Roles Of MAPKs/AP-1 Pathways In The Silica-induced TNF-α And TGF-β1 Expression In RAW264.7 Cells

Backgrond:Silicosis is a sort of destructive lung disease caused by inhalation of crystalline silica,and is characterized by interstitial pulmonary fibrosis.The early pathological processes of silicosis include: interstitial inflammation,assembling of alveolar macrophages(AM), damage and repair of alveolar epithelial cell.Among the cells participated in pulmonary fibrosis,the activated AM play an important role in the progress of silicosis.Macrophages are the initial source of inflammatory mediators produced in response to silica exposure that include oxygen radicals, leukotrienes,cytokines,and growth factors.These factors interact each other and compose complicated cytokine network which play important roles in pulmonary inflammation and fibrosis.Transforming growth factor-β1(TGF-β1) and tumor necrosis factor-α(TNF-α) are the most key cytokines.TGF-β1 has the function of chemotaxis and mitogen activation on fibroblasts and stimulates the expression of extracellular matrix andα-smooth muscle actin(α-SMA),which can change fibroblast phenotype and regulate the proliferation of alveolar typeâ…¡epithelial cells.One of the TNF-α's function is to induce AM produce TGF-β1.Our previous studies have proved that SiO₂ can induce overexpression of TGF-β1 and TNF-αin RAW264.7 cells.Mitogen-activated protein kinase(MAPKs) are activated in response to a variety of extracellular stimuli and play complex roles in the regulation of different fundamental cellular processes,such as cell proliferation,differentiation and apoptosis.Signal transduction pathways affecting target genes are via nuclear transcription factors.The phosphorylation of MAPKs leads to the activation of transcription factors(NF- K B,AP-1,Erg-1),which regulate the expression of downstream target genes.Some studies have demonstrated that SiO₂ can induce activation of MAPK and AP-1,so we hypothesis that MAPKs/ activator protein-1(AP-1) play important roles in the silica-induced TNF-αand TGF-β1 expression in macrophages,which remains to be determined.Objective:The present study was to investigate the role of MAPKs/ AP-1 signaling pathways in silica-induced TNF-αand TGF-β1 expression in macrophages.Methods:Murine macrophage cells(RAW264.7) were cultured and then stimulated with 100μg/ml SiO₂ for indicated times.We use Western blotting to assay AP-1(c-jun/c-fos) in nuclear protein and the phosphorylation of MAPKs(p38 kinase,Erk and JNK).SB203580, PD98059 and Curcumin were added into medium to prevent the activiy of p38 kinase,Erk and AP-1,respectively.C-Jun dominant negative mutant(TAM67) was transfected into cells to prevent AP-1 activity.AP-1 DNA binding activity was measured by electrophoretic mobility shift assay(EMSA).The expression of TNF-αand TGF-β1 were detected by RT-PCR and ELISA.Results:(1) SiO₂ activated p38 kinase and Erk,but not JNK in RAW264.7 cells.(2) Erk inhibitor(PD98059) and p38 kinase inhibitor (SB203580) inhibited silica-induced Erk and p38 kinase activity, respectively.The induction of TGF-β1 and TNF-αby SiO₂ was suppressed by 50μM PD98059,but not 20μM SB203580.(3) AP-1 DNA binding activity induced by SiO₂ increased after 3h and decreased after 6h.AP-1 DNA binding activity induced by SiO₂ was abolished by PD98059,not by SB203580.(4) AP-1 inhibitor Curcumin inhibited silica-induced AP-1(c-jun/c-fos) expression in nuclear protein.Curcumin downregulated the expression of TGF-β1 and TNF-αinduced by silica. (5)TAM67 inhibited the silica-induced AP-1 DNA binding activity, TNF-αand TGF-β1 expression.Conclusion:SiO₂ induced TGF-β1 and TNF-αexpression in RAW264.7 cells. SiO₂ induced the activation of MAPKs/AP-1 pathways in RAW264.7 cells.Our study is the first report to demonstrate that Erk/AP-1 signal pathways play important roles in silica-induced expression of TGF-β1 and TNF-αin RAW264.7 cells.Our study is the first report to Curcumin inhibited the expression of TGF-β1 and TNF-αin RAW264.7 cells.It provides new therapeutic options for silicosis.