Introduction

Chronic obstructive pulmonary disease (COPD), one of the major leading causes of death worldwide [1, 2], is characterized by expiratory airflow limitation and parenchymal emphysema [3]. Exposure to cigarette smoke (CS) represents the most important risk factor for the development of COPD; however, only ~15% of smokers develop the disease [4]. Increased cellular senescence is a major feature of aging and has been widely implicated in the pathogenesis of COPD by impairing cell repopulation and by aberrant cytokine secretion, the so-called senescence-associated secretory phenotype [57]. Hence, mapping the molecular mechanisms for CS-induced acceleration of cellular senescence may offer clues into COPD pathogenesis. Although the regulatory mechanisms for cellular senescence are complex and incompletely understood, recent advances, support the notion that a homeostatic balance of energy status and removal of damaged intracellular components through autophagic degradation are critical for the prevention of cellular senescence induced by CS exposure [68].

Autophagy, a lysosomal degradation pathway, occurs continuously at basal levels, during the homeostatic turnover of cytoplasmic components required to meet metabolic demands. Autophagy begins with the formation of an autophagosome, which fuses with the lysosomal membrane to deliver its contents, such as toxins and damaged cellular components, for degradation [9]. During autophagosome formation, the microtubule-associated protein light chain 3 I (LC3-I) is conjugated to phosphatidylamine to form LC3-phosphatidylamine, termed LC3-II. LC3-II then translocates to the autophagosome membrane, the process of which is essential for autophagosome formation [9, 10]. Therefore, a decrease in LC3-I and increase in LC3-II levels are markers reflecting the activation of autophagy. Recent studies have shown that increased autophagy occurs in the lungs of patients with COPD and in lung cells of mouse exposed to CS [11, 12]. However, the regulatory mechanisms for autophagy are also complex and incompletely understood, especially in the setting of CS exposure, and we noted Klotho gene with antisenescence property.

Mice with homozygous disruption of the Klotho gene exhibit multiple age-related disorders that are observed in humans, including skin atrophy, ectopic calcification, osteoporosis, and atherosclerosis [13, 14]. On the other hand, overexpression of Klotho in mice extends the lifespan independent of food intake and growth [15]. The Klotho gene is expressed primarily in the distal tubules of the kidney, the choroid plexus in the brain, the parathyroid, the testis, and the ovary [13, 1618]. The functions of Klotho have been investigated in several reports. Klotho inhibits insulin/IGF-1 signaling and increases resistance to oxidative stress [19, 20]. Our recent study also found that Klotho is decreased in alveolar macrophages of lungs of smokers and patients with COPD as well as in response to CS exposure in vitro in alveolar macrophages [21]. In our study, we indicated that cigarette smoking might promote the airway inflammation through the inhibition of Klotho by alveolar macrophages in COPD formation. However, the role of Klotho on CS-mediated autophagy is not known. Therefore, we hypothesized that Klotho plays an important role in CS-mediated autophagy in alveolar macrophages. In the present study, we sought to determine the effect of CS on the induction of autophagy in alveolar macrophages in vitro, as well as the involvement of Klotho in autophagy regulation. Our study implicates that Klotho is a key mediator of cigarette smoke-induced autophagy, and thereby demonstrates a critical role for Klotho in COPD pathogenesis.

Methods

Cell Culture

Murine alveolar macrophage cell line MH-S (CRL-2019) cells, were obtained from American Type Culture Collection (ATCC, Manassas, VA) and were cultured according to the ATCC prescription. The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. Fresh media was added every 2–3 days. The cells were starved with the medium with 1% FBS 24 h before use.

Preparation of Aqueous Cigarette Smoke Extract

The total particulate matter (TPM) content of Marlboro Red cigarette was 10 mg/cigarette, tar (10 mg/cigarette), and nicotine (0.8 mg/cigarette). A 10% cigarette smoke extract (CSE) was prepared by bubbling smoke from one cigarette into 10 mL of culture medium supplemented with 1% FBS at a rate of one cigarette per minute as described previously [2224], using a modification of the method described by Carp and Janoff [25]. The pH of the CSE was adjusted to 7.4 and was sterile filtered through a 0.45-m filter (25 mm Acrodisc; Pall, Ann Arbor, MI). The CSE preparation was standardized by monitoring the absorbance at 320 nm (optical density of 0.74). The spectral variations observed between different CSE preparations at 320 nm wavelength were found to be within the acceptable limits. CSE was freshly prepared for each experiment and diluted with culture medium containing 1% FBS immediately before use. Control medium was prepared by bubbling air through 10 mL of culture medium supplemented with 1% FBS, adjusting pH to 7.4, and sterile filtered as described for 10% CSE.

Transfection

MH-S cells were seeded on 6-well plates. For the Klotho knockdown experiments, the cells were transiently transfected with 20 μM klotho small-interfering RNA or negative control siRNA (NC siRNA) using LipofectamineTM 2000 transfection reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions.

Immunoblotting

Whole cell extracts were separated on a 6.5–12% sodium dodecyl sulfatepolyacrylamide gel by electrophoresis. Separated proteins were transferred onto nitrocellulose membranes (Amersham, Arlington Heights, IL), and blocked for 1 h at room temperature with 5% bovine serum albumin (BSA) (Sigma-Aldrich). The membranes were then incubated with the indicated primary antibody at 4 °C overnight. Membranes were washed in Tris-buffered saline, 0.1% Tween 20, and incubated with secondary antibody at 20 °C for 1 h. Equivalent loading of the gel was determined by quantification of protein as well as by reprobing membranes for GAPDH. ImageJ software (Version 1.41, National Institutes of Health, Bethesda, MD) was used for gel band quantitative densitometric analysis.

Statistical Analysis

Statistical analysis was performed using SPSS 17.0 software. Results among groups were analyzed by one-way ANOVA. Differences with probability (P) value less than 0.05 were considered statistically significant.

Results

Cigarette Smoke Extract (CSE) Induces Autophagy in Alveolar Macrophages

We investigated whether CSE could affect the induction of autophagy in alveolar macrophages. Treatment of MH-S cells with CSE caused a dose- and time-dependent increase in the conversion of LC3-I to LC3-II (Fig. 1). At the concentration of 0.5% CSE, approximately threefold increase in the amount of LC3-II/LC3-I was found as compared to controls (Fig. 1a). In addition, LC3-II/LC3-I was significantly increased after 72 h of CSE (0.1%) treatment (Fig. 1b).

Fig. 1
figure 1

CSE increases autophagy in MH-S cells. a MH-S cells were treated with indicated concentrations of CSE for 24 h. b MH-S cells were treated with 0.1% CSE for indicated time periods. Cell lysates were subjected to immunoblot analysis for the detection of LC3 levels. GAPDH was used as loading control. Quantification of results is presented as the amount of LC3-II normalized against LC3-I. Data are medians of 3 independent experiments performed in triplicate. *P < 0.05 and ***P < 0.001 compared with control values

Klotho Attenuates CSE-Induced Autophagy in Alveolar Macrophages

We recently reported that the levels of Klotho are decreased in response to CS exposure in the lungs of smokers and patients with COPD as well as in alveolar macrophages [21]. Based on this, we hypothesized that a decrease in Klotho levels in alveolar macrophages is involved in the induction of CS-induced autophagic response. To investigate the role of Klotho in CSE-induced autophagy, MH-S cells were pre-treated with Klotho (20, 200, or 2000 pmol/L) for 30 min, and then stimulated with CSE (0.5%) for 24 h. As shown in Fig. 2, CSE strongly increased the levels of LC3-II/LC3-I, while pretreatment with Klotho significantly inhibited CSE-induced autophagy.

Fig. 2
figure 2

Klotho attenuates CSE-induced autophagy. MH-S cells were treated with 0.5% CSE for 24 h, pretreatment with 20, 200, 2000 pM of Klotho protein for 30 min, respectively. Whole cell extracts were used for immunoblot analysis of LC3. GAPDH was used as loading control. Quantification of results is presented as the amount of LC3-II normalized against LC3-I. Data are medians of 3 independent experiments performed in triplicate. ***P < 0.001 compared with CSE; # P < 0.05 compared with control

Inhibition of Klotho Leads to Augmentation in CSE-Induced Autophagy in Alveolar Macrophages

To determine whether the decreased level of Klotho was associated with CSE-induced autophagy, cells were transiently transfected with negative control siRNA (NC siRNA) or Klotho siRNA for 72 h. As expected, CSE increased induction of autophagy and Knockdown of Klotho further increased autophagic activity (Fig. 3).

Fig. 3
figure 3

Klotho deficiency augments CSE-induced autophagy. The Klotho knockdown cells were stimulated with or without 0.5% CSE for 24 h, whole cell extracts were used for immunoblot analysis of LC3. GAPDH was used as loading control. Quantification of results is presented as the amount of LC3-II normalized against LC3-I. Data are medians of 3 independent experiments performed in triplicate. ***P < 0.001 compared with NC siRNA group; ### P < 0.001 compared with CSE

Cellular Signaling Involved in the Inhibitory Role of Klotho in Alveolar Macrophages

Next we investigated the expression of signaling molecules involved in alveolar macrophages autophagy induced by CSE. Exogenous Klotho protein significantly inhibited phosphorylation of several autophagy-related molecules. MH-S cells were stimulated with CSE in the presence or absence of Klotho (at 2000 pmol/L) for 30 min. Western blotting analysis showed that phospho-ERK, phospho-Akt, and phospho-IGF-1R markedly increased with CSE stimulation, whereas Klotho treatment significantly inhibited phospho-ERK, phospho-Akt, and phospho-IGF-1R in CSE-stimulated MH-S cells (Fig. 4). No differences were observed in the protein levels of total ERK and Akt between the 3 treatment groups.

Fig. 4
figure 4

Klotho inhibits phosphorylation of ERK, Akt, and IGF-1 proteins. MH-S cells were stimulated with 0.5% CSE in the presence or absence of Klotho (at 2000 pmol/L) for 30 min. Western blot analysis of protein and phosphorylation levels in MH-S cells. Data are 3 independent experiments performed in triplicate

Discussion

In this study, we report that Klotho inhibits CSE-induced autophagy via down-regulation of IGF-1R, Akt, and ERK phosphorylation in alveolar macrophages. Although the involvement of Klotho in COPD pathogenesis has been proposed without detailed mechanisms [21], our findings of decreased expression levels of Klotho in lung alveolar macrophages from COPD patients may support the notion that reduced Klotho is associated with COPD development through the enhancement of cellular senescence created by excessive autophagy during CS exposure.

Accumulating evidence indicates that autophagy plays a role in the pathogenic process in COPD. There is a significantly increased autophagic level in the lung tissue from COPD patients as compared with that of non-COPD subjects, suggesting that excessive autophagy may be crucial for COPD [26]. In our study, we demonstrated CSE-induced autophagy in a time- and concentration-dependent manner. This notion is consistent with a previous study, where increased autophagy was found in the clinical specimens of the lungs from patients with COPD relative to normal tissue, as evidenced by morphological and biochemical markers. Similar evidence of increased autophagy was also observed in the lungs of mice that were subjected to chronic inhalation of cigarette smoke [12]. CS exposure has been implicated in increased autophagy activation in COPD pathogenesis through enhanced apoptosis [27]. Therefore, decreased expression levels of Klotho in response to CS exposure appear to be associated with COPD development, whereas the detailed mechanism for the roles of Klotho in autophagy remains to be determined.

Autophagy is an adaptation pathway for cellular stress, including starvation, reactive oxygen species, endoplasmic reticulum stress, and microbe infection, and is hence generally considered to be a mechanism for cell survival [28]. However, there is functional cross-talk between autophagy and apoptosis, and increased autophagy may promote cell death in the setting of extraphysiologic conditions [29]. Intriguingly, increased autophagy in association with apoptosis induction has been demonstrated in COPD pathogenesis. LC3B has been proposed to regulate apoptosis in response to CSE, but a causal link between autophagy status and apoptosis induction remains uncertain [12, 26, 28, 30]. In our previous study [21], we have shown that Klotho activity was decreased in the lungs of smokers and patients with COPD as well as in alveolar macrophages exposed to CSE. Now we showed that Klotho inhibits CSE-induced autophagy in alveolar macrophages. So relative insufficiency of autophagic degradation may be a critical determination of cellular senescence in COPD pathogenesis, which can be attributed to reduced Klotho expression.

The signaling pathways involved in COPD pathogenesis of Klotho have, as yet, not been fully elucidated. Klotho has been found to inhibit insulin/IGF-1 signaling and, consequently, to induce cell apoptosis [3133]. Klotho regulates insulin/IGF-1 signaling by inhibiting tyrosine phosphorylation of the IGF-1 receptor [15]. Tyrosine phosphorylation of the IGF-1R is also involved in the activation of extracellular signal-regulated kinases (ERK) 1 and ERK2 and PI3K/Akt signaling [34]. In this study, we revealed that Klotho significantly inhibits IGF-1R, Akt, and ERK phosphorylation in alveolar macrophages. The PI3K–Akt pathway plays a pivotal role in the regulation of apoptosis in many cell types, and the ras–raf–MEK–ERK route has also been proposed to function in cell survival. We, therefore, propose that through inhibiting IGF-1 receptor phosphorylation, Klotho inhibits ERK and Akt activities and inhibits CSE-induced autophagy in alveolar macrophages.

Various cell types expressing IGF-1 are found within the lung, including alveolar macrophages, bronchial epithelial cells, and fibroblasts, hence participation of IGF-1 in lung disease pathogenesis has been widely reported [35, 36]. However, circulating IGF-1 levels and pathogenic involvement are still unknown in the context of COPD [37]. It may be attributed not only to the pleiotropic role of IGF-1 but also its bioavailability as influenced by IGF-1 binding proteins, which can potentiate or inhibit the biological activity of IGF-1 [38]. Recent studies have demonstrated that autophagy can be induced by reduced growth factor signaling. Growth factor signaling involved in the insulin/IGF-1–PI3K–Akt pathway has been shown to regulate cell autophagy through the insulin receptor [39]. In addition, activation of insulin/IGF signaling has been shown to suppress the autophagic–lysosomal pathway [40, 41]. Importantly, the Klotho protein functions as a circulating hormone that represses intracellular signals of insulin and IGF-1 [15, 42]. In this study, exogenous overexpression of the Klotho protein significantly inhibited IGF-1R, Akt, and ERK phosphorylation. This suggests that the signaling pathway of Klotho-IGF-1R/Akt may be responsible for autophagy.

Klotho and autophagy have not been implicated in cellular senescence and aging. Klotho has been shown to regulate aging and longevity in mammals [12], and CS also induces aging-like alterations in tissue and organ structure [43]. The failure in clearance of proteins due to decline of autophagy is associated with age-related pathogenesis such as neurodegenerative disease [44]. CS-induced autophagy is involved in the pathogenesis of CS-mediated lung age-related diseases, such as emphysema and COPD [45]. Emphysema and COPD are associated with the loss of regenerative capacity in lungs and cellular senescence aggravates adequate cell replacement by autophagy. Based on our data showing CS-mediated induction of autophagy via Klotho, it is tempting to speculate that Klotho is not only a key player in the regulation of autophagy but also involved in aging and cellular senescence in smokers. Our study highlights a central role for Klotho in CSE-induced autophagy and suggests that Klotho may serve as an ideal target for the development of novel therapeutic agents for COPD. The elucidation of this important interaction between Klotho and autophagy will advance our understanding of the pathogenic effect of COPD. Furthermore, a better understanding of the multiple pathways of autophagy and their impact on COPD pathogenesis may also facilitate the design of more specific therapies for the treatment of COPD.

Conclusion

In summary, our data show that CS induces autophagy in alveolar macrophages through the reduction in the level and activity of Klotho. We further showed that the Klotho-IGF-1R/Akt axis plays a pivotal role in the regulation of CS-induced autophagy, as evidenced by the studies using the pharmacological Klotho in response to CS. Therefore, optimal levels of autophagy induction achieved via Klotho modulation is a potential effective medical intervention for the prevention of accelerated cellular senescence, resulting in the amelioration of the tobacco smoking-related senescence-associated lung disease, COPD.