Idiopathic pulmonary fibrosis displays a condition in which the natural lung biological makeup is replaced by progress of lung structure aberrant remodeling, abundant accumulation of extracellular matrix and dramatic changes in the cell phenotype of both fibroblasts and alveolar epithelial cells [1
]. IPF, characterized by chronic, devastating and fibrotic interstitial pneumonia, contributes to the derangement of the respiratory function, eventually resulting in death. Therapeutic options are limited by a poor understanding of the underlying pathogenesis, although nintedanib and prifenidone could ameliorate the lung fibrosis to a certain extent [2
]. Herein, it is imminent to explore the underlying molecular basis of IPF and to furnish adequate theoretical foundations for the clinical treatment.
-acetyl-5-methoxytryptamine) is a neurohormone primarily secreted from the pineal gland [3
]. The functional effect of melatonin involves a wide spectrum of pathophysiological processes, including delaying senescence [5
], anti-tumorigenesis [6
], sleep melioration [7
] and immune adjustment [8
]. The function of melatonin is mainly mediated by its receptors, MT1 and MT2, classical G protein-coupled receptors [9
]. Furthermore, growing evidence suggests that melatonin exerts anti-fibrotic effects in the heart, kidney, lung, liver and other organs [4
]. During the pathogenesis of fibrosis, melatonin plays a protective role in initial injury to the organ, activation of effector cells (fibroblasts, myofibroblasts, and inflammatory cells), and accumulation of ECM. Martinez et al. [11
] demonstrated that melatonin inhibited the leptin-induced augmentation of Collagen1 and up-regulation of fibrotic markers, such as fibronectin, connective tissue growth factor (CTGF), and transforming growth factor β (TGF-β) in cardiac myofibroblasts. Das et al. [12
] found that melatonin mitigated hepatic fibrosis in mice by modulating hepatic stellate cell activation. Additionally, melatonin alleviated bleomycin (BLM)-induced pulmonary fibrosis via suppression of lipid peroxidation [13
]. Although an increasing number of studies have revealed the significant role of melatonin in the pathophysiological processes of pulmonary fibrosis, an exhaustive examination of the detailed mechanisms has not been presented.
Recent studies have shown that the Hippo signaling pathway plays critical roles in a variety of pivotal pathological processes, including organ growth control, cell proliferation, apoptosis, tissue regeneration and tumor suppression [14
]. Yes-associated protein (YAP), a key downstream effector of Hippo, has aroused great interest in studies of human diseases. Zhang et al. reported that OroxylinA ameliorated angiogenesis in liver fibrosis by inhibiting Hippo-YAP signaling [16
]. However, the effect of the Hippo pathway in IPF is still unclear. Recently, it was reported that GPCR signals participate in regulation of the Hippo signaling pathway [17
]. As mentioned above, melatonin signal transduction is mainly dependent on the function of MT1 and MT2. Therefore, we hypothesized that MT1 and MT2, which are classical GPCRs, activate the Hippo signal cascade to exert the anti-fibrotic function of melatonin during the process of IPF.
In this study, we found that melatonin attenuated TGF-β1-induced fibrogenesis in lung fibroblasts by activating the Hippo pathway and later promoting the nuclear translocation and increasing the inactivation and degradation of YAP1 in the cytoplasm. Furthermore, we also revealed that melatonin mitigated experimental pulmonary fibrosis in mice treated with BLM. This study elucidated the potential role of melatonin in the development of IPF, which may open therapeutic avenues for drug discovery and treatment of pulmonary fibrosis.
In the present study, we characterized the inhibitory effect of melatonin in BLM-induced pulmonary fibrosis and clarified the potent molecular mechanisms to provide an ideal explanation of this effect. Moreover, melatonin alleviated lung fibrosis in vivo and in vitro by binding to its receptor, and this anti-fibrotic effect was mitigated by up-regulation and functional activation of YAP1 (Figure 6
D). These findings suggest the therapeutic potential of melatonin for prevention and reversal of IPF.
Recently, emerging evidence has uncovered the significant role of melatonin in various pathophysiological processes [24
]. However, the role and underlying mechanism of melatonin in lung fibrosis are not well understood [4
], although some evidences have shown the effect of melatonin in lung fibrosis [25
]. Arslan et al. found that melatonin inhibited BLM-induced lung fibrosis in rats by suppressing oxidative stress [26
]. Consistent with this study, Yildirim et al. found that melatonin protected against lung fibrosis by suppressing catalase activity [13
]. A recent study from Zhao et al. revealed that melatonin attenuates BLM-induced lung fibrosis by inhibiting epithelial-mesenchymal transition via inhibition of endoplasmic reticulum stress [27
]. However, it is unclear whether melatonin exerts its anti-fibrotic role by regulating fibroblasts during lung fibrosis. As the most important effector cell, we found for the first time that melatonin inhibits the proliferation and migration of lung fibroblasts and mitigates fibroblast-myofibroblast transition during lung fibrosis. Meanwhile, melatonin has been shown to exert its biologic functions in a melatonin receptor-dependent or -independent manner [28
]. In this study, we found that luzindole, an inhibitor of melatonin receptors, strikingly blocked the anti-fibrotic effect of melatonin, suggesting that melatonin alleviate pulmonary fibrosis via a melatonin receptor-dependent pathway. Interestingly, the melatonin receptor agonists Neu-P11 [30
] and Tasimelteon [31
] exerted a function similar to that of melatonin. Therefore, whether Neu-P11 or Tasimelteon can participate in the process of lung fibrosis and exhibit a more significant effect merits further investigation.
Recently, an increasing number of evidences have demonstrated that YAP1 contribute to the initiation and evolution of fibrotic diseases [32
]. Whereas, subclinical function and mechanism of YAP1 in IPF have not been completely elucidated. Pan et al. discovered that Angiotensin II promote the collagen synthesis and cell proliferation in primary lung fibroblasts by increasing the YAP1 activity, ultimately leading to the progress of fibrosis [34
]. Liu et al. demonstrated that YAP/TAZ mediate mechanosignaling-induced fibroblast activation and lung fibrosis [35
]. Notably, in our previous studies, we illustrated that YAP1 contribute to the process of lung fibrosis. Overexpression of YAP1 enhanced the production of collagen and promoted the fibroblasts activation. In this study, we identified that forced expression of YAP1 abolished the inhibitory effect of melatonin in primary lung fibroblasts.
Recent studies have found that the Hippo pathway is regulated by G-protein-coupled receptor (GPCR) signaling [18
]. Yu et al found that GPCRs regulate the activation of the Hippo pathway [36
]. They proposed that G-coupled receptors stimulate Lats1/2 kinase and then increase YAP/TAZ phosphorylation. However, G12/13-, Gq/11-, and Gi/o-coupled receptors inhibited Lats1/2 kinase and promoted the dephosphorylation and nuclear localization of YAP1 [36
]. Consistent with this study, they also confirmed that activation of mutated Gq
promoted the occurrence and development of uveal melanoma by activating YAP [37
]. In addition, multiple studies have reported that other GPCRs, such as angiotensin II type 1 receptor (AT1R) and G protein-coupled estrogen receptor (GPER), fulfill their function in a variety of physiological and pathological conditions by disrupting the Hippo pathway [38
]. We supposed that melatonin inhibits lung fibrosis by regulating the activity and expression of the Hippo pathway through binding to melatonin receptors, which belong to the GPCR family. Interestingly, we found that melatonin inhibits the nuclear location and expression of YAP1, whereas this effect was nearly reversed by luzindole.
The present work revealed that melatonin alleviates lung fibrosis through YAP1 regulation. In a series of in vitro and in vivo experiments, we illuminated the critical role of YAP1 in the anti-fibrotic effect of melatonin. These findings indicate that administration of melatonin may be considered as a novel strategy for the treatment of lung fibrosis.
4. Materials and Methods
4.1. Experimental Pulmonary Fibrosis Model and Treatment
C57BL/6 mice (male; 6–8 weeks old) were obtained from Vital River Laboratory Animal Technology (Beijing, China). All animals were fed a chow diet and maintained in a 12-h light/12-h dark environment at 25 °C. In this work, the procedures for animal use were consistent with the regulations of the Ethics Committees of Harbin Medical University (No. 16520134, 1 March 2016) and conformed to the NRC Guide for the Care and Use of Laboratory Animals (2011, 8th ed.). To construct a pulmonary fibrosis model, bleomycin (BLM, Sigma-Aldrich Co., LLC, St. Louis, MO, USA) was injected intratracheally at a dose of 1.5 U/kg body weight.
Therefore, all the mice would be divided into three groups: Saline, BLM and BLM + Melatonin, there were six mice in each group. In BLM-induced pulmonary fibrosis animal model, melatonin (5mg/kg/d, Selleck, Shanghai, China) was intraperitoneally injected into mice for 21 days.
4.2. Isolation of Neonatal Mouse Lung Fibroblasts
Primary lung fibroblasts were isolated from the lungs of 1- to 3-day-old C57BL/6 mice. Neonatal mouse lung tissues were finely minced and placed together in 0.25% trypsin. After digestion for 90 min, the cell suspensions were centrifuged and resuspended in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS, BI), 100 U/mL penicillin and 100 μg/mL streptomycin. The cell mixture was seeded into culture flasks and incubated for 6 to 8 h to allow for preferential attachment of fibroblasts. Non-adherent and weakly attached cells were removed, and anchorage-dependent cells were incubated at 37 °C with 5% CO2.
4.3. Procedures for Cell Transfection
For cell transfection, lung fibroblasts were incubated with serum-free medium for 6 h. Lung fibroblasts were transfected with YAP1 using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). YAP1, was separately mixed with Opti-MEM® I Reduced Serum Medium (Gibco, NY, USA) for 5 min. Then, the two mixtures were combined and incubated at room temperature for 15 min. The combined mixture and Lipofectamine 2000 were added to the cell culture plate and incubated with cells at 37 °C with 5% CO2 for 36–48 h. In addition, in vitro experiments for lung fibroblasts transfection, Luzindole, a melatonin receptor inhibitor, was utilized at a dose of 100 nM/L.
4.4. Western Blot Analysis
Western blotting was performed as previously described [40
]. Total protein samples from snap-frozen lung tissues and cultured lung fibroblasts were prepared and transferred to nitrocellulose membranes (Pall Life Science, Waltham, MA, USA). The following primary antibodies were used; anti-Collagen 1 (14695-1-AP, Proteintech, Rosemont, IL, USA), anti-Fn1 (15613-1-AP, Proteintech), anti-YAP1 (13584-1-AP, Proteintech), anti-α-SMA (Ab7817, Abcam, Cambridge, MA, USA), anti-Twist1 (WL0109, Wanleibio, Dalian, China) and anti-β-actin (60008-1-AP, Proteintech), which was used as an internal control. The immunoreactivity was detected using an Odyssey Infrared Imaging System. The intensity of each of the blot bands was measured with Odyssey 3.0 software (Gene Company Limited, Hongkong, China).
4.5. Masson’s Trichrome Staining
The lungs of mice were rapidly dissected, immersed in 4% paraformaldehyde for a week and stained with Masson’s trichrome according to the manufacturer’s instructions to assess the degree of fibrosis. The fibrotic areas of tissues were examined under high power and scored in a total of 10 random fields per specimen. Digitized images were analyzed with Image-Pro-Plus 6.0 software. (Media Cybernetics, Inc., Rockville, MD, USA)
4.6. Immunohistochemistry Staining
Immunohistochemistry (IHC) staining was carried out as previously described [41
]. Mouse lung tissues were fixed with 4% paraformaldehyde for seven days, after being paraffin-embedded and sectioned. Primary antibodies against Fn1 and YAP1 were purchased from Proteintech (Rosemont, IL, USA). IHC was analyzed under a fluorescence microscope (DP80, Olympus, Tokyo, Japan).
4.7. SircolTM Soluble Collagen Assay
Collagen content assay was performed as previously described [19
]. Total samples were extracted from lung tissues of mice treated with Saline, BLM or BLM + Mel. According to the manufacturers’ instruction of SircolTM Soluble Collagen Assay (Biocolor, Northern Ireland, UK), the detection of soluble collagen was followed. The results were analysed by GraphPad Prism 5.0. (GraphPad Software, Inc., La Jolla, CA, USA)
4.8. Immunofluorescence Staining
Lung fibroblasts were treated with TGF-β1 (10 ng/mL, PeproTech, Rocky Hill, NJ, USA) or melatonin (400 μmol/L) or transfected with YAP1/sh-YAP1 (2 μg) for 40 h. Then, the cells were fixed for 30 min in 4% paraformaldehyde at room temperature and permeabilized with 0.2% Triton X-100 in PBS. After treatment with blocking buffer (PBS containing 50% goat serum albumin), the cells were incubated overnight at 4 °C with anti-α-SMA (1:200; Abcam) and anti-Vimentin (1:500; Cell Signalling Technology, Beverly, MA, USA). One day later, the cells were washed three times and incubated for 2 h at room temperature in the dark with FITC-conjugated goat anti-mouse/rabbit antibody (1:500, Alexa Fluor 488; Life Technologies, Waltham, MA, USA). Nuclei were stained with DAPI (Roche Molecular Biochemicals, Inc., Pleasanton, CA, USA) for 6 min. Immunofluorescence was analyzed under a fluorescence microscope (Nikon 80i, Tokyo, Japan).
4.9. Scratch Wound-Healing Assay
Cells were seeded on 6-well plates and incubated overnight at 37 °C at 5% CO2. Artificial wounds were made using 10-μL pipette tips (0 h), to generate a gap in the confluent cell layer. Cells were washed with PBS and fed with either control (serum-free) medium or medium containing different treatment combinations (TGF-β1, melatonin and luzindole). Phase-contrast microscopy images were observed at different time points using a Nikon TS100 microscope (Tokyo, Japan).
4.10. EdU Fluorescence Staining
Fluorescence staining with 5-ethynyl-2′-deoxyuridine (EdU) was used to detect newly synthesized DNA in lung fibroblasts after the corresponding treatments. All steps of the manufacturer′s instructions for a Cell-Light EdU DNA cell proliferation kit (RiboBio, Guangzhou, China) were followed. Images were obtained with a fluorescence microscope (Nikon 80i, Tokyo, Japan).
4.11. Quantitative RT-PCR
Total RNA samples were extracted from lung tissues of mice or cultured lung fibroblasts using TRIzol (Invitrogen, Carlsbad, CA, USA). As demonstrated in our previous work [40
], real-time RT-PCR was conducted on a 7500 Fast Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using Power SYBR®
Green Master Mix (Applied Biosystems). The comparative threshold cycle (Ct
) method was used to calculate the expression of fibrotic-related genes (Colα1, Colα3, et al.) β-Actin was used as the internal control.
4.12. Statistical Analysis
All data are presented as the mean ± SEM. One-way analysis of variance (ANOVA) followed by Dunnett’s test was used for multiple comparisons. A two-tailed value of p < 0.05 was considered to indicate a statistically significant difference. Statistical analyses were carried out using GraphPad Prism 5.0 and SPSS 14.0 software (Chicago, IL, USA).