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Carbohydrate sulfotransferase 3 (CHST3) overexpression promotes cartilage endplate-derived stem cells (CESCs) to regulate molecular mechanisms related to repair of intervertebral disc degeneration by rat nucleus pulposus.

ABSTRACT: To investigate the regulatory effect of carbohydrate sulfotransferase 3 (CHST3) in cartilage endplate-derived stem cells (CESCs) on the molecular mechanism of intervertebral disc degeneration after nucleus pulposus repair in rats. We performed GO and KEGG analysis of GSE15227 database to select the differential genes CHST3 and CSPG4 in grade Ⅱ, Ⅲ and Ⅳ intervertebral disc degeneration, IHC and WB to detect the protein profile of CHST3 and CSPG4, Co-IP for the interaction between CHST3 and CSPG4. Then, immunofluorescence was applied to measure the level of CD90 and CD105, and flow cytometry indicated the level of CD73, CD90 and CD105 in CESCs. Next, Alizarin red staining, Alcian blue staining and TEM were performed to evaluate the effects of CESCs into osteoblasts and chondroblasts, respectively, CCK8 for the cell proliferation of osteoblasts and chondroblasts after induction for different times; cell cycle of osteoblasts or chondroblasts was measured by flow cytometry after induction, and WB for the measurement of specific biomarkers of OC and RUNX in osteoblasts and aggrecan, collagen II in chondroblasts. Finally, colony formation was applied to measure the cell proliferation of CESCs transfected with ov-CHST3 or sh-CHST3 when cocultured with bone marrow cells, WB for the protein expression of CHST3, CSPG4 and ELAVL1 in CSECs, transwell assay for the migration of CESCs to bone marrow cells, TEM image for the cellular characteristics of bone marrow cells, and WB for the protein profile of VCAN, VASP, NCAN and OFD1 in bone marrow cells. CHST3 and CSPG4 were differentially expressed and interacted in grade Ⅱ, Ⅲ and Ⅳ intervertebral disc degeneration; CD73, CD90 and CD105 were lowly expressed in CESCs, osteogenic or chondroblastic induction changed the characteristics, proliferation, cell cycle and specific biomarkers of osteoblasts and chondroblasts after 14 or 21 days,; CHST3 affected the cell proliferation, protein profile, migration and cellular features of cocultured CESCs or bone marrow cells. CHST3 overexpression promoted CESCs to regulate bone marrow cells through interaction with CSPG4 to repair the grade Ⅱ, Ⅲ and Ⅳ intervertebral disc degeneration.

SUBMITTER: Guan Y

PROVIDER: S-EPMC8256370 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background contextLow back pain, affecting nearly 40% of adults, mainly results from intervertebral disc degeneration (IVDD), while the pathogenesis of IVDD is still not fully elucidated. Recently, some researches have revealed that necroptosis, a programmed necrosis, participated in the progression of IVDD, nevertheless, the underlying mechanism remains unclear.PurposeTo study the mechanism of necroptosis of Nucleus Pulposus (NP) cells in IVDD, focusing on the role of MyD88 signaling.Study designThe expression and co-localization of necroptotic indicators and MyD88 were examined in vivo, and MyD88 inhibitor was applied to determine the role of MyD88 signaling in necroptosis of NP cells in vitro.MethodsHuman disc specimens were collected from patients receiving diskectomy for lumbar disc herniation (LDH) or traumatic lumbar fractures after MRI scanning. According to the Pfirrmann grades, they were divided into normal (Grades 1, 2) and degenerated groups (4, 5). Tissue slides were prepared for immunofluorescence to assess the co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 histologically. The combination of TNFα, LPS and Z-VAD-FMK was applied to induce necroptosis of NP cells. Level of ATP, reactive oxygen species (ROS), live-cell staining and electron microscope study were employed to study the role of MyD88 signaling in necroptosis of NP cells.ResultsIn vivo, the increased expression and co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 were found in NP cells of degenerated disc, while very l low fluorescence intensity in tissue of traumatic lumbar fractures. In vitro, the MyD88 inhibitor effectively rescued the necroptosis of NP cells, accompanied by increased viability, ATP level, and decreased ROS level. The effect of MyD88 inhibition on necroptosis of NP cells was further confirmed by ultrastructure of mitochondria shown by Transmission Electron Microscope (TEM).ConclusionOur results indicated that the involvement of MyD88 signaling in the necroptosis of NP cells in IVDD, which will replenish the pathogenesis of IVDD and provide a novel potential therapeutic target for IVDD.

Project description:Intervertebral disc (IVD) degeneration is a complicated physiological change involving cellular senescence, inflammation and the degradation of the extracellular matrix. Long non?coding RNAs (lncRNAs) have been identified as new players in IVD degeneration. The present study aimed to identify lncRNAs implicated in IVD degeneration via the regulation of cellular senescence. In the present study, nucleus pulposus (NP) cells isolated from moderately degenerated IVD tissues exhibited a senescent phenotype with increased senescence rates, detected by senescence?associated ??galactosidase (SA???gal) staining, and reduced growth and migratory abilities. Microarray and target prediction analyses identified 353 differentially expressed lncRNAs, and 251 cis? and 2,170 trans?acting targets in degenerated NP cells. Bioinformatic analyses revealed that these predicted targets were enriched in the regulation of response to DNA damage stimulus, positive regulation of cell cycle processes and interferon?? production. In addition, a network of the top 10 upregulated and top 10 downregulated lncRNA targets was constructed, and two trans?acting targets, C?C motif chemokine ligand 5 (CCL5) and polyribonucleotide nucleotidyltransferase 1 (PNPT1) involved in aging or senescence, and their corresponding lncRNAs, lnc?ST8SIA5?1:2 and lnc?HRK?2:1, were identified. Reverse transcription?quantitative PCR validation demonstrated that the two targets and two candidate lncRNAs were significantly upregulated in degenerated NP cells. Overexpression of lnc?HRK?2:1, with validated higher expression levels, in normal NP cells induced a senescent phenotype, with enhanced rates of senescence detected by SA???gal staining in cells, decreased growth and migratory abilities and improved expression levels of CCL5 and PNPT1. Collectively, these results suggested that upregulation of lnc?HRK?2:1 prompted NP cell senescence in IVD degeneration, which may be associated with increased expression levels of CCL5 and PNPT1.

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Carbohydrate sulfotransferase 3 (CHST3) overexpression promotes cartilage endplate-derived stem cells (CESCs) to regulate molecular mechanisms related to repair of intervertebral disc degeneration by rat nucleus pulposus.

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