Division of Pediatric Dentistry, Department of Dental Medicine, Karolinska Institutet, Huddinge, Sweden.
BMC Genomics. 2010 Apr 15;11:241. doi: 10.1186/1471-2164-11-241.
Prostaglandin E2 (PGE2) is involved in several chronic inflammatory diseases including periodontitis, which causes loss of the gingival tissue and alveolar bone supporting the teeth. We have previously shown that tumor necrosis factor alpha (TNFalpha) induces PGE2 synthesis in gingival fibroblasts. In this study we aimed to investigate the global gene expression profile of TNFalpha-stimulated primary human gingival fibroblasts, focusing on signal pathways related to the PGE2-synthesizing enzymes prostaglandin E synthases (PGES), as well as the upstream enzyme cyclooxygenase-2 (COX-2) and PGE2 production.
Microarray and western blot analyses showed that the mRNA and protein expression of the inflammatory induced microsomal prostaglandin E synthase-1 (mPGES-1) was up-regulated by the cytokine TNFalpha, accompanied by enhanced expression of COX-2 and increased production of PGE2. In contrast, the expression of the isoenzymes microsomal prostaglandin E synthase-2 (mPGES-2) and cytosolic prostaglandin E synthase (cPGES) was unaffected by TNFalpha treatment. Using oligonucleotide microarray analysis in a time-course factorial design including time points 1, 3 and 6 h, differentially expressed genes in response to TNFalpha treatment were identified. Enrichment analysis of microarray data indicated two positively regulated signal transduction pathways: c-Jun N-terminal kinase (JNK) and Nuclear Factor-kappaB (NF-kappaB). To evaluate their involvement in the regulation of mPGES-1 and COX-2 expression, we used specific inhibitors as well as phosphorylation analysis. Phosphorylation analysis of JNK (T183/Y185) and NF-kappaB p65 (S536) showed increased phosphorylation in response to TNFalpha treatment, which was decreased by specific inhibitors of JNK (SP600125) and NF-kappaB (Bay 11-7082, Ro 106-9920). Inhibitors of JNK and NF-kappaB also decreased the TNFalpha-stimulated up-regulation of mPGES-1 and COX-2 as well as PGE2 production.
In the global gene expression profile, the enrichment analysis of microarray data identified the two signal transduction pathways JNK and NF-kappaB as positively regulated by the cytokine TNFalpha. Inhibition of these TNFalpha-activated signal pathways reduced the expression of mPGES-1 and COX-2 as well as their end product PGE2 in gingival fibroblasts. The involvement of the signal pathways JNK and NF-kappaB in the regulation of PGE2 induced by TNFalpha may suggest these two pathways as possible attractive targets in the chronic inflammatory disease periodontitis.
前列腺素 E2 (PGE2) 参与多种慢性炎症性疾病，包括牙周炎，后者可导致牙龈组织和支持牙齿的牙槽骨丧失。我们之前已经表明，肿瘤坏死因子 alpha (TNFalpha) 可诱导牙龈成纤维细胞合成 PGE2。在这项研究中，我们旨在研究 TNFalpha 刺激的原代人牙龈成纤维细胞的全基因表达谱，重点关注与 PGE2 合成酶前列腺素 E 合酶 (PGES) 相关的信号通路，以及上游酶环加氧酶-2 (COX-2) 和 PGE2 产生。
微阵列和 Western blot 分析表明，细胞因子 TNFalpha 上调了诱导的微粒体前列腺素 E 合酶-1 (mPGES-1) 的 mRNA 和蛋白表达，同时 COX-2 表达增强，PGE2 产量增加。相比之下，同工酶微粒体前列腺素 E 合酶-2 (mPGES-2) 和细胞质前列腺素 E 合酶 (cPGES) 的表达不受 TNFalpha 处理的影响。通过包括 1、3 和 6 小时时间点的时间过程因子设计的寡核苷酸微阵列分析，鉴定了对 TNFalpha 处理有反应的差异表达基因。微阵列数据的富集分析表明存在两条正调控的信号转导通路：c-Jun N-末端激酶 (JNK) 和核因子-κB (NF-κB)。为了评估它们在 mPGES-1 和 COX-2 表达调控中的作用，我们使用了特异性抑制剂和磷酸化分析。JNK (T183/Y185) 和 NF-κB p65 (S536) 的磷酸化分析表明，TNFalpha 处理后磷酸化增加，特异性 JNK 抑制剂 (SP600125) 和 NF-κB 抑制剂 (Bay 11-7082、Ro 106-9920) 可降低磷酸化。JNK 和 NF-κB 的抑制剂也降低了 TNFalpha 刺激的 mPGES-1 和 COX-2 以及 PGE2 产生的上调。
在全基因表达谱中，微阵列数据的富集分析确定了两条信号转导通路 JNK 和 NF-κB 被细胞因子 TNFalpha 正向调控。这些 TNFalpha 激活的信号通路的抑制减少了牙龈成纤维细胞中 mPGES-1 和 COX-2 及其终产物 PGE2 的表达。JNK 和 NF-κB 信号通路在 TNFalpha 诱导的 PGE2 表达中的参与可能表明这两条通路是牙周炎等慢性炎症性疾病的潜在有吸引力的靶点。
