Tumor necrosis factor-alpha stimulates the expression of C-C chemokine ligand 2 gene in fibroblasts from the human nasal polyp through the pathways of mitogen-activated protein kinase.
BACKGROUND
Recruitment of macrophages is crucial to the pathogenesis of the nasal polyp (NP) because this disease is believed to be inflammation related. Information regarding the expression of C-C chemokine ligand 2 (CCL2), an essential modulator of monocyte chemotaxis in nasal polyp fibroblasts (NPFs), remains unavailable. In this study, the effects of tumor necrosis factor (TNF)-a on CCL2 expression in NPFs and the signaling pathway involved were investigated.


METHODS
Primary cultures of NPFs were established from NPs. The expressions of CCL2, c-Fos, and c-Jun mRNAs in NPF after TNF-a stimulation were detected by Northern blot. Western blot was used to examine the activation of mitogen-activated protein kinase (MAPK) signaling pathways. Activator protein (AP) 1/DNA interactions were evaluated by electrophoretic mobility shift assay (EMSA).


RESULTS
Northern blot showed that TNF-alpha stimulated CCL2 gene expression in NPFs. Significant increase of B-Raf, phosphorated MAPK including mitogen-activated ERK-activate kinase (MEK)1/2, extracellular signal-related kinase 1/2, and p38 were detected by Western blot. c-Fos and c-Jun mRNAs were induced by TNF-alpha, and PD98059 (MEK inhibitor) and SB203580 (p38 inhibitor) abolished the up-regulation of c-Fos. EMSA revealed that TNF-a increased AP-1/DNA binding, and PD98059 and SB203580 attenuated this reaction, possibly via reducing c-Fos synthesis. PD98059 and curcunmin (AP-1 inhibitor) markedly suppressed the TNF-alpha-induced CCL2 expression, whereas the effect of SB203580 was less noted.


CONCLUSIONS
TNF-alpha induces CCL2 transcription in NPFs. B-Raf/MEK/ERK signaling cascade and to a less extent the p38 pathway are responsible for c-Fos activation and the subsequent AP-1/DNA interaction leading to CCL2 expression.
Lin
Sze-Kwan
SK
Department of Dentistry, National Taiwan University Hospital, Taiwan.
Kok
Sang-Heng
SH
Shun
Chia-Tung
CT
Hong
Chi-Yuan
CY
Wang
Chih-Chiang
CC
Hsu
Ming-Che
MC
Liu
Chia-Ming
CM
D015152:Blotting, Northern; D015153:Blotting, Western; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000235:genetics* / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D016762:Genes, fos; D016758:Genes, jun; D006801:Humans; D048728:MAP Kinase Kinase Kinase 1; D048730:MAP Kinase Kinase Kinase 2; D020935:MAP Kinase Signaling System; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009298:Nasal Polyps / Q000235:genetics* / Q000378:metabolism*; D048493:Proto-Oncogene Proteins B-raf; D015398:Signal Transduction; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D048051:p38 Mitogen-Activated Protein Kinases
C111835:CCL2 protein, human; D018932:Chemokine CCL2; D018808:Transcription Factor AP-1; D014409:Tumor Necrosis Factor-alpha; D048493:Proto-Oncogene Proteins B-raf; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048728:MAP Kinase Kinase Kinase 1; D048730:MAP Kinase Kinase Kinase 2