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.

Keywords: apoptotic bodies; endplate chondrocytes; intervertebral disc degeneration; mineralization.

Figure 1

Oxidative stress induces mineralization in primary endplate chondrocytes from rat intervertebral discs (IVDs). Endplate chondrocytes were isolated from rats and treated with, or without, H₂O₂ at the indicated doses for 7 days. (A) Alizarin Red staining for calcium deposition in endplate chondrocytes. (B) Semi‐quantitative analysis of the mineralized nodule in endplate chondrocytes. (C) von Kossa staining. (D) The percentage of von Kossa‐positive cells. (E) ALP staining in endplate chondrocytes. (F) Semi‐quantitative analysis of alkaline phosphatase (ALP) activities in endplate chondrocytes. (G) Longitudinal analysis of ALP activities in endplate chondrocytes following treatment with 2.0 mM H₂O₂. (H) Quantitative real time polymerase chain reaction (RT‐PCR) analysis of the relative levels of ALP, RUNX2, OCN and COL‐I mRNA transcripts in endplate chondrocytes. Data are representative images (magnification x 100) or expressed as the mean ± SEM of each group of cells from three separate experiments. *P < 0.05, **P < 0.01 vs the control

Figure 2

Oxidative stress promotes the generation of main Abs in endplate chondrocytes. Endplate chondrocytes were treated with, or without, H₂O₂ for 4, 8, 12 and 24 hours and the extracellular vesicles (EVs) in the supernatants of cultured cells were purified. (A) The levels of different types of EVs in the supernatants of cultured cells were examined longitudinally for protein contents. (B) The percentages of each type of EVs in the supernatants of cultured cells following treatment with 2.0 mmol/L H₂O₂. (C) Transmission electron microscopy of H₂O₂‐treated endplate chondrocytes. Treated cell demonstrated the presence of Abs. (D) Scanning electron microscopy of H₂O₂‐treated endplate chondrocytes. (E) The relative levels of Histone H3, ARF6 and CD9 to the control GAPDH expression were determined by Western blot. Data are representative images or expressed as the mean ± SEM of each group of cells from three separate experiments. *P < 0.05, **P < 0.01 vs the control

Figure 3

Apoptotic bodies (Abs)_ are effectively endocytosed and co‐localized with COL‐II to promote mineralization in endplate chondrocytes. Endplate chondrocytes were treated with, or without, Abs at the indicated doses for indicated time periods or treated with, or without, H₂O₂ and/or Abs for 7 days. (A) Subcellular distribution of Abs in endplate chondrocytes was analysed using confocal microscopy after staining with anti‐collagen II. (B) Alizarin Red S staining for calcium deposition in endplate chondrocytes. (C) Semi‐quantitative analysis of the mineralized nodules in endplate chondrocytes. (D) von Kossa staining of phosphate contents in endplate chondrocytes. (E) The percentage of von Kossa‐positive cells. (F) Quantitative RT‐PCR analysis of the relative levels of ALP, RUNX2, OCN and COL‐I to the control GAPDH mRNA transcripts in endplate chondrocytes. Data are representative images (magnification x 100) or expressed as the mean ± SEM of each group of cells from three separate experiments. **P < 0.01 vs the control

Figure 4

Oxidative stress and Abs promote the PPi metabolism in endplate chondrocytes. Endplate chondrocytes were treated with, or without, 2 mmol/L H₂O₂ and/or Abs for 24 hours. (A) and (B) The levels of PPi and Pi in the supernatants of cultured cells were measured. Data are expressed as the mean ± SD of each group of cells from three separate experiments. **P < 0.01 vs the control

Figure 5

Oxidative stress and Abs modulate the expression of ENPP1, ANK and TNAP expression in endplate chondrocytes. Endplate chondrocytes were treated with, or without, H₂O₂ and/or Abs for 24 hours. (A‐C) The relative levels of ENPP1, ANK and TNAP mRNA transcripts in individual groups of cells were determined by quantitative RT‐PCR. Furthermore, endplate chondrocytes were transfected with plasmid for the ENPP1 or TNAP promoter‐controlled luciferase expression, together with plasmid for Renilla luciferase expression. Subsequently, the cells were treated with, or without, H₂O₂ and/or Abs for 24 hours. The relative levels of luciferase to Renilla luciferase activities were determined by Dual luciferase assays. (D‐E) The ENPP1 and TNAP promoter activities in individual groups of cells. Data are expressed as the mean ± SD of each group of cells from three separate experiments. **P < 0.01 vs the control

Figure 6

Schematic diagram illustrates the potential mechanisms by which oxidative stress induced the generation of Abs which might contribute to the mineralization by endplate chondrocytes. Abs increase PPi metabolism by modulating ENPP1, ANK and TNAP expression, contributing to the mineralization in endplate chondrocytes. Red arrows show that Abs may be partially engulfed by endplate chondrocytes