Mutually Antagonistic Effects Of Activin A And TNF-α On Regulation Of Fibroblasts Activation

Tumor necrosis factor-alpha(TNF-α),as a proinflammatory cytokine,can act on a variety of cells.Activin A,a member of the transforming growth factor-beta(TGF-β)superfamily,is an important cytokine in inducing tissue fibrosis,which can promote the activation of M2 type macrophage and inhibit that bacterial lipopolysaccharide(LPS)activated M1 type macrophages.Fibroblasts are important cells involved in inflammatory response,and main functional cells of tissue repair,whose abnormal activation may induce tissue scar formation or fibrosis.During the inflammatory response,the levels of TNF-α and activin A both increase in tissue.However,it is still unclear whether activin A plays an anti-inflammatory role as the natural antagonist of TNF-α,and whether activin A and TNF-α can synergistically regulate fibroblasts activation.Therefore,in the present study,L929 cells(murine fibroblast cell line),a kind of TNF-α target cells for activity assay,were selected to investigate the combined effects of activin A and TNF-α on the activation of mouse fibroblasts and the related mechanisms,and the primary cultured cells were utilized to further determined the combined regulation effects of activin A and TNF-α on the activities of human fibroblasts.Ⅰ.METHODS1.Detection of activities in mouse fibroblastsCCK-8 assay and RTCA were used to analyze the proliferation and adhesion of L929 cells.Flow cytometry was used to detect the cell apoptosis.ELISA was used to detect the secretory levels of IL-6 and TGF-β1.The mRNA and protein expression of extracellular matrix(ECM)and related proteases was detected by RT-PCR and Western blotting.The cell migration ability was analyzed by Transwell chamber assay and microfluidic technology.2.Activation mechanism of mouse fibroblastsFlow cytometry was used to analyze the intracellular calcium levels and the expression levels of membrane-associated receptors in L929 cells.RT-PCR and Western blotting were used to detect the mRNA and protein expression levels of Smad and MAPK signalings related molecules.3.Culture of human periodontal ligament fibroblastsHuman periodontal ligament cells(hPDLCs)were cultured in vitro by tissue explants adherent method,and identified by immunocytochemistry staining.4.Detection of activities in human fibroblastsThe proliferation and adhesion of hPDLCs were detected by CCK-8 assay and RTCA.The expression levels of IL-6 and TGF-β1 were detected by ELISA.The mRNA and protein expression of ECM and related proteases was detected by RT-PCR and Western blotting.The cell migration ability was analyzed by Transwell chamber assay and microfluidic technology.5.Activation mechanism of human fibroblastsRT-PCR and Western blotting were used to detect the mRNA and protein expression levels of Smad and MAPK signalings related molecules in hPDLCs.Ⅱ.RESULTS1.Effects of activin A and TNF-α on the activities of mouse L929 fibroblasts1.1 TNF-α inhibited the proliferation of L929 cells and induced cell apoptosis,while activin A antagonized the above effects of TNF-α.1.2 TNF-α induced the secretion of inflammatory mediators NO and IL-6 in L929 cells,but activin A inhibited this effect of TNF-α.1.3 Activin A promoted the secretion of TGF-β1 in L929 cells,while TNF-αinhibited TGF-β1 secretion of L929 cells induced by activin A.1.4 Activin A up-regulated the expression of FN,Col I and MMP-2 mRNA,but TNF-α down-regulated the high expression of FN and MMP-2 mRNA induced by activin A in L929 cells.TNF-α promoted MMP-9 mRNA expression,while activin A inhibited the increase of MMP-9 mRNA expression induced by TNF-α in L929 cells.In addition,Western blotting results confirmed that activin A up-regulated MMP-2protein expression in L929 cells,while TNF-α inhibited the increase of MMP-2protein expression induced by Activin A.TNF-α up-regulated MMP-9 protein expression,while activin A inhibited the high expression of MMP-9 protein induced by TNF-α in L929 cells.1.5 Activin A and TNF-α alone could enhance the adhesion ability of L929 cells,and the combination of them had synergistic action.1.6 Activin A induced L929 cells migration,while TNF-α inhibited the chemotactic migration of L929 cells to activin A.2.Mechanisms of activin A and TNF-α regulating the activation in mouse fibroblasts2.1 Activin A induced the increase of intracellular calcium level in L929 cells,while TNF-α could weakened the affect of activin A.2.2 Activin A had no effects on the expression of TNFR1 and TNFR2,but TNF-αdown-regulated the expression of ActRⅡA on L929 cell membrane.2.3 Activin A had no significant effects on the expression of Smad signaling related molecules,but up-regulated the expression level of p-ERK1/2 protein in L929 cells.TNF-α up-regulated the expression levels of p-ERK1/2 and p-JNK proteins,while the expression level of p-ERK1/2 protein in L929 cells co-treated with activin A plus TNF-α was significantly lower than that treated with TNF-α alone in L929 cells.2.4 ERK inhibitor FR180204 not only inhibited the increase of p-ERK1/2 protein level in L929 cells induced by activin A,but also significantly attenuated the migration of L929 cells induced by activin A.3.Effects of activin A and TNF-α on the activities of human fibroblasts3.1 Human periodontal ligament cells were successfully cultured by tissue explants adherence in vitro.Immunocytochemical staining results showed that anti-cytokeratin staining was negative and anti-vimentin staining was positive in hPDLCs,which was consistent with the characteristics of fibroblasts.3.2 TNF-α inhibited the proliferation and promoted the secretion of IL-6 in hPDLCs,while activin A antagonized the above effects of TNF-α.3.3 Activin A promoted the secretion of TGF-β1 in hPDLCs,while the effect could be inhibited by TNF-α.3.4 Activin A promoted the expression of FN,Col I and MMP-9 mRNA in hPDLCs,while TNF-α down-regulated the high expression of MMP-9 mRNA induced by activin A.Western blotting results also showed that activin A up-regulated MMP-9 expression in hPDLCs,while TNF-α down-regulated the increase of MMP-9protein expression induced by activin A.3.5 Activin A and TNF-α could enhance the adhesion ability of hPDLCs,and the combination of them had synergistic action.3.6 Activin A induced the migration of hPDLCs,while TNF-α suppressed the migration of hPDLCs induced by activin A.4.Mechanisms of activin A and TNF-α regulating the activation in human fibroblasts4.1 Activin A had no significant effects on the expression of Smad signaling related molecules in HPDLCs,while TNF-α down-regulated ActRⅡA expression.4.2 Activin A up-regulated the levels of p-ERK1/2 and p-p38 proteins in HPDLCs,while TNF-α antagonized the increase of ERK1/2 and p38 phosphorylation induced by activin A.Ⅲ.CONCLUSIONIn conclusion,activin A belonged to a novel fibroblast chemokine,and activin A and TNF-α had different regulatory effects on fibroblast activation.The combined effects of activin A and TNF-α on fibroblasts mainly showed mutually antagonistic action,which was mediated by ERK signaling instead of classical Smad signaling.The above results suggested that activin A might play an anti-inflammatory role as the natural antagonist of TNF-α during the active phase of inflammation,while TNF-αplayed an anti-fibrosis role by antagonizing activin A.Moreover,the balance between activin A and TNF-α action might be the key factor in affecting the activities of fibroblasts.This study not only revealed that the co-regulation effects of activin A and TNF-α on fibroblasts activities had important biological significance,but also provided new ideas and research bases for further exploring therapeutic drug targets for fibroblast-mediated diseases.