The Mechanism Study Of Inflammatory Cytokines Regulate Chondroitin Sulfate Synthases:Implications For Intervertebral Disc Degeneration

ObjectiveLow back pain(LBP)is the major complaint of the elderly populations,and it also significantly affects young people in recent years.LBP causes severe economic loss and social burden each year.Currently,intervertebral disc degeneration(IDD)is reckoned as the major cause of LBP.The main characteristics of IDD are loss of water content of the disc,and lowered disc height.Chondroitin sulfate(CS)proteoglycans which located in the nucleus pulposus(NP)of the disc are the major responsible molecules which maintain the water content of the disc.The biosynthesis of CS is conducted by a series of biosynthesis enzymes,and this process is poorly understood in IDD.CS was proved to be downregulated during IDD progression,but the mechanism is elusive.Inflammatory cytokines were reported as a major regulator in the extracellular matrix components of the interveterbral disc.As an important member of the extracellular matrix of the disc,CS might also involve in the regulation of inflammatory cytokines to extracellular matrix.Therefore,we designed the experiments concerning how CS was regulated in IDD,our preliminary data indicated that interleukin-1beta could downregulate the synthesis of CS,while TGF-beta could up-regulate the CS synthesis in NP cells.Then we hypothesize the inflammatory cytokines regulation on CS might through its effect on the CS biosynthesis enzymes.To illustrate this regulatory mechanism might shed lights on the future therapeutic approaches on IDD.MethodsThe expression of CS synthases in human NP tissue was tested by immunohistochemistry or RT-PCR.The CS contents in human NP tissue were measured by using immunofluorescence microscopy and DMMB assay.The potential target microRNAs were examined by using high throughput RNA sequencing technique.The expression of CS synthases in NP cells was tested by using RT-PCR,western-blot and dual-luciferase reporter assay.The sub-cellular location and expression of CS synthases were measured by using immunofluorescence microscopy.The shRNA was used for knock-down the target gene expression in NP cells.Western-blot and dualluciferase assay were applied to examine the promoter activity and the protein level of the signaling related genes.ResultsWe found that TGF-beta could increase the expression of CHSY-1 and CHPF,TGF-beta promoted CHSY-1 gene expression partially via canonical Smad pathway and majorly via MAPK-AP1/SP1 signaling pathways.Knockdown of CHSY-1 in rat NP cells abolished the induction effect of TGF-beta on CS contents.TGF-beta up-regulated CHPF gene expression via Smad pathway,MAPK and RhoA/ROCK signaling crosstalk also involved in this regulation.Knockdown of CHPF in rat NP cells abolished the induction effect of TGF-beta on CS contents.The above results indicated that CHSY-1 and CHPF play an important role in CS biosynthesis in NP cells.However,By using correlation analysis to examine the relationship between the gene expressions of CHSY-1,CHPF and TGF-beta,we found lower expressions of CHSY-1 and CHPF were accompanied with higher expression of TGF-beta.This inconsistency between human tissue and NP cells indicated there were other regulatory mechanism involved.We further tested the effect of pro-inflammatory cytokine IL-1beta on NP cells,we found that IL-1beta decreased the gene expressions of CHSY-1/2/3,combined with the evidence which the expression of IL-1beta in human NP tissue is high,we figured that under degeneration status,the destructive effect of IL-1beta might override the protective effect of TGF-beta,thus promote the disease status.By using high-throughput RNA sequencing technique,we found that the regulation of IL-1beta on CS glycotransferases was depended on the activation of microRNA-194 and microRNA-515,these results indicated that microRNAs also involved in the proinflammatory cytokine regulation on CS metabolism in NP cells.ConclusionThe reduction of CS is the leading cause of water contents loss in NP tissue,which results in IDD.The altered expression profile of CS synthases in IDD might be the major reason for the loss of CS content.Pro-inflammatory cytokine IL-1beta and anti-inflammatory cytokine TGF-beta participant in the regulation of CS synthases,the imbalance of pro-inflammatory cytokine and anti-inflammatory cytokine might be the cause of down-regulation of CS synthases gene expression and loss of CS synthesis.MAPK,Smad and RhoA/ROCK signaling pathways mediates the effect of TGF-beta on CS synthases gene expression,and in IDD status,these pathways might majorly respond to pro-inflammatory cytokines rather than antiinflammatory cytokines stimulation,this could be the reason why TGF-beta lost its ability to increase CS synthases gene expression in IDD status.Pro-inflammatory cytokine IL-1beta could significantly reduce the expression of CS synthases genes and the synthesis of CS,and this regulation is further mediated by microRNA194 and microRNA-515.The demonstration of these regulatory mechanisms might provide the evidence for the future therapeutic approaches on IDD.