The Function Of Integrin α₅β₁, Extracellular Signal-regulated Kinase And Protein Kinase C In The Process Of Fibronectin Fragments Degeneration Nucleus Pulposus Cells

IntroductionIntrevertebral disc degeneration is a complex process which could be influenced by many factors.Fibronectin is a large extracellular glycoprotein of disc that helps to organize the matrix protein,and the main membrane receptor of it isα₅β₁ integrin subunit in NP cells.Fibronectin can be disintegrated by MMPs to Fn-f.It was found that Fn-f accumulated in the disc during degeneration,and the accumulation of Fn-f in disc was proved an adverse factor to intervertebral disc.It was tested to inject Fn-f into intervertebral disc,and found Fn-f could induce a progressive degenerative process in discs.And if expose the NP cells to the Fn-f,it could be found the NP cells were degenerated.Integrins are a class of cell adhesion molecules that regulate interactions between a cell and its surrounding matrix.In many cell types,integrins participate in signalling events,controlling gene expression,cell cycle and apoptosis.The presence ofα₁,α₂,α₅,α_?,β₁,β₃ integrin subunits have been documented in human disc tissue.And their function was also proved,the integrin receptors were critical for intervertebral disc cells interactions with collagens(α₁β₁) and fibronectin(α₅β₁).And integrin participated in the process of transforming mechanic signal to chemical signal.The ERK_1/2 and PKC ware important signal transduction factors to regulate many cellular processes,and it was proved that the signals of Fn-f also were transmitted by ERK in many types of cells.More important is the ERK and PKC was proved to participate in chondrocyte signaling pathways,and it was certificated that the ERK and PKC transmited signals in the process of Fn-f mediated chondrocyte degeneration.And the ERK and PKC was also participated in the signaling pathways of intervertebral disc.ObjectiveTo determine the expressions ofα₁,α₂,α₅,α_v,β₁,β₃ integrin subunits in normal and herniated intervertebral discs of human.To determine the effects of Fn-f on the integrinα₅ andβ₁ subunits expression by NP cells,and to evaluate the activation of ERK and PKC during the process of Fn-f degenerating the NP cells.MethodsThe human nucleus pulposus specimens which were collected from operations were divided into 3 groups:normal discs(n=10),protrusion(n=15),extrusion(n=15). RT-PCR and immunoprecipitation were used to evaluate theα₁,α₅,α_v,β₁ andβ₃ integrin subunits mRNA and protein expressions.RT-PCR was also performed to measure the mRNA level of collagens and fibronectin.The NP cells cultured in alginate bead were exposed to the 110kd Fn-f,the expression of the integrinα₅β₁ subunit,MMP9,MMP13 and collagenâ…¡were determined by RT-PCR,immunoblotting and immunofluorescence.After that the expression of integfinα₅β₁ was silenced,to determine the function ofα₅β₁ subunit in the process of Fn-f degenerating NP cells.The phosphorylation of the ERK was determined after the action of Fn-f.Then the inhibitor of ERK1/2 and the inhibitor of PKC were added into the medium of NP cells,to determine the action of p-ERK and PKC.Results1,Expression of Integrin Subunits in the human nucleus pulposus Theα₁,α₅,α_v,β₁,β₃ integrin subunits were all expressed in normal and herniated nucleus pulposus.The expressions ofα₅ in extrusion and protrusion groups were 2.2 and 1.5 fold of normal group,the expressions ofβ₁ subunits in extrusion and protrusion groups were 2.5 and 1.6 fold of normal group.But as toα₁,α_v,β₃,their expressions had no difference among the discs.2,Fn-f leaded to NP cells degenerationFn-f exposure leaded to degeneration of NP cells and up-regulation of the integrinα₅β₁.Relative to controls,Fn-f decreased the collagenâ…¡gene expression,but increased the gene expression of integrinα_s andβ₁,MMP9,MMP13.And the phosphorylation of the ERK1/2 was significantly enhanced under the treatment of Fn-f.3,The function of integrinα₅β₁After the expression of integrinα₅β₁ was silenced,the Fn-f could not degenerate the NP cells,since the expression of MMP9,MMP13,collagenâ…¡and the phosphorylation of the ERK1/2 had no change.4,The function of ERK_1/2 and PKCThe PD98059 eliminated the effect of Fn-f on NP cells.And after the GF109203X was added to the medium,the degenerated function of Fn-f also was eliminated.DiscussionIn this experiment the expression of integrin subunits in normal and herniated discs was examined.And the result showed thatα₅ andβ₁ subunits were both increased expression in herniated discs,but the others had no difference.It could be estimated that during the process of intervertebral disc degeneration some adverse factors may affect the expression of integrins subunits.Risbud reported hypoxia could activate MAPK activity and regulate the integrins expression.And it was reported that mechanics was transducted across the cell surface by integrin to the cytoskeleton.It is significant that the expression of fibronectin and its receptorα₅β₁ had same tendency which was increase in our study.So it could be hypothesised that the fibronectin also stimulateα₅ andβ₁ expression in intervertebral disc.But theα_v,β₃ which is the other two receptors of fibronectin had no change in intervertebral disc,and theα₁,α₂ andα_v receptors of collagens also have no difference in intervertebral disc,though the expression of collagenâ… and collagenâ…¡had change in our research.These could be explained by the selective function of fibronectin in intervertebral disc,andα₅β₁ recepotr may be just the key point which fibronecin influences on.These results may be attributed to the interaction between cells and the ECM in the process of degeneration. But seeing that the degeneration of the intervertebral disc is a complex process,the detail principle focused on integrin should be invested continually.Fn-f are specific degradation products of the extracellular matrix of intervertebral disc,produced during proteolytic damage,have the potential to enhance the disc degeneration process.The Fn-f as a degradation product enhances the levels of the product themselves,as in a positive feedback loop.And after the expression ofα₅β₁ subunit was silenced,the effect of Fn-f on NP cells was depressed,which also certificated the integfinα₅β₁ was an important receptor of Fn-f in NP cells.It was found that the expression of integrinα₅β₁ was increased after the treatment of Fn-f, which means that the Fn-f raised the expression of its receptor-integrinα₅β₁.This is the feed back loop that is Fn-f increase the expression of integrinα₅β₁,more Fn-f combine with its receptor,so the effect of Fn-f on NP cells is enhanced.In this experiment,the signaling transduction of Fn-f mediated degeneration was investigated.The role of ERK1/2 and PKC were determined in the Fn-f mediated NP cells degeneration.We found the phosphorylation of the ERK1/2 was significantly enhanced under the treatment of Fn-f,and the PD98059 eliminated the effect of Fn-f on NP cells.We also proved the activation of ERK1/2 was through a PKC-dependent Pathway.The ERK was an important signal transduction factor to regulate many cellular processes,and it was proved that the signals of Fn-f also were transmitted by ERK in many types of cells^[17.18].More important is the ERK was proved to participate in chondrocyte signaling pathways,and it was certificated that the ERK transmited signals in the process of Fn-f mediated chondrocyte degeneration.And the ERK was also participated in the signaling pathways of intervertebral disc.The relationship between PKC and ERK was very close,and PKC was also included in the process of Fn-f mediated degeneration.We proved the ERK1/2 and PKC were participated in the signaling pathway.So the feed back loop is that after the Fn-f combined with integrinα₅β₁,the PKC Pathway was activated,the ERK1/2 was phosphorylated,so the transcription factor of nucleus was changed,the expression of integrinα₅β₁ and MMP was increased and the expression of collagenâ…¡was decreased.ConclusionThe expressions of integrinα₅ andβ₁ subunits were increased in herniated nucleus pulposus.Fn-f have the potential to enhance the NP cells degeneration,and to increase the expression of integrinα₅β₁ subunit.Fn-f have the potential to enhance the NP cells degeneration in a positive feedback loop.And the integrinα₅β₁ subunit,ERK and PKC were all included in the loop.