Cellular Immunology 295 (2015) 137–143
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1a,25-Dihydroxyvitamin D3 counteracts the effects of cigarette smoke in airway epithelial cells Ruhui Zhang a, Haijin Zhao a, Hangming Dong a, Fei Zou b, Shaoxi Cai a,⇑ a b
Department of Respiratory, Chronic Airways Diseases Laboratory, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China
a r t i c l e
i n f o
Article history: Received 5 December 2014 Revised 18 February 2015 Accepted 13 March 2015 Available online 18 March 2015 Keywords: 1a,25-Dihydroxyvitamin D3 Cigarette smoke extracts ERK Calpain-1 E-cadherin
a b s t r a c t Cigarette smoke extracts (CSE) alter calpain-1 expression via ERK signaling pathway in bronchial epithelial cells. 1a,25-dihydroxyvitamin D3 (1,25D3) inhibits cigarette smoke-induced epithelial barrier disruption. This study was aimed to explore whether the 1,25D3 counteracted the CSE effects in a human bronchial epithelial cell line (16HBE). In particular, transepithelial electrical resistance (TER) and permeability, expression and distribution of E-cadherin and b-catenin, calpain-1 expression, and ERK phosphorylation were assessed in the CSE-stimulated 16HBE cells. The CSE induced the ERK phosphorylation, improved the calpain-1 expression, increased the distribution anomalies and the cleaving of E-cadherin and bcatenin, and resulted in the TER reduction and the permeability increase. The 1,25D3 reduced these pathological changes. The 1,25D3 mediated effects were associated with a reduced ERK phosphorylation. In conclusion, the present study provides compelling evidences that the 1,25D3 may be considered a possible valid therapeutic option in controlling the cigarette smoke-induced epithelial barrier disruption. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Epithelial cells play an important role in maintaining physiological homeostasis of airway by providing physical and immunological barriers to inhaled pathogens [1,2]. A central component in regulating the mucosal barriers is E-cadherin that provides critical architectural structure and immunological function for airway epithelium through the regulation of epithelial junctions, proliferation, differentiation, and production of growth factors and proinflammatory mediators [3–5]. There are indisputable evidences that cigarette smoke (CS) impairs the airway epithelial barrier by decreasing the E-cadherin production [6–9] and is associated with an increased incidence of chronic obstructive pulmonary disease (COPD), asthma and bronchitis [10–13]. The CS severely impairs functions of airway epithelium include disturbing epithelial junctions and increasing mucosal permeability by inducing calpain activation [6,14], which has been described to cleave the E-cadherin and result in the epithelial barrier dysfunction [15,16]. These previous studies suggest that the CS plays an important role in regulation of calpain expression. However, the mechanisms by which the CS induces the calpain expression and
⇑ Corresponding author. Tel./fax: +86 20 61641571. E-mail address: caishaox@fimmu.com (S. Cai). http://dx.doi.org/10.1016/j.cellimm.2015.03.004 0008-8749/Ó 2015 Elsevier Inc. All rights reserved.
promotes the E-cadherin cleavage in the airway epithelium remain to be elucidated. The calpain is a regulator of the cytoskeleton and promotes the turnover of adhesion complexes by cleaving focal adhesion proteins such as the E-cadherin [17]. The calpain-1 is a main isoform of calpain and can effectively cleave full-length E-cadherin to 100 kDa fragment [18,19]. The E-cad100 fragment is unable to bind to other catenins that result in cellular dissociation [19,20]. The activation mechanisms of the calpain system remain unclear despite the efforts of many laboratories. Recently, some researches have shown that the calpain expression is associated with mitogen activated protein kinase (MAPK) pathway [19,21] and Vitamin D inhibits the E-cadherin cleavage following the calpain activation [22]. Furthermore, some previous studies also suggest that the Vitamin D plays a positive role in maintaining the integrity of epithelial barrier [23–25]. In light of these findings, we hypothesized that the MAPK pathway may regulate the CS-induced calpain expression and epithelial barrier defects, meanwhile, the active form of Vitamin D, 1a,25dihydroxyvitamin D3 (1,25D3) may inhibit these pathological changes in the airway epithelial cells. In our study, the 1,25D3 indeed protected airway epithelia against the CS-induced barrier dysfunction, which was associated with the 1,25D3 down-regulated ERK signaling pathway to attenuate the calpain-1 and maintain the E-cadherin.
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2. Materials and methods
2.6. Western Blot Analysis
2.1. 1,25D3 preparation
The treated cells were detached with trypsin, centrifuged and resuspended in lysis buffer, then added to the homogenized samples with protease inhibitor, PMSF and calcineurin inhibitors. Following 1 h of incubation at 4 °C, the samples were centrifuged at 12,000g for 15 min, after which the supernatants were collected and boiled with standard SDS sample buffer, then separated by 10% SDS gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk at room temperature for 2 h, and respectively incubated with the appropriate primary antibody at 4 °C overnight. After which the membranes were washed and incubated for 1 h in the appropriate peroxidaseconjugated secondary antibody. The immunoreactive bands were detected using the ECL detection system (Wejia Technology Co, China) according to the manufacturer’s instructions.
103 mol/L 1,25D3 solution was prepared by dissolving 1 mg 1,25D3 (molecular weight: 416.64, Sigma, USA) in 2.4 mL 95% ethanol, and stored at 20 °C in the dark until needed for used. 1 lL 103 mol/L 1,25D3 solution was diluted with 99lL RPMI1640 medium (105 mol/L 1,25D3 solution was prepared), 1 lL 105 mol/L 1,25D3 solution was diluted with 999 lL RPMI1640 medium (108 mol/L 1,25D3 solution was prepared). 2.2. Cigarette smoke extract (CSE) preparation The CSE was prepared in a way similar to what have been published previously [26,27]. Unfiltered research grade cigarettes were purchased from Shanghai Cigarette Factory (Shanghai, China). The smoke of one cigarette containing 9.3 mg tar and 0.7 mg nicotine was drawn into 10 mL sterile serum-free cell culture medium with peristaltic pump. The pump was set at an optimum speed to allow one cigarette to burn in approximately 15 min and resulting solution was considered 100% CSE. This solution was filtered through a 0.22 lm pore acrodisc syringe filter (Corning, USA) for sterilization and diluted, and then applied within 20 min after the preparation. 2.3. Cell culture and treatment A human bronchial epithelial cell line (16HBE, Shanghai Fuxiang Biological Technology Co. Ltd, ATCC, USA) was maintained in RPMI1640 medium with 10% fetal calf serum. The cells were then incubated at 37 °C in a 5% CO2 incubator. After cells reached complete confluence, they were passaged and seeded to proper culture plates at a density of 2.5 104 cells/cm2. When the cells reached 90% confluence, the medium was changed to serum freeRPMI1640 and the cells were treated according to the experimental plan. After a 24 h incubation at 37 °C, 5% CO2, the cells were harvested for cell lysate preparation. Cell viability was evaluated by MTT. All the experimental intervention did not affect cell viability (data not shown).
2.7. Immunofluorescence At the end of the treatment, the cells were fixed with 1% paraformaldehyde at room temperature for 10 min, washed with PBS for 30 min, incubated with 0.2% Triton X-100 in PBS for 10 min, and rinsed again with PBS. Cells were blocked with 5% skim milk in PBS for 2 h. All samples were subsequently incubated with monoclonal anti-E-cadherin (Santa Cruz Biotechnology, USA), anti-b-catenin (Santa Cruz Biotechnology, USA), and FITC (green)linked anti-rabbit IgG (Santa Cruz Biotechnology, USA), cell nuclei were stained with 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma–Aldrich, China). The labeled sections were viewed with fluorescence confocal microscopy (Olympus, Japan). 2.8. Statistical analysis Data analysis was performed using SPSS software (Version 13.0). The variables were presented as the means ± standard deviation. One-way ANOVA with Bonferroni post hoc tests were performed to compare the data of all groups. Quantitative data was compared between two groups using t-test. Significant differences were considered at P < 0.05.
2.4. Transepithelial electrical resistance measurements
3. Results
The cells were passaged and seeded to Corning Transwell room, maintained in medium with 10% fetal calf serum, and incubated until the cell monolayers formation, the cells were respectively treated according to the experimental plan and incubated for 24 h. Transepithelial electrical resistance (TER) was measured with a Millicell-Electrical Resistance System (Millipore, USA). Mean values were computed and recorded for each set of cells.
3.1. The 1,25D3 inhibited the TER reduction, the permeability increase, and the distribution anomalies and cleaving of E-cadherin and b-catenin induced by CSE in bronchial epithelial cells
2.5. Permeability measurements The cells were seeded on gelatin-coated Costar Transwell Clear membranes (Sigma Chemical Co, USA.) and cultured for 1–2 days until complete confluence was reached. The medium was changed to serum free-RPMI1640, the cells were treated according to the experimental plan and incubated for 24 h. The apical medium (luminal side) was replaced with 200 ll phenol red-free RPMI1640 containing 0.5 mg/mL fluorescein isothiocyanate-dextran (FITC-dextran) (Sigma Chemical Co, USA), and the basal medium (non-luminal side) was replaced with 800 ll phenol red-free RPMI1640 without FITC-dextran, incubated at 37 °C for 90 min. Samples were respectively analyzed by fluorimetry (excitation 492 nm; emission 530 nm). Epithelial permeability was expressed as percent leakage of FITC-dextran from apical to basolateral compartments.
The CSE is associated with the epithelial barrier disruption and the E-cadherin cleaving [6,8], furthermore, the 1,25D3 plays a positive role in the airway epithelia barrier function [25,28]. Therefore, in order to assess the role of 1,25D3 in the 16HBE cells exposed to the CSE. The 16HBE cells (n = 4) were respectively treated with culture medium (control group), 108 mol/L 1,25D3 [25], and 2.5% CSE [6] with or without 108 mol/L 1,25D3 for 24 h. In the treated 16HBE cells, the CSE resulted in the TER reduction (Fig. 1A), the permeability increase (Fig. 1B), and the distribution anomalies (Fig. 1C) and cleaving (Fig. 1D) of E-cadherin and b-catenin. Meanwhile, the 1,25D3 could inhibit these pathological changes. 3.2. The CSE resulted in the cleaving and distribution anomalies of E-cadherin and b-catenin by increasing the expression of calpain-1 in bronchial epithelial cells The CSE have been shown to induce the calpain activation [6], which has the function to cleave E-cadherin and b-catenin [15,16]. Therefore, in order to observe whether the CSE induce
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Fig. 1. 1,25D3 inhibited the TER reduction, the permeability increase, and the distribution anomalies and cleaving of E-cadherin and b-catenin induced by CSE in bronchial epithelial cells. 16HBE (n = 4) cells were respectively treated with culture medium, 108 mol/L 1,25D3, and 2.5% CSE with or without 108 mol/L 1,25D3 for 24 h, and then were used for assessing the TER, the permeability, and the distribution and expression of E-cadherin and b-catenin. (A) The TER was expressed as the percentage of control cells (Mean ± SD). ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group. (B) The permeability was expressed as the percentage of control cells (Mean ± SD). ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group. (C) The distribution of E-cadherin and b-catenin were shown by immunofluorescence. (D) The expression of E-cadherin and bcatenin were measured by Western Blot Analysis, ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group.
the calpain-1 expression and to define the role of the calpain-1 in the 16HBE cells. The 16HBE cells (n = 4) were respectively treated with culture medium (control group), 108 mol/L ALLM (sigma– Aldrich, USA) [29], and 2.5% CSE with or without 108 mol/L ALLM for 24 h. In the treated 16HBE cells, the CSE significantly increased the expression of calpain-1 as well as resulted in the cleaving and distribution anomalies of E-cadherin (the E-cad100 fragments were significantly increased) and b-catenin (Fig. 2A and B). Meanwhile, the ALLM (the inhibitor of calpain-1) not only reduced the expression of calpain-1, but also decreased the cleaving and the distribution anomalies of E-cadherin (the E-cad100 fragments were significantly decreased) and b-catenin.
The 16HBE cells (n = 6) were respectively treated with culture medium (control group), 2.5% CSE, 108 mol/L ALLM, 105 mol/L U0126 (CST, USA) [34], 2.5% CSE with 108 mol/L ALLM, and 2.5% CSE with 105 mol/L U0126 for 24 h. As the Fig. 4A and B shown, both the ALLM and the U0126 reduced the abnormity of TER and permeability induced by the CSE in the 16HBE cells, meanwhile, as depicted in the Fig. 4C, the ALLM (the inhibitor of calpain-1) only reduced the expression of calpain-1, however, the U0126 (the inhibitor of ERK) not only reduced the expression of P-ERK, but also decreased the expression of calpain-1.
3.3. The CSE induced the calpain-1 expression and resulted in the epithelial barrier disruption that was inhibited by the 1,25D3 via the ERK signaling pathway in bronchial epithelial cells
The airway epithelium is a physical barrier and plays an important role in regulating the underlying tissue response to external stimuli [35]. The epithelial barrier integrity is maintained by strong cell–cell adhesion mediated by particular junctions. These junctions include tight junction, adherens junctions and desmosomes, which are composed of a number of transmembrane proteins such as occludin, claudin, junctional adhesion molecule, E-cadherin, bcatenin and a-catenin [36]. In these proteins, E-cadherin-dependent adhesion is a prerequisite for the assembly of other specialized cell– cell junctions [37] and specifically regulates cellular signaling towards formation of other junctions [38,39]. E-cadherin has attracted much attention due to the important role of it in airway epithelium [40]. Recent studies show that the CS promotes the Ecadherin cleavage by inducing the calpain activation in the airway epithelium [6,14], the activated calpain cleave full-length E-cadherin to 100 kDa fragment [18,19]. The E-cad100 fragment is
Three well-characterized MAPK pathways are ERK, p38, and JNK signaling pathways [30], which are the main pathways of calpains activation [31–33]. In order to observe the signaling pathways of the CSE induce the calpain-1 expression and to assess the role of 1,25D3 in these signaling pathways. The 16HBE cells (n = 4) were respectively treated with culture medium (control group), 108 mol/L 1,25D3, and 2.5% CSE with or without 108 mol/L 1,25D3 for 24 h. In the treated 16HBE cells, the CSE significantly increased the expression of calpain-1, P-ERK, P-P38, and P-JNK, however, the 1,25D3 inhibited the abnormal expression of calpain-1 and P-ERK (Fig. 3). In order to further understand the relationship between the calpain-1 and the ERK signaling pathway,
4. Discussion
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Fig. 2. CSE resulted in the cleaving and distribution anomalies of E-cadherin and b-catenin by increasing the expression of calpain-1 in bronchial epithelial cells. 16HBE (n = 4) cells were respectively treated with culture medium, 108 mol/L ALLM, 2.5% CSE with or without 108 mol/L ALLM for 24 h, and then were used for assessing the expression of E-cadherin, b-catenin and calpain-1, and the distribution of E-cadherin and b-catenin. (A) The expression of E-cadherin, b-catenin and calpain-1 were measured by Western Blot Analysis, ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group. (B) The distribution of E-cadherin and b-catenin shown by immunofluorescence.
Fig. 3. 1,25D3 reduced the expression of calpain-1 and P-ERK induced by CSE in bronchial epithelial cells. 16HBE (n = 4) cells were respectively treated with culture medium, 108 mol/L 1,25D3, 2.5% CSE with or without 108 mol/L 1,25D3 for 24 h, and then were used for measure the expression of calpain-1, ERK, P38, and JNK by Western Blot Analysis, ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group.
unable to bind to b-, c-, and a-catenin that lead to the disruption of E-cadherin adhesion complex and event promote cellular dissociation [19,41]. Therefore, inhibition of the calpain activity is the key of restraining the CS damage ability in airway epithelium. The calpains are calcium-activated cysteine proteases and ubiquitously express in tissue [42]. Recent studies show that the Vitamin D protects the integrity of epithelial barrier by reducing the E-cadherin cleavage following the calpain activation [22–25]. Therefore, whether the Vitamin D inhibits the CS damage ability in the airway epithelium is worth investigation. In our experiment, the CSE resulted in the calpain-1 expression increase, the E-cadherin
cleavage (the E-cad100 fragments were increased), and the epithelial barrier disruption (TER reduction and permeability increase), meanwhile, the ALLM (the inhibitor of calpain-1) and the 1,25D3 inhibited these pathological changes. It prompted that the calpain-1 expression increase was a reason of the CSE resulted in the E-cadherin cleavage and the epithelial barrier disruption, and the 1,25D3 could inhibit the CSE damage ability in the airway epithelium. The calpains-1 is a main isoform of calpains and has been demonstrated to cleave the E-cadherin to E-cad100 in epithelial cells [18,19]. The calpain-1 activation is associated with MAPK
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Fig. 4. CSE increased the expression of calpain-1 by the ERK signaling pathway that resulted in the TER reduction and the permeability increase in bronchial epithelial cells. 16HBE (n = 6) cells were respectively treated with culture medium (control group), 2.5% CSE, 108 mol/L ALLM, 105 mol/L U0126, 2.5% CSE with 108 mol/L ALLM, and 2.5% CSE with 108 mol/L ALLM for 24 h, and then were used for assessing the TER, the permeability, and the expression of calpain-1 and ERK. (A) The TER was expressed as the percentage of control cells (Mean ± SD). ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group. (B) The permeability was expressed as the percentage of control cells (Mean ± SD). ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group. (C) The expression of calpain-1 and ERK were measured by Western Blot Analysis, ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group.
pathway [19,21]. The MAPK pathway is well known cell cycle regulator, three well-characterized MAPK pathways in the cells are ERK, JNK, and p38 MAPK signaling pathways [30]. ERK-MAPKs are activated by several extracellular stimuli, such as growth factors, cytokines and oxidative stress. JNKs or stress-activated protein kinases respond to different types of stress to the cells. The p38 MAPK is activated in response to inflammatory mediators. These different MAPK cascades show a high degree of functional specificity and can be activated after the ERK, JNK, and p38
phosphorylated [31,32,43]. In our experiment, the CSE significantly induced the expression of calpain-1, P-ERK, PP38, and P-JNK, however, the 1,25D3 inhibited the abnormal expression of calpain-1 and P-ERK. It suggested that the CSE might activate the ERK, JNK, and p38 MAPK signaling pathways and increase the calpain-1 expression, and the 1,25D3 might inhibited the ERK signaling pathway and decrease the calpain-1 expression. In order to further understand the relationship between the calpain-1 and the ERK signaling pathway, we treated the 16HBE cells
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exposed to the CSE with the inhibitor of calpain-1(ALLM) and the inhibitor of ERK(U0126), both the ALLM and the U0126 reduced the epithelial barrier disruption induced by the CSE in the 16HBE cells, meanwhile, the ALLM (the inhibitor of calpain-1) only reduced the expression of calpain-1, however, the U0126 (the inhibitor of ERK) not only reduced the expression of P-ERK, but also decreased the expression of calpain-1. It suggested that the ERK signaling pathway was the upstream regulator of calpain-1, the CSE might induce the calpain-1 expression via the ERK signaling pathway and resulted in the epithelial barrier disruption, and the 1,25D3 might inhibit the CSE damage effect in the airway epithelium by down-regulating the ERK signaling pathway. Recent studies have identified the positive role of Vitamin D in maintaining the integrity of epithelial barrier, however, there are still lacking of consensus on the mechanism of Vitamin D in the airway epithelium. Mulligan et al. demonstrated the 1,25VD3 reduced the proinflammatory effects of CS on human sinonasal epithelial cells [44], moreover, many researches have indentified that proinflammatory cytokines can lead to the epithelial barrier damage [45–47]. In our study, it is uncertain whether proinflammatory cytokines play a role in the 16HBE cells treated with CSE. Further researches are needed to answer this question. In general, the CSE induced the calpain-1 expression via the ERK signaling pathway that resulted in the epithelial barrier disruption by increasing the cleaving and the distribution anomalies of Ecadherin and b-catenin. The 1,25D3 could inhibit these pathological changes by down-regulating the ERK signaling pathway. However, several limitations needed to be noted in this study, we did not directly evaluate the role of 1,25D3 and the mechanisms by which it protects the epithelial barrier in vivo. Because of the difference between epithelial cells in vitro and in vivo, the results of this research may not be identical to the clinical situation. Therefore, we intend to further answer these questions by using samples from CSE model and primary human bronchial epithelial cells in the near future. References [1] S.R. White, Apoptosis and the airway epithelium, Allergy 12 (2011) 1–21 (Cairo). [2] M.I. Gomez, A. Prince, Airway epithelial cell signaling in response to bacterial pathogens, Pediatr. Pulmonol. 43 (2008) 11–19. [3] I.H. Heijink, P.M. Kies, H.F. Kauffman, Down-regulation of E-cadherin in human bronchial epithelial cells leads to epidermal growth factor receptor-dependent Th2 cell-promoting activity, Immunology 178 (2007) 7678–7685. [4] N.L. Mihai, R. Ivan, J. Walker, M.A. Kukuruzinska, Hypoglycosylated E-cadherin promotes the assembly of tight junctions through the recruitment of PP2A to adherens junctions, Exp. Cell Res. 316 (2010) 1871–1884. [5] C. Xiao, S.M. Puddicombe, S. Field, Defective epithelial barrier function in asthma, J. Allergy Clin. Immun. 128 (2011) 549–556. [6] I.H. Heijink, S.M. Brandenburg, D.S. Postma, A.J.M. Osterhout, Cigarette smoke impairs airway epithelial barrier function and cell–cell contact recovery, Eur. Respir. J. 39 (2012) 419–428. [7] R. Shaykhiev, F. Otaki, P. Bonsu, Cigarette smoking reprograms apical junctional complex molecular architecture in the human airway epithelium in vivo, Cell. Mol. Life Sci. 68 (2011) 877–892. [8] M.S. Rosanna, C.M. Marina, S.F. Nieves, V.G. Monica, E.M. Maria, Hyaluronan and Layilin mediate loss of airway epithelial barrier function induced by cigarette smoke by decreasing E-cadherin, J. Biol. Chem. 287 (2012) 42288– 42298. [9] M. Broekema, H.N.H. Ten, F. Volbeda, Airway epithelial changes in smokers but not in ex-smokers with asthma, Am. J. Respir. Crit. Care Med. 180 (2009) 1170– 1178. [10] B.A. Forey, A.J. Thornton, P.N. Lee, Systematic review with meta-analysis of the epidemiological evidence relating smoking to COPD, chronic bronchitis and emphysema, BMC Pulm. Med. 11 (2011) 1–61. [11] I. Proctor, V. Sharma, M. Khoshzaban, A. Winstanley, Does smoking kill? A study of death certification and smoking, J. Clin. Pathol. 65 (2012) 129–132. [12] R. Powell, D. Davidson, J. Divers, Genetic ancestry and the relationship of cigarette smoking to lung function and percent emphysema in four race/ethnic groups: a cross-sectional study, Thorax 68 (2013) 634–642. [13] K. Gangl, R. Reininger, D. Bernhard, Cigarette smoke facilitates allergen penetration across respiratory epithelium, Allergy 64 (2009) 398–405.
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1a,25-Dihydroxyvitamin D3 counteracts the effects of cigarette smoke in airway epithelial cells Ruhui Zhang a, Haijin Zhao a, Hangming Dong a, Fei Zou b, Shaoxi Cai a,⇑ a b
Department of Respiratory, Chronic Airways Diseases Laboratory, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou 510515, China
a r t i c l e
i n f o
Article history: Received 5 December 2014 Revised 18 February 2015 Accepted 13 March 2015 Available online 18 March 2015 Keywords: 1a,25-Dihydroxyvitamin D3 Cigarette smoke extracts ERK Calpain-1 E-cadherin
a b s t r a c t Cigarette smoke extracts (CSE) alter calpain-1 expression via ERK signaling pathway in bronchial epithelial cells. 1a,25-dihydroxyvitamin D3 (1,25D3) inhibits cigarette smoke-induced epithelial barrier disruption. This study was aimed to explore whether the 1,25D3 counteracted the CSE effects in a human bronchial epithelial cell line (16HBE). In particular, transepithelial electrical resistance (TER) and permeability, expression and distribution of E-cadherin and b-catenin, calpain-1 expression, and ERK phosphorylation were assessed in the CSE-stimulated 16HBE cells. The CSE induced the ERK phosphorylation, improved the calpain-1 expression, increased the distribution anomalies and the cleaving of E-cadherin and bcatenin, and resulted in the TER reduction and the permeability increase. The 1,25D3 reduced these pathological changes. The 1,25D3 mediated effects were associated with a reduced ERK phosphorylation. In conclusion, the present study provides compelling evidences that the 1,25D3 may be considered a possible valid therapeutic option in controlling the cigarette smoke-induced epithelial barrier disruption. Ó 2015 Elsevier Inc. All rights reserved.
1. Introduction Epithelial cells play an important role in maintaining physiological homeostasis of airway by providing physical and immunological barriers to inhaled pathogens [1,2]. A central component in regulating the mucosal barriers is E-cadherin that provides critical architectural structure and immunological function for airway epithelium through the regulation of epithelial junctions, proliferation, differentiation, and production of growth factors and proinflammatory mediators [3–5]. There are indisputable evidences that cigarette smoke (CS) impairs the airway epithelial barrier by decreasing the E-cadherin production [6–9] and is associated with an increased incidence of chronic obstructive pulmonary disease (COPD), asthma and bronchitis [10–13]. The CS severely impairs functions of airway epithelium include disturbing epithelial junctions and increasing mucosal permeability by inducing calpain activation [6,14], which has been described to cleave the E-cadherin and result in the epithelial barrier dysfunction [15,16]. These previous studies suggest that the CS plays an important role in regulation of calpain expression. However, the mechanisms by which the CS induces the calpain expression and
⇑ Corresponding author. Tel./fax: +86 20 61641571. E-mail address: caishaox@fimmu.com (S. Cai). http://dx.doi.org/10.1016/j.cellimm.2015.03.004 0008-8749/Ó 2015 Elsevier Inc. All rights reserved.
promotes the E-cadherin cleavage in the airway epithelium remain to be elucidated. The calpain is a regulator of the cytoskeleton and promotes the turnover of adhesion complexes by cleaving focal adhesion proteins such as the E-cadherin [17]. The calpain-1 is a main isoform of calpain and can effectively cleave full-length E-cadherin to 100 kDa fragment [18,19]. The E-cad100 fragment is unable to bind to other catenins that result in cellular dissociation [19,20]. The activation mechanisms of the calpain system remain unclear despite the efforts of many laboratories. Recently, some researches have shown that the calpain expression is associated with mitogen activated protein kinase (MAPK) pathway [19,21] and Vitamin D inhibits the E-cadherin cleavage following the calpain activation [22]. Furthermore, some previous studies also suggest that the Vitamin D plays a positive role in maintaining the integrity of epithelial barrier [23–25]. In light of these findings, we hypothesized that the MAPK pathway may regulate the CS-induced calpain expression and epithelial barrier defects, meanwhile, the active form of Vitamin D, 1a,25dihydroxyvitamin D3 (1,25D3) may inhibit these pathological changes in the airway epithelial cells. In our study, the 1,25D3 indeed protected airway epithelia against the CS-induced barrier dysfunction, which was associated with the 1,25D3 down-regulated ERK signaling pathway to attenuate the calpain-1 and maintain the E-cadherin.
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2. Materials and methods
2.6. Western Blot Analysis
2.1. 1,25D3 preparation
The treated cells were detached with trypsin, centrifuged and resuspended in lysis buffer, then added to the homogenized samples with protease inhibitor, PMSF and calcineurin inhibitors. Following 1 h of incubation at 4 °C, the samples were centrifuged at 12,000g for 15 min, after which the supernatants were collected and boiled with standard SDS sample buffer, then separated by 10% SDS gel electrophoresis and transferred to polyvinylidene fluoride membranes. The membranes were blocked with 5% skim milk at room temperature for 2 h, and respectively incubated with the appropriate primary antibody at 4 °C overnight. After which the membranes were washed and incubated for 1 h in the appropriate peroxidaseconjugated secondary antibody. The immunoreactive bands were detected using the ECL detection system (Wejia Technology Co, China) according to the manufacturer’s instructions.
103 mol/L 1,25D3 solution was prepared by dissolving 1 mg 1,25D3 (molecular weight: 416.64, Sigma, USA) in 2.4 mL 95% ethanol, and stored at 20 °C in the dark until needed for used. 1 lL 103 mol/L 1,25D3 solution was diluted with 99lL RPMI1640 medium (105 mol/L 1,25D3 solution was prepared), 1 lL 105 mol/L 1,25D3 solution was diluted with 999 lL RPMI1640 medium (108 mol/L 1,25D3 solution was prepared). 2.2. Cigarette smoke extract (CSE) preparation The CSE was prepared in a way similar to what have been published previously [26,27]. Unfiltered research grade cigarettes were purchased from Shanghai Cigarette Factory (Shanghai, China). The smoke of one cigarette containing 9.3 mg tar and 0.7 mg nicotine was drawn into 10 mL sterile serum-free cell culture medium with peristaltic pump. The pump was set at an optimum speed to allow one cigarette to burn in approximately 15 min and resulting solution was considered 100% CSE. This solution was filtered through a 0.22 lm pore acrodisc syringe filter (Corning, USA) for sterilization and diluted, and then applied within 20 min after the preparation. 2.3. Cell culture and treatment A human bronchial epithelial cell line (16HBE, Shanghai Fuxiang Biological Technology Co. Ltd, ATCC, USA) was maintained in RPMI1640 medium with 10% fetal calf serum. The cells were then incubated at 37 °C in a 5% CO2 incubator. After cells reached complete confluence, they were passaged and seeded to proper culture plates at a density of 2.5 104 cells/cm2. When the cells reached 90% confluence, the medium was changed to serum freeRPMI1640 and the cells were treated according to the experimental plan. After a 24 h incubation at 37 °C, 5% CO2, the cells were harvested for cell lysate preparation. Cell viability was evaluated by MTT. All the experimental intervention did not affect cell viability (data not shown).
2.7. Immunofluorescence At the end of the treatment, the cells were fixed with 1% paraformaldehyde at room temperature for 10 min, washed with PBS for 30 min, incubated with 0.2% Triton X-100 in PBS for 10 min, and rinsed again with PBS. Cells were blocked with 5% skim milk in PBS for 2 h. All samples were subsequently incubated with monoclonal anti-E-cadherin (Santa Cruz Biotechnology, USA), anti-b-catenin (Santa Cruz Biotechnology, USA), and FITC (green)linked anti-rabbit IgG (Santa Cruz Biotechnology, USA), cell nuclei were stained with 40 ,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma–Aldrich, China). The labeled sections were viewed with fluorescence confocal microscopy (Olympus, Japan). 2.8. Statistical analysis Data analysis was performed using SPSS software (Version 13.0). The variables were presented as the means ± standard deviation. One-way ANOVA with Bonferroni post hoc tests were performed to compare the data of all groups. Quantitative data was compared between two groups using t-test. Significant differences were considered at P < 0.05.
2.4. Transepithelial electrical resistance measurements
3. Results
The cells were passaged and seeded to Corning Transwell room, maintained in medium with 10% fetal calf serum, and incubated until the cell monolayers formation, the cells were respectively treated according to the experimental plan and incubated for 24 h. Transepithelial electrical resistance (TER) was measured with a Millicell-Electrical Resistance System (Millipore, USA). Mean values were computed and recorded for each set of cells.
3.1. The 1,25D3 inhibited the TER reduction, the permeability increase, and the distribution anomalies and cleaving of E-cadherin and b-catenin induced by CSE in bronchial epithelial cells
2.5. Permeability measurements The cells were seeded on gelatin-coated Costar Transwell Clear membranes (Sigma Chemical Co, USA.) and cultured for 1–2 days until complete confluence was reached. The medium was changed to serum free-RPMI1640, the cells were treated according to the experimental plan and incubated for 24 h. The apical medium (luminal side) was replaced with 200 ll phenol red-free RPMI1640 containing 0.5 mg/mL fluorescein isothiocyanate-dextran (FITC-dextran) (Sigma Chemical Co, USA), and the basal medium (non-luminal side) was replaced with 800 ll phenol red-free RPMI1640 without FITC-dextran, incubated at 37 °C for 90 min. Samples were respectively analyzed by fluorimetry (excitation 492 nm; emission 530 nm). Epithelial permeability was expressed as percent leakage of FITC-dextran from apical to basolateral compartments.
The CSE is associated with the epithelial barrier disruption and the E-cadherin cleaving [6,8], furthermore, the 1,25D3 plays a positive role in the airway epithelia barrier function [25,28]. Therefore, in order to assess the role of 1,25D3 in the 16HBE cells exposed to the CSE. The 16HBE cells (n = 4) were respectively treated with culture medium (control group), 108 mol/L 1,25D3 [25], and 2.5% CSE [6] with or without 108 mol/L 1,25D3 for 24 h. In the treated 16HBE cells, the CSE resulted in the TER reduction (Fig. 1A), the permeability increase (Fig. 1B), and the distribution anomalies (Fig. 1C) and cleaving (Fig. 1D) of E-cadherin and b-catenin. Meanwhile, the 1,25D3 could inhibit these pathological changes. 3.2. The CSE resulted in the cleaving and distribution anomalies of E-cadherin and b-catenin by increasing the expression of calpain-1 in bronchial epithelial cells The CSE have been shown to induce the calpain activation [6], which has the function to cleave E-cadherin and b-catenin [15,16]. Therefore, in order to observe whether the CSE induce
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Fig. 1. 1,25D3 inhibited the TER reduction, the permeability increase, and the distribution anomalies and cleaving of E-cadherin and b-catenin induced by CSE in bronchial epithelial cells. 16HBE (n = 4) cells were respectively treated with culture medium, 108 mol/L 1,25D3, and 2.5% CSE with or without 108 mol/L 1,25D3 for 24 h, and then were used for assessing the TER, the permeability, and the distribution and expression of E-cadherin and b-catenin. (A) The TER was expressed as the percentage of control cells (Mean ± SD). ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group. (B) The permeability was expressed as the percentage of control cells (Mean ± SD). ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group. (C) The distribution of E-cadherin and b-catenin were shown by immunofluorescence. (D) The expression of E-cadherin and bcatenin were measured by Western Blot Analysis, ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group.
the calpain-1 expression and to define the role of the calpain-1 in the 16HBE cells. The 16HBE cells (n = 4) were respectively treated with culture medium (control group), 108 mol/L ALLM (sigma– Aldrich, USA) [29], and 2.5% CSE with or without 108 mol/L ALLM for 24 h. In the treated 16HBE cells, the CSE significantly increased the expression of calpain-1 as well as resulted in the cleaving and distribution anomalies of E-cadherin (the E-cad100 fragments were significantly increased) and b-catenin (Fig. 2A and B). Meanwhile, the ALLM (the inhibitor of calpain-1) not only reduced the expression of calpain-1, but also decreased the cleaving and the distribution anomalies of E-cadherin (the E-cad100 fragments were significantly decreased) and b-catenin.
The 16HBE cells (n = 6) were respectively treated with culture medium (control group), 2.5% CSE, 108 mol/L ALLM, 105 mol/L U0126 (CST, USA) [34], 2.5% CSE with 108 mol/L ALLM, and 2.5% CSE with 105 mol/L U0126 for 24 h. As the Fig. 4A and B shown, both the ALLM and the U0126 reduced the abnormity of TER and permeability induced by the CSE in the 16HBE cells, meanwhile, as depicted in the Fig. 4C, the ALLM (the inhibitor of calpain-1) only reduced the expression of calpain-1, however, the U0126 (the inhibitor of ERK) not only reduced the expression of P-ERK, but also decreased the expression of calpain-1.
3.3. The CSE induced the calpain-1 expression and resulted in the epithelial barrier disruption that was inhibited by the 1,25D3 via the ERK signaling pathway in bronchial epithelial cells
The airway epithelium is a physical barrier and plays an important role in regulating the underlying tissue response to external stimuli [35]. The epithelial barrier integrity is maintained by strong cell–cell adhesion mediated by particular junctions. These junctions include tight junction, adherens junctions and desmosomes, which are composed of a number of transmembrane proteins such as occludin, claudin, junctional adhesion molecule, E-cadherin, bcatenin and a-catenin [36]. In these proteins, E-cadherin-dependent adhesion is a prerequisite for the assembly of other specialized cell– cell junctions [37] and specifically regulates cellular signaling towards formation of other junctions [38,39]. E-cadherin has attracted much attention due to the important role of it in airway epithelium [40]. Recent studies show that the CS promotes the Ecadherin cleavage by inducing the calpain activation in the airway epithelium [6,14], the activated calpain cleave full-length E-cadherin to 100 kDa fragment [18,19]. The E-cad100 fragment is
Three well-characterized MAPK pathways are ERK, p38, and JNK signaling pathways [30], which are the main pathways of calpains activation [31–33]. In order to observe the signaling pathways of the CSE induce the calpain-1 expression and to assess the role of 1,25D3 in these signaling pathways. The 16HBE cells (n = 4) were respectively treated with culture medium (control group), 108 mol/L 1,25D3, and 2.5% CSE with or without 108 mol/L 1,25D3 for 24 h. In the treated 16HBE cells, the CSE significantly increased the expression of calpain-1, P-ERK, P-P38, and P-JNK, however, the 1,25D3 inhibited the abnormal expression of calpain-1 and P-ERK (Fig. 3). In order to further understand the relationship between the calpain-1 and the ERK signaling pathway,
4. Discussion
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Fig. 2. CSE resulted in the cleaving and distribution anomalies of E-cadherin and b-catenin by increasing the expression of calpain-1 in bronchial epithelial cells. 16HBE (n = 4) cells were respectively treated with culture medium, 108 mol/L ALLM, 2.5% CSE with or without 108 mol/L ALLM for 24 h, and then were used for assessing the expression of E-cadherin, b-catenin and calpain-1, and the distribution of E-cadherin and b-catenin. (A) The expression of E-cadherin, b-catenin and calpain-1 were measured by Western Blot Analysis, ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group. (B) The distribution of E-cadherin and b-catenin shown by immunofluorescence.
Fig. 3. 1,25D3 reduced the expression of calpain-1 and P-ERK induced by CSE in bronchial epithelial cells. 16HBE (n = 4) cells were respectively treated with culture medium, 108 mol/L 1,25D3, 2.5% CSE with or without 108 mol/L 1,25D3 for 24 h, and then were used for measure the expression of calpain-1, ERK, P38, and JNK by Western Blot Analysis, ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group.
unable to bind to b-, c-, and a-catenin that lead to the disruption of E-cadherin adhesion complex and event promote cellular dissociation [19,41]. Therefore, inhibition of the calpain activity is the key of restraining the CS damage ability in airway epithelium. The calpains are calcium-activated cysteine proteases and ubiquitously express in tissue [42]. Recent studies show that the Vitamin D protects the integrity of epithelial barrier by reducing the E-cadherin cleavage following the calpain activation [22–25]. Therefore, whether the Vitamin D inhibits the CS damage ability in the airway epithelium is worth investigation. In our experiment, the CSE resulted in the calpain-1 expression increase, the E-cadherin
cleavage (the E-cad100 fragments were increased), and the epithelial barrier disruption (TER reduction and permeability increase), meanwhile, the ALLM (the inhibitor of calpain-1) and the 1,25D3 inhibited these pathological changes. It prompted that the calpain-1 expression increase was a reason of the CSE resulted in the E-cadherin cleavage and the epithelial barrier disruption, and the 1,25D3 could inhibit the CSE damage ability in the airway epithelium. The calpains-1 is a main isoform of calpains and has been demonstrated to cleave the E-cadherin to E-cad100 in epithelial cells [18,19]. The calpain-1 activation is associated with MAPK
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Fig. 4. CSE increased the expression of calpain-1 by the ERK signaling pathway that resulted in the TER reduction and the permeability increase in bronchial epithelial cells. 16HBE (n = 6) cells were respectively treated with culture medium (control group), 2.5% CSE, 108 mol/L ALLM, 105 mol/L U0126, 2.5% CSE with 108 mol/L ALLM, and 2.5% CSE with 108 mol/L ALLM for 24 h, and then were used for assessing the TER, the permeability, and the expression of calpain-1 and ERK. (A) The TER was expressed as the percentage of control cells (Mean ± SD). ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group. (B) The permeability was expressed as the percentage of control cells (Mean ± SD). ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group. (C) The expression of calpain-1 and ERK were measured by Western Blot Analysis, ⁄p < 0.05 vs the control group; #p < 0.05 vs the CSE group.
pathway [19,21]. The MAPK pathway is well known cell cycle regulator, three well-characterized MAPK pathways in the cells are ERK, JNK, and p38 MAPK signaling pathways [30]. ERK-MAPKs are activated by several extracellular stimuli, such as growth factors, cytokines and oxidative stress. JNKs or stress-activated protein kinases respond to different types of stress to the cells. The p38 MAPK is activated in response to inflammatory mediators. These different MAPK cascades show a high degree of functional specificity and can be activated after the ERK, JNK, and p38
phosphorylated [31,32,43]. In our experiment, the CSE significantly induced the expression of calpain-1, P-ERK, PP38, and P-JNK, however, the 1,25D3 inhibited the abnormal expression of calpain-1 and P-ERK. It suggested that the CSE might activate the ERK, JNK, and p38 MAPK signaling pathways and increase the calpain-1 expression, and the 1,25D3 might inhibited the ERK signaling pathway and decrease the calpain-1 expression. In order to further understand the relationship between the calpain-1 and the ERK signaling pathway, we treated the 16HBE cells
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exposed to the CSE with the inhibitor of calpain-1(ALLM) and the inhibitor of ERK(U0126), both the ALLM and the U0126 reduced the epithelial barrier disruption induced by the CSE in the 16HBE cells, meanwhile, the ALLM (the inhibitor of calpain-1) only reduced the expression of calpain-1, however, the U0126 (the inhibitor of ERK) not only reduced the expression of P-ERK, but also decreased the expression of calpain-1. It suggested that the ERK signaling pathway was the upstream regulator of calpain-1, the CSE might induce the calpain-1 expression via the ERK signaling pathway and resulted in the epithelial barrier disruption, and the 1,25D3 might inhibit the CSE damage effect in the airway epithelium by down-regulating the ERK signaling pathway. Recent studies have identified the positive role of Vitamin D in maintaining the integrity of epithelial barrier, however, there are still lacking of consensus on the mechanism of Vitamin D in the airway epithelium. Mulligan et al. demonstrated the 1,25VD3 reduced the proinflammatory effects of CS on human sinonasal epithelial cells [44], moreover, many researches have indentified that proinflammatory cytokines can lead to the epithelial barrier damage [45–47]. In our study, it is uncertain whether proinflammatory cytokines play a role in the 16HBE cells treated with CSE. Further researches are needed to answer this question. In general, the CSE induced the calpain-1 expression via the ERK signaling pathway that resulted in the epithelial barrier disruption by increasing the cleaving and the distribution anomalies of Ecadherin and b-catenin. The 1,25D3 could inhibit these pathological changes by down-regulating the ERK signaling pathway. However, several limitations needed to be noted in this study, we did not directly evaluate the role of 1,25D3 and the mechanisms by which it protects the epithelial barrier in vivo. Because of the difference between epithelial cells in vitro and in vivo, the results of this research may not be identical to the clinical situation. Therefore, we intend to further answer these questions by using samples from CSE model and primary human bronchial epithelial cells in the near future. References [1] S.R. White, Apoptosis and the airway epithelium, Allergy 12 (2011) 1–21 (Cairo). [2] M.I. Gomez, A. Prince, Airway epithelial cell signaling in response to bacterial pathogens, Pediatr. Pulmonol. 43 (2008) 11–19. [3] I.H. Heijink, P.M. Kies, H.F. Kauffman, Down-regulation of E-cadherin in human bronchial epithelial cells leads to epidermal growth factor receptor-dependent Th2 cell-promoting activity, Immunology 178 (2007) 7678–7685. [4] N.L. Mihai, R. Ivan, J. Walker, M.A. Kukuruzinska, Hypoglycosylated E-cadherin promotes the assembly of tight junctions through the recruitment of PP2A to adherens junctions, Exp. Cell Res. 316 (2010) 1871–1884. [5] C. Xiao, S.M. Puddicombe, S. Field, Defective epithelial barrier function in asthma, J. Allergy Clin. Immun. 128 (2011) 549–556. [6] I.H. Heijink, S.M. Brandenburg, D.S. Postma, A.J.M. Osterhout, Cigarette smoke impairs airway epithelial barrier function and cell–cell contact recovery, Eur. Respir. J. 39 (2012) 419–428. [7] R. Shaykhiev, F. Otaki, P. Bonsu, Cigarette smoking reprograms apical junctional complex molecular architecture in the human airway epithelium in vivo, Cell. Mol. Life Sci. 68 (2011) 877–892. [8] M.S. Rosanna, C.M. Marina, S.F. Nieves, V.G. Monica, E.M. Maria, Hyaluronan and Layilin mediate loss of airway epithelial barrier function induced by cigarette smoke by decreasing E-cadherin, J. Biol. Chem. 287 (2012) 42288– 42298. [9] M. Broekema, H.N.H. Ten, F. Volbeda, Airway epithelial changes in smokers but not in ex-smokers with asthma, Am. J. Respir. Crit. Care Med. 180 (2009) 1170– 1178. [10] B.A. Forey, A.J. Thornton, P.N. Lee, Systematic review with meta-analysis of the epidemiological evidence relating smoking to COPD, chronic bronchitis and emphysema, BMC Pulm. Med. 11 (2011) 1–61. [11] I. Proctor, V. Sharma, M. Khoshzaban, A. Winstanley, Does smoking kill? A study of death certification and smoking, J. Clin. Pathol. 65 (2012) 129–132. [12] R. Powell, D. Davidson, J. Divers, Genetic ancestry and the relationship of cigarette smoking to lung function and percent emphysema in four race/ethnic groups: a cross-sectional study, Thorax 68 (2013) 634–642. [13] K. Gangl, R. Reininger, D. Bernhard, Cigarette smoke facilitates allergen penetration across respiratory epithelium, Allergy 64 (2009) 398–405.
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