Signaling pathway for 2,3,7,8-tetrachlorodibenzo- p-dioxin-induced TNF-alpha production in differentiated THP-1 human macrophages

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), a prototypic halogenated aromatic hydrocarbon (HAH), is known as one of the most potent toxicants. At least a part of its toxic effects appears to be derived from its ability to induce TNF-alpha production. However, the signaling pathway of TCDD that leads to TNF-alpha expression has not been elucidated. In this study, we investigated the signaling mechanism of TCDD-induced TNF-alpha expression in PMA-differentiated THP-1 macrophages. TCDD induced both mRNA and protein expression of TNF-alpha in a dose- and time-dependent manner. Alpha-Naphthoflavone (NF), an aryl hydrocarbon receptor (AhR) inhibitor, prevented the TCDD-induced expression of TNF-alpha at both mRNA and protein levels. Genistein, a protein tyrosine kinase (PTK) inhibitor, and PD153035, an EGFR inhibitor, also blocked the increase of TNF-alpha expression by TCDD, indicating the role of EGFR in TCDD-induced TNF-alpha expression. On the other hand, PP2, a c-Src specific inhibitor, did not affect TCDD-induced TNF-alpha expression. EGFR phosphorylation was detected as early as 5 min after TCDD treatment. TCDD-induced EGFR activation was AhR-dependent since co-treatment with alpha-NF prevented it. ERK was found to be a downstream effector of EGFR activation in the signaling pathway leading to TNF-alpha production after TCDD stimulation. Activation of ERK was observed from 30 min after TCDD treatment. PD98059, an inhibitor of the MEK-ERK pathway, completely prevented the TNF-alpha mRNA and protein expression induced by TCDD, whereas inhibitors of JNK and p38 MAPK had no effect. PD153035, an EGFR inhibitor, as well as alpha-NF significantly reduced ERK phosphorylation, suggesting that ERK activation by TCDD was mediated by both EGFR and AhR. These results indicate that TNF-alpha production by TCDD in differentiated THP-1 macrophages is AhR-dependent and involves activation of EGFR and ERK, but not c-Src, JNK, nor p38 MAPK. A signaling pathway is proposed where TCDD induces sequential activation of AhR, EGFR and ERK, leading to the increased expression of TNF-alpha.

15-Deoxy-delta12,14-PGJ2inhibits IL-6-induced Stat3 phosphorylation in lymphocytes

15-deoxy-delta12,14-PGJ2(15d-PGJ2) is a natural ligand that activates the peroxisome proliferators-activated receptor (PPAR) gamma, a member of nuclear receptor family implicated in regulation of lipid metabolism and adipocyte differentiation. Recent studies have shown that 15d-PGJ2 is the potent anti-inflammatory agent functioning via PPARgamma-dependent and -independent mechanisms. Most postulated mechanisms for anti-inflammatory action of PPARgamma agonists are involved in inhibiting NF-kappaB signaling pathway. We examined the possibility that IL-6 signaling via the Jak-Stat pathway is modulated by 15d-PGJ2 in lymphocytes and also examined whether the inhibition of IL-6 signaling is dependent of PPARgamma. 15d-PGJ2 blocked IL-6 induced Stat1 and Stat3 activation in primary human lymphocytes, Jurkat cells and immortalized rheumatoid arthritis B cells. Inhibition of IL-6 signaling was induced rapidly within 15 min after treatment of 15d-PGJ2. Other PPARgamma-agonists, such as troglitazone and ciglitazone, did not inhibit IL-6 signaling, indicating that 15d-PGJ2 affect the IL-6-induced Jak-Stat signaling pathway via PPARgamma-independent mechanism. Although cycloheximide reversed 15d-PGJ2-mediated inhibition of Stat3 activation, actinomycin D had no effect on 15d-PGJ2-mediated inhibition of IL-6 signaling, indicating that inhibition of IL-6 signaling occur independent of de novo gene expression. These results show that 15d-PGJ2 specifically inhibit Jak-Stat signaling pathway in lymphocytes, and suggest that 15d-PGJ2 may regulate inflammatory reactions through the modulation of different signaling pathway other than NF-kappaB in lymphocytes.

Effects of TGF-beta, GM-CSF, and PDGF on Proliferation and Expression of Cytokine and Metalloproteinase Genes in Rheumatoid Synovial Cells / 대한면역학회지

To investigate effects of cytokines on rheumatoid synovial cells, proliferation and expression of cytokine and metalloproteinase genes were studied with the primary culture of rheumatoid synovial cells which was treated with TNF-alpha, GM-CSF, TGF-alpha, PDGF and IL-B. By [3H] thymidine incorporation assay, TGF-beta and PDGF increased proliferation of synovial cells by 1.5 and 2.5 folds respectively. Cytokine gene expression was assessed by RT-PCR. Rheumatoid synovial cells expressed constitutively TGF-beta and IL-B at a high level and IL-1B, GM-CSF, and MIP-1a at a relatively low level. TGF-beta, GM-CSF and PDGF increased IL-B expression. Expression of pro-inflammatory cytokines and chemokines was increased by GM-CSF and PDGF. Both GM-CSF and PDGF increased the expression of IL-1B, GM-CSF MIP-la and IL-8. In addition, GM-CSF enhanced expression of TNF-alpha. Stromelysin and collagenase are the major proteinases responsible for destruction ot joints in rheumatoid arthritis (RA). These genes were expressed constitutivefy in rheumatoid synovial cells. In summary, PDGF and GM-CSF may piay an important role by inducing or increasing expression of IL-1B, TGF-beta and PDGF by increasing proliferation of rheumatoid synovial cells.