Impaired TLR4 and HIF expression in cystic fibrosis bronchial epithelial cells downregulates hemeoxygenase-1 and alters iron homeostasis in vitro
Impaired TLR4 and HIF expression in cystic fibrosis bronchial epithelial cells downregulates hemeoxygenase-1 and alters iron homeostasis in vitro
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DOI：
10.1152/ajplung.00167.2014
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发表时间：
2014-11-15
影响因子：
4.9
通讯作者：
Henke, Markus O.
中科院分区：
文献类型：
--
作者：
Chillappagari, Shashi;Venkatesan, Shalini;Henke, Markus O.
血红素加氧酶-1（HO-1）是一种可诱导的热休克蛋白，在氧化应激、脂多糖（LPS）和缺氧等多种细胞损伤时表达上调。在这项研究中，我们研究了Toll样受体4（TLR 4），缺氧诱导因子1 α（HIF-1 α）和铁在囊性纤维化（CF）中HO-1表达的体外作用。对CFTR-/-和野生型小鼠的肺切片进行TLR 4、HO-1、铁蛋白和HIF-1 α的免疫组织化学分析。CFBE 41 o-和16 HBE 14 o-细胞系用于通过免疫印迹、免疫荧光、实时PCR、荧光素酶报告基因分析和铁定量进行体外分析。我们观察到CFBE 410-细胞系和CF小鼠中TLR 4、HIF-1 α、HO-1和铁蛋白减少。在16 HBE 14 o-中使用TLR 4-siRNA的敲低研究揭示了HO-1的显著降低，证实了TLR 4在HO-1下调中的作用。在16 HBE 14 o-细胞中使用锡原卟啉抑制HO-1导致铁水平增加，表明HO-1在铁积累中的可能作用。此外，使用铁螯合剂螯合过量的铁导致CFBE 41 o-和16 HBE 14 o-中缺氧反应元件反应增加，暗示铁在HIF-1 α稳定和HO-1中的作用。总之，我们的体外结果表明，多种调节因子，如TLR 4表面表达受损，细胞内铁增加，HIF-1 α降低，下调CFBE 410-细胞中HO-1的表达。
Hemeoxygenase-1 (HO-1), an inducible heat shock protein, is upregulated in response to multiple cellular insults via oxidative stress, lipopolysaccharides (LPS), and hypoxia. In this study, we investigated in vitro the role of Toll-like receptor 4 (TLR4), hypoxia-inducible factor 1 alpha (HIF-1 alpha), and iron on HO-1 expression in cystic fibrosis (CF). Immunohistochemical analysis of TLR4, HO-1, ferritin, and HIF-1 alpha were performed on lung sections of CFTR-/- and wild-type mice. CFBE41o- and 16HBE14o- cell lines were employed for in vitro analysis via immunoblotting, immunofluorescence, real-time PCR, luciferase reporter gene analysis, and iron quantification. We observed a reduced TLR4, HIF-1 alpha, HO-1, and ferritin in CFBE41o- cell line and CF mice. Knockdown studies using TLR4-siRNA in 16HBE14o- revealed significant decrease of HO-1, confirming the role of TLR4 in HO-1 downregulation. Inhibition of HO-1 using tin protoporphyrin in 16HBE14o- cells resulted in increased iron levels, suggesting a probable role of HO-1 in iron accumulation. Additionally, sequestration of excess iron using iron chelators resulted in increased hypoxia response element response in CFBE41o- and 16HBE14o-, implicating a role of iron in HIF-1 alpha stabilization and HO-1. To conclude, our in vitro results demonstrate that multiple regulatory factors, such as impaired TLR4 surface expression, increased intracellular iron, and decreased HIF-1 alpha, downregulate HO-1 expression in CFBE41o- cells.