Laboratory of Genomic Function and Pathophysiology, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, Fukuyama, Japan.
Clin Exp Pharmacol Physiol. 2021 Feb;48(2):260-269. doi: 10.1111/1440-1681.13416. Epub 2020 Oct 29.
Increased permeability of endothelial cells lining the blood vessels in the brain leads to vascular oedema and, potentially, to stroke. The tight junctions (TJs), primarily responsible for the regulation of vascular permeability, are multi-protein complexes comprising the claudin family of proteins and occludin. Several studies have reported that downregulation of the claudin domain containing 1 (CLDND1) gene enhances vascular permeability, which consequently increases the risk of stroke. However, the transcriptional regulation of CLDND1 has not been studied extensively. Therefore, this study aimed to identify the transcription factors (TFs) regulating CLDND1 expression. A luciferase reporter assay identified a silencer within the first intron of CLDND1, which was identified as a potential binding site of the myeloid zinc finger 1 (MZF1) through in silico and TFBIND software analyses, and confirmed through a reporter assay using the MZF1 expression vector and chromatin immunoprecipitation (ChIP) assays. Moreover, the transient overexpression of MZF1 significantly increased the mRNA and protein expression levels of CLDND1, conversely, which were suppressed through the siRNA-mediated MZF1 knockdown. Furthermore, the permeability of FITC-dextran was observed to be increased on MZF1 knockdown as compared to that of the siGFP control. Our data revealed the underlying mechanism of the transcriptional regulation of CLDND1 by the MZF1. The findings suggest a potential role of MZF1 in TJ formation, which could be studied further and applied to prevent cerebral haemorrhage.
血管内皮细胞的通透性增加导致血管水肿，并可能导致中风。紧密连接（TJ）主要负责调节血管通透性，是由 Claudin 家族蛋白和封闭蛋白组成的多蛋白复合物。几项研究报告称，下调 Claudin 结构域包含 1 基因（CLDND1）可增强血管通透性，从而增加中风的风险。然而，CLDND1 的转录调控尚未得到广泛研究。因此，本研究旨在确定调节 CLDND1 表达的转录因子（TF）。荧光素酶报告基因检测鉴定出 CLDND1 第一个内含子内的沉默子，通过计算机和 TFBIND 软件分析鉴定出该沉默子是髓样锌指 1（MZF1）的潜在结合位点，并通过使用 MZF1 表达载体和染色质免疫沉淀（ChIP）检测进行的报告基因检测得到证实。此外，MZF1 的瞬时过表达显著增加了 CLDND1 的 mRNA 和蛋白表达水平，相反，通过 siRNA 介导的 MZF1 敲低抑制了其表达。此外，与 siGFP 对照相比，在 MZF1 敲低时 FITC-葡聚糖的通透性增加。我们的数据揭示了 MZF1 对 CLDND1 转录调控的潜在机制。这些发现表明 MZF1 在 TJ 形成中的潜在作用，这可以进一步研究并应用于预防脑出血。
