Actinobacillus actinomycetemcomitans lipopolysaccharide induces interleukin-6 expression through multiple mitogen-activated protein kinase pathways in periodontal ligament fibroblasts
Actinobacillus actinomycetemcomitans lipopolysaccharide induces interleukin-6 expression through multiple mitogen-activated protein kinase pathways in periodontal ligament fibroblasts
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DOI：
10.1111/j.1399-302x.2006.00314.x
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发表时间：
2006-12-01
影响因子：
--
通讯作者：
Kirkwood, K. L.
中科院分区：
文献类型：
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作者：
Patil, C.;Rossa, C., Jr.;Kirkwood, K. L.
伴放线放线杆菌在侵袭性牙周炎的发病机制中起主要作用。A.伴放线菌是牙周组织内炎性细胞因子产生的关键因子。在这项研究中，我们确定了主要的丝裂原活化蛋白激酶（MAPK）信号通路诱导的A。在小鼠牙周韧带（mPDL）成纤维细胞系中，使用放线菌伴菌LPS、大肠杆菌LPS和白细胞介素-1 β（IL-1 β）。用免疫印迹法检测A. actinomycetemcomitans LPS、大肠杆菌E. coli LPS和IL-1 β。通过逆转录-聚合酶链反应检测IL-6 mRNA诱导，同时通过酶联免疫吸附试验（ELISA）定量蛋白水平。我们利用p38、ERK和JNK MAPK的生化抑制剂来鉴定IL-6表达所需的MAPK信号通路。另外使用含有MAPK激酶-3和-6（MKK-3/6）的显性负突变体构建体的稳定mPDL细胞系和p38无效突变小鼠胚胎成纤维细胞（MEF）细胞来证实生物化学抑制剂数据。用SB 203580阻断p38 MAPK可降低A. actinomycetemcomitans LPS、大肠杆菌E. coli LPS和IL-1 β的抑制率分别> 70%、> 95%和接近60%。IL-6 ELISA结果表明，阻断p38 MAPK可降低A. actinomycetemcomitans LPS、大肠杆菌E. coli LPS和IL-1 β的表达水平分别为60%、50%和70%。所有MAPK抑制剂均能显著降低A. actinomycetemcomitans LPS、大肠杆菌E. coliLPS和IL-1 β，而只有p38抑制剂始终降低A. actinomycetemcomitans LPS、大肠杆菌E. coli LPS和IL-1 β诱导IL-6 mRNA稳态水平。使用MKK-3/6显性阴性稳定mPDL细胞系证实p38 MAPK LPS诱导的IL-6表达的贡献。野生型和p38 α（-/-）MEF细胞提供了额外的证据支持p38 α MAPK在A.伴放线菌LPS刺激的IL-6。结果表明，E. coli LPS、IL-1 β和A.在mPDL细胞中，伴放线菌LPS需要通过MKK-3-p38 α ERK、JNK和p38 MAPK进行信号传导。
Actinobacillus actinomycetemcomitans plays a major role in the pathogenesis of aggressive periodontitis. Lipopolysaccharide (LPS) derived from A. actinomycetemcomitans is a key factor in inflammatory cytokine generation within periodontal tissues. In this study, we identify major mitogen-activated protein kinase (MAPK) signaling pathways induced by A. actinomycetemcomitans LPS, Escherichia coli LPS and interleukin-1 beta (IL-1 beta) in a murine periodontal ligament (mPDL) fibroblast cell line. Immunoblot analysis was used to assess the phosphorylated forms of p38, extracellular-regulated kinase (ERK) and c-jun N-terminal kinase (JNK) MAPK following stimulation with A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta. IL-6 mRNA induction was detected via reverse transcription-polymerase chain reaction, while protein levels were quantified via enzyme-linked immunosorbent assays (ELISA). We utilized biochemical inhibitors of p38, ERK and JNK MAPK to identify the MAPK signaling pathways needed for IL-6 expression. Additional use of stable mPDL cell lines containing dominant negative mutant constructs of MAPK kinase-3 and -6 (MKK-3/6) and p38 null mutant mouse embryonic fibroblast (MEF) cells were used to substantiate the biochemical inhibitor data. Blocking p38 MAPK with SB203580 reduced the induction of IL-6 mRNA by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta by > 70%, > 95% and similar to 60%, respectively. IL-6 ELISA indicated that blocking p38 MAPK reduced the IL-6 protein levels induced by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta by similar to 60%, similar to 50% and similar to 70%, respectively. All MAPK inhibitors significantly reduced the IL-6 protein levels induced by A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta whereas only p38 inhibitors consistently reduced the A. actinomycetemcomitans LPS, E. coli LPS and IL-1 beta induction of IL-6 mRNA steady-state levels. The contribution of p38 MAPK LPS-induced IL-6 expression was confirmed using MKK-3/6 dominant negative stable mPDL cell lines. Wild-type and p38 alpha(-/-) MEF cells provided additional evidence to support the role of p38 alpha MAPK in A. actinomycetemcomitans LPS-stimulated IL-6. Our results indicate that induction of IL-6 by E. coli LPS, IL-1 beta and A. actinomycetemcomitans LPS requires signaling through MKK-3-p38 alpha ERK, JNK and p38 MAPK in mPDL cells.