Klotho expression is reduced in COPD airway epithelial cells: effects on inflammation and oxidant injury

Abstract

COPD (chronic obstructive pulmonary disease) is associated with sustained inflammation, excessive injury, and accelerated lung aging. Human Klotho (KL) is an anti-aging protein that protects cells against inflammation and damage. In the present study, we quantified KL expression in the lungs of COPD patients and in an ozone-induced mouse model of COPD, and investigated the mechanisms that control KL expression and function in the airways. KL distribution and levels in human and mouse airways were measured by immunohistochemistry and Western blotting. The effect of CSE (cigarette smoke extract) on KL expression was detected in human bronchial epithelial cells. Moreover, the effect of KL on CSE-mediated inflammation and hydrogen peroxide-induced cellular injury/apoptosis was determined using siRNAs. KL expression was decreased in the lungs of smokers and further reduced in patients with COPD. Similarly, 6 weeks of exposure to ozone decreased KL levels in airway epithelial cells. CSE and TNFα (tumour necrosis factor α) decreased KL expression and release from airway epithelial cells, which was associated with enhanced pro-inflammatory cytokine expression. Moreover, KL depletion increased cell sensitivity to cigarette smoke-induced inflammation and oxidative stress-induced cell damage. These effects involved the NF-κB (nuclear factor κB), MAPK (mitogen-activated protein kinase) and Nrf2 (nuclear factor erythroid 2-related factor 2) pathways. Reduced KL expression in COPD airway epithelial cells was associated with increased oxidative stress, inflammation and apoptosis. These data provide new insights into the mechanisms associated with the accelerated lung aging in COPD development.

Keywords: bronchial epithelial cells; chronic obstructive pulmonary disease (COPD); cytokines; klotho protein; oxidative stress; smoke.

© 2015 Authors; published by Portland Press Limited.

Figures

Figure 1. Klotho expression is significantly reduced in the lung of patients with COPD

(A) KL immunostaining is markedly reduced in COPD patients compared with that in smoking control subjects and healthy non-smokers (original magnification ×100). (B) Representative Western blots were probed using an anti-KL antibody and normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as loading control. (C) The immunoblots of KL and the control GAPDH bands were quantified using a LiCOR image analyser and normalized to GAPDH. Results are means±S.D. of the normalized arbitrary scan values of 15 COPD patients, 22 healthy smokers and 22 healthy non-smoking controls. *P<0.05, **P<0.001, #P<0.05.

Figure 2. Klotho levels are reduced in the airway epithelium and transiently increased in BALF of ozone-induced emphysematous mice

(A–C) Immunohistochemical analysis of the expression of KL protein in airway epithelium of mice exposed to air or ozone for 1, 3 and 6 weeks (original magnification ×200). KL immunostaining is similar in 1- or 3-week ozone-exposed mice compared with air controls (A and B), but there was slightest staining for KL in airway epithelial cells of 6-week-exposed mice (C). (D) BALF was obtained from all mice 24 h after the last exposure to ozone and the secreted KL was determined by ELISA. There was a significant but transient increase in KL secretion in 3-week ozone-exposed groups (3.6-fold increase, P<0.05), but not in 1-week (1.1-fold increase) or 6-week (1.04-fold decrease) ozone-exposed animals. Results are means±S.E.M., *P<0.05.

Figure 3. Cigarette smoke extract (CSE) and TNFα reduce intracellular Klotho expression in an NF-κB-dependent manner

Cells were treated with 5% CSE for 24, 48 and 72 h, and KL protein was measured by Western blotting. Immunoblots were quantified using a LiCOR image analyser and normalized to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Similar results were seen at the KL mRNA level detected by RT–qPCR (A). Cultured 16HBE cells were exposed to TNFα (3, 30 and 300 ng/ml) for 24 h, and KL expression was determined by Western blotting (B). Quantification results are means±S.E.M. from three independent experiments (*P<0.05, ***P<0.001 compared with the control group). In some experiments, cells were pre-treated with the NF-κB inhibitor JSH23 (20 μM) for 30 min (C). (#P<0.01 compared with control and *P<0.05 compared with TNFα). Cells were exposed to medium alone (control, C) or TNFα (T, 30 ng/ml), or pre-treated with the NF-κB inhibitor JSH23 (20 μM) before TNFα stimulation (T+J) for 24 h and KL expression was determined by immunofluorescence (D). The green fluorescence in the left-hand panels indicates KL, the blue stain indicates the nucleus, and the merged panels are on the right. Immunocytochemistry results are representative of at least three independent experiments.

Figure 4. CSE and TNFα inhibit Klotho secretion and increase the release of primary pro-inflammatory cytokines in cultured human bronchial epithelial (16HBE) cells

The supernatant from the cells treated in (A) were collected and KL secretion detected by ELISA. In separate experiments, 16HBE cells were treated with CSE or TNFα, or both (C+T), for 72 h and KL release was measured by ELISA (B). (*P<0.05, **P<0.01 compared with the control group at 72 h). The 16HBE cells were treated as in (C) and the release of IL-1β was determined. (**P<0.01 compared with the control group at 24 h, #P<0.05, ###P<0.001 compared with the control group at 48 h, $$P<0.01, $$$P<0.001 compared with the control group at 72 h). Cells were cultured in medium containing 5% CSE, exogenous TNFα (30 ng/ml) or both for 24, 48 and 72 h (D). (**P<0.01 compared with the TNFα group at 48 h, ##P<0.01 compared with the TNFα group at 72 h). Results are means±S.E.M. for three independent experiments.

Figure 5. Klotho knockdown modulates epithelial cell survival and inflammatory gene expression

(A) Transfection of 16HBE cells with KL siRNAs (siRNA1–siRNA3) significantly down-regulated KL protein and mRNA expression at 24 h compared with control siRNA (siRNAc) treatment. (B) CCK-8 staining shows that KL knockdown slightly, but significantly, reduced cell viability compared with control siRNA. (C) KL-directed siRNAs (siRNA1–siRNA3) induced apoptosis 24 h after transfection as determined by flow cytometry. Representative scatter plots of untreated control cells (CON), siRNAc-treated cells (NC) and siRNA1–siRNA3-treated cells (KL1019, KL1278 and KL2071) are shown. Results in the histogram are means±S.E.M. for three independent experiments. #P<0.05 compared with the control group, *P<0.05 compared with control siRNAc. (D) IL6, MCP1 and IL8 mRNA expression significantly increased after KL knockdown. Results are means±S.E.M. from three independent experiments. (*P<0.05, **P<0.01, ***P<0.001).

Figure 6. Klotho knockdown modulates cigarette smoke-induced inflammation: role of intracellular signalling pathways

(A) 16HBE cells were treated with KL-directed siRNA3 for 8 h before cells were exposed to 5% CSE for a further 6 h and IL-8 release was detected and compared with siRNAc-pre-treated cells. (B) The effects of siRNA3 knockdown of KL on 5% CSE-induced MEK/ERK (p-ERK) and p38 MAPK (p-p38) activation was determined after 30 min. (C) In similar experiments, NF-κB [p-IκB (inhibitor of NF-κB) and IκBα (IκB kinase α)] activation and p65 nuclear translocation was determined by Western blot analysis after 5 min in nuclear extracts. The p65 nuclear translocation was also studied by immunofluorescence. Cells were untreated (a) or treated with control siRNA (b) or KL-directed siRNA3 (c) for 8 h before cells were exposed to 5% CSE for 5 min. The green fluorescence in the left-hand panels indicates NF-κB/p65 staining, the blue stain indicates the nucleus, and the merged panels are on the right. Results in the histogram as means±S.E.M. for three independent experiments. ##P<0.01, #P<0.05 compared with the control group; *P<0.05 compared with siRNAc in response to 5% CSE. (D) The effect of 30 min pre-treatment of NF-κB (JSH23, 20 μM), JNK (SP600125, 20 μM), p38 MAPK (SB203580, 20 μM) and MEK/ERK (PD98059, 20 μM) inhibitors on the enhanced mRNA expression of IL-8 observed with 5% CSE and siRNA3-induced KL knockdown. Results are means±S.E.M. for three independent experiments. *P<0.05, **P<0.01, ***P<0.001 compared with CSE+siRNAc+DMSO-treated cells, ##P<0.01 compared with CSE+siRNA+DMSO-treated cells, $P<0.05.

Figure 7. KL deficiency aggravates oxidative-stress-induced cellular injury

(A and B) Cells were transfected with control siRNA (siRNAc) and three KL-directed siRNAs (siRNA1–siRNA3) for 6–8 h. After KL knockdown, cells were treated with H2O2 (200 μM) for 24 h, and flow cytometry was performed to determine the intracellular ROS content using the fluorescent probe DCFH-DA (A) and the apoptotic ratio using annexin V–FITC double staining (B). Results in the histogram are means±S.E.M. for three independent experiments. #P<0.05 compared with the control, *P<0.05 compared with H2O2+siRNAc-treated cells. (C) In some experiments, the cells were subsequently exposed to 5% CSE, and cell viability was determined using the CCK-8 method after 24 h. Results are means±S.E.M. for three independent experiments. #P<0.05 compared with the control group, *P<0.05 compared with CSE+siRNAc-treated cells.

Figure 8. KL deficiency aggravates oxidative-stress-induced cellular injury partly via Nrf2 consumption in 16HBE cells

(A) The ability of H2O2 (200 μM) to activate stress kinases was strengthened and the antioxidant transcription factor Nrf2 was attenuated in siRNA3-pre-treated cells compared with siRNAc-pre-treated cells. 16HBE cells were transfected with control siRNA (siRNAc) and KL-directed siRNA3 for 6–8 h before these cells were treated with H2O2 (200 μM). After 30 min, cell extracts were collected to detect the phosphorylation of several stress kinases [ERK, p38 and PI3K (phosphoinositide 3-kinase)/PI3K-activated serine/threonine kinase Akt/PKB] by Western blotting. (B) Results are means±S.E.M. from three independent experiments (#P<0.05 compared with the control group, *P<0.05 compared with the H2O2 + siRNAc-treated cells). (C) The effect of treatments on Nrf2 expression and translocation, with (D) results shown as means±S.E.M. from three independent experiments (*P<0.05 compared with the H2O2+siRNAc-treated total cell extracts, ##P<0.01 compared with the H2O2+siRNAc-treated nuclear extracts). Con, control; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.