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Biomedicine & Pharmacotherapy 89 (2017) 983–990
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Original article
Claudin-1 regulates pulmonary artery smooth muscle cell proliferation through the activation of ERK1/2 Xiandong Cheng, Yi Wang, Huilong Chen, Yongjian Xu, Weining Xiong, Tao Wang* Department of Respiratory and Critical Care Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030, China
A R T I C L E I N F O
Article history: Received 19 December 2016 Received in revised form 17 January 2017 Accepted 1 February 2017 Keywords: TNF-a Pulmonary arterial hypertension Claudin-1 NF-kB Proliferation ERK1/2
A B S T R A C T
Tumor necrosis factor alpha (TNF-a), a crucial inflammatory cytokine, is involved in the pathogenesis of pulmonary arterial hypertension (PAH). TNF-a can induce claudin-1 (CLDN1) expression and CLDN1 has been reported to be associated with the regulation of cellular functions including cell proliferation, migration. Thus, we aimed to explore whether CLDN1 participated in the etiology of PAH. Our study showed that CLDN1 expression was markedly increased in the lungs of rats with monocrotaline(MCT)induced PAH, especially in the pulmonary arterial smooth muscle sections. We also found that CLDN1 expression in primary human PASMCs was up-regulated by TNF-a, and the Nuclear factor-kB (NF-kB) inhibitor BAY 11-7082 suppressed CLDN1 up-regulation by TNF-a. CLDN1 overexpression by adenoviral transduction promoted PASMCs proliferation, while knockdown of CLDN1 by siRNA inhibited TNFa-induced cell proliferation. Mechanistic studies revealed that CLDN1 regulated human PASMC proliferation through the activation of ERK1/2. Together, our findings indicate that up-regulation of CLDN1 promotes PASMC proliferation contributing to pulmonary arterial remodeling in PAH. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Pulmonary arterial hypertension (PAH) is a life-threatening vascular diseasecharacterized by a progressive increase in pulmonary vascular resistance that can lead to right ventricular hypertrophy and failure [1]. Pulmonary vascular remodeling typified by increased proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs) is an important pathological feature of PAH [2,3]. Despite the spectrum of therapeutic options for PAH over the past decade, available therapies remain essentially palliative [4]. Therefore, a better understanding of key regulatory mechanism involved in PAH development will help to design more effective approaches for PAH treatment. Disruption of tight junctions (TJs) is associated with altered paracellular permeability and has been shown to be a hallmark of many pathologic states [5,6]. Claudins are crucial structural and functional components of TJs. Moreover, claudins have been found to be closely associated with intracellular signaling mechanisms [7,8]. Claudin-1 (CLDN1), a 23-kd transmembrane protein, has 4
* Corresponding author. E-mail address: [email protected] (T. Wang). http://dx.doi.org/10.1016/j.biopha.2017.02.063 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
transmembrane domains with 2 extracellular loops in epithelial cells and may serve as a promising target for diagnosis and therapy of human cancer [9]. CLDN1 can also induce epithelial-mesenchymal transition and regulate cell invasion and migration in human liver cell [8,10]. TNF-a can induce CLDN1 expression and CLDN1 subsequently mediates TNF-a-induced cell migration in a variety of cells, such as human gastric cancer cells and lung carcinoma cells [11,12]. Inflammatory mediators emerged as major contributing factors in the pathogenesis of pulmonary hypertension [13,14]. TNF-a is an important proinflammatory cytokine and elevated serum levels of TNF-a are observed in PAH patients [15]. Generally, monocrotaline (MCT) treatment is a well-established animal model for studying PAH, and pulmonary TNF-a expression is increased in rats with MCT-induced PAH [16]. Fujita and his colleagues found that CLDN1 is localized to both the nucleus and cytoplasm in ASM cells, while localized to the cell surface in epithelial cells. They also show that TNF-a can induce CLDN1 expression in airway smooth muscle (ASM) cells and CLDN1 can promote airway remodeling in asthmatic subjects [17]. Nevertheless, whether TNF-a regulates CLDN1 expression in PASMCs and the roles of CLDN1 in pulmonary vascular remodeling and PAH are largely unknown. In the present study, we found that CLDN1 was highly expressed in pulmonary vascular smooth muscle in MCT-induced
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experimental PAH rats. We demonstrated that CLDN1 regulates pulmonary vascular remodeling through regulating PASMC proliferation, which would provide a pivotal point for therapeutic intervention of PAH. 2. Materials and methods 2.1. Chemical reagents and antibodies BAY 11-7082, an inhibitor of IkB-a phosphorylation, was purchased from Selleck chemicals (Houston, TX, USA). U0126, an MEK1/2 inhibitor, was purchased from Selleck chemicals (Selleck, USA). An recombinant human TNF-a was purchased from Peprotech incorporation (Peprotech, Rocky Hill, NJ). Rabbit phospho-IkBa, IkBa, phospho-ERK1/2, ERK1/2 antibodies were obtained from Cell Signaling Technology (Danvers, USA). Rabbit CLDN1 antibody was purchased from Abcam (Cambridge, MA, USA). 2.2. Animal model of PAH Male Sprague-Dawley rats weighing between 210 and 250 g were purchased from the Center of Medical Experimental Animals of Hubei Province (Wuhan, China). Sprague-Dawley rats were randomly assigned to monocrotaline (MCT) group and control group (n = 6 each group). Rats in MCT group received an intraperitoneal injection of MCT (60 mg/kg). Vehicle (0.9% NaCl)injected rats served as controls. The animals were studied 3 weeks after MCT administration. All protocols employed in this study were reviewed and approved by the Animal Care and Use Committee of Tongji Medical College.
2.3. Immunofluorescence staining Tissue sections were deparaffinized, rehydrated and then blocked with goat serum. The slides were first probed with rabbit CLDN1 antibody (1:100) and mouse alpha-SMA antibody (1:100), and were then stained with Dylight 488 goat anti-mouse IgG (1:200) and Dylight 549 goat anti-rabbit IgG (1:200) (Abbkine, CA, USA). Nuclei were counterstained with 40 , 6-diamidino-2- phenylindole (DAPI). Finally, the slides were analyzed under an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan). 2.4. Cell culture and treatment Human primary PASMCs were obtained from CHI Scientific, Inc (Jiangyin, China) and then cultured in Dulbecco's modified Eagle medium (DMEM)/F12 (Hycolone, Logan, USA) containing 10% fetal bovine serum (Gibco, New York, USA) in a humidified incubator at 37 C with 5% CO2. BAY11-7082 was dissolved with dimethyl sulfoxide (DMSO). Human PASMCs were pretreated with either the NF-kB inhibitor BAY 11-7082 or DMSO for 1 h before TNF-a stimulation. 2.5. Adenoviral transduction of PASMCs Human PASMCs were transduced with empty adenovirus (Ad-control) or adenovirus encoding CLDN1 (Ad-CLDN1). In brief, human PASMCs were cultured at 50–60% confluency and then transduced with adenoviruses in complete medium. After 24 h of incubation, the virus-containing medium was replaced by serumfree medium and incubated for another 24 h for starvation. Cells were then used for subsequent investigations. The transduction
Fig. 1. CLDN1 expression was increased in the lung specimens of MCT-induced PAH rats. (A) Relative CLDN1 expression in the lung specimens of PAH rats was determined by western blot analysis (n = 6, * p < 0.05 vs. control). (B) Immunofluorescence staining analysis of CLDN1 expression in the lung sections of PAH models. Nuclei were stained with DAPI (blue), CLDN1 was stained with Dylight 549 (red), and a-SMA was stained with Dylight 488 (green). Magnification: 200.
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Fig. 2. CLDN1 was up-regulated by TNF-a in human PASMCs.qRT-PCR (A) and western blot (C) analysis of relative CLDN1 expression after human PASMCs were stimulated with different concentrations of TNF-a. (n = 3; * p < 0.05 vs. treatment with vehicle). qRT-PCR (B) and western blot (D) analysis of relative CLDN1 expression After treatment with TNF-a (10 ng/ml) for different time. (n = 3; * p < 0.05 vs. time at 0 h).
efficiency was examined by quantitative RT-PCR (qRT-PCR) and western blot analysis, respectively. 2.6. siRNA transfection siRNA targeting CLDN1 (si-CLDN1, Forward: 50 -GUCAAUGCCAGGUACGAAUTT-30 ; Reverse: 50 -AUUCGUACCUGGCAUUGACTT30 ) was purchased from Fulengen Co. (Guangzhou, China). Scrambled siRNA (si-control) was used as the negative control. For siRNA transfection, human PASMCs were grown in complete medium without antibiotics for 24 h, and were thereafter transfected with si-CLDN1 or si-control (final concentration of siRNA was 50 nM) using Lipofectamine2000 (Invitrogen, Carlsbad, CA) according to the procedure recommended by the manufacturer. After 6 h of incubation, the medium was replaced. The transfection efficiency was examined by qRT-PCR and western blot analysis, respectively. 2.7. qRT-PCR(quantitative RT-PCR) Total RNA was reversely transcribed with PrimeScript RT Master Mix (TaKaRa Biotechnology, Dalian, China), and cDNA was amplified using the SYBR Premix Ex Taq (TliRNaseH Plus) (TaKaRa) on 7500 PCR real time PCR system (Applied Biosystem, USA). The fold changes of expression values were finally quantified and calculated using the 2DDCt method. The qRT-PCR primers used for human CLDN1 and elongation factor 1a (EF1a) were as previously described [17]. Human EF-1a: Forward
50 -CTGAACCATCCAGGCCAAAT-30 , Reverse 50 -GCCGTGTGGCAATCCAAT-30 ; Human CLDN1: Forward 50 -CAGTCAATGCCAGGTACGAATTT-30 , Reverse 50 -AAGTAGGGCACCTCCCAGAAG-30 . EF-1a was used as an endogenous control. All amplifications were conducted in triplicate at least. 2.8. Western blot analysis The cultured human PASMCs were washed twice with ice-cold PBS and lysed in RIPA lysis buffer containing protease and phosphatase inhibitors on ice. The quantitation of total protein was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). An equal amount of total protein was loaded and separated by SDS-PAGE. The protein was then electrophoretically transferred to PVDF membranes (Millipore). After being blocked, the membranes were probed with rabbit CLDN1 (1:2000), phosphoIkBa (1:1000), IkBa (1:1000), ERK1/2 (1:1000), phospho-ERK1/2 (1:1000) antibodies and mouse GAPDH antibody(1:4000), and then incubated with HRP-conjugated secondary antibodies (1:5000) for 1 h, followed by ECL detection (Thermo Fisher Scientific). Relative levels of proteins were quantified using the ChemiDoc XRS+ with Image Lab software (Bio-Rad, USA). GAPDH was used as endogenous control. 2.9. Cell counting kit-8 assay (CCK-8) CCK-8 was obtained from Dojindo Laboratory (Kumamoto, Japan). In brief, human PASMCs were seeded in 96-well culture
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plates (3 103 cells/well) and allowed to adhere overnight. Then cells were transduced with adenoviruses or transfected with siRNA. After transfection, cells were stimulated with recombinant human TNF-a (10 ng/ml). Then CCK-8 (10 ml) was added to each well, and cells were incubated for another 2 h at 37 C. The absorbance was recorded at 570 nm. For each detect, the total procedure was repeated 3 times. 2.10. 5-bromo-2-deoxyuridine (BrdU) incorporation assay Human PASMCs were transduced with adenovirus or transfected with siRNA as described above. The medium was replaced and the cells were cultured for a further 3 days. BrdU was then added to the culture medium and the cells were cultured for an additional 4 h. The cells on coverslips were fixed with 4% formalin and BrdU was detected with Alexa Fluor 488-conjugated anti-BrdU antibody (Sigma, USA).
2.11. Statistical analysis Values from multiple experiments are expressed as means SEM, and statistical analysis was performed by Student’s t-test for comparisons between 2 groups, and one-way analysis of variance (ANOVA) for comparisons among groups (>2). The difference was considered significant at P < 0.05. Graph generation and statistical analysis were performed with GraphPad Prism5. 3. Results 3.1. CLDN1 expression was markedly increased in the lung specimens of PAH model To explore the role of CLDN1 in PAH in vivo, we constructed the PAH model using rats induced by MCT. We found that CLDN1 expression in the lungs of PAH experimental model was substantially increased compared with normal controls (Fig. 1A).
Fig. 3. TNF-a promoted the expression of CLDN1 through the NF-kB signaling pathway. (A) Human PASMCs were stimulated with TNF-a at different time points, and the levels of p-IkBa and IkBa were determined by western blot (n = 3; * p < 0.05 vs. time at 0 min). (B) Human PASMCs were stimulated with TNF-a for 5 min after pretreatment with BAY11-7082 for 1 h, and the levels of p-IkBa and IkBa were detected by western blot (n = 3; *p < 0.05 vs. DMSO plus TNF-a treatment). qRT-PCR (C) and western blot (D) analysis of relative CLDN1 expression after TNF-a stimulation following pretreatment with BAY11-7082 for 1 h. (n = 3; * p < 0.05 vs. DMSO plus TNF-a treatment).
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Fig. 4. CLDN1 promoted human PASMC proliferation. qRT-PCR(A) and western blot analysis(B) of CLDN1 expression at 48 h after transduction (n = 3; * p < 0.05 vs. Ad-control). Cell proliferation was determined using CCK-8 assay(C) andBrdU incorporation assay (D) at 72 h after transduction (n = 3; * p < 0.05 vs. Ad-control).
Moreover, immunofluorescence staining showed that CLDN1 expression was markedly increased in pulmonary arterial smooth muscle (PASM) sections of PAH rats compared with that seen in PASM sections of control rats (Fig. 1B). 3.2. CLDN1 was up-regulated by TNF-a in primary human PASMCs To investigate whether TNF-a can induce the expression of CLDN1 in human PASMCs, CLDN1 expression was determined after cells were cultured together with TNF-a. The results showed that the CLDN1 expression at mRNA level was up-regulated by TNF-a (Fig. 2A and B). Moreover, similar results were obtained at protein level of CLDN1 expression by Western blot analysis (Fig. 2C and D). 3.3. TNF-a induced the expression of CLDN1 through the NF-kB signaling pathway TNF-a induced the phosphorylation and degradation of IkB-a in human PASMCs that reached the maximal level at 5 min after stimulation (Fig. 3A), which was markedly inhibited by the NF-kB inhibitor BAY 11-7082 (Fig. 3B). BAY 11-7082 was used to further determine the molecular signaling pathways involved in TNFa-induced CLDN1 expression in human PASMCs.As shown in Fig. 3C and D, TNF-a-induced CLDN1 expression was markedly inhibited by BAY 11-7082 at both mRNA and protein levels. Above results suggest that increase of CLDN1 induced by TNF-a is mediated by the NF-kB signaling pathway.
3.4. CLDN1 overexpression promoted human PASMC proliferation To demonstrate the effects of CLDN1 on the proliferation of PASMC, human PASMCs were transduced with adenovirus expressing CLDN1 (Ad-CLDN1). The results showed that, compared with Ad-control group, CLDN1 expression was remarkably upregulated after transduction with Ad-CLDN1 at both mRNA level (Fig. 4A) and protein level (Fig. 4B). Moreover, CCK-8 assay (Fig. 4C) and BrdU incorporation assay (Fig. 4D) both showed that the proliferation ability of CLDN1-transduced human PASMCs was markedly elevated compared with that of the control cells. 3.5. CLDN1 knockdown decreased human PASMC proliferation We also used siRNA targeting CLDN1 (si-CLDN1) to knock down the expression of CLDN1, thus to further verify the effects of CLDN1 on PASMC proliferation. Our results showed that transfection of siCLDN1 markedly decreased its mRNA level (Fig. 5A) and protein level (Fig. 5B) at 48 h after transfection, indicating CLDN1 expression was successfully knocked down by siRNA. We have found that the CLDN1 expression in human PASMCs was low without the stimulation of TNF-a and was significantly increased after TNF-a treatment (Fig. 2). Besides, TNF-a also significantly promoted the proliferation of human PASMC at 72 h after stimulation (Fig. 5C and D), while we also found that CLDN1 knockdown significantly inhibited TNF-a-induced PASMC proliferation by CCK-8 (Fig. 5C) and BrdU incorporation assay (Fig. 5D).
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Fig. 5. CLDN1 knockdown decreased human PASMCs proliferation. qRT-PCR (A) and western blot analysis(B) of CLDN1 expression after siRNA-transfected PASMCs were treated with TNF-a (10 ng/ml) for 48 h. (n = 3; *p < 0.05 vs. si-control plus TNF-a treatment). Cell proliferation was measured by CCK-8 assay (C) and BrdUincorporation assay (D) after human PASMCs were stimulated with TNF-a for 72 h post-transfection. (n = 3; * p < 0.05 vs. control; # p < 0.05 vs. si-control plus TNF-a treatment).
3.6. CLDN1 promoted human PASMCs proliferation through the activation of ERK1/2 ERK1/2 mediates intracellular signaling associated with PASMC proliferation [18,19]. To investigate whether ERK1/2 activation was the possible mechanism involved in CLDN1-induced cell proliferation, human PASMCs were up-regulated by transduction with adenovirus or knockdown with siRNA, and then ERK1/2 phosphorylation was measured by western blot analysis. The results showed that ERK1/2 phosphorylation in CLDN knockdown cells was less than that in control siRNA-treated cells (Fig. 6A and B). Conversely, the significant increase of phspho-ERK1/2 was observed in Ad-CLDN1-treated cells compared with that in Adcontrol-treated cells (Fig. 6A and C). Meanwhile, treatment with the MEK1/2 inhibitor U0126 (10uM) markedly inhibited the phosphorylation of ERK1/2 induced by CLDN1 (Fig. 6A and C). Furthermore, treatment with U0126also significantly diminished human PASMC proliferation up-regulated by CLDN1 (Fig. 6D). 4. Discussion CLDN1 has been studied in variety of cells and diseases, however, the role of CLDN1 in human PASMCs and PAH has not been fully investigated. In the present study, we found that CLDN1 was highly increased in pulmonary vascular smooth muscle in PAH rats. More importantly, CLDN1 overexpression promoted human PASMC proliferation through the activation of ERK1/2, while knockdown of CLDN1 had opposite effect. The expression of CLDN1 in the lungs of PAH model or patients has not been clear. In our study, we showed the increased expression of CLDN1 in the lungs of rats with MCT-induced
experimental PAH and immunofluorescence staining further showed CLDN1 expression is increased in pulmonary vascular smooth muscle sections of PAH rats. These results indicate that CLDN1 may regulate PASMC proliferation and play a critical role in pulmonary vascular remodeling in PAH. Increasing evidence has suggested that inflammation plays an important role in the development of PAH in both human and experimental animal models [20]. TNF-a, a key inflammatory cytokine, has participated in pathological processes of many diseases, such as asthma and COPD [21,22]. Elevated serum levels of TNF-a were associated with survival in idiopathic and familial PAH patients [15]. Higher TNF-a level were also observed in COPD patients with pulmonary hypertension compared with patients without PAH [23]. Accordingly, it was also found that TNF-a plays an important role in PAH animal models. Overexpression of TNF-a has been shown to result in severe pulmonary hypertension and emphysema in mice [24,25]. Pulmonary TNF-a expression is increased in rats with MCT-induced PAH [16]. The TNF-a antagonist etanercept has been found to prevent and reverse MCT-induced pulmonary hypertension. In our study, we found that TNF-a treatment resulted in the up-regulation of CLDN1 in human PASMCs. Interestingly, there are potential NF-kB binding sites in the promoter region of human CLDN1 gene, which may be involved in TNF-a-induced CLDN1 expression [26]. To demonstrate it, human PASMCs were pretreated with the NF-kB inhibitor BAY 117082 before TNF-a stimulation. The results suggested that TNF-a induced the expression of CLDN1 in human PASMCs through the NF-kB signaling pathway. Our current findings confirm the relationship between TNF-a and CLDN1 in PASMCs. The proliferation of PASMC is considered an important pathological change leading to vascular remodeling in the
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Fig. 6. CLDN1 promoted human PASMC proliferation through the activation of ERK1/2. (A) p-ERK1/2 and ERK1/2 in human PASMCs were determined by western blot analysis. (B) After siRNA transfection, human PASMCs were treated with TNF-a (10 ng/ml) for 48 h. ERK1/2 phosphorylation was normalized to the basal level of si-control-transfected cells (n = 3; * p < 0.05 vs. si-control plus TNF-a treatment). (C) Human PASMCs were pretreated with or without U0126 (10 uM), and then transduced with Ad-CLDN1 for 48 h. ERK1/2 phosphorylation was normalized to the basal level of cells tansduced with Ad-control (n = 3; * p < 0.05 vs. Ad-control; #p < 0.05 vs. Ad-CLDN1). (D) As described in C, human PASMCs were transduced with Ad-CLDN1 for 72 h, cell proliferation was measured by BrdUincorporation assay (n = 3; * p < 0.05 vs. Ad-control; #p < 0.05 vs. AdCLDN1).
development of PAH. Several studies have provided evidence that CLDN1 expression is related to cell proliferation. Fujita et al. demonstrated that CLDN1 promoted the proliferation of human airway smooth muscle cells and exacerbated airway remodeling in asthmatic subjects [17]. In our study, CLDN1 overexpression promoted the proliferation of human PASMC and knockdown of CLDN significantly inhibited TNF-a-induced cell proliferation, implying that CLDN1 is a critical regulator of PASMC proliferation. Increased activation of ERK1/2 has been shown as a key mechanism involved in PASMC proliferation. It has been reported that CLDN1 can activate the c-Abl-ERK signaling pathway in human liver cells to induce epithelial-mesenchymal transition [8]. Similarly, we found that CLDN1 induced the phosphorylation of ERK1/2, while knockdown of CLDN1 had the opposite effect. Moreover, the MEK1/2 inhibitor U0126 suppressed the phosphorylation of ERK1/2 and human PASMC proliferation up-regulated by CLDN1. Although the role of CLDN1 in the regulation of PASMC proliferation has not been fully investigated, our findings suggest that the activation of ERK1/2 is involved in the process of CLDN1mediated human PASMC proliferation. In summary, our present study unveils a crucial role that CLDN1 plays in PASMC proliferation and pulmonary vascular remodeling in PAH. In addition, we demonstrate that TNF-a induces the expression of CLDN1 in human PASMCs through the NF-kB signaling pathway and CLDN1 overexpression promotes PASMC proliferation via the phosphorylation and activation of ERK1/2. The present observations further imply an important role that TNF-a plays in the pathogenesis of PAH and raise the possibility that CLDN1 may serve as a vital target for therapeutic intervention.
Conflicts of interest The authors declare no conflicts of interest. Funding This study was supported by National Natural Science Foundation of China (No. 81170049, 81470252, 81170021, 81570024). Author contributions Tao Wang, WeiningXiong, Yongjian Xu and Xiandong Cheng designed the experiments. Xiandong Cheng, Yi Wang and Huilong Chen performed the experiments. Tao Wang andXiandong Chenganalyzed the data and wrote the manuscript. All authors read and approved the manuscript. References [1] N.F. Voelkel, J. Gomez-Arroyo, A. Abbate, H.J. Bogaard, M.R. Nicolls, Pathobiology of pulmonary arterial hypertension and right ventricular failure, Eur. Respir. J. 40 (2012) 1555–1565. [2] M. Mandegar, Y.B. Fung, W. Huang, C.V. Remillard, L.J. Rubin, J.X.J. Yuan, Cellular and molecular mechanisms of pulmonary vascular remodeling: role in the development of pulmonary hypertension, Microvasc. Res. 68 (2004) 75–103. [3] P. Crosswhite, Z. Sun, Molecular mechanisms of pulmonary arterial remodeling, Mol. Med. 20 (2014) 191–201. [4] M. Humbert, H.A. Ghofrani, The molecular targets of approved treatments for pulmonary arterial hypertension, Thorax 71 (2016) 73–83.
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Original article
Claudin-1 regulates pulmonary artery smooth muscle cell proliferation through the activation of ERK1/2 Xiandong Cheng, Yi Wang, Huilong Chen, Yongjian Xu, Weining Xiong, Tao Wang* Department of Respiratory and Critical Care Medicine, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030, China
A R T I C L E I N F O
Article history: Received 19 December 2016 Received in revised form 17 January 2017 Accepted 1 February 2017 Keywords: TNF-a Pulmonary arterial hypertension Claudin-1 NF-kB Proliferation ERK1/2
A B S T R A C T
Tumor necrosis factor alpha (TNF-a), a crucial inflammatory cytokine, is involved in the pathogenesis of pulmonary arterial hypertension (PAH). TNF-a can induce claudin-1 (CLDN1) expression and CLDN1 has been reported to be associated with the regulation of cellular functions including cell proliferation, migration. Thus, we aimed to explore whether CLDN1 participated in the etiology of PAH. Our study showed that CLDN1 expression was markedly increased in the lungs of rats with monocrotaline(MCT)induced PAH, especially in the pulmonary arterial smooth muscle sections. We also found that CLDN1 expression in primary human PASMCs was up-regulated by TNF-a, and the Nuclear factor-kB (NF-kB) inhibitor BAY 11-7082 suppressed CLDN1 up-regulation by TNF-a. CLDN1 overexpression by adenoviral transduction promoted PASMCs proliferation, while knockdown of CLDN1 by siRNA inhibited TNFa-induced cell proliferation. Mechanistic studies revealed that CLDN1 regulated human PASMC proliferation through the activation of ERK1/2. Together, our findings indicate that up-regulation of CLDN1 promotes PASMC proliferation contributing to pulmonary arterial remodeling in PAH. © 2017 Elsevier Masson SAS. All rights reserved.
1. Introduction Pulmonary arterial hypertension (PAH) is a life-threatening vascular diseasecharacterized by a progressive increase in pulmonary vascular resistance that can lead to right ventricular hypertrophy and failure [1]. Pulmonary vascular remodeling typified by increased proliferation and apoptosis resistance of pulmonary artery smooth muscle cells (PASMCs) is an important pathological feature of PAH [2,3]. Despite the spectrum of therapeutic options for PAH over the past decade, available therapies remain essentially palliative [4]. Therefore, a better understanding of key regulatory mechanism involved in PAH development will help to design more effective approaches for PAH treatment. Disruption of tight junctions (TJs) is associated with altered paracellular permeability and has been shown to be a hallmark of many pathologic states [5,6]. Claudins are crucial structural and functional components of TJs. Moreover, claudins have been found to be closely associated with intracellular signaling mechanisms [7,8]. Claudin-1 (CLDN1), a 23-kd transmembrane protein, has 4
* Corresponding author. E-mail address: [email protected] (T. Wang). http://dx.doi.org/10.1016/j.biopha.2017.02.063 0753-3322/© 2017 Elsevier Masson SAS. All rights reserved.
transmembrane domains with 2 extracellular loops in epithelial cells and may serve as a promising target for diagnosis and therapy of human cancer [9]. CLDN1 can also induce epithelial-mesenchymal transition and regulate cell invasion and migration in human liver cell [8,10]. TNF-a can induce CLDN1 expression and CLDN1 subsequently mediates TNF-a-induced cell migration in a variety of cells, such as human gastric cancer cells and lung carcinoma cells [11,12]. Inflammatory mediators emerged as major contributing factors in the pathogenesis of pulmonary hypertension [13,14]. TNF-a is an important proinflammatory cytokine and elevated serum levels of TNF-a are observed in PAH patients [15]. Generally, monocrotaline (MCT) treatment is a well-established animal model for studying PAH, and pulmonary TNF-a expression is increased in rats with MCT-induced PAH [16]. Fujita and his colleagues found that CLDN1 is localized to both the nucleus and cytoplasm in ASM cells, while localized to the cell surface in epithelial cells. They also show that TNF-a can induce CLDN1 expression in airway smooth muscle (ASM) cells and CLDN1 can promote airway remodeling in asthmatic subjects [17]. Nevertheless, whether TNF-a regulates CLDN1 expression in PASMCs and the roles of CLDN1 in pulmonary vascular remodeling and PAH are largely unknown. In the present study, we found that CLDN1 was highly expressed in pulmonary vascular smooth muscle in MCT-induced
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experimental PAH rats. We demonstrated that CLDN1 regulates pulmonary vascular remodeling through regulating PASMC proliferation, which would provide a pivotal point for therapeutic intervention of PAH. 2. Materials and methods 2.1. Chemical reagents and antibodies BAY 11-7082, an inhibitor of IkB-a phosphorylation, was purchased from Selleck chemicals (Houston, TX, USA). U0126, an MEK1/2 inhibitor, was purchased from Selleck chemicals (Selleck, USA). An recombinant human TNF-a was purchased from Peprotech incorporation (Peprotech, Rocky Hill, NJ). Rabbit phospho-IkBa, IkBa, phospho-ERK1/2, ERK1/2 antibodies were obtained from Cell Signaling Technology (Danvers, USA). Rabbit CLDN1 antibody was purchased from Abcam (Cambridge, MA, USA). 2.2. Animal model of PAH Male Sprague-Dawley rats weighing between 210 and 250 g were purchased from the Center of Medical Experimental Animals of Hubei Province (Wuhan, China). Sprague-Dawley rats were randomly assigned to monocrotaline (MCT) group and control group (n = 6 each group). Rats in MCT group received an intraperitoneal injection of MCT (60 mg/kg). Vehicle (0.9% NaCl)injected rats served as controls. The animals were studied 3 weeks after MCT administration. All protocols employed in this study were reviewed and approved by the Animal Care and Use Committee of Tongji Medical College.
2.3. Immunofluorescence staining Tissue sections were deparaffinized, rehydrated and then blocked with goat serum. The slides were first probed with rabbit CLDN1 antibody (1:100) and mouse alpha-SMA antibody (1:100), and were then stained with Dylight 488 goat anti-mouse IgG (1:200) and Dylight 549 goat anti-rabbit IgG (1:200) (Abbkine, CA, USA). Nuclei were counterstained with 40 , 6-diamidino-2- phenylindole (DAPI). Finally, the slides were analyzed under an Olympus BX51 fluorescence microscope (Olympus, Tokyo, Japan). 2.4. Cell culture and treatment Human primary PASMCs were obtained from CHI Scientific, Inc (Jiangyin, China) and then cultured in Dulbecco's modified Eagle medium (DMEM)/F12 (Hycolone, Logan, USA) containing 10% fetal bovine serum (Gibco, New York, USA) in a humidified incubator at 37 C with 5% CO2. BAY11-7082 was dissolved with dimethyl sulfoxide (DMSO). Human PASMCs were pretreated with either the NF-kB inhibitor BAY 11-7082 or DMSO for 1 h before TNF-a stimulation. 2.5. Adenoviral transduction of PASMCs Human PASMCs were transduced with empty adenovirus (Ad-control) or adenovirus encoding CLDN1 (Ad-CLDN1). In brief, human PASMCs were cultured at 50–60% confluency and then transduced with adenoviruses in complete medium. After 24 h of incubation, the virus-containing medium was replaced by serumfree medium and incubated for another 24 h for starvation. Cells were then used for subsequent investigations. The transduction
Fig. 1. CLDN1 expression was increased in the lung specimens of MCT-induced PAH rats. (A) Relative CLDN1 expression in the lung specimens of PAH rats was determined by western blot analysis (n = 6, * p < 0.05 vs. control). (B) Immunofluorescence staining analysis of CLDN1 expression in the lung sections of PAH models. Nuclei were stained with DAPI (blue), CLDN1 was stained with Dylight 549 (red), and a-SMA was stained with Dylight 488 (green). Magnification: 200.
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Fig. 2. CLDN1 was up-regulated by TNF-a in human PASMCs.qRT-PCR (A) and western blot (C) analysis of relative CLDN1 expression after human PASMCs were stimulated with different concentrations of TNF-a. (n = 3; * p < 0.05 vs. treatment with vehicle). qRT-PCR (B) and western blot (D) analysis of relative CLDN1 expression After treatment with TNF-a (10 ng/ml) for different time. (n = 3; * p < 0.05 vs. time at 0 h).
efficiency was examined by quantitative RT-PCR (qRT-PCR) and western blot analysis, respectively. 2.6. siRNA transfection siRNA targeting CLDN1 (si-CLDN1, Forward: 50 -GUCAAUGCCAGGUACGAAUTT-30 ; Reverse: 50 -AUUCGUACCUGGCAUUGACTT30 ) was purchased from Fulengen Co. (Guangzhou, China). Scrambled siRNA (si-control) was used as the negative control. For siRNA transfection, human PASMCs were grown in complete medium without antibiotics for 24 h, and were thereafter transfected with si-CLDN1 or si-control (final concentration of siRNA was 50 nM) using Lipofectamine2000 (Invitrogen, Carlsbad, CA) according to the procedure recommended by the manufacturer. After 6 h of incubation, the medium was replaced. The transfection efficiency was examined by qRT-PCR and western blot analysis, respectively. 2.7. qRT-PCR(quantitative RT-PCR) Total RNA was reversely transcribed with PrimeScript RT Master Mix (TaKaRa Biotechnology, Dalian, China), and cDNA was amplified using the SYBR Premix Ex Taq (TliRNaseH Plus) (TaKaRa) on 7500 PCR real time PCR system (Applied Biosystem, USA). The fold changes of expression values were finally quantified and calculated using the 2DDCt method. The qRT-PCR primers used for human CLDN1 and elongation factor 1a (EF1a) were as previously described [17]. Human EF-1a: Forward
50 -CTGAACCATCCAGGCCAAAT-30 , Reverse 50 -GCCGTGTGGCAATCCAAT-30 ; Human CLDN1: Forward 50 -CAGTCAATGCCAGGTACGAATTT-30 , Reverse 50 -AAGTAGGGCACCTCCCAGAAG-30 . EF-1a was used as an endogenous control. All amplifications were conducted in triplicate at least. 2.8. Western blot analysis The cultured human PASMCs were washed twice with ice-cold PBS and lysed in RIPA lysis buffer containing protease and phosphatase inhibitors on ice. The quantitation of total protein was determined using a BCA protein assay kit (Pierce, Rockford, IL, USA). An equal amount of total protein was loaded and separated by SDS-PAGE. The protein was then electrophoretically transferred to PVDF membranes (Millipore). After being blocked, the membranes were probed with rabbit CLDN1 (1:2000), phosphoIkBa (1:1000), IkBa (1:1000), ERK1/2 (1:1000), phospho-ERK1/2 (1:1000) antibodies and mouse GAPDH antibody(1:4000), and then incubated with HRP-conjugated secondary antibodies (1:5000) for 1 h, followed by ECL detection (Thermo Fisher Scientific). Relative levels of proteins were quantified using the ChemiDoc XRS+ with Image Lab software (Bio-Rad, USA). GAPDH was used as endogenous control. 2.9. Cell counting kit-8 assay (CCK-8) CCK-8 was obtained from Dojindo Laboratory (Kumamoto, Japan). In brief, human PASMCs were seeded in 96-well culture
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plates (3 103 cells/well) and allowed to adhere overnight. Then cells were transduced with adenoviruses or transfected with siRNA. After transfection, cells were stimulated with recombinant human TNF-a (10 ng/ml). Then CCK-8 (10 ml) was added to each well, and cells were incubated for another 2 h at 37 C. The absorbance was recorded at 570 nm. For each detect, the total procedure was repeated 3 times. 2.10. 5-bromo-2-deoxyuridine (BrdU) incorporation assay Human PASMCs were transduced with adenovirus or transfected with siRNA as described above. The medium was replaced and the cells were cultured for a further 3 days. BrdU was then added to the culture medium and the cells were cultured for an additional 4 h. The cells on coverslips were fixed with 4% formalin and BrdU was detected with Alexa Fluor 488-conjugated anti-BrdU antibody (Sigma, USA).
2.11. Statistical analysis Values from multiple experiments are expressed as means SEM, and statistical analysis was performed by Student’s t-test for comparisons between 2 groups, and one-way analysis of variance (ANOVA) for comparisons among groups (>2). The difference was considered significant at P < 0.05. Graph generation and statistical analysis were performed with GraphPad Prism5. 3. Results 3.1. CLDN1 expression was markedly increased in the lung specimens of PAH model To explore the role of CLDN1 in PAH in vivo, we constructed the PAH model using rats induced by MCT. We found that CLDN1 expression in the lungs of PAH experimental model was substantially increased compared with normal controls (Fig. 1A).
Fig. 3. TNF-a promoted the expression of CLDN1 through the NF-kB signaling pathway. (A) Human PASMCs were stimulated with TNF-a at different time points, and the levels of p-IkBa and IkBa were determined by western blot (n = 3; * p < 0.05 vs. time at 0 min). (B) Human PASMCs were stimulated with TNF-a for 5 min after pretreatment with BAY11-7082 for 1 h, and the levels of p-IkBa and IkBa were detected by western blot (n = 3; *p < 0.05 vs. DMSO plus TNF-a treatment). qRT-PCR (C) and western blot (D) analysis of relative CLDN1 expression after TNF-a stimulation following pretreatment with BAY11-7082 for 1 h. (n = 3; * p < 0.05 vs. DMSO plus TNF-a treatment).
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Fig. 4. CLDN1 promoted human PASMC proliferation. qRT-PCR(A) and western blot analysis(B) of CLDN1 expression at 48 h after transduction (n = 3; * p < 0.05 vs. Ad-control). Cell proliferation was determined using CCK-8 assay(C) andBrdU incorporation assay (D) at 72 h after transduction (n = 3; * p < 0.05 vs. Ad-control).
Moreover, immunofluorescence staining showed that CLDN1 expression was markedly increased in pulmonary arterial smooth muscle (PASM) sections of PAH rats compared with that seen in PASM sections of control rats (Fig. 1B). 3.2. CLDN1 was up-regulated by TNF-a in primary human PASMCs To investigate whether TNF-a can induce the expression of CLDN1 in human PASMCs, CLDN1 expression was determined after cells were cultured together with TNF-a. The results showed that the CLDN1 expression at mRNA level was up-regulated by TNF-a (Fig. 2A and B). Moreover, similar results were obtained at protein level of CLDN1 expression by Western blot analysis (Fig. 2C and D). 3.3. TNF-a induced the expression of CLDN1 through the NF-kB signaling pathway TNF-a induced the phosphorylation and degradation of IkB-a in human PASMCs that reached the maximal level at 5 min after stimulation (Fig. 3A), which was markedly inhibited by the NF-kB inhibitor BAY 11-7082 (Fig. 3B). BAY 11-7082 was used to further determine the molecular signaling pathways involved in TNFa-induced CLDN1 expression in human PASMCs.As shown in Fig. 3C and D, TNF-a-induced CLDN1 expression was markedly inhibited by BAY 11-7082 at both mRNA and protein levels. Above results suggest that increase of CLDN1 induced by TNF-a is mediated by the NF-kB signaling pathway.
3.4. CLDN1 overexpression promoted human PASMC proliferation To demonstrate the effects of CLDN1 on the proliferation of PASMC, human PASMCs were transduced with adenovirus expressing CLDN1 (Ad-CLDN1). The results showed that, compared with Ad-control group, CLDN1 expression was remarkably upregulated after transduction with Ad-CLDN1 at both mRNA level (Fig. 4A) and protein level (Fig. 4B). Moreover, CCK-8 assay (Fig. 4C) and BrdU incorporation assay (Fig. 4D) both showed that the proliferation ability of CLDN1-transduced human PASMCs was markedly elevated compared with that of the control cells. 3.5. CLDN1 knockdown decreased human PASMC proliferation We also used siRNA targeting CLDN1 (si-CLDN1) to knock down the expression of CLDN1, thus to further verify the effects of CLDN1 on PASMC proliferation. Our results showed that transfection of siCLDN1 markedly decreased its mRNA level (Fig. 5A) and protein level (Fig. 5B) at 48 h after transfection, indicating CLDN1 expression was successfully knocked down by siRNA. We have found that the CLDN1 expression in human PASMCs was low without the stimulation of TNF-a and was significantly increased after TNF-a treatment (Fig. 2). Besides, TNF-a also significantly promoted the proliferation of human PASMC at 72 h after stimulation (Fig. 5C and D), while we also found that CLDN1 knockdown significantly inhibited TNF-a-induced PASMC proliferation by CCK-8 (Fig. 5C) and BrdU incorporation assay (Fig. 5D).
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Fig. 5. CLDN1 knockdown decreased human PASMCs proliferation. qRT-PCR (A) and western blot analysis(B) of CLDN1 expression after siRNA-transfected PASMCs were treated with TNF-a (10 ng/ml) for 48 h. (n = 3; *p < 0.05 vs. si-control plus TNF-a treatment). Cell proliferation was measured by CCK-8 assay (C) and BrdUincorporation assay (D) after human PASMCs were stimulated with TNF-a for 72 h post-transfection. (n = 3; * p < 0.05 vs. control; # p < 0.05 vs. si-control plus TNF-a treatment).
3.6. CLDN1 promoted human PASMCs proliferation through the activation of ERK1/2 ERK1/2 mediates intracellular signaling associated with PASMC proliferation [18,19]. To investigate whether ERK1/2 activation was the possible mechanism involved in CLDN1-induced cell proliferation, human PASMCs were up-regulated by transduction with adenovirus or knockdown with siRNA, and then ERK1/2 phosphorylation was measured by western blot analysis. The results showed that ERK1/2 phosphorylation in CLDN knockdown cells was less than that in control siRNA-treated cells (Fig. 6A and B). Conversely, the significant increase of phspho-ERK1/2 was observed in Ad-CLDN1-treated cells compared with that in Adcontrol-treated cells (Fig. 6A and C). Meanwhile, treatment with the MEK1/2 inhibitor U0126 (10uM) markedly inhibited the phosphorylation of ERK1/2 induced by CLDN1 (Fig. 6A and C). Furthermore, treatment with U0126also significantly diminished human PASMC proliferation up-regulated by CLDN1 (Fig. 6D). 4. Discussion CLDN1 has been studied in variety of cells and diseases, however, the role of CLDN1 in human PASMCs and PAH has not been fully investigated. In the present study, we found that CLDN1 was highly increased in pulmonary vascular smooth muscle in PAH rats. More importantly, CLDN1 overexpression promoted human PASMC proliferation through the activation of ERK1/2, while knockdown of CLDN1 had opposite effect. The expression of CLDN1 in the lungs of PAH model or patients has not been clear. In our study, we showed the increased expression of CLDN1 in the lungs of rats with MCT-induced
experimental PAH and immunofluorescence staining further showed CLDN1 expression is increased in pulmonary vascular smooth muscle sections of PAH rats. These results indicate that CLDN1 may regulate PASMC proliferation and play a critical role in pulmonary vascular remodeling in PAH. Increasing evidence has suggested that inflammation plays an important role in the development of PAH in both human and experimental animal models [20]. TNF-a, a key inflammatory cytokine, has participated in pathological processes of many diseases, such as asthma and COPD [21,22]. Elevated serum levels of TNF-a were associated with survival in idiopathic and familial PAH patients [15]. Higher TNF-a level were also observed in COPD patients with pulmonary hypertension compared with patients without PAH [23]. Accordingly, it was also found that TNF-a plays an important role in PAH animal models. Overexpression of TNF-a has been shown to result in severe pulmonary hypertension and emphysema in mice [24,25]. Pulmonary TNF-a expression is increased in rats with MCT-induced PAH [16]. The TNF-a antagonist etanercept has been found to prevent and reverse MCT-induced pulmonary hypertension. In our study, we found that TNF-a treatment resulted in the up-regulation of CLDN1 in human PASMCs. Interestingly, there are potential NF-kB binding sites in the promoter region of human CLDN1 gene, which may be involved in TNF-a-induced CLDN1 expression [26]. To demonstrate it, human PASMCs were pretreated with the NF-kB inhibitor BAY 117082 before TNF-a stimulation. The results suggested that TNF-a induced the expression of CLDN1 in human PASMCs through the NF-kB signaling pathway. Our current findings confirm the relationship between TNF-a and CLDN1 in PASMCs. The proliferation of PASMC is considered an important pathological change leading to vascular remodeling in the
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Fig. 6. CLDN1 promoted human PASMC proliferation through the activation of ERK1/2. (A) p-ERK1/2 and ERK1/2 in human PASMCs were determined by western blot analysis. (B) After siRNA transfection, human PASMCs were treated with TNF-a (10 ng/ml) for 48 h. ERK1/2 phosphorylation was normalized to the basal level of si-control-transfected cells (n = 3; * p < 0.05 vs. si-control plus TNF-a treatment). (C) Human PASMCs were pretreated with or without U0126 (10 uM), and then transduced with Ad-CLDN1 for 48 h. ERK1/2 phosphorylation was normalized to the basal level of cells tansduced with Ad-control (n = 3; * p < 0.05 vs. Ad-control; #p < 0.05 vs. Ad-CLDN1). (D) As described in C, human PASMCs were transduced with Ad-CLDN1 for 72 h, cell proliferation was measured by BrdUincorporation assay (n = 3; * p < 0.05 vs. Ad-control; #p < 0.05 vs. AdCLDN1).
development of PAH. Several studies have provided evidence that CLDN1 expression is related to cell proliferation. Fujita et al. demonstrated that CLDN1 promoted the proliferation of human airway smooth muscle cells and exacerbated airway remodeling in asthmatic subjects [17]. In our study, CLDN1 overexpression promoted the proliferation of human PASMC and knockdown of CLDN significantly inhibited TNF-a-induced cell proliferation, implying that CLDN1 is a critical regulator of PASMC proliferation. Increased activation of ERK1/2 has been shown as a key mechanism involved in PASMC proliferation. It has been reported that CLDN1 can activate the c-Abl-ERK signaling pathway in human liver cells to induce epithelial-mesenchymal transition [8]. Similarly, we found that CLDN1 induced the phosphorylation of ERK1/2, while knockdown of CLDN1 had the opposite effect. Moreover, the MEK1/2 inhibitor U0126 suppressed the phosphorylation of ERK1/2 and human PASMC proliferation up-regulated by CLDN1. Although the role of CLDN1 in the regulation of PASMC proliferation has not been fully investigated, our findings suggest that the activation of ERK1/2 is involved in the process of CLDN1mediated human PASMC proliferation. In summary, our present study unveils a crucial role that CLDN1 plays in PASMC proliferation and pulmonary vascular remodeling in PAH. In addition, we demonstrate that TNF-a induces the expression of CLDN1 in human PASMCs through the NF-kB signaling pathway and CLDN1 overexpression promotes PASMC proliferation via the phosphorylation and activation of ERK1/2. The present observations further imply an important role that TNF-a plays in the pathogenesis of PAH and raise the possibility that CLDN1 may serve as a vital target for therapeutic intervention.
Conflicts of interest The authors declare no conflicts of interest. Funding This study was supported by National Natural Science Foundation of China (No. 81170049, 81470252, 81170021, 81570024). Author contributions Tao Wang, WeiningXiong, Yongjian Xu and Xiandong Cheng designed the experiments. Xiandong Cheng, Yi Wang and Huilong Chen performed the experiments. Tao Wang andXiandong Chenganalyzed the data and wrote the manuscript. All authors read and approved the manuscript. References [1] N.F. Voelkel, J. Gomez-Arroyo, A. Abbate, H.J. Bogaard, M.R. Nicolls, Pathobiology of pulmonary arterial hypertension and right ventricular failure, Eur. Respir. J. 40 (2012) 1555–1565. [2] M. Mandegar, Y.B. Fung, W. Huang, C.V. Remillard, L.J. Rubin, J.X.J. Yuan, Cellular and molecular mechanisms of pulmonary vascular remodeling: role in the development of pulmonary hypertension, Microvasc. Res. 68 (2004) 75–103. [3] P. Crosswhite, Z. Sun, Molecular mechanisms of pulmonary arterial remodeling, Mol. Med. 20 (2014) 191–201. [4] M. Humbert, H.A. Ghofrani, The molecular targets of approved treatments for pulmonary arterial hypertension, Thorax 71 (2016) 73–83.
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