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Caspase-8 and Caspase-9 Functioned Differently at Different Stages of the Cyclic Stretch-Induced Apoptosis in Human Periodontal Ligament Cells

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Caspase-8 and Caspase-9 Functioned Differently at Different Stages of the Cyclic Stretch-Induced Apoptosis in Human Periodontal Ligament Cells RESEARCH ARTICLE Caspase-8 and Caspase-9 Functioned Differently at Different Stages of the Cyclic Stretch-Induced Apoptosis in Human Periodontal Ligament Cells Yaqin Wu1,2☯, Dan Zhao1,2☯, Jiabao Zhuang1,2, Fuqiang Zhang1,2, Chun Xu1,2* 1 Department of Prosthodontics, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China, 2 Shanghai Key Laboratory of Stomatology & Shanghai Research Institute of Stomatology, a11111 Shanghai, China ☯ These authors contributed equally to this work. * [email protected] Abstract OPEN ACCESS Citation: Wu Y, Zhao D, Zhuang J, Zhang F, Xu C Background (2016) Caspase-8 and Caspase-9 Functioned Differently at Different Stages of the Cyclic Stretch- Human periodontal ligament (PDL) cells underwent apoptosis after mechanical stretch load- Induced Apoptosis in Human Periodontal Ligament ing. However, the exact signalling pathway remains unknown. This study aimed to elucidate Cells. PLoS ONE 11(12): e0168268. doi:10.1371/ how the apoptotic caspases functioned in the cyclic stretch-induced apoptosis in human journal.pone.0168268 PDL cells. Editor: Ferenc Gallyas, Jr., University of PECS Medical School, HUNGARY Received: October 1, 2016 Materials and Methods Accepted: November 29, 2016 In the present study, 20% cyclic stretch was selected to load the cells for 6 or 24 h. The fol- lowing parameters were analyzed: apoptotic rates, the protein levels of caspase-3, -7, -8 Published: December 12, 2016 and -9 and the activities of caspase-8 and -9. Subsequently, the influences of caspase-8 Copyright: © 2016 Wu et al. This is an open access and caspase-9 inhibitors on the apoptotic rate and the protein level of the activated cas- article distributed under the terms of the Creative Commons Attribution License, which permits pase-3 were assessed as well. unrestricted use, distribution, and reproduction in any medium, provided the original author and Results source are credited. The apoptotic rates increased in response to cyclic stretch, but the cells entered different Data Availability Statement: All relevant data are within the paper and its Supporting Information apoptotic stages after 6 and 24 h stretches. Caspase-3, -7, -8 and -9 were all activated after files. stretch loading. The stretch-induced apoptosis and the protein level of the activated cas- Funding: This work was supported by National pase-3 were inhibited after inhibiting both caspase-8 and caspase-9 in both 6 and 24 h Natural Science Foundation of China (grant stretched cells and after inhibiting caspase-9 in 24 h stretched cells. numbers 31470903, 31270991, 30900282); Shanghai Pujiang Program (grant number 13PJD021); Science and Technology Commission Conclusion of Shanghai (grant numbers 10QA1404200, Caspase-8 and -9 functioned differently at different apoptotic stages in human PDL cells 08411961500, 07ZR14070); Shanghai Summit & Plateau Disciplines; Shanghai Leading Academic after cyclic stretch. Discipline Project (grant numbers S30206-sms02, PLOS ONE | DOI:10.1371/journal.pone.0168268 December 12, 2016 1 / 15 Caspase-8 and Caspase-9 in Cyclic Stretch-Induced Apoptosis T0202). The funders had no role in study design, data collection and analysis, decision to publish, or Introduction preparation of the manuscript. Periodontal ligament (PDL) is a fibrous structure interfacing the tooth with its surrounding Competing Interests: The authors have declared bone [1]. PDL cells sensitively perceive mechanical stimuli from normal mastication, occlusal that no competing interests exist. trauma or orthodontic forces [2]. There are growing evidences showing that the maintenance of periodontal health requires for physiological mechanical stimulations [3], while aberrant mechanical stimulations can be associated with pathological changes and cell death in peri- odontal tissue [4]. Apoptosis has been defined as a type of programmed cell death [2, 5], and can be conducted in two classic signalling pathways: the extrinsic pathway and the intrinsic pathway [6]. The extrinsic pathway is a caspase-dependent pathway and is extensively used to describe the pro- cess of apoptosis induced by extracellular stimulations which are recognized and propagated by the specific membrane receptors. The intrinsic pathway involves a mitochondrion-centered control mechanism via either a caspase-dependent pathway or a caspase-independent path- way, and can be triggered by multiple intracellular stimulations [7]. The extrinsic pathway is mediated by caspase-8 while the intrinsic pathway can be initiated through caspase-9, and both pathways trigger apoptosis through the cleavage of the downstream executioner proteins, caspase-3 and -7 [8]. Mechanical stress can lead to a series of biological responses in periodontal ligament, including alterations of cellular behavior, gene expression and protein production [9, 10]. Sev- eral studies revealed cell apoptosis in PDL under orthodontic forces in animal experiments in vivo [11, 12]. In our previous studies, cell death via apoptosis was also found in human PDL cells in response to cyclic stretching [13, 14]. The early and the late apoptosis were induced notably after 6 and 24 h 20% cyclic stretch loading, respectively [13, 14]. Wang et al. also reported that apoptosis of certain extent was induced by 20% cyclic stretching force, with a higher apoptotic rate after 24 h stretch than that after 6 h stretch [15]. Meanwhile, our recent study uncovered that apoptosis in human PDL cells after cyclic stretch occurred through the activation of caspase-3 via caspase-9 [14]. The differential expression of some force-sensing genes with regard to apoptosis, such as FAS and so on, was also detected [16]. Results of previous researches indicated that different duration of cyclic stretches induced apoptosis of different stages in human PDL cells, and the stretch-induced apoptosis involved a complicated signalling pathway. However, the precise mechanism underlying the stretch- induced apoptotic pathway in human PDL cells is unknown. The present study aimed to pro- vide novel insight into the roles of caspase-8 and caspase-9 at different stages of the stretch- induced apoptosis in in vitro cultured human PDL cells. We hope the findings in this study will help us better understand the mechanism of the force-driven apoptosis in PDL cells. Materials and Methods Cell culture The experimental protocol is approved by the Ethics Committee of Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. The relevant Judgement's reference num- ber: [2008]17. Human PDL tissues were harvested from extracted premolars of teenagers (11± 13 years old) undergoing orthodontic treatments. Written informed consents were obtained from their parents. Human PDL cells were prepared in vitro according to the protocols described previously [17, 18]. High glucose DMEM (HyClone, Logan, Utah, USA), FBS (Gibco, Carlsbad, CA, USA) and antibiotics (HyClone) were applied to cell culture. Pieces of human PDL tissues were incubated in the DMEM supplemented with 20% (v/v) FBS and five- fold reinforced antibiotics (500 U/mL penicillin and 500 μg/mL streptomycin). When cells PLOS ONE | DOI:10.1371/journal.pone.0168268 December 12, 2016 2 / 15 Caspase-8 and Caspase-9 in Cyclic Stretch-Induced Apoptosis grew out from the tissue pieces and reached 80%-90% confluence, they were passaged and cul- tured in DMEM with 10% (v/v) FBS and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin). Human PDL cells were cultured in a 37ÊC humidified incubator with 5% CO2 in air. Cells at passage 4 to 6 were used for the subsequent experiments. Loading of cyclic stretch The CSU was applied to stretch the human PDL cells, as described in our recent studies previ- ously [13, 14, 16±18]. The CSU is comprised of three components, a strainer, a controller and a PC. Cells were passaged onto a cell culture dish (diameter 60 mm), with an elastic silicon rubber membrane (Q7-4750, Dow Corning Co., Midland, MI, USA) in the bottom. The flexi- ble membrane was deformed by the spherical cap moving up and down repeatedly, and there- fore, the attached cells were stretched. The controller and the PC controlled all changes in the stretch strain and the movement of the spherical cap. The human PDL cells were passaged at a concentration of 1.5 ×106 cells per dish onto the flexible-bottomed cell culture dishes. When the cells achieved 70%-80% confluence, they were subjected to the cyclic stretch with 20% strain for 6 or 24 h at a frequency of 6 cycles/min (5 s stretch and 5 s relaxation). PDL cells cultured in a similar condition but without stretch load- ing served as non-stretching controls. Yamaguchi et al. reported that stretching strain less than 24% is appropriate to mimic the strain confronted by human PDL cells in vivo [19]. Addition- ally, our recent studies reported that the early and the late apoptosis were apparently enhanced in response to 6 and 24 h 20% stretch strains, respectively [13, 14]. Therefore, the cultured cells were stretched by cyclic stretch of 20% strain for 6 or 24 h in the present study. The loading frequency of 6 cycles/min (5 s stretch and 5 s relaxation) was the same as that in our previous studies [18]. For experiments of apoptosis analysis, caspases activity analysis and Western Blot analysis, three independent treatments were carried out in every group. Morphological observation At the end of the stretch loading, the cells were observed under an inverted phase-contrast light microscope (Leica DMRIRB, Bensheim, Germany) to determine the morphological changes after cyclic stretch. Analysis of apoptosis by flow cytometry Apoptotic rate was detected by annexin V/PI double staining method using an FITC Annexin V Apoptosis Detection Kit (Becton Dickinson, San Jose, CA, USA), according to the manufac- turer's instruction. The supernatant and the cells of the sample were collected and were centri- fuged in 1000 r/min for 5 min to get the sediment. After re-suspension in Binding Buffer, the cells were stained with FITC Annexin V and PI for 15 min at room temperature in darkness.
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