Klotho, an antiaging molecule, attenuates oxidant-induced alveolar epithelial cell mtDNA damage and apoptosis

Abstract

Alveolar epithelial cell (AEC) apoptosis and inadequate repair resulting from "exaggerated" lung aging and mitochondrial dysfunction are critical determinants promoting lung fibrosis. α-Klotho, which is an antiaging molecule that is expressed predominantly in the kidney and secreted in the blood, can protect lung epithelial cells against hyperoxia-induced apoptosis. We reasoned that Klotho protects AEC exposed to oxidative stress in part by maintaining mitochondrial DNA (mtDNA) integrity and mitigating apoptosis. We find that Klotho levels are decreased in both serum and alveolar type II (AT2) cells from asbestos-exposed mice. We show that oxidative stress reduces AEC Klotho mRNA and protein expression, whereas Klotho overexpression is protective while Klotho silencing augments AEC mtDNA damage. Compared with wild-type, Klotho heterozygous hypomorphic allele (kl/+) mice have increased asbestos-induced lung fibrosis due in part to increased AT2 cell mtDNA damage. Notably, we demonstrate that serum Klotho levels are reduced in wild-type but not mitochondrial catalase overexpressing (MCAT) mice 3 wk following exposure to asbestos and that EUK-134, a MnSOD/catalase mimetic, mitigates oxidant-induced reductions in AEC Klotho expression. Using pharmacologic and genetic silencing studies, we show that Klotho attenuates oxidant-induced AEC mtDNA damage and apoptosis via mechanisms dependent on AKT activation arising from upstream fibroblast growth factor receptor 1 activation. Our findings suggest that Klotho preserves AEC mtDNA integrity in the setting of oxidative stress necessary for preventing apoptosis and asbestos-induced lung fibrosis. We reason that strategies aimed at augmenting AEC Klotho levels may be an innovative approach for mitigating age-related lung diseases.

Keywords: Klotho; alveolar epithelial cell; mitochondrial DNA damage; oxidative stress; pulmonary fibrosis.

Fig. 1.

Oxidative stress depletes Klotho expression in several types of alveolar epithelial cells (AECs). A: AEC Klotho protein expression following amosite asbestos (5–25 µg/cm²) or H₂O₂ (100–200 µM) exposure for 24 h. GAPDH is a loading control. A densitometric analysis using GAPDH as a loading control from three experiments is shown. B: secreted AEC Klotho level following asbestos or H₂O₂ exposure for 24 h. C: A549 and MLE-12 cell mRNA expression for Klotho following asbestos exposure as assessed by quantitative PCR (qPCR). Values are represented as means ± SE; *P < 0.05 vs. control; n = 3.

Fig. 2.

Klotho overexpression and recombinant Klotho prevent MLE-12 cell mitochondrial DNA (mtDNA) damage and intrinsic apoptosis exposure to asbestos or H₂O₂. Klotho overexpression (A–C) and recombinant Klotho (D–F) blocks oxidative stress-induced cleaved caspase-9, mtDNA damage, and intrinsic apoptosis caused by amosite asbestos (25 µg/cm²) or H₂O₂ (200 μM) for 24 h in MLE-12 cells. A: Klotho overexpression is measured by anti-His antibody. Cleaved caspase-9 in total cell lysate was assessed by Western blot analysis showing representative figure. A densitometric analysis using GAPDH as a loading control from three experiments is shown. B: mtDNA damage was performed by qPCR-based measurement using isolated whole genomic DNA from each condition. C: intrinsic apoptosis was detected by ELISA assay by using cell death detection kit. D: cleaved caspase 9 (CC-9) and GAPDH in total cell lysate were assessed by Western blot analysis; shown is a representative figure and a densitometric quantification of CC-9/GAPDH expression normalized to control with PBS. E: oxidant-induced MLE-12 cell mtDNA damage by qPCR-based measurement. F: intrinsic apoptosis by ELISA assay using cell death detection kit were measured as described. Values are represented as means ± SE; *P < 0.05 vs. control and +P < 0.05 vs. empty vector or PBS vehicle ASB/H₂O₂; n = 3.

Fig. 3.

Klotho deficiency augments asbestos-induced AEC mtDNA damage and intrinsic apoptosis. Control siRNA or Klotho siRNA were transfected to MLE-12 cells for 48 h and were exposed to amosite asbestos (25 µg/cm²) or H₂O₂ (200 μM) for 24 h. Cleaved caspase-9 in total cell lysate as well as Klotho expression were assessed by Western blot analysis showing representative figures and a densitometric analysis using GAPDH as a loading control (A), and mtDNA damage was performed by qPCR-based measurement using isolated whole genomic DNA from each condition (B); intrinsic apoptosis was detected by ELISA assay by using cell death detection kit (C). Values are represented as means ± SE; *P < 0.05 vs. control and +P < 0.05 vs. scramble; n = 3.

Fig. 4.

Asbestos reduces serum and AT2 cell Klotho levels as compared with TiO₂ in wild-type (WT) mice. Serum (A) and primary AT2 cells (B) were isolated from WT mice at 3 wk after intratracheal instillation with TiO₂ or crocidolite asbestos. C: primary AT2 cells were isolated from WT mice, then exposed to amosite asbestos or H₂O₂. ELISA was performed to detect Klotho level at each condition. Data are presented as means ± SE; *P < 0.05 vs. TiO₂ or control; n = 3 mice per group.

Fig. 5.

kl/+ Mice exposed to asbestos presents the increase of lung fibrosis and AT2 cell mtDNA damage compared with WT. After intratracheal instillation with TiO₂ or crocidolite asbestos for 3 wk, serial lung sections from age-matched WT and kl/+ mice were subject to Masson’s trichrome stain (A), lung fibrosis scores (B), and lung collagen levels (C). A: representative histology is shown from four to nine mice in WT and kl/+ mice after treatment. B: the fibrosis score = (severity: 0–4) × (extent: 1–3). *P < 0.05 vs. TiO₂, +P < 0.05 vs. WT+crocidolite asbestos; n = 3–4. C: lung collagen levels as assessed by Sircol assay. *P < 0.05 vs. TiO₂, +P < 0.05 vs. WT+crocidolite asbestos; n = 3–9. D: mtDNA damage was assessed by a fluorescent-based PCR mtDNA damage assay using genomic DNA of primary AT2 cells isolated from the lungs of WT and kl/+ mice 3 wk after IT instillation of TiO₂ or crocidolite asbestos. Graph expressed as the ratio of lesion frequency per fragment as compared with WT AT2 cells exposed to TiO₂. *P < 0.05 vs. TiO₂, +P < 0.05 vs. WT + crocidolite asbestos, and #P < 0.05 vs. kl/+ mice+crocidolite asbestos; n = 3.

Fig. 6.

Mitochondrial catalase and its mimetic, EUK-134, protects oxidant-induced Klotho reduction in serum from WT and MCAT mice as well as MLE-12 cells. A: serum were collected from WT and MCAT mice at 3 wk after intratracheal instillation with TiO₂ or crocidolite asbestos, and Klotho level was measured by ELISA. B and C: confluent MLE-12 cells were incubated with EUK-134 (20 μM) for 3 h, and then were exposed to amosite asbestos (25 µg/cm²) or H₂O₂ (200 μM) for 24 h. Cell lysates from each group were shown to Klotho level performed with Western blot analysis showing representative image with the densitometric analysis of Klotho/GAPDH relative to untreated control (B) and ELISA (C). Data are presented as means ± SE; *P < 0.05 vs. WT+TiO₂ or PBS+control; n = 3 mice or MLE-12 cells per group.

Fig. 7.

The Klotho/fibroblast growth factor receptor (FGFR)/AKT axis regulates oxidative stress-induced AEC mtDNA damage and apoptosis. A: effect of signaling pathway inhibitors on oxidant-induced AEC mtDNA damage. MLE-12 cells were preincubated with an AKT inhibitor (GSK690693, 10 µM), ERK inhibitor (U0125, 10 µM), JNK inhibitor (SP600125, 5 µM), or p38 MAPK inhibitor (SB203580, 5 µM) for 6 h and exposed to amosite asbestos (25 µg/cm²) or H₂O₂ (200 µM) for 24 h. mtDNA damage was performed by qPCR-based measurement (*P < 0.05 vs. DMSO). B: phospho-AKT is inhibited in asbestos-exposed MLE-12 cells. MLE-12 cells were exposed to amosite asbestos (5 or 25 µg/cm²) for 1 or 3 h. Cell extracts were isolated, and phospho-AKT level was evaluated by Western blot analysis showing representative image and semiquantitative analyzed graph (*P < 0.05 vs. control; n = 3). C and D: AKT1 silencing increases mtDNA damage and blocks the protective effects of recombinant Klotho in oxidant-induced MLE-12 cells. Cells were transfected with siRNA targeted to AKT1, and then recombinant Klotho was preincubated for 3 h and exposed to amosite asbestos (25 µg/cm²) or H₂O₂ (200 µM) for 24 h. mtDNA damage was performed by qPCR-based measurement using isolated whole genomic DNA from each condition. Values are represented as means ± SE; *P < 0.05 vs. Scr/control and +P < 0.05 siAKT vs. siAKT+reKlotho; n = 3. E: FGFR1, but not IGFR, silencing blocks reKlotho’s protective effects against oxidant-induced AEC mtDNA damage. MLE-12 cells were transfected with siRNA targeted to IGFR or FGFR1, and then recombinant Klotho was preincubated for 3 h and exposed to amosite asbestos (25 µg/cm²) or H₂O₂ (200 µM) for 24 h. mtDNA damage was performed by qPCR-based measurement using isolated whole genomic DNA from each condition. Values are represented as means ± SE; *P < 0.05 vs. Scr/control, +P < 0.05 siIGFR vs. siIGFR+reKlotho; n = 3. F: hypothetical model by which Klotho prevents oxidant-induced AEC mtDNA damage, apoptosis, and lung fibrosis.