缺氧诱导人肺成纤维细胞中preadipocyte 因子 1 通过 ERK/PEA3/c-Jun 通路表达。
Hypoxia-induced preadipocyte factor 1 expression in human lung fibroblasts through ERK/PEA3/c-Jun pathway.
机构信息
Gradual Institute of Medical Sciences, College of Medicine, Taipei Medical University, 250 Wu-Hsing Street, Taipei, 11031, Taiwan.
Division of Pulmonary Medicine, Department of Internal Medicine, School of Respiratory Therapy, Wan Fang Hospital, Taipei Medical University, 250 Wu-Hsing Street, Taipei, 11031, Taiwan.
出版信息
Mol Med. 2021 Jul 6;27(1):69. doi: 10.1186/s10020-021-00336-w.
BACKGROUND
Several studies have reported that hypoxia plays a pathological role in severe asthma and tissue fibrosis. Our previous study showed that hypoxia induces A disintegrin and metalloproteinase 17 (ADAM17) expression in human lung fibroblasts. Moreover, preadipocyte factor 1 (Pref-1) is cleaved by ADAM17, which participates in adipocyte differentiation. Furthermore, Pref-1 overexpression is involved in tissue fibrosis including liver and heart. Extracellular signal-regulated kinase (ERK) could active downstram gene expression through polyoma enhancer activator 3 (PEA3) phosphorylation. Studies have demonstrated that PEA3 and activator protein 1 (AP-1) play crucial roles in lung fibrosis, and the Pref-1 promoter region contains PEA3 and AP-1 binding sites as predicted. However, the roles of ERK, PEA3, and AP-1 in hypoxia-stimulated Pref-1 expression in human lung fibroblasts remain unknown.
METHODS
The protein expression in ovalbumin (OVA)-induced asthmatic mice was performed by immunohistochemistry and immunofluorescence. The protein expression or the mRNA level in human lung fibroblasts (WI-38) was detected by western blot or quantitative PCR. Small interfering (si) RNA was used to knockdown gene expression. The collaboration with PEA3 and c-Jun were determined by coimmunoprecipitation. Translocation of PEA3 from the cytosol to the nucleus was observed by immunocytochemistry. The binding ability of PEA3 and AP-1 to Pref-1 promoter was assessed by chromatin immunoprecipitation.
RESULTS
Pref-1 and hypoxia-inducible factor 1α (HIF-1α) were expressed in the lung sections of OVA-treated mice. Colocalization of PEA3 and Fibronectin was detected in lung sections from OVA-treated mice. Futhermore, Hypoxia induced Pref-1 protein upregulation and mRNA expression in human lung fibroblasts (WI-38 cells). In 60 confluent WI-38 cells, hypoxia up-regulated HIF-1α and Pref-1 protein expression. Moreover, PEA3 small interfering (si) RNA decreased the expression of hypoxia-induced Pref-1 in WI-38 cells. Hypoxia induced PEA3 phosphorylation, translocation of PEA3 from the cytosol to the nucleus, PEA3 recruitment and AP-1 binding to the Pref-1 promoter region, and PEA3-luciferase activity. Additionally, hypoxia induced c-Jun-PEA3 complex formation. U0126 (an ERK inhibitor), curcumin (an AP-1 inhibitor) or c-Jun siRNA downregulated hypoxia-induced Pref-1 expression.
CONCLUSIONS
These results implied that ERK, PEA3, and AP-1 participate in hypoxia-induced Pref-1 expression in human lung fibroblasts.
背景
多项研究表明，缺氧在严重哮喘和组织纤维化中起病理性作用。我们之前的研究表明，缺氧可诱导人肺成纤维细胞中 A 型分解素金属蛋白酶 17（ADAM17）的表达。此外，前脂肪细胞因子 1（Pref-1）可被 ADAM17 切割，而 ADAM17 参与脂肪细胞分化。此外，Pref-1 的过表达与包括肝和心脏在内的组织纤维化有关。细胞外信号调节激酶（ERK）可通过多瘤增强子激活物 3（PEA3）磷酸化激活下游基因表达。研究表明，PEA3 和激活蛋白 1（AP-1）在肺纤维化中发挥关键作用，并且 Pref-1 启动子区域包含预测的 PEA3 和 AP-1 结合位点。然而，ERK、PEA3 和 AP-1 在人肺成纤维细胞中缺氧刺激 Pref-1 表达中的作用尚不清楚。
方法
通过免疫组织化学和免疫荧光检测卵清蛋白（OVA）诱导的哮喘小鼠中的蛋白表达。通过 Western blot 或定量 PCR 检测人肺成纤维细胞（WI-38）中的蛋白表达或 mRNA 水平。使用小干扰（si）RNA 敲低基因表达。通过共免疫沉淀确定 PEA3 和 c-Jun 的协作。通过免疫细胞化学观察 PEA3 从细胞质向细胞核的易位。通过染色质免疫沉淀评估 PEA3 和 AP-1 与 Pref-1 启动子的结合能力。
结果
OVA 处理的小鼠肺组织中表达 Pref-1 和缺氧诱导因子 1α（HIF-1α）。OVA 处理的小鼠肺组织中检测到 PEA3 和纤维连接蛋白的共定位。此外，缺氧诱导人肺成纤维细胞（WI-38 细胞）中 Pref-1 蛋白的上调和 mRNA 表达。在 60 个汇合的 WI-38 细胞中，缺氧上调 HIF-1α 和 Pref-1 蛋白表达。此外，PEA3 siRNA 降低了 WI-38 细胞中缺氧诱导的 Pref-1 表达。缺氧诱导 PEA3 磷酸化、PEA3 从细胞质向细胞核易位、PEA3 募集和 AP-1 结合到 Pref-1 启动子区域，以及 PEA3-荧光素酶活性。此外，缺氧诱导 c-Jun-PEA3 复合物形成。U0126（ERK 抑制剂）、姜黄素（AP-1 抑制剂）或 c-Jun siRNA 下调缺氧诱导的 Pref-1 表达。
结论
这些结果表明，ERK、PEA3 和 AP-1 参与人肺成纤维细胞中缺氧诱导的 Pref-1 表达。
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