CILP Is Regulated By Tension Stimulation And Affects The Synthesis Of Matrix Phenotype

BackgroundLow back pain(LBP)is one of the main disorders that has drew attention of scientists for its threat to people’s health.70-80% of individuals in the world have experienced LBP at some time in their lives,of which a large percentage underwent surgeries,casting a heavy economic burden on families and society.Generally,Intervertebral disc degeneration(IDD)is regarded as the main cause contributing to the initiation and progress of LBP.With respect to the complicated pathology in IDD associated with susceptible genes,excessive mechanical loading,increased cellular senescence,nutrition failure and so on,more studies are required to identify critical factors that play important roles in IDD and cast a detailed knowledge of the mechanism in IDD.Cartilage intermediate layer protein(CILP)is restrictively distributed in articular cartilage and intervertebral discs(IVDs).Previous studies have shown an increased expression of CILP in aging discs and revealed the correlation of a functional single nucleotide polymorphism(SNP)in 1184 allele of CILP gene with the incidence of disc diseases,which indicates the possible involvement of CILP in IDD.Further,it has been validated that the polymorphism in 1184 allele increases the affinity of CILP for transforming growth factor-beta(TGF-β),handicaping the interaction of TGF-β with its receptor,suggesting that CILP may be involved in IDD by interfering in the interaction of some growth factors with resident cells.Similarly,CILP is also able to decrease the pro-anabolism effect of IGF-1 on chondrocyte and indirectly promote PPi supersaturation in bovine aging and osteoarthritis cartilage.Moreover,in transgenic mice over-expressing CILP,a low signal was detected by MRI at early stage,indicating an early degeneration initiated in IVDs,which suggests the ability of CILP to launch the initiation of degeneration.In summary,the irregularly expressed CILP in IDD may not just be a landmark for degeneration,but also contribute to the progress as an accelerator.However,it still remains unknown how CILP is involved in IDD.The pathology of IDD is complex associated with increasing cell autophagy,senescence,and invasion of inflammatory mediators,charactered by up-regulation of catabolic enzymes and down-regulation of matrix phenotype,which lead to the imbalance between the anabolism and catabolism in extracellular matrix(ECM)and bring about the disrupt in ECM homeostasis finally.The failure in ECM homeostasis give rise to the disorder in the macro-environment around resident cells,accelerating the degeneration through exposing resident cells to altered pH and abnormal osmotic pressure.What is worse,the alteration in ECM deteriorates the mechanical environment around the resident cells,which increases the expression of matrix proteases in NP cells and exert an inhibition on the synthesis of matrix phenotype such as Agg and Col II.In a word,the homeostasis of ECM play a pivotal role in maintaining the structure and function of IVDs and the disrupt of ECM homeostasis is the major event responsible for the progress of IDD.Hence,it is imperative to reveal whether CILP expression exert an influence on the homeostasis of ECM,which help to further confirm the pro-degeneration role of CILP in IDD.Meanwhile,it still remains unknown how CILP expression is up-regulated in degenerative IVDs.Previous studies have shown that TGF-β and BMP-2 are able to promote CILP synthesis and forms an negative feedback mechanism with the inhibitory effect by CILP.However,With the decreased NP cells in NP tissues and declined secretion of growth factors by resident cells in degenerative IVDs,the up-regulation of CILP is unlikely to be an result of increased TGF-β or BMP-2.Hence,it still remains unknown how the expression of CILP is increased and maintained at a high level in IDD.With respect to the causes involved in the pathology of IDD.Mechanical stimuli should be the critical factor affecting the metabolism of NP cells.Previous studies have shown mechanical stimuli,including tension,compression and shear force,exert diverse effects on the metabolism of NP cells,which are relied on the strength,duration and frequency.Further,mechanical stress has a great impact on the homeostasis of ECM and abnormal mechanical loading is regarded as one of the main causes accelerating the progress of ECM degeneration.Hence,it is profound to investigate whether CILP expression can be regulated by mechanical stress,which may help to explain the irregularly expressed CILP in IDD.Through settling those issues,the pro-degeneration role of CILP in IDD will be further confirmed,and we will get a primary knowledge of regulatory mechanism underlying the up-regulation of CILP in IDD.Methods1.NP tissues were collected from patients whose IVDs were removed for traumatic fracture or severe IDD in Xinqiao hospital in 2016,which were grouped into mild degeneration group(Pfirrmann grade ≤III)and severe degeneration group(Pfirrmann grade >III);The difference in CILP expression between the two groups was confirmed by immunohistochemistry and integrated optical density(IOD).2.NP cells were treated with cyclic tensile strain in different duration and strength by using Flexcell 5000 CTM.RT-qPCR and western blotting were used to detect the expression of CILP and the main matrix phenotype(Agg and Col II).;3.NP cells were treated with recombinant CILP in various concentrations for 72 h,then RT-qPCR and western bloting were used to detect the expression of the main matrix phenotype;Meanwhile,NP cells were transfected with CILPsiRNA for 48 h,then RT-qPCR and western bloting were used to detect the expression of the main matrix phenotype.Taken together,it will be revealed whether CILP exert an influence on the homeostasis of ECM.Results1.The expression of CILP in severe degenerative discs is obviously increased compared with mild ones;2.5% tensile strain exerts an insignificant influence on CILP expression;10% tensile strain markedly inhibits CILP expression while up-regulates the level of main matrix phenotype;20% tensile strain increases CILP expression and Smad3 phosphorylation level significantly,and the up-regulation of CILP is obviously suppressed while NP cells are pretreated with Smad3 inhibitor(S1S3);3.Transfection with CILP siRNA for 48 h significantly decreases CILP expression,meanwhile,the synthesis of aggrecan and collagen II are increased obviously.In addition,treating NP cells with recombinant CILP in high concentration(1000ug/ml)for 72 h markedly suppresses the expression of aggrecan and collagen II.Conclusion1.CILP is differently expressed in IVDs with distinct degeneration and increased along the progress of degeneration.The result in our study is in consistent with previous studies concerning degeneration-associated factors in degenerative IVDs.Previous studies have shown the up-regulation of CILP in Osteoarthritis(OA)tissues and confirmed the pro-inflammatory effect of CILP on chondrocyte cells.With respect to those studies,it is reasonable to speculate that up-regulation of CILP in NP tissues is not only an indicator for IDD,it may contribute to the initiation and progress of IDD as well as in cartilage tissues.2.Mechanical stress regulate CILP expression in a time-dependent and strength-dependent manner:(a).Proper tensile strain significantly increases the synthesis of matrix phenotype(Agg and Col II),while exerts an suppression on CILP expression,which is in compliance with the situation in health IVD,the result may help to explain why CILP is especially but lowly expressed in non-degenerative discs.(b).Excessive strain significantly increases CILP expression in mRNA and protein level,which is contrary to the former result induced by a weaker strain,implying that mechanical stimuli exerts an diverse regulation on CILP expression relying on the strength.Further,combining with previous results,excessive strain on NP cells resulted from degeneration in ECM may be one of the main causes contributing to the irregular expression of CILP in IDD.(C).Pretreating NP cells with Smad3 inhibitor significantly suppress the up-regulation of CILP by 20% CTS,implying that abnormal mechanical stimuli contribute to promotion on CILP expression via activating Smads pathway.Moreover,previous studies have shown Smad signal pathway mediates the up-regulation by TGF-β via activating the enhancer in CILP gene and MAPK signal pathway is involved in the process via the cross-talk with Smad pathway.Given that MAPK pathway is also the main signal pathway mediating the regulation of mechanical stimuli on NP cells,it suggests Smad signal pathway is the common way regulating CILP expression and mechanical strain regulate CILP expression via the cross-talk between MAPK and Smad pathways.3.CILP,which is synthesized and secreted into ECM by NP cells,is able to inhibit the expression of main matrix phenotype.Our study is the first to confirm the pro-degradation effect on ECM homeostasis by CILP.Combined with our former results confirming the increased CILP expression in IDD,Our study backs up the hypothesis that irregularly expressed CILP contribute to IDD by accelerating the degradation of ECM.Moreover,As our study confirmed the mechanic-mediated regulation on CILP expression and the inhibitory effect of CILP on matrix phenotype,it is reasonable to speculate that CILP may mediate the regulation on ECM by mechanical stress,which need more studies to validate.