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      Autophagy in spinal ligament fibroblasts: evidence and possible implications for ossification of the posterior longitudinal ligament

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          Abstract

          Background

          The molecular mechanisms of ossification of the posterior longitudinal ligament (OPLL) remain to be elucidated. The aim of the present study was to investigate the autophagy of spinal ligament fibroblasts derived from patients with OPLL and to examine whether autophagy-associated gene expression was correlated with the expression of osteogenic differentiation genes.

          Methods

          Expression of autophagy-associated genes was detected in 37 samples from 21 OPLL patients and 16 non-OPLL patients. The correlation of autophagy-associated gene expression and the expression of osteogenic differentiation genes was analyzed by Pearson’s correlation. The expression of autophagy-associated genes of ligament fibroblasts was assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunofluorescence. The incidence of autophagy was assessed by flow cytometry. After knockdown using small interfering RNA targeting Beclin1, the expression of osteogenic differentiation genes were compared in spinal ligament fibroblasts.

          Results

          In clinical specimens, mRNA expression levels of microtubule-associated protein 1 light chain 3 and Beclin1 were higher in the OPLL group compared with the non-OPLL group. Pearson correlation analysis demonstrated that Beclin1 expression was positively correlated with expression of osteocalcin (OCN) ( r = 0.8233, P < 0.001), alkaline phosphatase, biomineralization associated (ALP) ( r = 0.7821, P < 0.001), and collagen type 1 (COL 1) ( r = 0.6078, P = 0.001). Consistently, the upregulation of autophagy-associated genes in ligament fibroblasts from patients with OPLL were further confirmed by western blotting and immunofluorescence. The incidence of autophagy was also increased in ligament fibroblasts from patients with OPLL. Furthermore, knockdown of Beclin1 led to a decrease in the expression of OCN, ALP, and COL 1 by 63.2% ( P < 0.01), 52% ( P < 0.01), and 53.2% ( P < 0.01) in ligament fibroblasts from patients with OPLL, respectively.

          Conclusions

          Beclin1-mediated autophagy was involved in the osteogenic differentiation of ligament fibroblasts and promoted the development of OPLL.

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          Most cited references25

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          A role for NBR1 in autophagosomal degradation of ubiquitinated substrates.

          Autophagy is a catabolic process where cytosolic cellular components are delivered to the lysosome for degradation. Recent studies have indicated the existence of specific receptors, such as p62, which link ubiquitinated targets to autophagosomal degradation pathways. Here we show that NBR1 (neighbor of BRCA1 gene 1) is an autophagy receptor containing LC3- and ubiquitin (Ub)-binding domains. NBR1 is recruited to Ub-positive protein aggregates and degraded by autophagy depending on an LC3-interacting region (LIR) and LC3 family modifiers. Although NBR1 and p62 interact and form oligomers, they can function independently, as shown by autophagosomal clearance of NBR1 in p62-deficient cells. NBR1 was localized to Ub-positive inclusions in patients with liver dysfunction, and depletion of NBR1 abolished the formation of Ub-positive p62 bodies upon puromycin treatment of cells. We propose that NBR1 and p62 act as receptors for selective autophagosomal degradation of ubiquitinated targets.
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            Estradiol inhibits osteoblast apoptosis via promotion of autophagy through the ER-ERK-mTOR pathway.

            Estradiol could protect osteoblast against apoptosis, and apoptosis and autophagy were extensively and intimately connected. The aim of the present study was to test the hypothesis that autophagy was present in osteoblasts under serum deprivation and estrogen protected against osteoblast apoptosis via promotion of autophagy. MC3T3-E1 osteoblastic cells were cultured in a serum-free and phenol red-free minimal essential medium (α-MEM). Ultrastructural analysis, lysosomal activity assessment and monodansycadaverine (MDC) staining were employed to determine the presence of autophagy, and real time PCR was used to evaluate the expression of autophagic markers. Meanwhile, the osteoblasts were transferred in a serum-free and phenol red-free α-MEM containing either vehicle or estradiol. Apoptosis and autophagy was assessed by using the techniques of real-time PCR, Western blot, immunofluorescence assay, and flow cytometry. The possible pathway through which estrogen promoted autophagy in the serum-deprived osteoblasts was also investigated. Real-time PCR demonstrated the expression of LC3, beclin1 and ULK1 genes in osteoblasts under serum deprivation, and immunofluorescence assay verified high expression of proteins of these three autophagic bio-markers. Lysosomes and autolysosomes accumulated in the cytoplasm of osteoblasts were also detected under transmission electron microscopy, MDC staining and lysosomal activity assessment. Meanwhile, estradiol significantly decreased the expression of proteins of the bio-markers of apoptosis, and at the same time increased the expression of proteins of the bio-markers of autophagy in the serum-deprived osteoblasts. Furthermore, the estradiol-promoted autophagy in serum-deprived osteoblasts could be blocked by estrogen receptor (ER) antagonist (ICI 182780), and estradiol failed to rescue the cells pretreated with an inhibitor of vacuolar ATPase (bafilomycin A) from apoptosis. Serum deprivation resulted in apoptosis through activation of Caspase-3 and induced autophagy through inhibition of phospho-mammalian target of rapamycin (p-mTOR). Both 3-methyladenine (3MA) and U0126 led to increase of apoptosis in osteoblasts with serum deprivation. Estradiol failed to over-ride the inhibitory effect of 3MA on phosphorylation of AKT but directly led to dephosphorylation of mTOR and upregulation of LC3 protein expression. However, the estradiol-enhanced LC3 protein expression was significantly suppressed by U0126 through inhibition of phosphorylation of extracellular signal-regulated kinase (ERK). Estradiol rescued osteoblast apoptosis via promotion of autophagy through the ER-ERK-mTOR pathway.
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              Autophagy in rat annulus fibrosus cells: evidence and possible implications

              Introduction Programmed cell death of intervertebral disc (IVD) cells plays an important role in IVD degeneration, but the role of autophagy, a closely related cell death event, in IVD cells has not been documented. The current study was designed to investigate the effect of interleukin (IL)-1β on the occurrence of autophagy of rat annulus fibrosus (AF) cells and the interrelationship between autophagy and apoptosis. Methods Rat AF cells were isolated and exposed, in tissue cultures with or without serum, to IL-1β in different concentrations for 24 hours. Ultrastructural analysis, flow cytometry and lysosomal activity assessment were performed after the in vitro treatment to determine the presence and levels of autophagy. The mRNA expression of autophagy-related proteins (Beclin-1, Bcl-2 and microtubule associated protein 1 light chain 3 (LC3)) were evaluated using real-time PCR. 3-methyladenine (3-MA), a PI3K inhibitor, was used to determine the interaction between autophagy and apoptosis via the suppression of autophagy. Results Autophagy was detected in rat AF cells under serum starvation condition by transmission electron microscopy. PCR and flow cytometry results showed that IL-1β enhanced the autophagy-induction effect of serum deprivation in a dose-dependent manner. However, IL-1β alone failed to induce autophagy in AF cells cultured without serum starvation. When autophagy was suppressed by 3-MA, the apoptosis incidence was increased. Serum supplement also partly reversed the autophagy incidence without affecting the apoptosis incidence in the same cells. Conclusions IL-1β up-regulates serum deprivation-induced autophagy of AF cells in a dose-dependent manner. Autophagy may represent a protective mechanism against apoptosis in AF cells and IVD degeneration.
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                Author and article information

                Contributors
                yuesjtu@outlook.com
                Linzunwen83@126.com
                chenjiangwei1031@126.com
                dingding6704@163.com
                maoweiwei@189.cn
                shisheng273@163.com
                nywylbr1971@163.com
                Journal
                J Orthop Surg Res
                J Orthop Surg Res
                Journal of Orthopaedic Surgery and Research
                BioMed Central (London )
                1749-799X
                22 October 2020
                22 October 2020
                2020
                : 15
                : 490
                Affiliations
                [1 ]GRID grid.284723.8, ISNI 0000 0000 8877 7471, Department of Orthopaedics, The Fifth Affiliated Hospital, , Southern Medical University, ; No. 566 Congcheng Avenue, Conghua District, Guangzhou, 510900 People’s Republic of China
                [2 ]GRID grid.260463.5, ISNI 0000 0001 2182 8825, Department of Orthopedic Surgery, The First Affiliated Hospital, , Nanchang University, ; No. 17, Yongwaizheng Street, Nanchang, 330006 Jiangxi People’s Republic of China
                [3 ]GRID grid.412987.1, ISNI 0000 0004 0630 1330, Department of Stomatology, , Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, ; 1665, Kongjiang Road, Shanghai, 200092 People’s Republic of China
                [4 ]GRID grid.412987.1, ISNI 0000 0004 0630 1330, Department of Pediatric Neurosurgery, , Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, ; 1665, Kongjiang Road, Shanghai, 200092 People’s Republic of China
                [5 ]GRID grid.24516.34, ISNI 0000000123704535, Department of Orthopedics, Shanghai Tenth People’s Hospital, School of Medicine, , Tongji University, ; 301 Middle Yanchang Road, Shanghai, 200072 People’s Republic of China
                Author information
                http://orcid.org/0000-0001-7632-9871
                Article
                2017
                10.1186/s13018-020-02017-6
                7579890
                33092625
                d175e378-f03a-43fd-8c7e-6126afa654ee
                © The Author(s) 2020

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                : 17 April 2020
                : 14 October 2020
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;
                Award ID: 81501196
                Categories
                Research Article
                Custom metadata
                © The Author(s) 2020

                Surgery
                autophagy,ossification of the posterior longitudinal ligament,fibroblast,osteogenic differentiation

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