Cigarette smoke induces mucin hypersecretion and inflammatory response through the p66shc adaptor protein-mediated mechanism in human bronchial epithelial cells.

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Molecular Immunology xxx (2015) xxx–xxx

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Cigarette smoke induces mucin hypersecretion and inflammatory response through the p66shc adaptor protein-mediated mechanism in human bronchial epithelial cells J. Yang a , H.M. Yu b , X.D. Zhou a,c,∗ , H.P. Huang c , Zh. Han c , V.P. Kolosov d , J.M. Perelman d a

Division of Respiratory Medicine, Second Affiliated Hospital, Chongqing Medical University, Chongqing, China Division of Geriatrics Medicine, First Affiliated Hospital, Chongqing Medical University, Chongqing, China c Division of Respiratory Medicine, Affiliated Hospital of Hainan Medical University, Haikou, China d Far Eastern Scientific Center of Physiology and Pathology of Respiration, Siberian Branch, Russian Academy of Medical Sciences, Russian Federation b

a r t i c l e

i n f o

Article history: Received 19 August 2015 Received in revised form 2 November 2015 Accepted 3 November 2015 Available online xxx Keywords: p66Shc Mitochondria ROS NF-␬B Inflammation MUC5AC

a b s t r a c t The p66Shc adaptor protein is a newly recognized mediator of mitochondrial dysfunction and might play a role in cigarette smoke (CS)-induced airway epithelial cell injury. CS can induce an excessive amount of reactive oxygen species (ROS) generation, which can cause mitochondrial depolarization and injury through the oxidative stress-mediated Serine36 phosphorylation of p66Shc. The excessive production of ROS can trigger an inflammatory response and mucin hypersecretion by enhancing the transcriptional activity of pro-inflammatory cytokines and mucin genes. Therefore, we speculate that p66Shc plays an essential role in airway epithelial cell injury and the process of mucin generation in CS-induced chronic inflammatory airway diseases. Our present study focuses on the role of p66Shc in ROS generation, and on the resulting mitochondrial dysfunction, inflammatory response and mucus hypersecretion in CS-stimulated human bronchial epithelial cells (16HBE). We found that CS disturbed the mitochondrial function by increasing the level of phosphorylated p66Shc in these cells and that the effects were significantly reduced by silencing p66Shc. Conversely, the ectopic overexpression of wild-type p66Shc enhanced these effects. We also found that high levels of ROS inhibited FOXO3a transcriptional activity, which led to NF-␬B activation. Subsequently, activated NF-␬B promoted pro-inflammatory cytokine production and mucin hypersecretion. Thus, manipulating p66Shc might offer a new therapeutic modality with which to treat chronic inflammatory airway diseases. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Chronic obstructive pulmonary disease (COPD) is a leading cause of morbidity and mortality in both developed and developing countries (Asia Pacific Roundtable Group, 2005). This condition is characterized by chronic airway inflammation and mucus hypersecretion. These characteristics can be exacerbated by inflammatory mediators, cytokines, pathogens, cold temperatures and cigarette smoke extract (Chung, 2005; Shao et al., 2004; Li et al., 2011). It has long been known that exposure to CS triggers an inflammatory cascade in the airways that results in damage to the lung epithelium due to increased oxidative stress (Wu et al., 2014).

∗ Corresponding author at: No. 74, Linjiang Road, Yuzhong District, Chongqing 400010, China. Fax: +86 23 63711527. E-mail address: [email protected] (X.D. Zhou).

Cigarette smoke contains various chemicals including ROS such as superoxide (O2•− ) and hydroxyl radicals (• OH), which are present in high concentrations in the gaseous phase. However, the gaseous-phase ROS in CS are essentially unable to enter the cells and are thus not capable of entering the circulation. Nonetheless, CS-induced systemic oxidative stress inevitably causes tissue injury and sustains systemic inflammation, resulting in the development and progression of COPD (Remels et al., 2007; van der Toorn et al., 2007). These studies suggest that the intracellular generation of ROS may play an important role in the pathophysiology of COPD. p66Shc is a longevity adaptor protein, which is involved in the oxidative stress-induced various inflammatory diseases (Camici et al., 2007; Arany et al., 2008, 2013). The ShcA gene encodes three adaptor proteins (p66Shc, p52Shc, and p46Shc), which share a phosphotyrosine binding domain (PTB), a collagen homology domain (CH1), and a C-terminal Src homology 2 domain (SH2).

http://dx.doi.org/10.1016/j.molimm.2015.11.002 0161-5890/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Yang, J., et al., Cigarette smoke induces mucin hypersecretion and inflammatory response through the p66shc adaptor protein-mediated mechanism in human bronchial epithelial cells. Mol. Immunol. (2015), http://dx.doi.org/10.1016/j.molimm.2015.11.002

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p46Shc and p52Shc are products of alternative translation initiation sites within the same transcript, whereas p66Shc contains a unique N-terminal collagen homology domain (CH2) that includes Ser36. The phosphorylation of the Ser36 site is crucial for the oxidative stress response under physiological conditions (El-Shemerly et al., 1997; Yang and Horwitz, 2000). Recently, one mechanism by which p66Shc promotes oxidative stress was shown to be that oxidative stress causes the phosphorylation of p66Shc at its Ser36 residue, which facilitates its binding to the mitochondria, and within them, to cytochrome c. This binding induces mitochondrial ROS release by diverting oxidized cytochrome c, which in turn results in the opening of the mitochondrial permeability transition pore (PTP) and subsequent injury (Giorgio et al., 2005; Gonzalez et al., 2007). The amount of ROS within the cell is finely modulated by enzymatic and nonenzymatic antioxidant defenses such as superoxide dismutases (SODs), catalase (CAT), glutathione peroxidase (GPx), and glutathione. Physiological ROS levels play important roles as second messengers within the intracellular signaling process. Thus, an exacerbation of ROS production or insufficient scavenging has been demonstrated to impair many biological processes, including endothelial or epithelial function in several pathological contexts. On the one hand, CS may increase the intracellular levels of reactive oxygen species (ROS) via phosphorylation of the Ser36 residue of p66Shc in epithelial cells or other types of cells (Magenta et al., 2014). On the other hand, p66Shc inhibits the FOXO3a transcription factor, causing a decrease in the expression of the ROS scavenging enzymes (Nemoto and Finkel, 2002). In addition, some studies have shown that ROS overexpression causes the phosphorylation of p66Shc and leads to a further increase in the cell injury through a mitochondrial-dependent mechanism (Pinton and Rizzuto, 2008). Subsequently, this increased intracellular ROS may activate various ROS-sensitive signaling pathways, such as the mitogen-activated protein kinases (MAPKs), and a number of downstream transcriptional factors, such as nuclear factor-␬B (NF␬B), and may ultimately promote inflammatory gene expression (Moretto et al., 2009; Cheng et al., 2009). Based on the accumulated evidence, we speculate that p66Shc plays a crucial role in mitochondrial ROS production. It is well known that ROS upregulate the expression of redox-sensitive pro-inflammatory cytokines (e.g., IL-6, IL-8 and TNF-␣) and transcription factors (e.g., NF-␬B and AP-1) (Yang et al., 2006). Therefore, there is considerable support for the hypothesis that p66Shc is involved in the development of COPD through ROS–NF␬B pathway. The goal of the current study was to explore the hypothesis that CS induces mucus hypersecretion and the inflammatory response through the p66Shc-mediated mechanism in human bronchial epithelial cells.

carbamate (PDTC), a specific NF-␬B activity inhibitor: International Laboratory, USA. 2.2. Preparation of CSE Cigarette smoke extract (CSE) was prepared as previously described (Gu et al., 2015). Briefly, Kentucky 2R4F researchreference filtered cigarettes (The Tobacco Research Institute, University of Kentucky, Lexington, KY) were smoked via a peristaltic pump. Just before the experiments, the filters were cut from the cigarettes. Each cigarette was smoked for 5 min, leaving a 17mm butt. The smoke from four cigarettes was bubbled through 50 ml of cell growth medium or mitochondrial respiration buffer, and this solution was considered 100% strength CSE. 2.3. Cell culture The 16HBE human airway epithelial cells were obtained from a segment of a human airway and immortalized using an origindefective SV40 (Gruenert et al., 1995). They were cultured at 37 ◦ C in RPMI 1640 medium supplemented with 10% fetal bovine serum (Hyclone) and 100 units/ml of penicillin/streptomycin. The cells were maintained in 75-cm2 flasks at 37 ◦ C in an air-ventilated and humidified incubator maintained at 5% CO2 . The culture media was renewed every 2–3 days, and the cells were maintained between 90 and 95% confluency and were passaged every 2–4 days by trypsinization. 2.4. Cell viability analysis at various concentrations of CSE The 16HBE cells were seeded at a density of 2 × 104 cells/cm2 onto Costar brand Transwellplate inserts with a diameter of 12 mm in RPMI 1640 mediumsupplemented with 10% fetal calf serum, streptomycin (100 ␮g/ml), penicillin (100 ␮g/ml) and HEPES (25 mM) at 37 ◦ C in a 5% CO2 /95% air, water-jacketed incubator. After the cells reached confluence, they were exposed to various concentrations of CSE (0–30%) for 24 h. The viability of cells was evaluated using a conventional 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction assay, as previously described. Briefly, the MTT solution (final concentration, 0.5 mg/ml) was added to each well. The cells were then incubated for 4 h at 37 ◦ C. After this incubation, 150 ␮l of DMSO was added to each well for 15 min at room temperature, and the absorbance at 490 nm was measured using an ELISA reader (TECAN SunriseTM Remote, Austria). 2.5. Flow cytometric analysis of cell death

2. Materials and methods 2.1. Materials The human bronchial epithelial cell line (16HBE) was purchased from the Experimental Medical Research Center of Guangzhou Medical College (Guangzhou, Guangdong, China). The sources of the other supplies and reagents were as follows: Roswell Park Memorial Institute (RPMI) 1640 medium and fetal bovine serum (FBS): Elastin Products Company, Owensville, MO; anti-phospho-Ser36 p66Shc and anti-p66Shc: Alexis Biochemicals/Axxora, San Diego, CA); phospho-FOXO3a (Thr 32) and FoxO3a: Millipore Corp, Billerica, MA; anti-MUC5AC, anti-NF-␬B p65, anti-NF-␬B, anti-I␬B␣, anti-phospho-I␬B␣, anti-cytochrome and anti-␤-actin antibodies: Cell Signaling Technology, Beverly, MA, USA; N-acetylcysteine (NAC) (≥99% purity grade), a ROS scavenger and pyrrolidine dithio-

The cell surface exposure of phosphatidylserine and plasma membrane disruption were evaluated using staining with annexinV-FITC and propidium iodide according to the manufacturer’s instructions (IQ Products, Groningen, The Netherlands). The cells were analyzed by flow cytometry (Calibur, Becton Dickinson Medical Systems, Heidelberg, Germany). 2.6. Analysis of intracellular ROS generation 16HBE cells were plated in 6-well plates and were exposed to various concentrations of CSE (0–20%) for 24 h. H2 DCFDA solution (final concentration, 10 ␮mol/l) was subsequently added to each well. The plates were incubated for 20 min at 37 ◦ C. After the incubation, the relative content of ROS in cells was measured by using a fluorescence microplate reader (Becton-Dickinson, San Jose, CA, USA).




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Fig. 1. The effect of cigarette smoke extract (CSE) on human bronchial epithelial cells and their mitochondrial function (A–D). 16HBE cells were stimulated with a range of concentrations of CSE (0–30%) for 24 h. The viability of cells was assessed using MTT (A), and the apoptosis rate of cells was analyzed by flow cytometry (B). The mitochondrial membrane potential (MMP) (C) and intracellular ATP levels (D) are shown. The cells viability gradually decreased and the apoptosis rate increased with increasing concentration of CSE (0–30%) for 24 h. Similarly, MMP and production of ATP both also declined in a concentration-dependent manner. The data shown are representative of three independent experiments and are presented as the means ± SDs (n = 3). * p < 0.05 and ** p < 0.01 versus the control (Con).

2.7. Measurement of mitochondrial function 2.7.1. Detection of mitochondrial membrane potential (MMP) The 16HBE cells were stained with 5 ␮g/ml of the JC-1 probe (Molecular Probes, Leiden, The Netherlands) according to the manufacture’s instructions. The loaded cells were stimulated for 24 h with various concentrations of CSE (0–30%) at 37 ◦ C. In addition, the isolated mitochondria were stained with 0.2 ␮g/ml JC-1 probe for

10 min at 37 ◦ C. State III respiration was reached after the addition of 5 mM sodium succinate and 1 mM ADP. The mitochondria were stimulated for 15 min at 37 ◦ C with various concentrations of CSE. The cells and isolated mitochondria were monitored with an excitation wavelength of 485 nm through a 590-nm band-pass filter in an FL600 fluorescent plate reader (Bio-Tek Instruments, Abcoude, The Netherlands).


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Fig. 2. CSE augments p66Shc expression in a concentration-dependent manner in 16HBE cells (A–D). 16HBE cells were stimulated with a range of concentrations of CSE (0–20%) for 24 h. The levels of the p66Shc mRNA and protein were measured using reverse transcriptase-polymerase chain reaction/real-time PCR and western blotting, respectively. CSE induced the expression of p66Shc mRNA (A and B) and protein (C and D) in a concentration-dependent manner. The data shown are representative of three independent experiments and are presented as the means ± SDs (n = 3). * p < 0.05 and ** p < 0.01 versus the control (Con).

Fig. 3. The expression of p66Shc in 16HBE cells (A–D). 16HBE cells were transfected with specific siRNA targeting for p66Shc (sip66Shc), wild-type p66Shc (WT) or nontargeted siRNA (siNT), as described in Section 2. After transfection, the cells were treated with CSE (20%) for 24 h to measure the expression of p66Shc mRNA and protein. The gene expression of p66Shc was assessed using reverse transcriptase-polymerase chain reaction (A) and real-time PCR (B). The data represent the fold increase in expression of p66Shc relative to GAPDH values as the means ± SDs of the 2−Ct values (n= 3). The expression of the p66Shc protein was measured using western blotting (C). The results are expressed as the relative ratio to ␤-actin expression (D). The positive control was 20% CSE alone. The data shown are representative of three independent experiments and are presented as the means ± SDs (n = 3). ∗ p < 0.05, ** p < 0.01 and # p > 0.05 versus only CSE; ˆp < 0.01 versus sip66Shc.

2.7.2. Measurement of ATP The intracellular ATP levels of these cells were assessed after treatment with Triton X-100. To measure the mitochondrial ATP, the isolated mitochondria (final protein concentration of 100 ␮g/ml) were resuspended in respiration buffer. The experiments were performed in state III respiration. Various concentrations of CSE were examined with DNP (final concentration

of 20 ␮M) serving as the negative control. The mitochondria were incubated for 15 min at 37 ◦ C. At the end of the incubation period, the ATP synthesis was stopped by freezing the samples in −196 ◦ C nitrogen. The ATP levels were measured using the Enliten ATP assay from Promega (Leiden, The Netherlands) in a Berthold microplate luminometer (Berthold Detection Systems, Pforzheim, Germany).




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Fig. 4. The effect of altering the expression of p66Shc levels on cell viability, apoptosis and mitochondrial function (A–D). 16HBE cells were transfected with a specific siRNA targeting for p66Shc (sip66Shc), wild-type p66Shc (WT) or non-targeted siRNA (siNT), as described in Section 2. After transfection, the cells were treated with CSE (20%) for 24 h to measure the viability of cells (A), apoptosis rate (B), mitochondrial membrane potential MMP (C) and intracellular ATP levels (D). The positive control was 20% CSE alone. The data shown are representative of three independent experiments and are presented as the means ± SDs (n = 3). * p < 0.05 and ** p < 0.01 versus CSE alone; ˆ p < 0.01 versus sip66Shc; # p > 0.05 versus siNT.

2.8. ELISA for IL-8, IL-6, TNF-˛ and MUC5AC proteins IL-8, IL-6, TNF-␣ and MUC5AC proteins in the biological samples were separately testing using enzyme-linked immunosorbant assays (ELISAs) in a 96-well format according to the manufac-

turer’s directions (IL-8, IL-6, TNF-␣ were purchased from R&D Systems, Inc.; MUC5AC was purchased from Yuanye, Shanghai, China). The quantities were measured spectrophotometrically at Abs 450 nm with wavelength correction set to Abs 550 nm. All samples were diluted to be within the middle 60% of the 8-point




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immunoprecipitation, 500 ␮g of the total cell lysate was incubated with the appropriate primary antibody overnight at 4 ◦ C using the Catch and Release v2.0 reversible immunoprecipitation system (Millipore, Charlottesville, VA). The immunoprecipitated proteins were resolved by SDS-PAGE as described above. In the immunoprecipitation studies, negative control IgGs (1:1000) were also used to determine cross reactivity.

2.11. Cell transfection

Fig. 5. The effect of expression of p66Shc levels on intracellular ROS production. 16HBE cells were transfected with specific siRNA targeting for p66Shc (sip66Shc), wild-type p66Shc (WT) or non-targeted siRNA (siNT), as described in Section 2. After transfection, the cells were treated with CSE (20%) for 24 h to measure the levels of intracellular ROS production. The data shown are representative of three independent experiments and are presented as the means ± SDs (n = 3). * p < 0.05 and ** p < 0.01 versus CSE alone; ˆ p < 0.01 versus sip66Shc; # p > 0.05 versus siNT.

standard curve, and the concentrations were calculated from the log-transformed absorbance values, as recommended. In addition to standard curves, every plate contained an independent calibrator sample that tested within the range provided. 2.9. Real-time PCR analysis The total RNA was extracted from the 16HBE cells treated with the various stimuli using RNAiso plus solution (Takara; Dalian, Liaoning, CHN). The total RNA was used to synthesize the cDNA using the PrimeScript 1st Strand cDNA synthesis kit (Takara) according to the manufacturer’s instructions. The cDNA was used for the real-time PCR reaction. All primers were synthesized by Sangon (Shanghai, CHN) as follows: MUC5AC (forward) 5 -TCCGGCCTCATCTTCTCC3 and (reverse) 5 -ACTTGGGCACTGGTGCTG-3 ; p66Shc (forward) 5 -ACTACCCTGTGTTCCTTCTTTC-3 and (reverse) 5 TCGGTGGATTCCTGAGATACTGT-3 . We performed quantitative real-time PCR with a Bio-Rad iCycler real time system using the SYBR Green qPCR Master mix from SABiosciences Fredrick, MD). All primers were synthesized by Takara. GAPDH mRNA was also amplified as an internal control. The relative abundance of the specific mRNA species was quantified using the comparative 2−Ct methods. 2.10. Immunoprecipitation and immunoblotting After the various treatments, the cells were homogenized in a RIPA lysis buffer containing protease inhibitors. The protein concentrations of the lysates were estimated using a BCA Protein Assay Kit (Beyotime, Beijing, China). From each sample, 30 ␮l of protein was separated using 6-10% SDS-PAGE (Beyotime) and transferred onto PVDF membrane (Sigma). After incubation with 5% non-fat dried milk in PBST, the PVDF membranes were incubated with the primary antibody (anti-phospho-Ser36p66shc, anti-p66shc, phospho-FOXO3a (Thr 32), FoxO3a, anti-NF-␬B p65, anti-I␬B␣, anti-phospho-I␬B␣, anti-cytochrome or anti-␤-actin antibodies (all were diluted 1:1000) at 4 ◦ C overnight. Following three washes in PBST, the membranes were incubated with corresponding HRPconjugated secondary antibodies (1:5000) at 37 ◦ C for 1 h. Specific protein complexes were revealed by enhanced chemiluminescence (ECL), and densitometric quantification of bands was performed using the Quantity-One software (Bio-Rad, Hercules, CA, USA). For

The roles of p66shc and its Ser36 phosphorylation/cytochrome c binding in ROS production, mitochondrial depolarization and cell injury were determined using p66shc-knockdown 16HBE cells. The plasmids or the siRNAs were used: the wild-type p66Shc plasmid, the S36A mutant of p66Shc plasmid (Arany et al., 2008), the HA-tagged cytochrome c-binding mutant (W134F) p66Shc plasmid (Arany et al., 2010) and p66Shc-specific siRNA (Kisilow et al., 2002) were synthesized by Integrated DNA Technology (Coralville, IA). The promoter activity of p66Shc was determined after transfecting the cells with a p66shc-promoter-luciferase reporter (Kim et al., 2008). The plasmids were transiently transfected into the cells using Lipofectamine 2000 (Invitrogen, Grand Island, NY) in six-well plates.

2.12. Statistical analysis Each experiments were performed three times with at least six replicate wells in each independent experiment. The analyses were performed with SPSS statistics 17.0 software. The results are shown as the means ± SD. Any differences were evaluated for statistical significance using Student’s t-test or a one-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc analysis. p values less than 0.05 were regarded as statistically significant.

3. Results 3.1. CSE induces mitochondrial dysfunction in a concentration-dependent manner A previous study confirmed that CSE directly induces mitochondrial dysfunction by inhibiting complex I and II activities and decreasing the mitochondrial membrane potential, mitochondrial oxygen consumption and production of ATP and that one of consequences is lung epithelial cell injury (van der Toorn et al., 2007). p66Shc is a key regulator of the mitochondrial production of oxidative stress and plays an important role in the pathogenesis of various chronic diseases (Arany et al., 2010; Chintapalli et al., 2007). We examined whether CSE caused airway epithelial cell injury by inducing mitochondrial dysfunction and augmenting p66Shc expression. We first examined the 16HBE cells viability and mortality using the MTT assay and flow cytometry, respectively. Fig. 1A and B shows that the cell viability gradually decreased and the apoptosis increased after a 24 h exposure to increasing concentrations of CSE (0–30%). Then, we evaluated the mitochondrial function as indicated by the mitochondrial membrane potential and the production of ATP, as shown in Fig. 1C and D. Both of these parameters also declined in a concentration-dependent manner. Low concentrations of CSE (0–20%) had no cytotoxic effect on the cell proliferation or apoptosis: the growth and apoptosis rates were 80.5–99.8% and 2.93–10.67%, respectively. Higher concentrations of CSE (25–30%) markedly reduced the cell viability and increased cell apoptosis. Therefore, the optimal concentration of CSE for subsequent studies was determined to be 20%.




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Fig. 6. The binding of p66Shc to cytochrome c requires the phosphorylation of p66Shc at Ser36 (A and B). The 16HBE cells were transfected with the Ser36 phosphorylationdeficient (S36A) or cytochrome c-binding of p66Shc mutant (W134F) plasmid and exposed to 20% CSE as previously described. The cell lysates were immunoprecipitated with an anti-cytochrome c antibody and immunoblotted with an anti-p66Shc antibody, as described in Section 2. The same blot was re-hybridized with an anti-cytochrome c antibody after stripping. A representative example is shown in (A), while the densitometric analyses of the p66Shc/cytochrome c ratios are shown in (B), ** p < 0.01 versus only CSE. Binding of p66shc to cytochrome c is necessary for CSE-induced mitochondrial dysfunction (C). The content of intracellular ROS, mitochondrial membrane potential MMP and intracellular ATP levels were determined. The results are expressed as percentage of their levels in the different stimulated cells, 20% CSE as the positive control, all of which are shown in (C). The data shown are representative of three independent experiments and are presented as the means ± SDs (n = 3). * p < 0.05 and ** p < 0.01 versus CSE alone.

3.2. p66Shc mediated CSE-induced airway epithelial cell injury and the exacerbation of ROS production It is well known that p66Shc is a key regulator for the oxidative stress-mediated pathogenesis of various inflammation diseases (Arany et al., 2013) and therefore, might play a role in the CSEinduced cellular responses. In parallel with the findings described above, we observed a significant increase in the p66Shc mRNA and protein within the experimental concentration range for 24 h (Fig. 2A–D). The expression of p66Shc gene and protein (including p-p66Shc) were increased by 9.08-, 5.05- and 4.11-fold, respectively, in response to the 20% CSE stimulation (Fig. 2A and B), which suggests that p66Shc may be involved in the CSE-induced cellular responses. Next, we performed experiments using p66Shc-WT and p66Shc-siRNA. As shown in Fig. 3A–D, the levels of the p66Shc gene and protein (including p-p66Shc) in the p66Shc-WT-treated cells were increased by 1.49-, 1.34- and 1.19-fold, respectively, in response to 20% CSE stimulation. Whereas transfection with p66Shc-siRNA yielded 3.71-, 8.89- and 9.0-fold decrease in the initial expression for the gene and protein, respectively. The next experiment investigated whether the p66Shc expression was associated with CSE-induced exacerbation of airway epithelial cell injury and ROS production. This result showed that the p66Shc-WT dramatically decreased cell viability (Fig. 4A) and increased cell apoptosis (Fig. 4B). Similarly, p66Shc-WT significantly suppressed the mitochondrial function (Fig. 4C and D) and ROS production (Fig. 5), whereas knockdown of p66Shc significantly inhibited these phenomena. These results implied that the extent of oxidative injury and ROS generation depends on the levels of p66Shc and, more specifically, that the CSE-induced increase in p66Shc may be responsible for CSE-induced airway epithelial cell injury.

3.3. CSE-induced airway epithelial cell injury and exacerbation of ROS production that requires Serine36 phosphorylation and cytochrome c binding to p66Shc The experiment described above showed that the levels of p66Shc phosphorylation were also gradually increased in concentration-dependent manner at 24 h within the experimental dose range (Fig. 2). In addition, earlier studies have shown that p66Shc phosphorylation at Ser36 triggers a cascade of events that lead to an increase in ROS production (Cosentino et al., 2008; Pinton et al., 2007) because the Ser36-phosphorylated p66Shc transfers into the mitochondria (Orsini et al., 2004), where it may be responsible for the deleterious effects of p66Shc. In addition, p66Shc binding to the mitochondria and to cytochrome c is known to be the critical step in ROS release, inhibiting p66Shc phosphorylation at Ser36 will impair cytochrome c-binding. Therefore, to determine whether the Ser36 phosphorylation of p66shc is required for p66shc to bind to cytochrome c and interaction between p66shc and cytochrome c is needed to cause adverse effects of CSE on cell injury and ROS production. For this experiment, the 16HBE cells were transfected with the S36A mutant of the p66Shc plasmid to impair Ser36 phosphorylation and the cytochrome c-binding (W134F) mutants of p66Shc to assesse the levels of cytochrome c (a mitochondrial marker). As shown in Fig. 6A and B, the levels of p66Shc and cytochrome c expression were significantly inhibited by S36A mutant and W134F, respectively. For another, the ROS production were inhibited by the S36A and W134F mutants, whereas, levels of mitochondria membrane potential (MMP) and ATP were enhanced under different conditions (Fig. 6C). These results were important in the demonstration that CSE induced the airway epithelial cell injury and ROS generation depending on Ser36 phosphorylation of p66shc and its subsequent binding to cytochrome c.




Fig. 7. The involvement of p66Shc, ROS and NF-␬B in the CSE-induced increases in cell apoptosis, mitochondrial injury. The cells were pretreated with ROS scavenger N-acetylcysteine (NAC, 5 mM, 24 h) or transfected with specific siRNA targeting for p66Shc (sip66Shc) or with wild-type p66Shc (WT) prior to stimulation with 20% CSE. The apoptosis rate of the cells was analyzed by flow cytometry (A). The levels of intracellular ROS production, mitochondrial membrane potential (MMP) and intracellular ATP levels are shown (B). The transfection of the cells with wild-type p66Shc (WT) specifically augmented cell apoptosis and mitochondrial injury, whereas PDTC, NAC and p66Shc silencing (sip66Shc) inhibited the effects. The data shown are representative of three independent experiments and are presented as the means ± SDs (n = 3). * p < 0.05 and ** p < 0.01 versus CSE alone; ˆ p < 0.01 versus sip66Shc.

3.4. p66Shc mediates CSE-induced inflammatory reponse and mucus hypersecretion by activating the ROS/NF-B signaling pathway and inhibiting FOXO3a activity Using real-time PCR and western blot analysis, we clearly detected high expression of p66Shc in CSE-treated 16HBE cells

(Fig. 2). Recently, some studies have reported that oxidative stress induces p66Shc phosphorylation, causing FOXO3a inactivation and a decrease of FOXO3a-RelA/p65 interaction, following increase of NF-␬B activity (Guo et al., 2009). To determine whether the expression of p66Shc was associated with ROS generation, FOXO3a and NF-␬B activity. 16HBE cells were transfected with p66Shc-siRNA




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Fig. 8. The effects of p66Shc on the nuclear FOXO3a and NF-␬B activities in 16HBE cells. (A) The cells were pretreated with ROS scavenger N-acetylcysteine (NAC, 5 mM, 24 h) or transfected with specific siRNA targeting for p66Shc (sip66Shc) or with wild-type p66Shc (WT) prior to stimulation with 20% CSE. Phosphorylated FOXO3a (FOXO3a-pT32) was collected from nuclei and measured by immunoprecipitation (IP), and the total FOXO3a was collected from cytoplasm and nuclei and detected by immunoblotting (IB). (B) Densitometric analysis of FOXO3a-pT32 proteins expression. The total protein ratios expressed as fold increase relative to FOXO3a (* p < 0.05 and ** p < 0.01 versus CSE alone; ˆ p < 0.01 versus sip66Shc). (C–D) The cells were pretreated with ROS scavenger N-acetylcysteine (NAC, 5 mM, 24 h), the specific inhibitor of NF-␬B (PDTC, 100 ␮M, 30 min) or transfected with wild-type p66Shc (WT) or sip66Shc prior to stimulation with 20% CSE. Phosphorylated NF-␬B (NF-␬B p65) and I␬B␣ (p-I␬B␣) were collected from nuclei and measured by immunoprecipitation (IP). The total proteins were collected from cytoplasm and nuclei and detected by immunoblotting (IB). (E) Densitometric analysis of NF-␬B p65 or p-I␬B␣ protein expression. The total protein ratios are expressed as the fold increase relative to NF-␬B or I␬B␣. (* p < 0.05 and ** p < 0.01 versus CSE alone; ˆ p < 0.01 versus sip66Shc). The data shown are representative of three independent experiments and are presented as the means ± SDs (n = 3).

or p66Shc-WT and subsequently exposed to 20% CSE. Transfection with p66Shc-siRNA caused significantly decrease of cell apoptosis and inhibition of CSE-induced ROS production and mitochondrial dysfunction (Fig. 7A and B). Similarly, transfection with p66ShcsiRNA had inhibitory effects on the expression levels of NF-␬B p65 (Fig. 8C) and phosphorylated I␬B␣ (Fig. 8D). However, p66ShcsiRNA enhanced the expression of phosphorylated FOXO3a (T32) (Fig. 8A). Conversely, transfection of p66Shc-WT showed a significant increased in the ROS production and cell injury (Fig. 7A and B). Furthermore, the expression levels of phosphorylated FOXO3a (T32) was inhibited and the NF-␬B activity was enhanced by p66Shc-WT (Fig. 8A, C and D). These data suggested that the levels of p66Shc may be closely related to ROS generation, FOXO3a and NF-␬B activity. NF-␬B is a ubiquitous transcription factor known to enhance inflammatory response and mucin gene transcriptional activity by binding to their promoters (Preciado et al., 2008). Often ROS has been shown to influence the DNA binding properties of the NF-␬B proteins themselves (Hirota et al., 1999). In other word, ROS/NF␬B signaling pathway may play an important role in CSE-induced inflammatory reponse and mucus hypersecretion. In our experiments, we have shown that ROS production, FOXO3a and NF-␬B activity were mediated by the expression levels of p66Shc. Thus, we deduced that p66Shc could play an important part in mucous hypersecretion in respiratory diseases. To confirm whether p66Shc mediated CSE-induced inflammatory reponse and mucus generation. The abundance of IL-6, IL-8, TNF-␣ and MUC5AC protein were assessed by ELISA (Fig. 9A and D), the changes of MUC5AC gene were also detected by RT-PCR (Fig. 9B and C). As expected, the expression of IL-6, IL-8, TNF-␣ and MUC5AC were significantly increased in 16HBE cells transfected with p66Shc-WT. Transfection of p66ShcsiRNA had an inhibitory effect on these inflammatory cytokines and mucin expression.

Next to explore the molecular mechanisms that p66Shc mediated inflammatory reponse and mucus hypersecretion, the cell apoptosis rate and the levels of ROS, MMP, ATP, FOXO3a and NF␬B were analyzed. Fig. 7A and B have shown that NAC and PDTC clearly attenuated the CSE-induced cell apoptosis, ROS production and mitochondrial injury. Similarly, NF-␬B p65 and p-I␬B␣ activation were also inhibited by NAC or PDTC(Fig. 8C and D), however, FOXO3a activation was enhanced by NAC or PDTC(Fig. 8A). Also as expected, expression of IL-6, IL-8, TNF-␣ and MUC5AC were attenuated by NAC or PDTC, in addition, the changes of these inflammatory cytokines and MUC5AC were consistent with p66Shc (Fig. 9A–D). Recently, some studies have reported that the levels of phosphorylated p66Shc be regulated by the content of intracellular ROS and NF-␬B activity (Almeida et al., 2010). In turn, to further confirmed whether the levels of ROS and NF-␬B activity affect the expression of p66Shc and p-p66Shc. Cells were stimulated with NAC or PDTC pretreatment, the changes of p66Shc and p-p66Shc were analyzed by Western blot, As shown in Fig. 10, NAC and PDTC significantly inhibited CSE-induced the expression of p66Shc and p-p66Shc. It is important to note that p66Shc expression is required for the levels of intracellular ROS and NF-␬B activity. Taken together, these results strongly suggest that p66Shc mediates CSE-induced inflammatory reponse and mucus hypersecretion depending on ROS/NF-␬B signaling pathway and FOXO3a activity; Otherwise, the levels of intracellular ROS and NF-␬B activity affect the expression of p66Shc. 4. Disscussion Cigarette smoke is the major risk factor for COPD, causing chronic airway inflammation. Because, long-term cigarette smoke exposure induces mitochondrial structural and functional changes




Fig. 9. The involvement of p66Shc, ROS and NF-␬B in the CSE-induced increases in inflammatory response and MUC5AC secretion. The cells were pretreated with ROS scavenger N-acetylcysteine (NAC, 5 mM, 24 h) or transfected with specific siRNA targeting for p66Shc (sip66Shc) or with wild-type p66Shc (WT) prior to stimulation with 20% CSE. The pro-inflammatory cytokines (IL-6, IL-8 and TNF-␣) were analyzed by ELISA (A). The MUC5AC mRNA levels were measured by reverse transcriptase-polymerase chain reaction (B), and real-time PCR (C) and the protein levels by ELISA (D). The transfection of the cells with wild-type p66Shc (WT) specifically enhanced the inflammation and MUC5AC secretion (middle bar), whereas PDTC, NAC and p66Shc silencing (sip66Shc) inhibited the effects. The data shown are representative of three independent experiments and are presented as the means ± SDs (n = 3). * p < 0.05 and ** p < 0.01 versus CSE alone; ˆ p < 0.01 versus sip66Shc.

in COPD epithelium, which result in damage to in the airway barrier function (Hoffmann et al., 2013; Ning et al., 2013). The consequence of this injury is a significant increase in the lung and airway epithelial cell apoptosis and mucous cell hyperplasia, which are assumed to be directly involved in emphysema and sputum oversecretion (Preciado et al., 2008). Recently, some studies have indicated that the mitochondria are crucially involved in the regulation of these cell death processes by the releasing proapoptotic mediators (e.g., cytochrome c, apoptosis-inducing factor). Furthermore, the mitochondria are the major source of intracellular ROS, and an excessive mitochondrial ROS generation is believed to be a key contributor to the airway barrier dysfunction (van der Toorn et al., 2007). Indeed, CSE adversely affects mitochondrial function and mitochondrial ROS production (Banzet et al., 1999; Yang et al., 2007). The adaptor protein p66Shc is one of the key regulators of the mitochon-

drial production of ROS. Animal and cell studies have demonstrated that ischemia/reperfusion (I/R)-induced oxidative stress and acute kidney injury (AKI) are associated with increases in the expressoin of p66shc, which is responsible for the increases in the mitochondrial ROS production and the resulting renal cell injury (Arany et al., 2008). Furthermore, Arany et al. (2011) have confirmed that CS augments the IR-AKI-dependent Ser36 phosphorylation and binding of p66Shc to cytochrome c through CS-induced intracellular ROS that originate in the mitochondria. Thus, we hypothesized that p66Shc may be associated with airway epithelial cell injury and the resulting inflammatory response in smoking-induced chronic airway inflammatory diseases. This study was undertaken to gain a theoretical basis for our hypothesis. In this study, the data showed that stimulating cells with CSE caused a significant increase in the expression of p66Shc




Fig. 10. The effects of intracellular ROS production and NF-␬B activity on CSEinduced p66Shc expression. The cells were pretreated with the ROS scavenger N-acetylcysteine (NAC, 5 mM, 24 h) or the specific inhibitor of NF-␬B (PDTC, 100 ␮M, 30 min) prior to stimulation with 20% CSE. The total protein was collected from 16HBE cells, and western blotting analysis was performed to demonstrate the presence of p66Shc or phosphorylated p66Shc (p-p66Shc) in these cells (A). Densitometric analysis of p66Shc or p-p66Shc protein expression. The total protein ratios are expressed as the fold increase relative to ␤-actin (B) (* p < 0.05 and ** p < 0.01 versus CSE alone). The data shown are representative of three independent experiments and are presented as the means ± SDs (n = 3).

and its phosphorylation in a concentration-dependent manner, whereas the cell viability, mitochondrial membrane potential and ATP production gradually decreased. Furthermore, the CSE-induced adverse effects were inhibited by knockdown of p66Shc and enhanced by transfection with wild-type p66Shc. These data also provided evidence that p66Shc is involved in the airway epithelial cell injury. To date, some studies have reported that p66Shc modulates ROS production by three mechanisms restricted to the plasma membrane, the nucleus, and the mitochondria, respectively. At the plasma membrane, p66Shc promotes RAC1 activation and triggers NADPH membrane oxidase-dependent ROS production. In addition, the activation of RAC1 provides a positive feedback to p66Shc (Khanday et al., 2006). The nuclear mechanism is associated with forkhead box sub-group O (FOXO) transcription factors. Specifically, phosphorylated p66Shc translocates from the cytosol to the nucleus resulting in the inhibition of the activity of the FOXO transcription factors (especially FOXO3a). One consequence of this inhibition is that the expression of ROS-scavenging enzymes CAT and manganese superoxide dismutase (MnSOD) is noticeably decreased (Nemoto and Finkel, 2002). Finally, p66Shc is serinephosphorylated through the activation of oxidative stress-induced protein kinase C␤II (PKC␤II) and isomerized by the peptidylprolyl cis/trans isomerase PIN-1 (Pinton et al., 2007). This isomerization allows the dephosphorylation of the Ser-36 residue by the serine threonine phosphatase PP2A and induces the translocation of p66Shc from the cytosol into the mitochondrial intermembrane space. One consequence of p66Shc binding to cytochrome c is that the cytochrome acts as an oxidoreductase and generates ROS. In turn, these ROS further increase the levels of p66Shc phosphorylation and trigger the mitochondrial route of apoptosis, which results in an enhancement of mitochondrial ROS production. It is worth

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noting that the mitochondrial ROS production induced by p66Shc further increases intracellular ROS levels, thus generating a positive control loop (Giorgio et al., 2005). The mitochondria are the main site of ROS generation, and cigarette smoke disrupts the mitochondrial function by decreasing the capacity of mitochondria for ATP synthesis, leading to cellular dysfunction. Our important series of experiments showed that overexpression of p66Shc distinctly enhanced ROS production, whereas knocking down p66Shc or the Ser36A mutant of p66Shc attenuated the effect. Furthermore, the degree of cell injury was consistent with the levels of p66Shc expression and ROS production. The results demonstrated that the CSE-induced intracellular ROS mainly originated in the mitochondria. Excessive mitochondrial ROS production is deleterious to cellular function by the causing a depolarization of the mitochondria and impeding of ATP production. Importantly, p66Shc was involved in this process and Ser36 phosphorylation was pivotal for mitochondrial ROS production. Recent work has reported that p66Shc generates mitochondrial ROS utilizing the mitochondrial electron transport chain by oxidizing of cytochrome c, p66Shc is translocated into the mitochondrial intermembrane space, a process which requires Ser36 phosphorylation (Orsini et al., 2004). Our studies further revealed that CSE increased the binding of the p66Shc, but not the Ser36A mutant, to cytochrome c. In contrast, the Ser36A form was similar to binding mutant of p66Shc (W134F) in its ability to bind cytochrome c. Therefore, the increased binding of p66Shc to cytochrome c plays a role in the ROS-dependent airway epithelial cell injury, as the Ser36A mutant decreased the levels of Ser36-phosphorylated p66Shc, which in turn led to a decrease in the p66Shc available in the mitochondria. Subsequently, the decreased binding of p66Shc to cytochrome c resulted in an increase in ROS production. NF-␬B proteins are a family of transcription factors that are of central importance in inflammation and immunity (Hayden and Ghosh, 2008; Vallabhapurapu and Karin, 2009). Earlier studies have revealed that cigarette smoke products activate NF-␬B in macrophages, human lung epithelial cells and squamous cell lines, which leads to a chronic airway inflammatory response and mucus hypersecretion (Shishodia et al., 2003; Gensch et al., 2004). Although we have confirmed that CSE caused excessive mitochondrial ROS production and airway epithelial cell injury, it is unclear whether the subsequent inflammatory response and mucin overexpression were associated with the interaction between NF-␬B and excessive ROS generation. In this context, ROS have been reported to both activate and to repress NF-␬B signalling. To demonstrate that CSE caused the subsequent inflammatory response and mucin overexpression through the ROS/NF-␬B pathway, 16HBE cells were pre-treated with NAC or PDTC before the CSE exposure. As predicted, NAC clearly attenuated the CSE-induced ROS production and pro-inflammatory mediator expression. the NF-␬B activity was also inhibited by NAC. Furthermore, we showed that the levels of MUC5AC gene and protein expression were both significantly decreased by the NAC pretreatment. Similarly, the NF-␬B inhibitor PDTC decreased these adverse effects. These data demonstrated that the CSE-induced ROS generation triggers inflammatory response and mucin overexpression by activating NF-␬B signaling. However, the findings that the level of phosphorylated FOXO3a expression was higher following NAC pretreatment than with CSE stimulation alone and that the level of MnSOD expression was consistent with phosphorylated FOXO3a were unexpected (date not shown). We also found that NAC and PDTC inhibited the Ser36 phosphorylation of p66Shc. These additional results suggested that ROS-activated NF-␬B signalling may be involved in the phosphorylation of FOXO3a, at least in part. Moreover, the ROS-induced inactivation of the FOXO3a activity inhibited the interaction of FOXO3a with RelA/p65, and thus augmenting the binding of NF-␬B




proteins to DNA in nuclei. In addition, the excessive ROS production would further aggravate the intracellular oxidative stress. This would then induced PKC␤ activation and phosphorylation of p66Shc, which would then be recognized by Pin-1 and the transferred from the cytosol to the mitochondria. Thus, this positive feedback between p66Shc and ROS would ultimately be expected to lead to intracellular oxidative stress (DeMarchi et al., 2013). In summary, the data from this study show a novel role for p66Shc in the regulation of CSE-induced human airway epithelial cell injury, inflammatory response and mucus hypersecretion. The expression of p66Shc was significantly increased in human airway epithelial cells following CSE exposure, which induced excessive intracellular ROS production and mitochondrial dysfunction. The excessive ROS triggers the inflammatory response and mucus hypersecretion in part by activating NF-␬B signaling and inhibiting FoxO3a activity. Thus, the p66Shc-mediated mechanism may have clinical implication for the prevention/amelioration of the oxidative stress-associated lung inflammatory response and mucus hypersecretion in chronic smokers.

Competing interests The authors declare that they have no competing interests toward any aspect of the work described in this paper.

Acknowledgments This work was supported by grant from the National Nature Science Foundation of China (No. 81100003 and No. 81200005), and China–Russia Cooperation Research Foundation (No. 31211120168).

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