Mechanism Of Intervertebral Disc Degeneration Research Articles

MiR-486-5p Inhibits Inflammatory Response, Matrix Degradation and Apoptosis of Nucleus Pulposus Cells through Directly Targeting FOXO1 in Intervertebral Disc Degeneration.

microRNA-486-5p (miR-486-5p) and forkhead box protein O1 (FOXO1) play an important role in the development of intervertebral disc degeneration (IDD). However, their molecular mechanisms in IDD remain unknown. qRT-PCR assay was used to identify miR-486-5p expression in nucleus pulposus (NP) cells. In-vitro transfection, CCK-8, flow cytometry and luciferase reporter assay were used to validate the role and relationship of miR-486-5p and FOXO1 in lipopolysaccharides (LPS)-stimulated NP cells. qRT-PCR and Western blot were used to measure the expression levels of inflammatory cytokines, matrix degrading enzymes, and extracellular matrix (ECM)-related genes. miR-486-5p expression was significantly down-regulated, while FOXO1 expression was up-regulated in LPS-treated NP cells (P<0.001). miR-486-5p over-expression repressed LPS-induced expressions of inflammatory cytokines (IL-1β, IL-6 and TNF-α) and matrix degrading enzymes (MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5), and promoted the expressions of LPS-inhibited ECM-related genes (Aggrecan and Collagen II) (all P<0.001). In addition, miR-486-5p over-expression protected NP cells against LPS-induced apoptosis. However, inhibition of miR-486-5p led to the opposite effects. Mechanically, FOXO1 was a direct target gene of miR-486-5p. Over-expressed FOXO1 aggravated LPS-induced injury, and antagonized protection effects of miR-486-5p. miR-486-5p can inhibit inflammatory response, ECM degradation and apoptosis in NP cells by directly targeting FOXO1, which may contribute to the biological therapy of IDD.

TGF-β Initiates β-Catenin-Mediated CTGF Secretory Pathway in Old Bovine Nucleus Pulposus Cells: A Potential Mechanism for Intervertebral Disc Degeneration.

ABSTRACTWe have recently demonstrated that overexpression of Smurf2 under the control of type II collagen alpha 1 (Col2a1) promoter induces an intervertebral disc degeneration phenotype in Col2a1‐Smurf2 transgenic mice. The chondrocyte‐like cells that express type II collagen and Smurf2 in the transgenic mouse discs are prone to degenerate. However, how the chondrocyte‐like cells contribute to disc degeneration is not known. Here, we utilized primary old bovine nucleus pulposus (NP) cells as substitutes for the chondrocyte‐like cells in Col2a1‐Smurf2 transgenic mouse discs to identify mechanism. We found that 35% of the cells were senescent; TGF‐β treatment of the cells induced a rapid moderate accumulation of β‐catenin, which interacted with connective tissue growth factor (CTGF/CCN2) in the cytoplasm and recruited it to the membrane for secretion. The TGF‐β‐initiated β‐catenin‐mediated CTGF secretory cascade did not occur in primary young bovine NP cells; however, when Smurf2 was overexpressed in young bovine NP cells, the cells became senescent and allowed this cascade to occur. These results suggest that Smurf2‐induced disc degeneration in Col2a1‐Smurf2 transgenic mice occurs through activation of CTGF secretory pathway in senescent disc cells. © 2018 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

High amplitude and low frequency cyclic mechanical strain promotes degeneration of human nucleus pulposus cells via the NF-κB p65 pathway.

Disc degeneration alters the structure and function of intervertebral discs and is the basis of spinal degenerative diseases. To establish the molecular mechanism of intervertebral disc degeneration caused by mechanical strain, this study examined the effects of different amplitude (3%, 9%, 19%) cyclic mechanical strain (CMS) at a low frequency (0.01 Hz) on the secretion of cartilage extracellular matrix, expression of inflammatory cytokines and catabolic proteases, and activation of NF-κB signaling pathway in human nucleus pulposus cells. We also investigated effects of low frequency and high amplitude (19%) CMS on degeneration of human nucleus pulposus cells in the presence or absence of p65 inhibitor, p65 silencing shRNA, or p65 overexpression. While 3% CMS did not significantly decrease aggrecan or type II collagen expression, or increase TNF-α, IL-1β, IL-6 expression, 9% and 19% CMS showed the significant effects. Low frequency and high amplitude (19%) CMS was found to promote p65 activation in human nucleus pulposus cells, and IL-1β was found to promote p65 nuclear translocation though IκB kinase phosphorylation. Furthermore, degeneration process of nucleus pulposus cells was found attenuated in the presence of p65 inhibitor or p65 silencing shRNA, but promoted with p65 overexpression. These data suggest that high amplitude and low frequency CMS could promote degeneration of human nucleus pulposus cells significantly via the NF-κB p65 pathway. Our findings have uncovered the effect of CMS on human nucleus pulposus cell degeneration and have identified a previously unknown intrinsic underlying mechanism.

Effects of plasma vaporization ablation on expression of inflammatory factors in rabbit model of intervertebral disc degeneration

Intervertebral Disc Degeneration (IDD) is one common disease presented as wrist pain. Plasma vaporization ablation is a novel method treating IDD. This study utilized vaporization ablation on rabbit IDD model, whose expression of inflammatory factors was analysed along with the correlation with plasma vaporization ablation. Rabbit IDD model was generated by puncture approach and treated with plasma vaporization ablation. Control group received no treatment after surgery. MRI scanning was performed on rabbit intervertebral disc. Serum and intervertebral disc tissue samples were collected and tested for expression of Tumor Necrosis Factor α (TNF-α), Interleukin-2 (IL-2) and IL-4 by Enzyme Linked Immunosorbent Assay (ELISA) or Immunohistochemistry (IHC) methods. 2 w after puncture, rabbit had rupture of intervertebral disc fibrous ring, plus change of low-strength T2 signal but not in T1 signal. 4 w after surgery, model rats had further decreased intervertebral disc T2 signals. Both serum and intervertebral disc levels of TNF-α, IL-2 and IL-4 were significantly elevated in model group, and were gradually decreased with elongated treatment (p<0.05). Plasma vaporization ablation can decrease expression of TNF-α, IL-2 and IL-4 in both serum and intervertebral disc tissues, probably forming one mechanism of IDD.

Expression and Role of Sphingosine 1-Phosphate Receptors in Intervertebral Disc Degeneration

Background: e mechanism of intervertebral disc degeneration is unclear, inflammation may play an important role in it. Sphingosine 1-phosphate (S1P) is an important lipid mediator, has been confirmed to be implicated in many inflammation processes.Purpose to study the expression and role of S1PRs in the intervertebal disc (IVD) degeneration to enhance understanding of disc degeneration. Methods Degenerated and normal IVD were harvested from patients with IVD degeneration via surgery operation. The degenerated grade of each IVD specimen was assessed by Histological method. Expression of S1P receptor subtypes in each specimen was evaluated using real-time PCR, immunohistochemistry, and western blotting. The effect of S1PR on inflammation induced by interleukin-1β in nucleus pulposus (NP) cells was also assessed by real time PCR and western blot. Results The nucleus pulposus mainly expressed theS1PR1/2/3, and the expression decreased in the severe degenerated nucleus pulposus cells. The ligand, S1P, inhibited the up-regulation of matrix metallopeptidase-3(MMP-3) and ADAM metallopeptidase with thrombospondin type 1 motif 4(ADAMTS4) induced by IL-1β. Conclusions The results show that an the expression of S1PRs in degenerative discs is down-regulated as degeneration, and S1P can inhibit the inflammation response induced by IL-1β in NP cells, implicating that S1P/S1PR may contribute to IVD degeneration.

Caveolin‑1 regulates oxidative stress‑induced senescence in nucleus pulposus cells primarily via the p53/p21 signaling pathway invitro.

Previous studies have indicated that cellular senescence is a critical underlying mechanism of intervertebral disc degeneration. However, the precise mechanism by which cellular senescence accelerates disc degeneration has not been fully elucidated. Caveolin-1 has recently emerged as an important regulator of cellular senescence. Therefore, the aim of the present study was to investigate whether caveolin-1 is involved in nucleus pulposus (NP) cellular senescence during oxidative stress. PCR was used to detect caveolin-1 mRNA expression and protein expression was detected by western blotting. Caveolin-1 expression at the mRNA and protein levels was markedly increased following treatment with tert-butyl hydroperoxide, and an increase in premature senescence was observed, as determined by senescence-associated β-galactosidase staining and the decline of cellular proliferative ability. In addition, caveolin-1 gene expression was successfully knocked down by lentivirus-mediated RNA interference, which exerted a protective effect against the cellular senescence induced by oxidative stress. Notably, p53 and p21 protein expression, though not p16 protein expression, decreased with caveolin-1 silencing. The results suggested that caveolin-1 may be involved in NP cellular senescence during oxidative stress in vitro, mainly via the p53/p21 signaling pathway. Thus, caveolin-1 may represent a novel therapeutic target for the prevention of intervertebral disc degeneration.

Role of miR-155 in the regulation of MMP-16 expression in intervertebral disc degeneration.

ABSTRACTThe molecular mechanisms of intervertebral disc degeneration (IDD) remain elusive. We found that miR‐155 is down‐regulated in degenerative nucleus pulposus (NP), and more severe degeneration is correlated with higher matrix metallopeptidase 16 (MMP‐16) expression. MMP‐16 also degraded matrix aggrecan. Here, we addressed the in vivo miR‐155‐mediated pathological impact on IDD using a classic puncture mouse model. Lentiviral upregulated‐miR‐155 or downregulated‐miR‐155 was transduced into the discs of C57 mice, which was validated by real‐time polymerase chain reaction (real‐time PCR) and in situ hybridization. Immunohistochemistry and western blotting revealed that up‐regulation of miR‐155 resulted in down‐regulation of MMP‐16 and an increase in aggrecan and collagen type II in mouse NP; whereas, down‐regulation of miR‐155 resulted in up‐regulation of MMP‐16 and a decrease in aggrecan in mouse NP. Radiographic and histological analysis showed that the up‐regulation of miR‐155 attenuated IDD, while down‐regulation of miR‐155 resulted in the deterioration of IDD. These findings indicate that decreased miR‐155 contributed to the up‐regulation of MMP‐16 in vivo, and MMP‐16 further degraded aggrecan and collagen type II, leading to the dehydration and degeneration of discs. Our findings revealed a therapeutic role for miR‐155 in IDD. © 2017 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 35:1323–1334, 2017.

Oxidative damage induces apoptosis and promotes calcification in disc cartilage endplate cell through ROS/MAPK/NF-κB pathway: Implications for disc degeneration

Cartilage endplate (CEP) cell calcification and apoptosis play a vital role in the intervertebral disc degeneration (IVDD). Oxidative stress is a key factor in inducing programmed cell death and cartilage calcification. However, the cell death and calcification of cartilage endplate cells under oxidative stress have never been described. The present study investigated the apoptosis and calcification in the cartilage endplate cell under oxidative stress induced by H2O2 to understand the underlying mechanism of IVDD. The cartilage endplate cells isolated from human lumbar discs were subjected to different concentrations of H2O2 for various time periods. The cell viability was determined by CCK-8 assay, whereas Western blot, immunofluorescence, and Alcian blue, Alizarin red, and Von Kossa staining evaluated the apoptosis and calcification. The level of mitochondria-specific reactive oxygen species (ROS) was quantified with an oxygen radical-sensitive probe—MitoSOX. The potential signaling pathways were investigated by Western blot after the addition of N-acetyl-l-cysteine (NAC). We found that the oxidative stress induced by H2O2 increased the apoptosis and subsequently the calcification in the cartilage endplate cells through the ROS/p38/ERK/p65 pathway. The apoptosis and the calcification of the cartilage endplate cells induced by H2O2 can be abolished by NAC. These results suggested that regulating the apoptosis and the calcification in the cartilage endplate cells under oxidative stress should be advantageous for the survival of cells and might delay the process of disc degeneration.

Autophagy Is a Protective Response to the Oxidative Damage to Endplate Chondrocytes in Intervertebral Disc: Implications for the Treatment of Degenerative Lumbar Disc.

Low back pain (LBP) is the leading cause of disability in the elderly. Intervertebral disc degeneration (IDD) was considered as the main cause for LBP. Degeneration of cartilaginous endplate was a crucial harmful factor during the initiation and development of IDD. Oxidative stress was implicated in IDD. However, the underlying molecular mechanism for the degeneration of cartilaginous endplate remains elusive. Herein, we found that oxidative stress could induce apoptosis and autophagy in endplate chondrocytes evidenced by western blot analysis, flow cytometry, immunofluorescence staining, GFP-LC3B transfection, and MDC staining. In addition, we also found that the apoptosis of endplate chondrocytes was significantly increased after the inhibition of autophagy by bafilomycin A1 shown by flow cytometry. Furthermore, mTOR pathway upstream autophagy was greatly suppressed suggested by western blot assay. In conclusion, our study strongly revealed that oxidative stress could increase autophagy and apoptosis of endplate chondrocytes in intervertebral disc. The increase of autophagy activity could prevent endplate chondrocytes from apoptosis. The autophagy in endplate chondrocytes induced by oxidative stress was mTOR dependent. These findings might shed some new lights on the mechanism for IDD and provide new strategies for the treatments of IDD.

The matrikine N-acetylated proline-glycine-proline induces premature senescence of nucleus pulposus cells via CXCR1-dependent ROS accumulation and DNA damage and reinforces the destructive effect of these cells on homeostasis of intervertebral discs

Intervertebral disc (IVD) cell senescence is a recognized mechanism of intervertebral disc degeneration (IDD). Elucidating the molecular mechanisms underlying disc cell senescence will contribute to understanding the pathogenesis of IDD. We previously reported that N-acetylated proline-glycine-proline (N-Ac-PGP), a matrikine, is involved in the process of IDD. However, its roles in IDD are not well understood. Here, using rat nucleus pulposus (NP) cells, we found that N-Ac-PGP induced premature senescence of NP cells by binding to CXCR1. N-Ac-PGP induced DNA damage and reactive oxygen species accumulation in NP cells, which resulted in activation of the p53-p21-Rb and p16-Rb pathways. Moreover, the RT2 profiler PCR array showed that N-Ac-PGP down-regulates the expression of antioxidant genes in NP cells, suggesting a decline in the antioxidants of NP cells. On the other hand, N-Ac-PGP up-regulated the expression of matrix catabolic genes and inflammatory genes in NP cells. Concomitantly, N-Ac-PGP reinforced the destructive effects of senescent NP cells on the homeostasis of the IVDs in vivo. Our study suggests that N-Ac-PGP plays critical roles in the pathogenesis of IDD through the induction of premature senescence of disc cells and via the activation of catabolic and inflammatory cascades in disc cells. N-Ac-PGP also deteriorates the redox environment of disc cells. Hence, N-Ac-PGP is a new potential therapeutic target for IDD.

The Use of Stem Cells in the Treatment of Intervertebral Disc Degeneration

The paper presents a review of current data on the use of stem cells in the treatment of intervertebral disc degeneration. Acute spinal pain is often a consequence of the pathology affecting the intervertebral disc. Many applied therapeutic techniques do not provide effective results as expected because most of them address symptoms, but do not treat the underlying disease. We have outlined current findings on the molecular mechanisms of intervertebral disc degeneration, analyzed international experimental studies demonstrating the feasibility of a stem cell therapy for intervertebral disc degeneration. The conducted studies reported on the clinical application of mesenchymal stem cells or stem cells derived from adipose, synovium, and bone marrow tissue. The most pressing and undetermined issues that require further experimental and clinical studies are indicated and defined in the article.

MyD88-dependent Toll-like receptor 4 signal pathway in intervertebral disc degeneration.

Lower back pain (LBP) is a common and remitting problem. One of the primary causes of LBP is thought to be degeneration of the intervertebral disc (IVD). The aim of the present study was to investigate the role of the myeloid differentiation primary-response protein 88 (MyD88)-dependent Toll-like receptor 4 (TLR4) signal pathway in the mechanism of IVD degeneration. IVD nucleus pulposus cells isolated and cultured from the lumbar vertebrae of Wistar rats were stimulated by various doses of lipopolysaccharide (LPS; 0.1, 1, 10 and 100 µg/ml) to simulate IVD degeneration. Cells were rinsed and cultured in serum-free Dulbecco's modified Eagle's medium/F12. Reverse transcription-quantitative polymerase chain reaction was used to determine the levels of TLR4, MyD88, tumor necrosis factor α (TNFα), and interleukin-1β (IL-1β) mRNA expression after 1, 3, 6, 9 and 12 h of incubation. Additionally, western blot and enzyme-linked immunosorbent assay analyses were used to determine the levels of TLR4, MyD88, TNFα, and IL-1β protein expression after 24, 48 and 72 h of incubation. The levels of TLR4, MyD88, TNFα and IL-1β mRNA all increased in the cells stimulated by 10 µg/ml LPS at 3, 6 and 9 h (all P<0.001). Furthermore, the levels of TLR4, MyD88, TNFα and IL-1β protein all increased at 24, 48 and 72 h (all P<0.001). Additionally, the mRNA and protein levels of TLR4, MyD88, TNFα and IL-1β increased significantly in the cells stimulated by 1, 10 and 100 µg/ml LPS compared with the control group, and reached a peak in the 10 µg/ml LPS group (all P<0.001). These results suggest that the MyD88-dependent TLR4 signal pathway is a target pathway in IVD degeneration. This pathway is time phase- and dose-dependent, and when activated can lead to the release of inflammatory factors that participate in IVD degeneration.

Potential Role of lncRNAs in Contributing to Pathogenesis of Intervertebral Disc Degeneration Based on Microarray Data.

BackgroundOur study intended to identify potential long non-coding RNAs (lncRNAs) and genes, and to elucidate the underlying mechanisms of intervertebral disc degeneration (IDD).Material/MethodsThe microarray of GSE56081 was downloaded from the Gene Expression Omnibus database, including 5 human control nucleus pulposus tissues and 5 degenerative nucleus pulposus tissues, which was on the basis of GPL15314 platform. Identification of differentially expressed lncRNAs and mRNAs were performed between the 2 groups. Then, gene ontology (GO) and pathway enrichment analyses were performed to analyze the biological functions and pathways for the differentially expressed mRNAs. Simultaneously, lncRNA-mRNA weighted coexpression network was constructed using the WGCNA package, followed by GO and KEGG pathway enrichment analyses for the genes in the modules. Finally, the protein-protein interaction (PPI) network was visualized.ResultsA total of 135 significantly up- and 170 down-regulated lncRNAs and 2133 significantly up- and 1098 down-regulated mRNAs were identified. Additionally, UBA52 (ubiquitin A-52 residue ribosomal protein fusion product 1), with the highest connectivity degree in PPI network, was remarkably enriched in the pathway of metabolism of proteins. Eight lncRNAs – LINC00917, CTD-2246P4.1, CTC-523E23.5, RP4-639J15.1, RP11-363G2.4, AC005082.12, MIR132, and RP11-38F22.1 – were observed in the modules of lncRNA-mRNA weighted coexpression network. Moreover, SPHK1 in the green-yellow module was significantly enriched in positive regulation of cell migration.ConclusionsLncRNAs LINC00917, CTD-2246P4.1, CTC-523E23.5, RP4-639J15.1, RP11-363G2.4, AC005082.12, MIR132, and RP11-38F22.1 were differentially expressed and might play important roles in the development of IDD. Key genes, such as UBA52 and SPHK1, may be pivotal biomarkers for IDD.

Identification of the potential molecular targets for human intervertebral disc degeneration based on bioinformatic methods.

The present study aimed to explore potential molecular targets and gain further insights into the mechanism of intervertebral disc degeneration (IDD) progression. Microarray datasets of GSE19943, GSE15227 and GSE34095 were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) in 3 IDD specimens compared with 3 controls in GSE34095, DEGs in 7 grade III and 3 grade IV samples compared with 5 grade II samples in GSE19943, and differentially expressed miRNAs in 3 degenerated samples compared with 3 controls in GSE15227 were screened. Grade III‑ and IV‑specific networks were constructed and grade‑specific genes were extracted. The network features were analyzed, followed by Gene Ontology (GO) enrichment analysis and pathway enrichment analysis of grade‑specific genes and DEGs identified in GSE34095. Furthermore, miRNA‑pathway interactions were analyzed using Fisher's exact test. Tumor protein p53 (TP53) was a hub gene in the grade III‑specific network and ubiquitin C (UBC) was identified to be a hub gene in the grade IV‑specific network. Six significant features were identified by grade‑specific network topology analysis. Grade‑specific genes and DEGs were involved in different GO terms and pathways. Differentially expressed miRNAs were identified to participate in 35 pathways, among which 6 pathways were significantly enriched by DEGs, including apoptosis. The present study identified that key genes (TP53 and UBC) and miR‑129‑5p may participate in the mechanism of IDD progression. Thus, they may be potential therapeutic targets for IDD.

Genetic and functional studies of the intervertebral disc: a novel murine intervertebral disc model.

Intervertebral disc (IVD) homeostasis is mediated through a combination of micro-environmental and biomechanical factors, all of which are subject to genetic influences. The aim of this study is to develop and characterize a genetically tractable, ex vivo organ culture model that can be used to further elucidate mechanisms of intervertebral disc disease. Specifically, we demonstrate that IVD disc explants (1) maintain their native phenotype in prolonged culture, (2) are responsive to exogenous stimuli, and (3) that relevant homeostatic regulatory mechanisms can be modulated through ex-vivo genetic recombination. We present a novel technique for isolation of murine IVD explants with demonstration of explant viability (CMFDA/propidium iodide staining), disc anatomy (H&E), maintenance of extracellular matrix (ECM) (Alcian Blue staining), and native expression profile (qRT-PCR) as well as ex vivo genetic recombination (mT/mG reporter mice; AdCre) following 14 days of culture in DMEM media containing 10% fetal bovine serum, 1% L-glutamine, and 1% penicillin/streptomycin. IVD explants maintained their micro-anatomic integrity, ECM proteoglycan content, viability, and gene expression profile consistent with a homeostatic drive in culture. Treatment of genetically engineered explants with cre-expressing adenovirus efficaciously induced ex vivo genetic recombination in a variety of genetically engineered mouse models. Exogenous administration of IL-1ß and TGF-ß3 resulted in predicted catabolic and anabolic responses, respectively. Genetic recombination of TGFBR1fl/fl explants resulted in constitutively active TGF-ß signaling that matched that of exogenously administered TGF-ß3. Our results illustrate the utility of the murine intervertebral disc explant to investigate mechanisms of intervertebral disc degeneration.

Macrophage Migration Inhibitory Factor Inhibits the Migration of Cartilage End Plate-Derived Stem Cells by Reacting CD74

Introduction Macrophage migration inhibitory factor (MIF) is a multifunctional cytokine that regulates inflammatory reactions and the pathophysiology of many inflammatory diseases. Intervertebral disc (IVD) degeneration is characterized by an inflammatory reaction, but the potential role of MIF in IVD degeneration has not yet been determined. Recent studies have shown that MIF and its receptor, CD74, are involved in regulating the migration of human mesenchymal stem cells (MSCs). Thus, MIF might impair the ability of mesenchymal stem cells (MSCs) to home to injured tissues. Our previous studies indicated that cartilage endplate-derived stem cells (CESCs) as a type of MSCs exist in human degenerate IVDs. Here, we investigate the role of MIF in regulating the migration of CESCs. Material and Method CESCs were isolated and identified. Results We have shown that MIF was distributed in human degenerate IVD tissues and was subject to regulation by the proinflammatory cytokine tumor necrosis factor-α. Furthermore, in-vitro cell migration assays revealed that nucleus pulposus (NP) cells inhibited the migration of CESCs in a number-dependent manner, and enzyme-linked immunosorbent assays revealed that the amount of MIF in conditioned medium was significantly increased as a function of increasing cell number. In addition, recombinant human MIF inhibited the migration of CESCs in a dose-dependent manner. CESCs migration was restored when an antagonist of MIF, (S, R)-3(4-hydroxyphenyl)-4, 5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), was added. Finally, a CD74 activating antibody (CD74Ab) was used to examine the effect of CD74 on CESCs motility and inhibited the migration of CESCs in a dose-dependent manner. Conclusion We have identified and characterized a novel regulatory mechanism governing cell migration during IVD degeneration. The results will benefit understanding of another possible mechanism for IVD degeneration, and might provide a new method to repair degenerate IVD by enhancing CESCs migration to degenerated NP tissues to exert their regenerative effects. Disclosure of Interest None declared

Role of nuclear factor of activated T-cells in regulating ADAMTS -4 promoter activity in nucleus pulposus cells

Objective To observe the regulatory effect of nuclear factor of activated T-cells( NFAT)-1,4,5 on ADAMTS-4 promoter activity in nucleus pulposus cells,so as to discuss the underlying molecular mechanism of intervertebral disc degeneration. Methods The ADAMTS-4-pβ-gal-Basic was inserted into PGL3-Basic vector after double digestion by the restriction enzyme XhoⅠ and HindⅢ. The purified PGL3-ADAMTS-4 plasmid was obtained through screening. Rat nucleus pulposus cells were cultured in vitro and,via Lipofectamine2000 transfection reagent,transfected with NFAT-1,NFAT-4,NFAT-5 or DN-TonEBP expressing plasmids with or without appropriate backbone vector and 175 ng ADAMTS-4 promoter. The normal cells were taken as controls and transfected cells were taken as treatment group. Dual-LuciferaseTMreporter assay system was used to detect the effect of NFAT-1,4,5( TonEBP) on ADAMTS-4 promoter activity. Results We successfully constructed PGL3-ADAMTS-4 plasmid and identified two NFAT-Bind elements in the promoter. Compared with the control group,ADAMTS-4 promoter activity was significantly inhibited in NFAT-1 group( P 0. 05). While the activities of ADAMTS-4 promoter in NFAT-4,NAFT-5 and DN-TonEBP groups showed no significant changes( P 0. 05). Conclusion NFAT-1 can regulate ADAMTS-4 expression,which may play a role in modulating aggrecan content in the intervertebral disc physiology and / or intervertebral disc degeneration,providing a new strategy for biological treatment of intervertebral disc degeneration.

Cell death in intervertebral disc degeneration

Degeneration of intervertebral disc (IVD) is mainly a chronic process of excessive destruction of the extracellular matrix (ECM), and also is thought to be the primary cause of low back pain. Presently, however, the underlying mechanism of IVD degeneration is still not elucidated. Cellular loss from cell death has been believed to contribute to the degradation of ECM and plays an important role in the process of IVD degeneration, but the mechanisms of cell death in degenerated IVD remain unclear. Apoptosis, a very important type of IVD cell death, has been considered to play a crucial role in the process of degeneration. Autophagy, a non-apoptosis death type of programmed cell death, has been considered extensively involved in many pathological and physiological processes, including the degenerative diseases. Thus, the research on cell death in IVD degeneration has become a new focus recently. In this review, by analyzing the available literature pertaining to cell death in IVD and discussing the inducing factors of IVD degeneration, NP cells and ECM in IVD degeneration, apoptotic signal transduction pathways involved in IVD cell death, the relationship of cell death with IVD degeneration and potential therapeutic strategy for IVD degeneration by regulating cell death, we conclude that different stimuli induce cell death in IVD via various signal transduction pathways, and that cell death may play a key role in the degenerative process of IVD. Regulation of cell death could be a potential and attractive therapeutic strategy for IVD degeneration.

Apoptosis in TMJ Disc Diseases

The apoptosis is the programmed cell death, a distinct process compared to cellular necrosis, which plays an important role in both human embryonic development and adult tissue homeostasis. Apoptosis represents a physiological form of cell death. The role of apoptosis is to remove harmful, damaged or unwanted cells without inducing inflammatory response by the release of cell contents [1]. Apoptosis is activated through two principal signaling pathways: intrinsic and extrinsic, both of which are potential anticancer therapeutic targets. In contrast to necrosis, which is a form of cell death resulting from an acute cellular stress or trauma, apoptosis is a death orderly and regular, does not induce inflammation, requires energy (ATP) and generally lead to an advantage during the body life. Besides its importance as a biological phenomenon, it has acquired a huge medical value, since imperfect process of apoptosis covers many diseases. Excessive activity can cause trouble by apoptotic cell loss (see, for example neurodegenerative diseases such as Parkinson’s disease), while a weak apoptosis may involve uncontrolled cell growth in malignancy mechanism [2]. Apoptosis is the most common mechanism by which the body eliminates damaged or unneeded cells, without local inflammation from leakage of cell contents. Cells that are undergoing apoptosis exhibit a characteristic pattern of morphologic changes, including cell shrinkage, condensation, fragmentation of the nucleus and bubbling of the plasma membrane, known as “blebbing,” and chromatin condensation and nucleosomal fragmentation [1,3]. Apoptosis functions through two main, alternative pathways: death receptor-mediated (or extrinsic) and mitochondria-dependent (or intrinsic). The former pathway is initiated by ligation of specific death receptors by their ligands. The main death receptors—Fas and tumour necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) receptors R1 and R2—induce cell death following ligation with Fas ligand (FasL) or TRAIL, respectively [4-7]. Ligation of TRAIL R1 by TNFα also induces apoptosis after inhibition of nuclear factor kappa-B (NF-kB). Fas ligation by FasL is followed by recruitment of FADD (Fas-associated via death domain) and subsequently of caspase 8. This process gives rise to caspase 8 activation, which can be inhibited by the anti-apoptotic molecule FLICE inhibitory protein (Flip). Caspase 8 induces apoptosis by directly activating caspase 3 or by cleaving bid (BH3 interacting domain death agonist), resulting in mitochondrial dysfunction and subsequent release of cytochrome c and activation of caspases 9 and 3. Caspase 3 promotes the typical apoptosis features, including DNA fragmentation and cell death in several tissues [8,9]. It is unclear whether repetitive loading and apoptosis are quantitatively related in any type of cartilage. Several studies have demonstrated the crucial function of apoptotic mechanisms in intervertebral disc degeneration, and the involvement of apoptosis in various conditions associated with intervertebral disc degeneration has been thoroughly explored [10]. However, apoptosis has been documented to have a central role not only in intervertebral disc degeneration [10]. Although a number of features of temporomandibular joint (TMJ) disc degeneration have been thoroughly studied, data on the involvement of apoptotic mechanisms and their mediators are few and quite recent; indeed most of the research conducted on disc apoptosis has focused on the intervertebral disc. TMJ disc degeneration is believed to be a consequence of mechanical and biological events affecting the equilibrium between matrix synthesis and degradation. According to this hypothesis loss of cellularity, collagen fibre fragmentation and TMJ disc tears and clefts would all depend on excessive apoptosis, a situation where collagen fibre degradation is not offset by synthesis of new fibres because of apoptosis-induced cell loss. The findings correlating TMJ disc internal derangement and apoptosis largely come from animal models [11]. However, our group has extensively analyzed programmed cell death in human TMJ discs with and without internal derangement [12,13]. We have advanced Corresponding author.

Macrophage migration inhibitory factor inhibits the migration of cartilage end plate-derived stem cells by reacting with CD74.

BackgroundMacrophage migration inhibitory factor (MIF) is a multifunctional cytokine that regulates inflammatory reactions and the pathophysiology of many inflammatory diseases. Intervertebral disc (IVD) degeneration is characterized by an inflammatory reaction, but the potential role of MIF in IVD degeneration has not been determined. Recent studies have shown that MIF and its receptor, CD74, are involved in regulating the migration of human mesenchymal stem cells (MSCs); Thus, MIF might impair the ability of mesenchymal stem cells (MSCs) to home to injured tissues. Our previous studies indicated that cartilage endplate (CEP)-derived stem cells (CESCs) as a type of MSCs exist in human degenerate IVDs. Here, we investigate the role of MIF in regulating the migration of CESCs.Methods and FindingsCESCs were isolated and identified. We have shown that MIF was distributed in human degenerate IVD tissues and was subject to regulation by the pro-inflammatory cytokine TNF-α. Furthermore, in vitro cell migration assays revealed that nucleus pulposus (NP) cells inhibited the migration of CESCs in a number-dependent manner, and ELISA assays revealed that the amount of MIF in conditioned medium (CM) was significantly increased as a function of increasing cell number. Additionally, recombinant human MIF (r-MIF) inhibited the migration of CESCs in a dose-dependent manner. CESCs migration was restored when an antagonist of MIF, (S, R)-3(4-hydroxyphenyl)-4, 5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1), was added. Finally, a CD74 activating antibody (CD74Ab) was used to examine the effect of CD74 on CESCs motility and inhibited the migration of CESCs in a dose-dependent manner.ConclusionsWe have identified and characterized a novel regulatory mechanism governing cell migration during IVD degeneration. The results will benefit understanding of another possible mechanism for IVD degeneration, and might provide a new method to repair degenerate IVD by enhancing CESCs migration to degenerated NP tissues to exert their regenerative effects.