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Apoptotic bodies from endplate chondrocytes enhance the oxidative stress-induced mineralization by regulating PPi metabolism.

ABSTRACT: This study aimed to investigate the role of apoptotic bodies (Abs) from the oxidative stressed endplate chondrocytes in regulating mineralization and potential mechanisms. Endplate chondrocytes were isolated from rats and treated with H2O2 to induce oxidative stress. The calcium deposition for matrix mineralization in the cells was examined by histological staining. The expression levels of calcification-related genes in individual groups of cells were determined by quantitative real time-PCR (qRT-PCR). Subsequently, extracellular vesicles (EVs) were purified and characterized. The effect of treatment with H2O2 and/or Abs on the mineralization, extracellular PPi metabolism and related gene expression were determined. Oxidative stress significantly increased the mineralization and promoted the generation of main Abs from endplate chondrocytes. Abs were effectively endocytosed by endplate chondrocytes and co-localized with collagen (COL)-II in the cytoplasm, which enhanced the mineralization, alkaline phosphatase (ALP), osteocalcin (OCN), Runt-related transcription factor 2 (RUNX2) and COL-I expression in endplate chondrocytes. Furthermore, treatment either H2O2 or Abs significantly decreased PPi, but increased Pi production and treatment with both further enhancing the changes in endplate chondrocytes. Similarly, treatment either H2O2 or Abs significantly decreased the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), and ankylosis protein (ANK) expression and ENPP1 promoter activity, but increased the tissue-nonspecific alkaline phosphatase (TNAP) expression and TNAP promoter activity in endplate chondrocytes. Oxidative stress promoted the generation of Abs, which might enhance the oxidative stress-mediated mineralization in endplate chondrocytes by regulating the PPi metabolism.

SUBMITTER: Yuan FL

PROVIDER: S-EPMC6484318 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Apoptotic bodies from endplate chondrocytes enhance the oxidative stress-induced mineralization by regulating PPi metabolism.

Yuan Feng-Lai FL Yuan Feng-Lai FL Xu Rui-Sheng RS Ye Jun-Xing JX Zhao Ming-Dong MD Ren Li-Jun LJ Li Xia X

Journal of cellular and molecular medicine 20190320 5

This study aimed to investigate the role of apoptotic bodies (Abs) from the oxidative stressed endplate chondrocytes in regulating mineralization and potential mechanisms. Endplate chondrocytes were isolated from rats and treated with H2O2 to induce oxidative stress. The calcium deposition for matrix mineralization in the cells was examined by histological staining. The expression levels of calcification-related genes in individual groups of cells were determined by quantitative real time-PCR (q ...[more]

PMID: 30892812

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Project description:MicroRNAs (miRNAs) are known to play critical roles in many cellular processes including those regulating skeletal development and homeostasis. A previous study from our group identified differentially expressed miRNAs in the developing human growth plate. Among those more highly expressed in hypertrophic chondrocytes compared to progenitor chondrocytes was miR-138, therefore suggesting a possible role for this miRNA in regulating chondrogenesis and/or endochondral ossification. The goal of this study was to determine the function of miR- 138 in regulating osteogenesis by using human osteoarthritic dedifferentiated chondrocytes (DDCs) as source of inducible cells. We show that over-expression of miR-138 inhibited osteogenic differentiation of DDCs in vitro. Moreover, cell shape was altered and cell proliferation and possibly migration was also suppressed by miR-138. Given alterations in cell shape, closer analysis revealed that F-actin polymerization was also inhibited by miR-138. Computational approaches showed that the small GTPase, RhoC, is a potential miR-138 target gene. We pursued RhoC further given its function in regulating cell proliferation and migration in cancer cells. Indeed, miR-138 over-expression in DDCs resulted in decreased RhoC protein levels. A series of rescue experiments showed that RhoC over-expression could attenuate the inhibitory actions of miR-138 on DDC proliferation, F-actin polymerization and osteogenic differentiation. Bone formation was also found to be enhanced within human demineralized bone scaffolds seeded with DDCs expressing both miR-138 and RhoC. In conclusion, we have discovered a new mechanism in DDCs whereby miR-138 functions to suppress RhoC which subsequently inhibits proliferation, F-actin polymerization and osteogenic differentiation. To date, there are no published reports on the importance of RhoC in regulating osteogenesis. This opens up new avenues of research involving miR-138 and RhoC pathways to better understand mechanisms regulating bone formation in addition to the potential use of DDCs as a cell source for bone tissue engineering. © 2018 The Authors. JBMR Plus is published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research.

Project description:BackgroundOxidative stress has a vital role in the early stages of vitiligo. Autoantigens released from apoptotic melanocytes (MC) under oxidative stress are involved in the presentation and recognition of antigens. However, the transport of autoantigens to the cell surface and their release to the extracellular environment are still unclear. Apoptotic bodies (ABs) have always been considered as a key source of immunomodulators and autoantigens. Yet, the role of ABs in the immune mechanism of vitiligo is still unknown.PurposeTo explore whether MC's autoantigens translocate into ABs during oxidative stress-induced apoptosis and study the molecular mechanisms underlying autoantigen migration and AB formation.MethodsPIG3V (an immortalized human vitiligo melanocyte cell line) were treated with H2O2, and ABs were separated. Transmission electron microscopy, flow cytometry, Western blot, mass spectrometry, and other methods were used to determine the relocation of specific antigens in PIG3V cells to ABs. After pretreatment with specific inhibitors (Rho kinase (Y-27632), myosin light chain kinase (MLCK, ML-9), pan-caspase (zVAD-FMK), and JNK (SP600125)), the pathway of autoantigen translocation into ABs and the formation of apoptotic bodies were determined.ResultsWhen treated with 0.8 mM H2O2, ABs were released from these cells. Autoantigens such as tyrosinase-related protein 1 (TYRP-1) and cleavage nuclear membrane antigen Lamin A/C (Asp230) were concentrated in ABs. The expression of autoantigens and the formation of ABs increased in a time- and dose-dependent manner after treatment with H2O2, while the application of specific inhibitors inhibited the formation of apoptotic bodies, i.e., the expression of antigens.ConclusionVitiligo autoantigens translocate into ABs in the process of apoptosis induced by oxidative stress. The cytoskeletal protein activation pathway and the JNK-related apoptosis pathway are involved in the transport of autoantigens and the formation of ABs. ABs may be the key bridge between MC cell apoptosis and cellular immunity.

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Apoptotic bodies from endplate chondrocytes enhance the oxidative stress-induced mineralization by regulating PPi metabolism.

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Apoptotic bodies from endplate chondrocytes enhance the oxidative stress-induced mineralization by regulating PPi metabolism.

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