Zhaomin Zheng

TNF-α and IL-1β Promote a Disintegrin-like and Metalloprotease with Thrombospondin Type I Motif-5-mediated Aggrecan Degradation through Syndecan-4 in Intervertebral Disc

Background: Disc degeneration is characterized by elevated levels of cytokines, TNF-α and IL-1β and an increase in aggrecan-rich matrix degradation. Results: Cytokine-dependent syndecan-4 expression regulate ADAMTS-5/aggrecanse2 activation in disc cells. Conclusion: Syndecan-4 may play a key role in pathogenesis of degenerative disc disease. Significance: Syndecan-4 may offer a potential therapeutic target for controlling progression of degenerative disc disease. Elevated levels of TNF-α, IL-1β and a resultant increase in ADAMTS (a disintegrin-like and metalloprotease with thrombospondin type I motifs) expression is seen during disc degeneration. However, if these pro-inflammatory cytokines control ADAMTS activity is not definitively known. The goal of the investigation was to study if TNF-α and IL-1β regulate syndecan-4 (SDC4) expression, and if SDC4 was responsible for promoting aggrecan degradation through controlling ADAMTS activity in nucleus pulposus cells of the intervertebral disc. Cytokine treatment increased SDC4 expression and promoter activity. Use of inhibitor, SM7368 and co-transfections with IκBα, RelA/p50 showed that NF-κΒ regulated both basal and cytokine-dependent SDC4 transcription. SDC4 promoter harboring RelA binding site mutation was unresponsive to the cytokines. Moreover, cytokines failed to increase SDC4 promoter activity in RelA-null cells. Cytokines increased ADAMTS-4/5 expression and aggrecan degradation and promoted SDC4 interaction with ADAMTS-5. Treatment with heparinase-III and p-nitrophenyl-β-d-xylopyranoside (PNPX), an inhibitor of heparan sulfate synthesis and transfection with SDC4-shRNA partially blocked cytokine mediated aggrecan degradation. Analysis of human tissues showed increased aggrecan degradation with a concomitant increase in SDC4 and ADAMTS-5 protein expression with severity of disc disease. Likewise, SDC4, TNF-α, IL-1β, ADAMTS-4, and ADAMTS-5 mRNA expression increased in degenerate tissues. We conclude that in nucleus pulposus, TNF-α and IL-1β regulate SDC4 expression, which plays a key role in pathogenesis of degenerative disc disease by promoting aggrecan degradation by ADAMTS-5.

Inflammatory Cytokines Induce NOTCH Signaling in Nucleus Pulposus Cells IMPLICATIONS IN INTERVERTEBRAL DISC DEGENERATION

Background: The regulation of NOTCH signaling under inflammatory conditions in the nucleus pulposus is unknown. Results: Expression of select NOTCH pathway genes, including NOTCH2 and NOTCH signaling, is regulated by IL-1β and TNF-α. Conclusion: Inflammatory cytokines promote NOTCH signaling in disc. Significance: NOTCH signaling may play a role in pathogenesis of disc disease. The objective of the study was to investigate how inflammatory cytokines, IL-1β, and TNF-α control NOTCH signaling activity in nucleus pulposus (NP) cells. An increase in expression of selective NOTCH receptors (NOTCH1 and -2), ligand (JAGGED2), and target genes (HES1, HEY1, and HEY2) was observed in NP cells following cytokine treatment. A concomitant increase in NOTCH signaling as evidenced by induction in activity of target gene HES1 and HEY1 promoters and reporter 12xCSL was seen. Moreover, treatment increased activity of a 2-kb NOTCH2 promoter. Treatment of cells with NF-κB and MAPK inhibitors abolished the inductive effect of cytokines on NOTCH2 promoter and its expression. Gain and loss-of-function studies confirmed the inductive effect of p65 on NOTCH2 promoter activity. In contrast, p50 blocked the cytokine induction of promoter activity. Supporting promoter studies, lentiviral delivery of sh-p65, and sh-IKKβ significantly decreased cytokine dependent change in NOTCH2 expression. Interestingly, MAPK signaling showed an isoform-specific control of NOTCH2 promoter; p38α/β2/δ, ERK1, and ERK2 contributed to cytokine dependent induction, whereas p38γ played no role. Analysis of human NP tissues showed that NOTCH1 and -2 and HEY2 expression correlated with each other. Moreover, expression of NOTCH2 and IL-1β as well as the number of cells immunopositive for NOTCH2 significantly increased in histologically degenerate discs compared with non-degenerate discs. Taken together, these results explain the observed dysregulated expression of NOTCH genes in degenerative disc disease. Thus, controlling IL-1β and TNF-α activities during disc disease may restore NOTCH signaling and nucleus pulposus cell function.

LIM mineralization protein-1 suppresses TNF-α induced intervertebral disc degeneration by maintaining nucleus pulposus extracellular matrix production and inhibiting matrix metalloproteinases expression

Imbalanced metabolism of Nucleus pulposus (NP) extracellular matrix (ECM) is closely correlated to Intervertebral Disc Degenerative Disease. LIM mineralization protein‐1 (LMP‐1) has been proven to induce sulfated glycosaminoglycan (sGAG) production in NP and have an anti‐inflammatory effect in pre‐osteoclast. However, whether it has any effect on the NP ECM production and degradation under inflammatory stimulation has not been studied. In the current study, a TNF‐α induced cell model was established in vitro. Lentivirus encoding LMP‐1 (LV‐LMP‐1) and short heparin LMP‐1 (LV‐shLMP‐1) were constructed to overexpress and knockdown LMP‐1 expression in NP cells. LMP‐1 mRNA level was regulated in a dose‐dependent manner after transfection. LV‐LMP‐1 increased whereas LV‐shLMP‐1 decreased collagen II, aggrecan, versican expression, and sGAG production. LV‐LMP‐1 abolished while LV‐shLMP‐1 aggravated TNF‐α mediated down‐regulation of the above matrix genes via ERK1/2 activation. Moreover, LV‐LMP‐1 abrogated TNF‐α induced MMP‐3 and MMP‐13 expression via inhibiting p65 translocation and MMP‐3 and MMP‐13 promoter activity. These results indicated that LMP‐1 had an ECM production maintenance effect under inflammatory stimulation. This effect was via up‐regulation of matrix genes expression at least partially through ERK1/2 activation, and down‐regulation of MMPs expression through NF‐κB inhibition.

Both Expression of Cytokines and Posterior Annulus Fibrosus Rupture Are Essential for Pain Behavior Changes Induced by Degenerative Intervertebral Disc: An Experimental Study in Rats

The aim of this study was to analyze the relationship between intervertebral disc degeneration and low back pain (LBP). Rat L4/5 disc degeneration model was established by annular puncture using a 0.4 mm needle anteriorly or posteriorly. In both anterior and posterior puncture models, magnetic resonance imaging (MRI) and histological analyses revealed marked disc degeneration 2 weeks after puncture. Cytokine expression was up‐regulated in different level in nucleus pulposus (NP) from 3 days after puncture. Pain behavioral tests indicated that the anterior disc puncture did not induce pain behavior changes, whereas the posterior disc puncture resulted in mechanical allodynia from 1 day to 21 days after injury. Besides, cytokine expression was significantly increased in dorsal root ganglion (DRG) at 1 and 2 weeks after posterior puncture, but not after the anterior puncture. These findings indicate the NP of the degenerative disc expresses different levels of inflammatory cytokines, and posterior disc puncture produced mechanical allodynia. The expression phase of cytokines in the NP was accordance with mechanical hyperalgesia in the posterior disc puncture model. Both expression of cytokines and posterior annulus fibrosus (AF) rupture in degenerative intervertebral disc are essential for pain behavior changes.

TGF-β1 antagonizes TNF-α induced up-regulation of matrix metalloproteinase 3 in nucleus pulposus cells: role of the ERK1/2 pathway

Abstract Tumor necrosis factor-α (TNF-α) has been shown to have a catabolic effect on intervertebral disc degeneration (IVDD), including increasing MMP3 expression and subsequent extracellular matrix (ECM) degradation. In contrast, transforming growth factor-β1 (TGF-β1) has an anabolic effect on nucleus pulposus (NP) cells. However, the anti-catabolic effect of TGF-β1 under inflammatory condition is unknown. The aim of this study was to demonstrate whether TGF-β1 can reverse TNF-α-induced MMP3 increase in NP cells and to further investigate the underlying mechanisms. The transcriptional activity, gene expression, and protein levels of MMP3 were measured by luciferase reporter assay, qRT-PCR and western blot, respectively. TNF-α increased MMP3 expression in rat NP cells time and dose dependently. TGF-β1 could abolish TNF-α-mediated up-regulation of collagen I and MMP3 expression, and down-regulate aggrecan and collagen II expression. The ERK1/2 signaling pathway was activated after exposure to TGF-β1. Treatment with ERK1/2 inhibitors (PD98059 and U0126) abolished the antagonistic effect of TGF-β1 on TNF-α mediated catabolic responses. These findings provide novel evidence supporting the anti-catabolic role of TGF-β1 in IVDD, which is important for the potential clinical application of TGF-β1 in disc degenerative disorders.

Enrichment of committed human nucleus pulposus cells expressing chondroitin sulfate proteoglycans under alginate encapsulation

OBJECTIVE Intervertebral disc (IVD) degeneration is associated with a malfunction of the nucleus pulposus (NP). Alginate culturing provides a favorable microenvironment for the phenotypic maintenance of chondrocyte-like NP cells. However, NP cells are recently evidenced to present heterogeneous populations, including progenitors, fibroblastic cells and primitive NP cells. The aim of this study is to profile the phenotypic changes of distinct human NP cells populations and describe the dynamic expression of chondroitin sulfate glycosaminoglycans (CS-GAGs) in extended alginate encapsulation. METHOD Non-degenerated (ND-NPC) and degenerated (D-NPC) NP cells were expanded in monolayers, and subject to 28-day culture in alginate after serial passaging. CS-GAG compositional expression in monolayer-/alginate-cultured NP cells was evaluated by carbohydrate electrophoresis. Cellular phenotypic changes were assessed by immunologic detection and gene expression analysis. RESULTS Relative to D-NPC, ND-NPC displayed remarkably higher expression levels of chondroitin-4-sulfate GAGs over the 28-day culture. Compared with monolayer culture, ND-NPC showed increased NP marker expression of KRT18, KRT19, and CDH2, as well as chondrocyte markers SOX9 and MIA in alginate culture. In contrast, expression of fibroblastic marker COL1A1, COL3A1, and FN1 were reduced. Interestingly, ND-NPC showed a loss of Tie2+ but gain in KRT19+/CD24+ population during alginate culture. In contrast, D-NPC showed more consistent expression levels of NP surface markers during culture. CONCLUSION We demonstrate for the first time that extended alginate culture selectively enriches the committed NP cells and favors chondroitin-4-sulfate proteoglycan production. These findings suggest its validity as a model to investigate IVD cell function.

Matrix metalloproteinase 12 is an indicator of intervertebral disc degeneration co-expressed with fibrotic markers

OBJECTIVE Recent evidence suggests a role of fibrogenesis in intervertebral disc (IVD) degeneration. We aim to explore if fibrotic genes may serve as IVD degeneration indicators, and if their expression is associated with myofibroblast activity. DESIGN Transcriptional expression of fibrosis markers (COL1A1, COL3A1, FN1, HSP47, MMP12, RASAL1) were analyzed in degenerated (D) and non-degenerated (ND) human nucleus pulposus (NP) and annulus fibrosus (AF) cells, along with traditional (SOX9, ACAN) and newly established degeneration markers (CDH2, KRT19, KRT18, FBLN1, MGP, and COMP). Protein expression was investigated by immunohistochemistry in human IVDs, and in rodent IVDs undergoing natural ageing or puncture-induced degeneration. Co-expression with myofibroblast markers was examined by double staining on human and rat specimens. Disc degeneration severity and extent of fibrosis were determined by histological scoring and picrosirius red staining respectively. RESULTS Human D-NP showed more intensive staining for picrosirius red than ND-NP. Among the genes examined, D-NP showed significantly higher MMP12 expression along with lower KRT19 expression. Protein expression analysis revealed increased MMP12(+) cells in human D-IVD. Histological scoring indicated mild degeneration in the punctured rat discs and discs of ageing mouse. Higher MMP12 positivity was found in peripheral NP and AF of the degenerative rat discs and in NP of the aged mice. In addition, human D-NP and D-AF showed increased α-SMA(+) cells, indicating enhanced myofibroblast activity. MMP12 was found co-expressed with α-SMA, FSP1 and FAP-α in human and rat degenerative IVDs. CONCLUSIONS Our study suggests that in addition to a reduced KRT19 expression, an increased expression of MMP12, a profibrotic mediator, is characteristic of disc degenerative changes. Co-expression study indicates an association of the increased MMP12 positivity with myofibroblast activity in degenerated IVDs. Overall, our findings implicate an impact of MMP12 in disc cell homeostasis. The precise role of MMP12 in IVD degeneration warrants further investigation.

LIM Mineralization Protein-1 Enhances Bone Morphogenetic Protein-2–Mediated Osteogenesis Through Activation of ERK1/2 MAPK Pathway and Upregulation of Runx2 Transactivity

LIM mineralization protein‐1 (LMP‐1) is an intracellular regulator of bone formation. Upregulation of bone morphogenetic proteins (BMPs) and stabilization of BMP/Smad signaling have been proven to be the key mechanisms through which LMP‐1 enhances osteogenesis. However, how LMP‐1 regulates BMPs expression and related bone formation remains unclear. In this study, a LMP‐1–induced osteogenesis cell model was used to study the molecular action of LMP‐1 on BMP‐2 expression and bone formation. The results show that overexpression of LMP‐1 significantly increases, whereas downregulation of endogenous LMP‐1 decreases BMP‐2 expression and bone formation. Antagonism of BMP‐2 with noggin or short hairpin BMP‐2 significantly attenuates the osteoinductive effect of LMP‐1, suggesting that the osteoinductive effect of LMP‐1 is mediated by BMP‐2. LMP‐1 regulation of BMP‐2 is found to occur at the transcription level using a luciferase reporter assay with a reporter construct containing a BMP‐2 promoter. A promoter deletion assay reveals that –1000/–500 bp is the key regulated region by LMP‐1. A Runx2‐binding site is then located at –934/–920 bp and confirmed by luciferase assay using a reporter construct containing repeats of this Runx2‐binding site and the site‐directed mutagenesis analysis. Overexpression of LMP‐1 significantly increases Runx2 expression. Downregulation of Runx2 expression significantly decreases BMP‐2 promoter activity and BMP‐2 expression. A ChIP assay demonstrates that LMP‐1 increases the interaction between Runx2 and BMP‐2 promoter. A luciferase reporter assay using the OSE2 promoter containing a Runx2‐binding site confirms that Runx2 transactivity can be upregulated by LMP‐1. Moreover, inhibiting the activation of different pathways with specific pathway inhibitors reveals that ERK1/2 MAPK activation is essential for LMP‐1–induced upregulation of Runx2 transactivity and subsequent BMP‐2 expression. In conclusion, our novel findings describe a positive regulatory effect of LMP‐1 on BMP‐2 expression and BMP‐2–mediated osteogenesis. This effect occurs through activation of ERK1/2 pathway and subsequent upregulation of Runx2 transactivity.

TNF-α and TGF-β1 regulate Syndecan-4 expression in nucleus pulposus cells: role of the mitogen-activated protein kinase and NF-κB pathways

Abstract Syndecan-4 is emerging as an important player in cell interaction with the extracellular environment and has been shown to be involved in the progression of intervertebral disc degeneration. However, the mechanism of syndecan-4 regulation by TNF-α and the role of TGF-β1 in regulating syndecan-4 expression remain poorly understood in nucleus pulposus (NP) cells. The aim of this study was to investigate these mechanisms. We exposed NP cells to TNF-α and the gene, protein expression, and promoter activity levels of syndecan-4 were measured by qPCR, western blotting, and the luciferase reporter assay, respectively. The activation of the MAPK and NF-κB pathways was detected using western blot analysis. Syndecan-4 expression in rat NP cells was increased by TNF-α, but this was neither time nor dose dependent in response to TNF-α. ERK1/2, JNK, and NF-κB pathways were activated following TNF-α treatment. Treatment with ERK1/2 and NF-κB inhibitors decreased the up-regulation of syndecan-4 by TNF-α. However, JNK inhibition showed no effect on syndecan-4 expression induced by TNF-α. TNF-α mediated up-regulation of syndecan-4 was antagonized by TGF-β1. This study provided evidence for the differential regulation by MAPK and NF-κB pathways in the over-expression of syndecan-4 promoted by TNF-α in NP cells. Our results demonstrate that TGF-β1 exerts anabolic effects on intervertebral discs by inhibiting the expression of syndecan-4.

Role of SHOX2 in the development of intervertebral disc degeneration

Intervertebral disc (IVD) degeneration is the most common cause of low back pain, which affect 80% of the population during their lives, with heavy economic burden. Many factors have been demonstrated to participate in IVD degeneration. In this study, we investigated the role of short stature homeobox 2 (SHOX2) in the development of IVD degeneration. First, we detected the expression of SHOX2 in different stages of human IVD degeneration; then explored the role of SHOX2 on nucleus pulposus (NP) cells proliferation and apoptosis, finally we evaluated the effect of SHOX2 on the production of extracellular matrix in NP cells. Results showed that the expression of SHOX2 is mainly in NP compared with AF tissues, its expression decreased with the severity of human IVD degeneration. TNF‐α treatment led to dose‐ and time‐dependent decrease in SHOX2 mRNA, protein expression and promoter activity in NP cells. The silencing of SHOX2 inhibited NP cells proliferation and induced NP cells apoptosis. Finally, SHOX2 silencing led to decreased aggrecan and collagen II expression, along with increased ECM degrading enzymes MMP3 and ADAMTS‐5 in NP cells. In summary, our results indicated that SHOX2 plays an important role in the process of IVD degeneration, and might be a protective factor for IVD degeneration. Further studies are required to confirm its exact role, and clarify the mechanism.