury, acute myocardial infarction, and chronic kidney disease (Torres et al., 2007; Pavik et al., 2013), which are partly ameliorated by endogenous Klotho restoration or exogenous

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Klotho replacement. These data suggest that the upregulation of renal Klotho expression is a potential anti-aging strategy. However, the molecular

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mechanisms underlying the regulation of renal Klotho expression remain unclear.

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Resveratrol is a natural polyphenol primarily found in grapes and red wine

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(Nonomura et al., 1963) that offers various benefits for common age-related diseases, such as obesity, diabetes, cancer, and neurodegenerative and

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cardiovascular diseases, because of its anti-aging and antioxidant properties (Baur and Sinclair, 2006; Smoliga et al., 2011). Recent studies have expanded

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its role in kidney diseases, reporting that resveratrol exerts renoprotective

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effects through antiinflammatory and antioxidant activities in both acute kidney injury- and streptozotocin-induced diabetic rats. The anti-aging, antioxidant,

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and renoprotective effects of resveratrol overlap with the functions of Klotho, suggesting that resveratrol is a candidate drug that enhances renal Klotho expression.

The molecular effects of resveratrol have been suggested to be complex

mechanisms involving multiple signaling pathways (Kovacic and Somanathan, 2010). Resveratrol activates Sirtuin 1 (Sirt1), an NAD+-dependent protein

deacetylase that is a key regulator of lifespan in several model organisms under metabolic stress (Chung et al., 2010). Moreover, resveratrol exerts an anti-aging effect by modulating various transcriptional factors, such as activating transcription factor 3 (ATF3) and activator protein 1 (AP-1), which 6

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typically form a complex involved in regulating cell apoptosis and survival (Whitlock et al., 2011; de la Lastra and Villegas, 2005). This study investigated the molecular mechanisms through which

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resveratrol regulates renal Klotho expression. We hypothesized that resveratrol enhances renal Klotho expression by recruiting the transcription

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factors ATF3 and c-Jun, a member of the AP-1 family. We revealed a clear link between resveratrol and the transcriptional regulation of Klotho by showing resveratrol

upregulates

renal

Klotho

expression

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that

through

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ATF3/c-Jun-complex-mediated transcriptional activation in vitro and in vivo. Klotho upregulation may be one of the molecular mechanisms responsible for

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the anti-aging properties of resveratrol.

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2. Material and methods 2.1. Cell culture The rat renal epithelial cell line NRK-52E (BCRC60086) was purchased from

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the Bioresource Collection and Research Center of the Food Industry Research and Development Institute in Taiwan. Cells were grown in

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Dulbecco’s Modified Eagle’s medium supplemented with 10% fetal bovine serum, 150 U/mL of penicillin, and 150 mg/mL of streptomycin. The cells were

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incubated at 37 °C in 5% CO2 and 95% air. Confluent cells were detached

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using a 0.05% trypsin/0.02% ethylenediaminetetraacetic acid (EDTA) solution and subcultured to the second passage in 24-well culture plates. To examine

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the effect of resveratrol on these cells, the cells were treated with the indicated amount of resveratrol for 24 h or with 40 μM resveratrol for the indicated

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duration.

2.2. RNA isolation and semiquantitative RT-PCR

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RNA was extracted from kidney tissues and cultured NRK-52E cells by means of homogenization in a TRIzol reagent (Invitrogen, CA, USA). The obtained total RNA was subjected to DNase treatment to prevent contamination with genomic DNA. Subsequently, 2 μg of total RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA). The obtained cDNA was used to determine Klotho, ATF3, and c-Jun mRNA levels by using RT-PCR. The expression level of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an

internal control to normalize differences in RNA extraction and reverse transcription efficiency. Table 1 shows the primer sequences used for 8

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amplification, which was performed using a GeneAmp PCR System 9700 thermocycler (Applied Biosystems, Foster City, CA, USA). The PCR products were analyzed by performing electrophoresis using 1.2% agarose gels and

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visualized after subjecting the products to ethidium bromide staining and ultraviolet irradiation. Densitometric analysis of band intensity was performed

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using ImageJ (NIH, USA). The relative level of Klotho mRNA expression was

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determined by normalizing the band intensity of Klotho to that of GAPDH.

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2.3. Immunoblotting analysis

Kidney and renal epithelial cells were incubated with an RIPA buffer [10 mM

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Tris-HCl (pH 7.2), 150 mM NaCl, 0.1% SDS, 1% sodium deoxycholate, 1% Triton X-100, and 5 mM EDTA] containing protease inhibitor cocktails (Sigma)

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and a phosphatase inhibitor mixture (PhosSTOP; Roche Applied Science,

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Indianapolis, IN, USA) at 4 °C for 20 min. The protein extracts were separated using SDS-PAGE and transferred onto a polyvinylidine difluoride membrane

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(Millipore, Billerica, MA, USA). The expression of expected proteins was detected using rat anti-Klotho monoclonal (1:1000; Alpha Diagnostic), rabbit anti-ATF3 polyclonal (1:500; Santa Cruz Biotechnology), rabbit anti-p-c-Jun monoclonal (1:1000; Cell Signaling), rabbit anti-c-Jun monoclonal (1:1000; Epitomics), mouse anti-GAPDH monoclonal (1:10,000; BD Millipore), and mouse anti-β-actin monoclonal (1:10,000; Sigma) antibodies. Subsequently,

blots were incubated with the appropriate secondary antibodies labeled with horseradish peroxidase. Immunoreactive proteins were detected using the chemiluminescence method (Pierce, Rockford, IL, USA). The intensity of immunopositive bands was quantified by performing pixel-density scanning 9

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and computed calculation using Molecular Analyst software (Bio-Rad Laboratories, Hercules, CA, USA). The average pixel density of an area devoid of bands was subtracted from the values obtained for bands of interest to

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normalize average pixel density against the background. Relative expression was determined by dividing the normalized average pixel density of the bands

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2.4. Transient transfection and luciferase reporter assays

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of interest by the average pixel density of the GAPDH band.

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The cells in each well were transfected with jetPEI (PolyPlus-transfection, France) according to the manufacturer’s protocol, and total DNA was adjusted

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to 1.0 μg by adding empty vectors. Luciferase assays were performed using the Promega luciferase assay kit. The measurements were expressed

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numerically as relative light units. Luciferase activities are shown as the mean

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of three independent experiments.

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2.5. Immunocytochemistry

The NRK-52E cells were grown on glass coverslips under EtOH or resveratrol (40 μM) treatment for 24 h. The cells were then fixed in 4% paraformaldehyde

for 10 min and permeabilized with 0.1% Triton X-100 for 10 min. The slides were washed three times with phosphate-buffered saline (PBS) and blocked with 2% BSA in PBS for 60 min. The slides were then incubated with rabbit anti-ATF3 polyclonal and mouse anti-p-c-Jun monoclonal antibodies (1:100) overnight at 4 °C, and were subsequently incubated with an Alexa Fluor 488 or Alexa Fluor 594 antibody (Invitrogen) for 2 h at room temperature. The nuclei were counterstained with DAPI for 10 min. The cells were mounted with 10

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Vectashield (Vector Laboratories, Burlingame, CA, USA), and images were acquired using a Leica DM2500 fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). Five digital images of each field of view were

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captured. The integrated optical density was measured by performing computer analysis using Image-Pro Plus Version 3.0 software (Media

2.6. Coimmunoprecipitation extracts

containing

equivalent

amounts

of

protein

were

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Nuclear

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Cybernetics, Silver Spring, MD, USA).

immunoprecipitated in a lysis buffer containing a polyclonal antibody against

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ATF3 overnight at 4 °C. Protein A–Sepharose beads (GE Healthcare) were added to the immunoprecipitated mixture for 2 h before being washed three

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times with buffer A. The entire immunoprecipitate was then suspended in an

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SDS-PAGE sample buffer, boiled, and loaded onto an SDS-PAGE gel. The separated proteins were transferred to a nitrocellulose membrane. The blot

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was probed with rabbit anti-ATF3 and anti-p-c-Jun antibodies, and was then probed with appropriate secondary antibodies and detected using the Clean-Blot IP Detection Reagent (Thermo Scientific, Rockford, IL, USA)

according to the manufacturer’s instructions.

2.7. Plasmids

The full-length ATF3 coding sequence was amplified using PCR and cloned into the pSG5.HA (hemaglutinin) vector at the EcoRI and XhoI sites. The mutant plasmid ATF3 (∆80–100) was constructed by deleting ATF3 residues 80–100 by using site-directed mutagenesis. Wild-type (WT) and mutant 11

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(TAM67) c-Jun were cloned into the pCMV vector, as described previously (Wang et al., 2011). TAM67 contained a deletion in the N-terminal domain (amino acids 3–122) of c-Jun. Expression vectors for Flag.Sirt1 WT and an

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H363Y mutant were obtained from Addgene (Cambridge, MA, USA) (Brunet et al., 2004). The human Klotho promoter (2.8 kb upstream of the transcription site)

was

amplified

by

conducting

using

the

and

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5-CACAATAGGAAAGAGAACGCGTGGGAAGG-3

PCR

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start

5-GCCCTCGAGGAAGGTGCCCTG-3 primer pair. Genomic DNA of HEK293

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cells was used as a template for amplification. After electrophoresis on an agarose gel, the PCR products were purified using the QIAquick Gel Extraction

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Kit (Qiagen, Valencia, CA, USA). The purified PCR products were then digested overnight at 37 °C with MluI and XhoI (Fermentas, Lithuania) and

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ligase (Roche, Germany).

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ligated into the pGL3 luciferase reporter vector (Promega) by using T4 DNA

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2.8. Chromatin immunoprecipitation assay Chromatin immunoprecipitation (ChIP)

assays were performed using

NRK-52E cells that were or were not treated with resveratrol (40 μM) for 24 h. The cells were cross-linked with 1% formaldehyde at 37 °C for 15 min as described previously. Immunoprecipitation (IP) was performed using 1 μg of an

anti-ATF3, anti-c-Jun, or antinormal mouse IgG antibody with rotation at 4 °C overnight. The following day, the chromatin–antibody complexes were eluted from the solution by incubation with 40 μL of salmon sperm DNA-saturated 50% protein A/G–Sepharose beads at 4 °C for 2 h. The beads were harvested and washed as described previously. Cross-linking was reversed by heating at 12

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65 °C overnight, followed by treatment with 100 μg/μL of proteinase K at 50 °C for 1 h. DNA was extracted using a gel extraction kit (Geneaid, Taiwan) and dissolved in 60 μL of water. Table 1 shows the primer pairs specific for the

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ATF3- and AP1-responsive elements.

short-hairpin-RNA

(TRCN0000072223)

(shRNA)-mediated

and

ATF3

knockdown,

(TRCN0000013572)

LacZ

shRNA-containing

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For

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2.9. Generation of ATF3 knockdown clones

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lentiviral vectors were obtained from the National RNAi Core Facility (Academia Sinica, Taiwan) and prepared according to standard protocols. In

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brief, HEK293T cells were cotransfected with LacZ and ATF3 shRNAs, and the virus-containing medium was collected 48 h after transfection. To generate

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stably transduced shRNA clones, human renal carcinoma cells (RCCs; i.e.,

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ACHN cells) were infected with a shRNA-containing lentivirus (multiplicity of infection = 3) in media containing polybrene (8 μg/mL). The cells were treated

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with puromycin 48 h after infection (final concentration, 0.75 μg/mL), and puromycin-resistant clones were selected and pooled.

2.10. Statistical analyses

Data are expressed as the mean ± SEM. Differences between the groups were analyzed using the unpaired t test. P < 0.05 indicated significance.

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3. Results 3.1. Resveratrol induces renal Klotho expression in vivo and in vitro To investigate whether resveratrol regulates renal Klotho expression, we

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examined Klotho mRNA and protein levels in kidney tissues obtained from C57BL/6 mice. The mice were intraperitoneally injected with resveratrol for 1

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week. A significant increase in Klotho mRNA levels (supplementary Figs. S1A and B) and corresponding increases in Klotho protein levels (supplementary

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Figs. S1C and D) were observed in the kidney after 1 week of resveratrol

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treatment.

To confirm this in vivo observation further, we treated the rat renal

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epithelial cell line NRK-52E with resveratrol to determine its effect on the regulation of renal Klotho expression in vitro. Semiquantitative RT-PCR and

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immunoblotting analysis revealed that resveratrol induced renal Klotho gene

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and protein expression in a dose- and time-dependent manner (Fig. 1).

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3.2. Resveratrol induces the upregulation and nuclear translocation of activating transcription factor 3 and c-Jun To delineate the signaling pathways involved in the resveratrol-induced upregulation of renal Klotho expression, we first analyzed the sequences of the

5′-flanking region of Klotho gene promoters in humans, rats, and mice. We determined that the ATF3- and AP-1-responsive elements were highly conserved among these species, according to an analysis conducted using Genomatix–MatInspector software (Fig. 2A). In addition, we determined the effects of resveratrol on the renal expression of ATF3 and c-Jun, which functionally interacts with ATF3 (Kiryu-Seo et al., 2008). In addition to Klotho 14

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expression, a progressive increase in ATF3 and c-Jun mRNA expression was observed in the NRK-52E cells exposed to increasing resveratrol doses (Fig. 2B). Accordingly, we determined that resveratrol dose-dependently increased

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the levels of ATF3 as well as the phosphorylated form of c-Jun (p-c-Jun) (Fig. 2C). The immunofluorescence analysis confirmed that the number and

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intensity of ATF3 and c-Jun staining were increased and colocalized as Klotho

staining was increased in NRK-52E cells stimulated by resveratrol (Figs. 2D

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and E).

3.3. Resveratrol stimulates activating-transcription-factor-3-mediated induction

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of Klotho promoter activity

To elucidate the functional role of ATF3 in regulating Klotho expression, we

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constructed a WT ATF3 and mutant-type ATF3 with one DNA-binding region

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deletion, ATF3 (∆80–100). In this experiment, ATF3 (∆80–100) played a dominant-negative role in regulating Klotho mRNA expression (Fig. 3A,

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compare lanes 4–6 with lanes 1–3) and reduced the stimulatory effect of resveratrol (Fig. 3A, compare lanes 10–12 with lanes 7–9). We observed a similar functional role of ATF3 (∆80–100) in the Klotho promoter reporter assay.

The WT ATF3 could enhance the activity of a 2.8-kb Klotho promoter fragment

up to 2.7-fold and acted synergistically with resveratrol, thus enhancing the activity of the promoter fragment 5.2-fold (Fig. 3B, compare histograms 1–6, closed columns). In this reporter system, the ATF3 (∆80–100) mutant reduced the

activity

of

the

endogenous

Klotho

promoter

and

suppressed

resveratrol-mediated induction of promoter activity (Fig. 3B, compare histograms 1–6, open columns). 15

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We further examined the functional effect of endogenous ATF3 on Klotho mRNA expression in human RCCs, ACHN cells, by silencing ATF3 gene expression. Consistent with the data obtained from rat renal cells, resveratrol

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induced the expression of Klotho and ATF3 genes and proteins (Fig. 3C, compare lanes 1–5). The levels of resveratrol-induced Klotho gene expression

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were significantly reduced when ATF3 was downregulated in the ACHN cells (Figs. 3C and D, compare lanes 6–10). These results suggested that

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resveratrol-induced Klotho expression is dependent on the DNA-binding

Resveratrol

upregulates

Klotho

expression

through

the

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3.4.

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activities of endogenous ATF3.

activating-transcription-factor-3/c-Jun-complex-mediated pathway

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To investigate whether ATF3 and c-Jun act synergistically in regulating Klotho

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expression, the NRK-52E cells were transiently transfected with WT c-Jun alone or together with dominant-negative mutant c-Jun (TAM67) expression

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vectors or ATF3. Subsequently, the cells were cultured in the presence or absence of resveratrol. In the absence of resveratrol, c-Jun induced Klotho mRNA expression, which was abolished by TAM67 cotransfection (Figs. 4A and B, compare lanes 4–6 with lanes 1–3), and resveratrol augmented the stimulatory effect of c-Jun overexpression on Klotho mRNA expression (Figs.

4A and B, compare lanes 10–12 with lanes 7–9). In the absence of resveratrol, cotransfection of a combination of ATF3 and c-Jun caused an additional increase in Klotho transcription in the NRK-52E cells compared with the cells transfected with ATF3 or c-Jun alone (Figs. 4C and D, compare lanes 1–6). However, in the presence of resveratrol, cotransfection of a combination of 16

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ATF3 and c-Jun exerted an effect on Klotho transcription comparable to that observed in the cells transfected with ATF3 or c-Jun alone (Figs. 4C and D,

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compare lanes 7–12).

3.5. Activating transcription factor 3 and c-Jun physically interact and directly

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bind to the Klotho promoter through resveratrol stimulation

ATF3 usually activates transcription after it heterodimerizes with the

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transcriptional factor c-Jun, a member of the AP-1 family. To elucidate whether

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ATF3 and p-c-Jun physically interact, we performed reciprocal Co-IP assays in resveratrol-treated NRK-52E cells by using antibodies against ATF3 and c-Jun.

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As shown in Figure 5, ATF3 and c-Jun were coimmunoprecipitated with each other and resveratrol upregulated ATF3 and p-c-Jun as well as the

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ATF3/p-c-Jun complex in the NRK-52E cells (Fig. 5A, lanes 1–4). In support of

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this finding, immunofluorescence double staining revealed that a substantial amount of ATF3 colocalized with p-c-Jun in the nucleus of the NRK-52E cells

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after resveratrol stimulation (Fig. 5B). To confirm the direct binding of ATF3 and c-Jun to the Klotho promoter after resveratrol treatment, a ChIP assay was

performed using the NRK-52E cells. IP of the chromatin/protein complex with anti-ATF3 or anti-c-Jun antibodies yielded two bands corresponding to the ATF3- and AP-1-responsive elements (190 and 180 bp, respectively) within the Klotho promoter (Fig. 5C), suggesting that both ATF3 and c-Jun directly bind to

the promoter.

3.6. Resveratrol induces Sirt1 expression involved in regulating Klotho mRNA, not activating transcription factor 3 or c-Jun mRNA expression 17

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We first assessed the effect of resveratrol on endogenous Sirt1 mRNA expression. Our resveratrol dose- and time-course data revealed that resveratrol induced Sirt1 (Figs. 6A and B) as well as ATF3 and c-Jun

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expression in NRK-52E cells (Fig. 2). In addition, we investigated whether Sirt1 could induce Klotho mRNA expression and the role of Sirt1 deacetylase

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activity in NRK-52E cells through one dominant-negative and enzyme-dead Sirt1 mutant (H363Y) (Fig. 6C). The data indicated that WT Sirt1 induced

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Klotho mRNA in a dose-dependent fashion (Fig. 6C, compare lanes 1–4;

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P < 0.05). The Sirt1 mutant (H363Y) produced two effects: One effect was an inductive effect and the other effect was a dominant-negative effect (compare

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lanes 1 and 5 or 6; P < 0.05 and P < 0.01, respectively), suggesting that Sirt 1 plays a functional role in the induction of Klotho mediated through

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deacetylase-dependent and independent pathways. Exogenously transfected

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Sirt1 did not enhance resveratrol-induced Klotho mRNA expression, regardless of whether it was a wild type or mutant (compare lanes 7–11),

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whereas a large amount of the Sirt1 mutant (H363Y) significantly expressed its dominant-negative effect on resveratrol-induced Klotho mRNA expression, but

not on resveratrol-induced ATF3 or c-Jun mRNA expression (compare lanes 7 and 12; P < 0.05).

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4. Discussion Resveratrol is well known for its anti-aging benefits exerted through the competitive inhibition of cAMP-specific phosphodiesterases, leading to

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increased NAD+ levels and Sirt1 activity (Chung et al., 2010; Park et al., 2012). Other signaling mechanisms involved in its anti-aging effects include Notch,

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phosphatidylinositol 3-kinase/Akt, mitogen-activated protein kinase, and c-Jun N-terminal kinase (JNK)/AP-1 (Fulda and Debatin, 2006). Moreover,

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resveratrol exerts antioxidant effects by modulating various transcription

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factors, such as AP1, nuclear factor-kappa B, signal transducer and activator of transcription 3, hypoxia-inducible factor 1α, β-catenin, and peroxisome

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proliferator-activated receptor γ (PPAR-γ) (de la Lastra and Villegas, 2005; Hai and Hartman, 2001; Harikumar and Aggarwal, 2008). In this paper, we

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propose another possible signaling mechanism, the ATF3/c-Jun complex, by

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showing a molecular link between resveratrol and the anti-aging gene Klotho. Resveratrol activated renal Klotho transcription in vivo and in vitro. The

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upregulation of Klotho gene expression by resveratrol may partially explain its anti-aging properties.

ATF3, a member of the ATF/c-AMP response element binding family of

transcription factors, is a crucial mediator of cellular stress response signaling. ATF3 participates in a broad spectrum of biological processes, including apoptosis, cell cycle regulation, cell motility, tumor growth (Thompson et al., 2009), TGF-β signaling (Yin et al., 2010), angiogenesis (Ameri et al., 2007),

endoplasmic reticulum stress, immune response, and nerve injury and repair (Hai and Hartman, 2001). ATF3 can be induced rapidly by various stress signals, such as elevated temperature, cytokines, hypoxia, DNA damage, 19

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oxidative stress, and endoplasmic reticulum stress (Chen et al., 1996; Hai et al., 1999) as well as by treating cells with antitumorigenic compounds, including indole-3-carbinol, conjugated linoleic acid, epicatechin gallate,

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tolfenamic acid, and a PI3 kinase inhibitor (Lee et al., 2010, 2006; Cho et al., 2007; Yamaguchi et al., 2006). Polyphenol resveratrol is an anticancer nutrient

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that induces apoptosis by upregulating ATF3 expression in human colorectal cancer cells (Whitlock et al., 2011). In accordance with previous studies, we

us

determined that resveratrol stimulates renal ATF3 expression both in vivo and

an

in vitro, suggesting that ATF3 participates in the downstream signaling cascades induced by resveratrol in the kidney.

M

ATF3 usually represses gene transcription as a homodimer. However, it can activate transcription when heterodimerized with other bZIP proteins, such members

of

the

AP-1

family

(e.g.,

c-Jun

and

c-Fos),

d

as

te

CCAAT/enhancer-binding proteins, and gadd153/CHOP10 (Chen et al., 1996; van Dam and Castellazzi, 2001). The ATF3/c-Jun dimer may play a role in cell

Ac ce p

cycle progression (Taub, 1996) and can induce heat shock protein 27 expression, which may suppress apoptosis in neuronal cells through Akt activation and MEKK1-JNK inhibition (Nakagomi et al., 2003). However, under resveratrol stimulation, cotransfection of c-Jun with ATF3 did not exert an additional effect on Klotho transcription, possibly because c-Jun cotransfection

prominently increased resveratrol-induced ATF3 protein expression, eliciting the negative feedback suppression of Klotho transcription by increasing ATF3

dimerization (Liang et al., 1996). In addition, resveratrol augmented both ATF3 and c-Jun expression, potentially causing transcriptional squelching. In addition to ATF3 and c-Jun, recent studies have demonstrated the 20

Page 20 of 44

Hsu et al.

presence of PPAR-γ and Egr-1-binding sites within the Klotho promoter; these binding sites may mediate the induction of Klotho transcription by PPAR-γ agonists and the epidermal growth factor, respectively (Choi et al., 2010;

ip t

Zhang et al., 2008). By contrast, the inflammatory cytokines tumor necrosis factor α (TNF-α) and TNF-like weak inducer of apoptosis reduce renal Klotho

cr

transcription by increasing RelA binding to the Klotho promoter (Moreno et al., 2011). Moreover, the overexpression of p16 and p53 tumor suppressor

us

proteins reduces Klotho promoter activity in HEK293 cells (Turan and Ata,

an

2011). The G395A polymorphism located in the promoter region of the human Klotho gene was recently reported to be significantly associated with some

M

aging processes, such as the decrease in bone mineral density in women, atherosclerotic coronary artery disease, and cognitive impairment in elderly

d

people (Kawano and Kawaguchi, 2004; Deary et al., 2005; Rhee et al., 2006;

te

Shimoyama et al., 2009). These data suggest that modulating Klotho promoter activity is a crucial molecular mechanism in regulating Klotho expression.

Ac ce p

In addition to its anti-aging properties, Klotho exhibits diverse biological

functions, including renoprotective effects (Satoh et al., 2012; Sugiura et al., 2010). In previous studies, exogenous supplementation with recombinant Klotho or adenovirus-mediated Klotho gene delivery ameliorated renal

damage and reduced cellular apoptosis in an ischemic kidney injury model (Sugiura et al., 2010) as well as attenuated renal fibrosis by suppressing the TGF-β1-related pathway in a unilateral ureteral obstruction model (Satoh et al., 2012). Thus, stimulating endogenous Klotho production may be an alternative method for treating kidney diseases. Recent studies have determined that both resveratrol and ATF3 exert renoprotective effects similar to Klotho in 21

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Hsu et al.

experimental acute kidney injury models (Li et al., 2010). These data support the contention that resveratrol, ATF3, and Klotho are functionally connected and participate in the same signaling pathway.

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Resveratrol is a pharmacological activator of Sirt1 for upregulating antioxidant defense mechanisms and attenuates aging- and exercise-induced

cr

oxidative damage as well as mitochondrial dysfunction (Csiszar et al., 2009; Jackson et al., 2010, 2011; Kim et al., 2011). Our data suggested that

us

resveratrol induced ATF3 and c-Jun mRNA as well as Sirt1 mRNA. The

an

deacetylase activity of Sirt1 may participate in the induction effect of resveratrol on Klotho mRNA expression, but not in the effects on ATF3 or

M

c-Jun mRNA because the H363Y dominant-negative Sirt1 exerts no suppression effect (Fig. 6). Previous studies have indicated that Sirt1 may

d

reduce c-Fos/c-Jun acetylation induced by p300, thus inhibiting the

te

transcriptional activity of AP-1 (Zhang et al., 2010) or enhancing the activity of a hepatitis B virus core promoter by targeting transcription factor AP-1 (Ren et

Ac ce p

al., 2014). In addition, Sirt1 can deacetylate Egr-1; the enzymatic activity of Sirt1 is not required for inhibiting Egr-1-driven transcription of the Sirt1 promoter (Pardo and Boriek, 2012). Future studies can investigate the functional role of Sirt1 in regulating ATF3 when resveratrol facilitates ATF3 transcriptional regulation through Egr-1 (Whitlock et al., 2011). However, the current findings suggest that resveratrol exerts distinctive effects on Sirt1 and the ATF3/c-Jun complex in regulating Klotho gene expression.

22

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5. Conclusions This study revealed that resveratrol increases the transcription of the anti-aging Klotho gene in the kidney. After resveratrol treatment, the

ip t

transcription factors ATF3 and c-Jun functionally interact and coordinately transactivate the Klotho gene through their binding motif within the Klotho

cr

promoter. These results suggest that Klotho upregulation through the ATF3/c-Jun complex is one of the molecular mechanisms underlying the

us

anti-aging properties of resveratrol. Thus, resveratrol is a potential therapeutic

an

intervention for aging- or oxidative stress-associated disorders characterized

Ac ce p

te

d

M

by Klotho downregulation.

23

Page 23 of 44

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Acknowledgments This work was supported by grants provided by the National Science Council, Taiwan, Republic of China (NSC 99-2628-B-016-002-MY3 and NSC Tri-Service

General

Hospital

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102-2314-B-016-006-MY3),

(TSGH-C100-011-015-S02 and TSGH-C101-006-011-015-S02), and the

cr

Ministry of National Defense-Medical Affairs Bureau (MAB 101-31). No

financial support was received for conducting the research and preparation of

Ac ce p

te

d

M

an

us

the article. The authors declare no conflicts of interest.

24

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Figure legends Fig. 1. Effects of resveratrol treatment on Klotho mRNA and protein expression in rat epithelial cells. Representative semiquantitative RT-PCR analysis of

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Klotho mRNA in NRK-52E cells stimulated using the indicated resveratrol dose for 24 h (A) and 40 μM resveratrol for the indicated duration (B). The levels of

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Klotho mRNA are expressed as the percentage of GAPDH mRNA levels relative to those detected in the untreated control. Representative

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immunoblots of total protein lysates isolated from the NRK-52E cells exposed

an

to the indicated resveratrol dose for 24 h (C) and 40 μM resveratrol for the indicated duration (D) were labeled with antibodies against Klotho or β-actin.

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Klotho protein expression levels were quantified using computer-assisted densitometric analysis and presented as the percentage of β-actin protein

d

expression levels relative to those detected in the untreated control. Data are

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untreated control.

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presented as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01 versus the

Fig. 2. Effects of resveratrol treatment on ATF3 and c-Jun expression in

NRK-52E cells. (A) Schematic diagram of the predicted AP1- and ATF3-responsive elements within the human Klotho promoter region, as

analyzed using Genomatix–MatInspector software. Representative RT-PCR (B) and immunoblots (C) for ATF-3 and c-Jun expression in NRK-52E cells treated with the indicated resveratrol dose for 24 h. Target gene and protein expression levels were quantified using computer-assisted densitometric analysis and are presented as the percentage of GAPDH mRNA or β-actin

protein expression levels relative to those detected in the untreated control. 33

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Data are presented as the mean ± SEM (n = 3). *P < 0.05 versus the untreated control. Colocalization of Klotho with ATF3 or p-c-Jun in the NRK-52E cells in the presence of the vehicle or 40 μM resveratrol for 24 h is shown.

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Representative immunofluorescence images showing double staining for Klotho (red) and ATF3 (green) (D) or c-Jun (green) (E). The merged images

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are shown in the rightmost panel. Nuclei were visualized using DAPI staining

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(blue). p-c-Jun, phosphorylated c-Jun. Original magnification, 200.

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Fig. 3. Resveratrol stimulates ATF3-mediated induction of Klotho promoter activity. (A) NRK-52E cells were cotransfected with the indicated amount of the

M

WT ATF3 expression plasmid in the presence of a 0.5-µg empty control vector or a DNA-binding null ATF3 mutant [ATF3 (∆80–100)] expression plasmid for

d

24 h. The transfected cells were then treated with the vehicle (lanes 1–6) or 40

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μM resveratrol (lanes 7–12) for 12 h. The effects of ATF3 on Klotho mRNA expression were analyzed using RT-PCR. Cell lysates were subjected to

Ac ce p

western blotting to monitor the levels of ATF3 and the loading control (GAPDH). (B) The NRK-52E cells were cotransfected with a 0.25-μg phKL-Luc reporter and the indicated amount of the WT ATF3 (close columns) or ATF3 (∆80–100)

(open columns) expression plasmid. After 24-h transient transfection, the cells were treated with the vehicle (lanes 1–3) or 20 μM resveratrol (lanes 4–6) for

12 h, and the Klotho promoter luciferase activity was subsequently. The numbers placed above the columns indicate the luciferase activity relative to an index of 1 (for the empty control vector alone). (C) Stable transfectants of ACHN cells expressing ATF3 siRNA (ACHN/shATF3) and LacZ siRNA (ACHN/shLacZ) were generated and treated with the indicated resveratrol 34

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dose for 24 h. Total cell lysates subjected to western blotting were probed with an antibody against ATF3 or GAPDH. (D) The expression levels of Klotho mRNA were normalized against those of GAPDH mRNA. Data are presented

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as the mean ± SEM (n = 3). *P < 0.05 versus ACHN/shLacZ treated with the vehicle; #P > 0.05 versus ACHN/shLacZ treated with the same dosage of

cr

resveratrol.

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Fig. 4. Resveratrol induces Klotho expression in NRK-52E cells through the

an

ATF3/c-Jun-complex-mediated signaling pathway. (A) NRK-52E cells were cotransfected with the indicated amount of the WT c-Jun expression plasmid in

M

the presence of a 0.5-μg empty control vector or dominant-negative c-Jun (TAM67) expression plasmid for 24 h. The transfected cells were then treated

d

with the vehicle (lanes 1–6) or 40 μM resveratrol (lanes 7–12) for 12 h. (C) The

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NRK-52E cells were cotransfected with the indicated amount of WT ATF3, c-Jun, or a combination of both. After 24-h transient transfection, the cells were

Ac ce p

treated with the vehicle (lanes 1–6) or 40 μM resveratrol (lanes 7–12) for 12 h.

The effects of ATF3, c-Jun, or a combination of both on Klotho mRNA expression were analyzed using RT-PCR. In addition, cell lysates were subjected to western blotting to monitor the levels of ATF3, p-c-Jun, c-Jun, and the loading control (GAPDH). (B and D) The expression levels of Klotho mRNA

were normalized against those of GAPDH mRNA and are presented as percentages of those detected in the untreated empty control vector. Data are presented as the mean ± SEM (n = 3). *P < 0.05, **P < 0.01 versus the untreated empty control vector.

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Fig. 5. Resveratrol increased the binding of the ATF3/p-c-Jun complex to the Klotho promoter. (A) Cell lysates obtained from NRK-52E cells that were or were not treated with resveratrol were immunoprecipitated with an anti-ATF3,

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anti-c-Jun, or IgG control antibody. The pull-down immunocomplexes were subjected to western blotting with an anti-ATF3 or anti-p-c-Jun antibody. (B)

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Colocalization of ATF3 and p-c-Jun in the nuclei of NRK-52E cells that were or

were not treated with 40 μM resveratrol for 24 h. Double-labeling

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immunocytochemical staining for anti-ATF3 (red) and anti-p-c-Jun (green)

an

antibodies and merged images of both proteins are shown. Nuclei were visualized using DAPI staining (blue). Original magnification, 200. (C) After

M

the NRK-52E cells were treated with the vehicle (lanes 1, 3, 5, and 7) or 40 μM resveratrol (lanes 2, 4, 6, and 8) for 24 h, a ChIP assay was performed as

d

described in the Materials and Methods section. Cell lysates were cross-linked

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with formaldehyde and immunoprecipitated using anti-ATF3, anti-c-Jun, and negative control IgG antibodies. The eluted DNAs were analyzed using PCR to

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identify the ATF3- and AP1-responsive elements located within the Klotho proximal promoter region. The cyclin D1 promoter, which contains an

ATF3-binding site, was used as a positive control.

Fig. 6. Effects of resveratrol treatment on Sirt1 mRNA expression in rat

epithelial cells. Representative semiquantitative RT-PCR analysis of Sirt1 mRNA in NRK-52E cells stimulated using the indicated resveratrol dose for 24 h (A) and 40 μM resveratrol for the indicated duration (B). The levels of Sirt1 mRNA are expressed as the percentage of GAPDH mRNA levels relative to those detected in the untreated control. Data are presented as the mean ± 36

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Hsu et al.

SEM (n = 3). *P < 0.05 versus the untreated control. (C) NRK-52E cells were transfected with the indicated amount of the WT Sirt1 or dominant-negative Sirt1 (H363Y) expression plasmid DNA and a 0.9-μg vector alone. The

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transfected cells were then treated with the vehicle (lanes 1–6) or 40 μM resveratrol (lanes 7–12) for 24 h. The effects of Sirt1 on Klotho, ATF3, and

cr

c-Jun mRNA were analyzed using RT-PCR. The expression levels of Klotho,

ATF3, and c-Jun mRNA were normalized against those of GAPDH mRNA and

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presented as percentages of those detected in the untreated empty control

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M

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vector. Data are presented as the mean ± SEM (n = 3).

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Table 1. Primer sequences used for RT-PCR and ChIP assay

Gene

Forward, 5′-3′

Reverse, 5′-3′

assay

GGGACATTTCCCTGTGACTTTG

GGGAGGTCTCCGTACTTGAA

RT-PCR

mKlotho

CAATGGCTTTCCTCCTTTAC

AACACAGGTTTGCGTAGTCT

RT-PCR

rKlotho

AAATGGCTGGTTTGTCTCGGG

GCAACAACTCCTTGTCCTGACTC

RT-PCR

m,rATF3

CAGGCCAGGTCTCTGCCT

CTGCTTAGCTCTGCAATGTTCCTTC

RT-PCR

m,rc-Jun

CGAGAGCGCTCCGTGAGTGA

GGACTGGAGGAACGAGGCGTT

RT-PCR

hGAPDH

CAAGATCATCAGCAATGCCT

AGGGATGATGTTCTGGAGAG

RT-PCR

mGAPDH

ACTCCACTCACGGCAAATTC

CCTTCCACAATGCCAAAGTT

RT-PCR

rGAPDH

ATGGGAAGCTGGTCATCAAC

m,rSirt1

TAGATACCTTGGAGCAGGTTG

M

an

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cr

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hKlotho

CCACAGTCTTCTGAGTGGCA

d

CAGTAATTTCTGAAAGCTTTACAGGG RT-PCR

te

rKL_ATF3 ATCTCAGGACGGAGGGCATGGT TGATTATCCAGATAAGGCGCCGC rKL_AP1

ATGGCTTCTCAGGTTGTGGT

AATCTTTCAGACGACATCCCT

ChIP ChIP

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h, human; m, mouse; r, rat

RT-PCR

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Figure 2

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Figure 3

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Figure 4

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Figure 5

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