All animal experiments were performed with approval from Tokai University animal study institutional review board.
Reagents and plasmids
Plasmids were kindly provided by Dr. Raymond Poon (Hospital for Sick Children, University of Toronto, Toronto, Ontario, Canada) (plasmid encoding wild-type (WT) β-catenin and the backbone), Ilsa Rovira (Cardiology Branch, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD, USA) (plasmid encoding Flag-tagged full-length Klotho) , Dr. Young Han Lee (Institute of Biomedical Science and Technology, Konkuk University Hospital, Seoul, South Korea) (plasmids encoding luciferase-tagged Klotho constructs, Del-1, Del-2, Del-3, and Del-4) , Dr. Michael C. Naski (University of Texas Health Science Center at San Antonio, San Antonio, TX, USA) (plasmid encoding luciferase-tagged aggrecan) , Dr. Sean A. McCarthy (University of Michigan, Ann Arbor, MI, USA) (plasmid encoding Dickkopf-1 (Dkk1), Dkk2, Dkk3, and Dkk4 expression vectors or backbone vector), and Dr. Yoshihiko Yamada (Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, Bethesda, MD, USA) (plasmid encoding luciferase-tagged type-II collagen) . The constitutively active (CA)-GSK3β (no. 14754) and dominant-negative (DN) GSK3β (no. 14755) were purchased from Addgene (Cambridge, MA, USA). Topflash (optimal Tcf-binding site) and Fopflash (a promoter with a mutated Tcf-binding site) were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY, USA). β-Catenin small interfering RNA (siRNA) (no. sc-29209) and control siRNA duplexes were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). In the Klotho constructs, the TCF/LEF binding motifs within the klotho promoter were analyzed by using the Web-based tool for predicting transcription factor-binding sites in DNA sequences, Transcription Element Search System (TESS). As an internal transfection control, we used the empty vector pGL4.74 (Promega, Fitchburg, WI, USA) containing the Renilla reniformis luciferase genes. The amount of transfected plasmid, the pretransfection period after seeding, and the posttransfection period before harvesting were optimized for rat nucleus pulposus cells by using the pSVβ-galactosidase plasmid (Promega). We used 6-bromoindirubin-3'-oxime (BIO) (no. 361550; Calbiochem, San Diego, CA, USA) to examine the activity of the Wnt signaling activity. BIO is a cell-permeable, highly potent, selective, reversible, and ATP-competitive specific inhibitor of GSK-3α/β activity . Recombinant Klotho protein (α-Klotho) (no. 100-53) was purchased from Peprotech (Rocky Hill, NJ, USA).
Isolation of intervertebral disc cells
In total, 64 (32 female and 32 male) 12-week-old Sprague-Dawley rats were used for this study. None of the variables differed between females and males. Nucleus pulposus cells were isolated by using methods reported by Hiyama et al., 2007 . In brief, the rats were euthanized by injection of an excess of pentobarbital sodium (100 mg/kg) (Nembutal; Abbott Laboratories, Abbott Park, IL, USA). The spinal column was removed under aseptic conditions, and the lumbar intervertebral discs were separated under microscopy. The gel-like nucleus pulposus was separated from the annulus fibrosus. The obtained nucleus pulposus tissue was digested in a mixture of 0.01% trypsin and allowed to digest at 37°C for 15 minutes. The digested tissue was passed through a cell strainer (BD Biosciences, San Jose, CA, USA) with a pore size of 100 μm and was washed twice with phosphate-buffered saline (PBS; Gibco, Invitrogen, Carlsbad, CA, USA). The isolated cells were maintained in Dulbecco modified Eagle medium (DMEM; Gibco, Invitrogen) and 10% fetal bovine serum (FBS; Gibco, Invitrogen) supplemented with antibiotics (1% penicillin/streptomycin) at 37°C in a humidified atmosphere of 5% CO2. When confluent, the nucleus pulposus cells were harvested and subcultured in 10-cm dishes. Cells were then counted and plated at the appropriate density. Because cells obtained from the rat intervertebral disc tissues were variable in morphology until passage 2 to 3, we used low-passage (< 3) cells cultured in monolayers for all experiments.
Cell culture in hypoxia
To induce hypoxia, nucleus pulposus cells were cultured in a mixture of 2% O2, 5% CO2, and 93% N2 for 24 hours. The plating density chosen was dependent on the requirements of the individual assays. The concentration of oxygen and the duration of the incubation period chosen for this study were based on the in vitro studies of Risbud et al.
 and on information generated on the oxemic status of the disc in vivo.
Nucleus pulposus cells were plated in flat-bottom 96-well plates (5 × 103 cells/well) and maintained in conditions of normoxia or hypoxia for 24 hours. After this treatment, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100 (vol/vol) in PBS for 10 minutes, blocked with PBS containing 5% FBS, and incubated overnight at 4°C with antibodies against β-catenin (1:200 dilution; Cell Signaling Technology, Danvers, MA, USA). After washing, the cells were incubated with anti-rabbit IgG Alexa Fluor 594 (red) and/or anti-goat Alexa Fluor 488 secondary (green) antibodies (Invitrogen) at a dilution of 1:50 and 10 μM 4',6-diamidino-2-phenylindole (DAPI) for 1 hour at room temperature for nuclear staining. The samples were observed with a fluorescence microscope connected to a digital imaging system. Similar experiments were performed to assess the expression of anti-rabbit-Klotho (1:200 dilution; Abcam, Cambridge, UK) and anti-goat-Klotho (1:50 dilution; Santa Cruz Biotechnology). Negative controls without the primary antibody were prepared.
Freshly isolated rat spines were immediately fixed in 4% paraformaldehyde in PBS, decalcified, embedded in paraffin, sectioned, and assessed by histology and then immunostaining. Sagittal sections were deparaffinized in xylene, rehydrated through a graded ethanol series, and stained with hematoxylin. To localize Klotho, sections were incubated with anti-Klotho antibody (ab75023; Abcam) in 2% bovine serum albumin (BSA) in PBS at a dilution of 1:200 at 4°C overnight. After thoroughly washing the sections, the bound primary antibody was detected with a biotinylated universal secondary antibody (Vector Laboratories, Burlingame, CA, USA) at a dilution of 1:20 for 10 minutes at room temperature. Sections were incubated with a streptavidin/peroxidase complex for 5 minutes and washed with PBS, and the color was developed by using 3'-3-diaminobenzidine (DAB) (VECTASTAIN Universal Quick Kit; Vector Laboratories) and peroxidase DAB substrate kit (Vector), according to the manufacturer's protocol. Negative controls without the primary antibody (anti-Klotho) were prepared.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay
To measure disc-cell proliferation, a modified MTT assay was carried out as previously described . In brief, exponentially growing nucleus pulposus cells were seeded in 24-well plates at 1.5 × 104 cells/well. After treatment with Klotho (100 ng/ml), MTT diluted in serum-free DMEM was added to the culture medium to a final concentration of 0.5 mg/ml. No data showed the physiological protein concentration of Klotho in the nucleus pulposus cells. We therefore chose to use the concentration of Klotho protein (100 ng/ml) referred to in a previous study . At the end of the incubation period (2 hours at 37°C), the medium was removed, and the precipitated formazan crystals were solubilized in dimethyl sulfoxide (DMSO). Product formation was measured by reading the absorbance at 590 nm by using a microplate reader (Pharmacia).
Staining for senescence-associated β-galactosidase (β-gal)
To stain nucleus pulposus cells for senescence-associated β-gal activity, we used a cell-senescence histochemical staining kit (CS0030; Sigma-Aldrich, St. Louis, MO, USA) according to the previously described protocol . Cells were treated for 24 hours with or without 100 ng/ml Klotho. To quantify the results, a minimum of 100 cells spanning five different microscopy fields were scored for staining, and results represent the mean ± standard deviation (SD).
Cell-cycle analysis by flow cytometry
Nucleus pulposus cells were grown in 24-well plates under a humidified 5% CO2 atmosphere at 37°C. The seeding density was 5 × 104 cells/ml. The cells were allowed to adhere for 24 hours in medium containing 2% FBS. The culture medium in each flask was then replaced with medium containing 0.5% FBS. Klotho (100 ng/ml) was added to this medium as a concentrated stock solution dissolved in DMSO. After an additional incubation period of 24 or 48 hours, the cell-cycle distribution of the nucleus pulposus cells was analyzed with flow cytometry after DNA staining with propidium iodide by using the CycleTEST PLUS kit (BD Pharmingen, San Diego, CA, USA). CELLQuest (BD Pharmingen) and ModFit LT (BD Pharmingen) software packages were used for cell acquisition and analysis. Each plot represents the analysis of 10,000 events. The histograms present typical results, and the percentages of cells in G1, S, and G2/M cell-cycle phases are shown as the means of triplicate measurements. The results from three individual experiments are shown.
Total RNA extraction and real-time reverse transcriptase (RT)-PCR analysis
Total RNA was extracted from rat nucleus pulposus and annulus fibrosus cells or tissues by using the TRIzol RNA isolation protocol (Invitrogen). Before elution from the column, RNA was treated with RNase-free DNAse I. Total RNA (100 ng) was used as a template for the real-time PCR analyses. The cDNA was synthesized by the reverse transcription of mRNA by using the protocol described previously (26). The real-time PCR analyses were performed in triplicate by using 96-well plates with the Fast SYBR Green Master Mix (Applied Biosystems, Carlsbad, CA, USA). Two microliters of cDNA per sample was used as the template for real-time PCR: 1 μl of forward primer and 1 μl of reverse primer were added to 20 μl SYBR Green Master Mix. PCR reactions were performed in an Applied Biosystems 7500 Fast system, according to the manufacturer's instructions. All primers for β-catenin, Klotho, and aggrecan were synthesized by Takara Bio, Inc. (Tokyo, Japan): β-catenin (NCBI number: AF_121265.1): forward, 5'-GCCAGTGGATTCCGTACTGT-3' and reverse, 5'-GAGCTTGCTTTCCTGATTGC-3'; Klotho (NCBI number: NM_031336.1): forward, 5'-CGATGTTCGTGACAGCCAATG-3' and reverse, 5'-GTTGATGCCGTCCAACACGTAG-3'; and aggrecan (NCBI number: NM_022190.1): forward, 5'-TCCGCTGGTCTGATGGACAC-3' and reverse, 5'-CCAGATCATCACTACGCAGTCCTC-3'. To normalize each sample, a control gene (GAPDH) was used, and the arbitrary intensity threshold (Ct) of amplification was computed. The expression scores were obtained by the ΔΔCt calculation method.
Gene-suppression studies by small interfering RNA (siRNA)
We silenced β-catenin expression in nucleus pulposus cells by using small interfering RNA (siRNA) technology. In brief, nucleus pulposus cells were transferred to 24-well plates at a density of 6 × 104 cells/well 1 day before transfection. The next day, cells were treated with β-catenin siRNA or control siRNA duplexes at a final concentration of 100 to 500 ng by using Lipofectamine 2000. Cells also received Klotho promoter constructs and the pGL4.74 plasmid at the time of transfection. Six hours after transfection, the medium was replaced with complete growth medium, and the cells were allowed to recover for 18 hours. Cells were then cultured for 24 hours, and luciferase activity was measured.
Genomic DNA was extracted from cells not treated with BIO and BIO-treated cells by using a FastPure DNA Kit (Takara Bio), and bisulfite treatment of genomic DNA was performed by using MethylEasy Xceed (Human Genetic Signatures) and following the manufacturers' instructions. The bisulfite-treated DNA was amplified by PCR with Takara Taq Hot Start Version (Takara Bio) and T-Vector pMD20 (Takara Bio) for cytosine-phosphate-guanosine (CpG)-rich regions around the rat Klotho gene. PCR products amplified by using Takara LA Taq HS were subcloned into T-Vector pMD20, and sequence analysis was performed. All methylation studies were performed by bisulfite modification of DNA, which converts all unmethylated CpG sites to uracil-phosphate-guanosine, leaving methylated CpGs intact. The DNA was then amplified with the following gene-specific primers: Klotho, forward, 5'-TGATGTGGGGATATTTTAGGA-3', and reverse, 5'-CAACAAATACAACRACAACAAA-3'. After sequencing of the amplified DNA, methylated CpGs were identified with visual inspection of the sequencing traces in the electropherograms and by comparison with sequencing traces of BIO-treated cell gene sequences relative to the reference in the control cell sequence.
Western blotting analysis
After treatment, nucleus pulposus cells were immediately washed 3 times with ice-cold PBS and solubilized in lysis buffer. Proteins were prepared by using the CellLytic NuCLEAR extraction kit (Sigma-Aldrich). All wash buffers and the final resuspension buffer included × 1 protease inhibitor cocktail (Roche, Switzerland), NaF (5 mM), and Na3VO4 (200 μM). Total lysates (30 to 60 μg of protein/lane) were subjected to electrophoresis by using 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Bio-Rad, Hercules, CA, USA). The resolved proteins were transferred electrophoretically to nitrocellulose membrane "blots". The blots were blocked with 5% BSA in TBST (50 mM Tris, pH 7.6, 150 mM NaCl, 0.1% Tween 20) and incubated overnight at 4°C in 5% BSA in TBST with anti-β-catenin (1:1,000; Cell Signaling) or anti-Klotho (1:1,000; Abcam). Immunolabeling was detected with electrochemiluminescence reagent (Amersham Biosciences, Piscataway, NJ, USA). Quantification of Western blots was performed by using Image J pixel analysis (NIH Image software). Data from Western blots are presented as band density normalized to the loading control (actin).
Transfections and dual luciferase assay
Nucleus pulposus cells were transferred to 24-well plates at a density of 6 × 104 cells/well 1 day before transfection. In several experiments, cells were cotransfected with 100 to 500 ng of expression plasmids or the backbone vector, along with reporter plasmids. Lipofectamine 2000 (Invitrogen) was used as a transfection reagent. After 24 hours, cells were maintained in either the hypoxic or the normoxic condition. Twenty-four hours after treatment, the cells were harvested, and a Dual-Luciferase reporter assay system (Promega) was used for the sequential measurements of firefly and Renilla luciferase activities. The results were normalized for transfection efficiency and are expressed as a relative ratio of luciferase to pGL4.74 activities (denoted as relative activity). To check the transfection efficiency, nucleus pulposus cells were transfected with plasmid encoding green fluorescent protein; the transfection efficiency for nucleus pulposus cells was 60% to 70%. In addition, the overexpression or silencing efficiency of the targets was confirmed at the mRNA level or protein level. The luciferase activities and relative ratios were quantified by using a Turner Designs Luminometer Model TD-20/20 instrument (Promega).
Typically, data were compiled from at least three independent triplicate experiments, each performed on separate cultures and on separate occasions. We calculated and have displayed the responses as fold change (over the untreated control). Data are presented as mean ± SD. Comparisons of data between groups were performed by using the Student t test or ANOVA for assessing variance. Statistical significance (P < 0.05) is denoted with an asterisk.