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Inverse Agonist of Retinoid-Related Orphan Receptor-Alpha Prevents Apoptosis and Degeneration in Nucleus Pulposus Cells via Upregulation of YAP.

ABSTRACT: Intervertebral disc degenerative disease (IDD) is the most common degenerative spine disease, which leads to chronic low back pain and symptoms in the lower extremities. In this study, we found that RORα, a member of the retinoid-related orphan receptor family, is significantly elevated in nucleus pulposus tissue in IDD patients. The elevation of RORα is associated with increased apoptosis of nucleus pulposus (NP) cells. Therefore, we applicated a well-established inverse agonist of RORα, SR3335, to investigate its role in regulating NP cell metabolism and apoptosis. To further investigate the mechanism that SR3335 regulates the pathogenesis of IDD in vitro, tumor necrosis factor alpha (TNF-α) stimulation was used in human NP cells to mimic the hostile environment that leads to degeneration. We found that SR3335 treatment reversed the trend of increased apoptosis in NP cells induced by TNF-α treatment. Next, TNF-α treatment upregulated the expression of type II collagen and aggrecan and downregulated MMP13 (matrix-degrading enzyme matrix metalloproteinase 13) and ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4). However, these effects were reversed after SR3335 treatment. Furthermore, we find that SR3335 mediated the effect in NP cells by regulating the YAP signaling pathway, especially by affecting the phosphorylation state of YAP. In conclusion, the reduction of matrix degradation enzymes and apoptosis upon SR3335 treatment suggests that SR3335 is a promising drug in reversing the deleterious microenvironment in IDD patients.

SUBMITTER: Liang T

PROVIDER: S-EPMC8337132 | 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:INTRODUCTION: Synovial mesenchymal stem cells (MSCs) have high proliferative and chondrogenic potentials, and MSCs transplanted into the articular cartilage defect produce abundant extracellular matrix. Because of similarities between the articular cartilage and the intervertebral disc cartilage, synovial MSCs are a potential cell source for disc regeneration. Here, we examined the effect of intradiscal transplantation of synovial MSCs after aspiration of nucleus pulposus in rabbits. METHODS: The nucleus pulposus tissues of rabbit's intervertebral discs were aspirated to induce disc degeneration, and allogenic synovial MSCs were transplanted. At 2, 4, 6, 8, 16, 24 weeks postoperatively, we evaluated with imaging analyses such as X-ray and magnetic resonance imaging (MRI), and histological analysis. To investigate interaction between synovial MSCs and nucleus pulposus cells, human synovial MSCs and rat nucleus pulposus cells were co-cultured, and species specific microarray were performed. RESULTS: The existence of transplanted cells labeled with DiI or derived from green fluorescent protein (GFP)-expressing transgenic rabbits was confirmed up until 24 weeks. X-ray analyses demonstrated that intervertebral disc height in the MSC group remained higher than that in the degeneration group. T2 weighted MR imaging showed higher signal intensity of nucleus pulposus in the MSC group. Immunohistological analyses revealed higher expression of type II collagen around nucleus pulposus cells in the MSC group compared with even that of the normal group. In co-culture of rat nucleus pulposus cells and human synovial MSCs, species specific microarray revealed that gene profiles of nucleus pulposus were altered markedly with suppression of genes relating matrix degradative enzymes and inflammatory cytokines. CONCLUSIONS: Synovial MSCs injected into the nucleus pulposus space promoted synthesis of the remaining nucleus pulposus cells to type II collagen and inhibition of expressions of degradative enzymes and inflammatory cytokines, resulting in maintaining the structure of the intervertebral disc being maintained.

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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Inverse Agonist of Retinoid-Related Orphan Receptor-Alpha Prevents Apoptosis and Degeneration in Nucleus Pulposus Cells via Upregulation of YAP.

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