Regulation of oxidative stress by the anti-aging hormone klotho

Abstract

klotho is an aging suppressor gene and extends life span when overexpressed in mice. Klotho protein was recently demonstrated to function as a hormone that inhibits insulin/insulin-like growth factor-1 (IGF-1) signaling. Here we show that Klotho protein increases resistance to oxidative stress at the cellular and organismal level in mammals. Klotho protein activates the FoxO forkhead transcription factors that are negatively regulated by insulin/IGF-1 signaling, thereby inducing expression of manganese superoxide dismutase. This in turn facilitates removal of reactive oxygen species and confers oxidative stress resistance. Thus, Klotho-induced inhibition of insulin/IGF-1 signaling is associated with increased resistance to oxidative stress, which potentially contributes to the anti-aging properties of klotho.

FIGURE 1. Klotho increases resistance to oxidative stress

A, the effect of Klotho protein on lipid oxidation in cultured cells. HeLa cells left untreated (−) or treated (+) with 100 μm paraquat in the presence (+) or absence (−) of 200 pm recombinant Klotho protein were loaded with a fluorescent lipid oxidation probe C11-BODIPY^581/591. Yellow represents high oxidation and red represents low oxidation. A representative result from four independent experiments is shown. B–D, the effect of Klotho protein on cell death induced by oxidative stress. B, representative dot plots of CHO cells stained with annexin V-FITC and propidium iodide (PI). The cells were treated with 100 μm paraquat in the presence (right panel) or absence (left panel) of 200 pm Klotho protein. The bottom right quadrant represents early apoptotic cells. The top right and left quadrants represent late apoptotic and necrotic cells, respectively. The percentage counts of each quadrant are indicated. C, representative histograms of the CHO cells based on annexin V binding in the presence (red) and absence (blue) of 200 pm Klotho protein under the paraquat-induced oxidative stress. D, mean percentages of annexin V-positive and negative cells in the presence and absence of Klotho protein. The annexin V-negative cells virtually represent live cells under these experimental conditions, because necrotic cell counts were <4% (the top left quadrant in B). *, p < 0.01 by ANOVA (n = 4). E, urinary 8-OHdG levels in wild-type mice (WT) and EFmKL48 mice (EFmKL). *, p = 0.02 by ANOVA (n = 4). F, Kaplan-Meier analysis of survival after the paraquat challenge between age-matched male wild-type mice (WT) and EFmKL48 mice (EFmKL). p = 0.0019 by log-rank test.

FIGURE 2. Klotho protein reduces FOXO phosphorylation and promotes its nuclear translocation

A, the effect of Klotho protein on basal levels of Akt and FOXO phosphorylation. HeLa cells transfected with the FOXO1-myc expression vector were stimulated with various concentrations of Klotho protein as indicated for 30 min (left panels). Cell lysates were immunoblotted with anti-Akt antibody (Akt), anti-myc antibody (FOXO1), or antibody specific to phosphorylated Akt (p-Akt) or FOXO (p-FOXO1). To determine the time course, the cells were stimulated with 200 pm Klotho protein for various time periods as indicated (right panels). B, protein microarray analysis of Akt and FOXO1 phosphorylation. HeLa cells transfected with FOXO1-myc expression vector were incubated in the presence or absence of 200 pm Klotho protein for 30 min and then stimulated with 10 nm insulin or left untreated for 30 min. Cell lysates were arrayed on nitrocellulose-coated slides in quadricate in a serial dilution format as indicated. The slides were probed with anti-Akt antibody (Akt), anti-myc antibody (FOXO1), or antibody specific to phosphorylated Akt (p-Akt) or FOXO (p-FOXO1). Representative blots from four independent experiments are shown. C, Klotho reduces both basal and insulin-induced phosphorylation of all three FOXOs. HeLa cells transfected with the FOXO1-myc, FOXO3a-myc, or FOXO4-myc expression vector were treated as described for B. Cell lysates were analyzed in the same way as described for A. D, Klotho reduces insulin-induced phosphorylation of Akt and FOXO in mice. The lysates of muscles from KL^−/− mice (KL^−/−), wild-type (WT), and EFmKL48 mice (EFmKL) administered with insulin or saline were immunoblotted with anti-phospho-Akt antibody (p-Akt), anti-Akt antibody (Akt), anti-phosopho-FOXO antibody (p-FOXO), or anti-FOXO1 antibody (p-FOXO1). Representative blots from four independent experiments are shown. E, Klotho-induced nuclear translocation of FOXO1. HeLa cells transfected with the FOXO1-myc expression vector were incubated with or without 200 pm Klotho protein for 30 min and then stained with anti-myc antibody. F, Klotho-induced nuclear translocation of FOXO1 determined by immunoblotting. HeLa cells transfected with the FOXO1-myc expression vector were incubated with the indicated concentrations of Klotho protein for 30 min and then fractionated into cytoplasm and nuclear fractions. Each fraction was immunoblotted with anti-myc antibody (left panels). To determine the time course, the cells were incubated with 200 pm Klotho protein for the indicated time periods (right panels).

FIGURE 3. Klotho enhances FOXO transcriptional activity and increases SOD2 protein levels

A, measurement of FOXO transcriptional activity by using the luciferase reporter 3xFHRE-Luc (left panel), which contains three canonical FOXO-binding sites, or SOD2-Luc (right panel), which contains the human SOD2 gene promoter. HeLa cells were transfected with the reporter plasmids and incubated in the presence (+) or absence (−) of 400 pm Klotho protein before the luciferase assay. The error bars indicate S.D. *, p < 0.05 by ANOVA (n = 6). B, the effect of Klotho protein on FOXO-binding to the SOD2 promoter. HeLa cells were transfected with FOXO1-myc expression vector and then stimulated with 400 pm Klotho protein or left untreated in the presence or absence of 50 nm wortomannin before chromatin immunoprecipitation assay. Wortmannin treatment activates FOXO1 through inhibiting PI3K and serves as a positive control. A representative result from four independent experiments is shown. C, the effect of Klotho protein on SOD2 protein levels. HeLa cells were incubated with the indicated concentrations of Klotho protein for 16 h and subjected to immunoblot analysis using anti-SOD2 antibody (SOD2) and anti-actin antibody (Actin). D, quantification of relative amounts of SOD2 protein in cultured cells (COS, HeLa, CHO) by reverse-phase protein microarray. Upper left, microarray layout. The numbers indicate hours after Klotho stimulation (400 pm). The cell lysates were arrayed in duplicate in a serial dilution format as indicated. A representative blot with anti-SOD2 antibody from two independent experiments is shown. The graph shows a representative plot between spot signal intensity (y axis) and relative sample concentration (x axis, log scale) with a fitted curve and 99% confidence bands. The regression line at the half-maximal intensity (EC₅₀) is indicated. The amount of SOD2 protein in the sample was determined as the dilution factor at EC₅₀. The amount of actin was quantified in the same way for loading controls. The coefficient of variation was <0.9% and R² > 0.996 for all regression lines. E, a time course of Klotho-induced increase in SOD2 protein levels quantified by protein microarray analysis. COS, HeLa, and CHO cells were incubated with 400 pm Klotho protein for 6 or 16 h. The fold increase in the SOD2 protein amount from time 0 is indicated. All data were normalized to the actin level. The average of two independent experiments is shown. Each dilution was spotted in duplicate. F, Klotho overexpression increases SOD2 mRNA in muscle. Total RNA isolated from muscles of four 8-week-old male KL^−/− mice (KL^−/−), wild-type mice (WT), or EFmKL48 mice (EFmKL) were pooled, and 20-μg aliquots were loaded on each lane. G, Klotho overexpression increases SOD2 protein in mouse tissues. Muscle of 8-week-old male KL^−/− mice (KL^−/−), wild-type mice (WT), and EFmKL48 mice (EFmKL) were analyzed as described for C. H, quantification of SOD2 protein by the protein microarray analysis. Protein extracted from the hind limb muscle of 8-week-old male KL^−/− mice (KL^−/−), wild-type mice (WT), and EFmKL48 mice (EFmKL) was analyzed as in D. The error bars indicate S.D. *, p < 0.05 by ANOVA (n = 4). Each dilution was spotted in duplicate.