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ASIC1 and ASIC3 mediate cellular senescence of human nucleus pulposus mesenchymal stem cells during intervertebral disc degeneration.

ABSTRACT: Stem cell approaches have become an attractive therapeutic option for intervertebral disc degeneration (IVDD). Nucleus pulposus mesenchymal stem cells (NP-MSCs) participate in the regeneration and homeostasis of the intervertebral disc (IVD), but the molecular mechanisms governing these processes remain to be elucidated. Acid-sensing ion channels (ASICs) which act as key receptors for extracellular protons in central and peripheral neurons, have been implicated in IVDD where degeneration is associated with reduced microenvironmental pH. Here we show that ASIC1 and ASIC3, but not ASIC2 and ASIC4 are upregulated in human IVDs according to the degree of clinical degeneration. Subjecting IVD-derived NP-MSCs to pH 6.6 culture conditions to mimic pathological IVD changes resulted in decreased cell proliferation that was associated with cell cycle arrest and induction of senescence. Key molecular changes observed were increased expression of p53, p21, p27, p16 and Rb1. Instructively, premature senescence in NP-MSCs could be largely alleviated using ASIC inhibitors, suggesting both ASIC1 and ASIC3 act decisively upstream to activate senescence programming pathways including p53-p21/p27 and p16-Rb1 signaling. These results highlight the potential of ASIC inhibitors as a therapeutic approach for IVDD and broadly define an in vitro system that can be used to evaluate other IVDD therapies.

SUBMITTER: Ding J

PROVIDER: S-EPMC8064223 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

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.

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ASIC1 and ASIC3 mediate cellular senescence of human nucleus pulposus mesenchymal stem cells during intervertebral disc degeneration.

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