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.

Increased permeability of vascular endothelial cells in the brain is an underlying cause of stroke, which is associated with high mortality rates worldwide. Vascular permeability is regulated by tight junctions (TJs) formed by claudin family and occludin proteins. In particular, increased vascular permeability is associated with decreased claudin domain-containing 1 (CLDND1) expression, which belongs to the TJs family. We previously reported that myeloid zinc finger 1 (MZF1) acts as an activator of CLDND1 expression by binding to its first intron. Several transcription factors regulate transcription by acting on the promoter regions of target genes. However, transcription factors acting on the promoter of CLDND1 are not completely elucidated. Thus, we focused on the promoter region of human CLDND1 to identify factors that could regulate its transcription. Reporter analysis of CLDND1 promoter region revealed an enhancer in the-742/-734 region with MZF1 and specificity protein 1 (SP1) binding sites. Chromatin immunoprecipitation assays confirmed that both MZF1 and SP1 could bind to CLDND1 enhancer region. MZF1 overexpression significantly increased CLDND1 expression, whereas overexpression of SP1 had no effect. Moreover, the identified enhancer region exhibited stronger transcriptional and binding capacity than the first intron. Thus, CLDND1 expression is more strongly regulated by competitive action of MZF1 and SP1 binding to the promoter-enhancer region than the first intron silencer region. These results provide novel insights for the development of potential therapies and preventive strategies for stroke in the future.

Claudin proteins are intercellular adhesion molecules. Increased claudin domain-containing 1 (CLDND1) expression is associated with the malignant transformation of estrogen receptor-negative breast cancer cells with low sensitivity to hormone therapy. Abnormal CLDND1 expression is also implicated in vascular diseases. Previously, we investigated the regulatory mechanism underlying CLDND1 expression and identified a strong enhancer region near the promoter. In silico analysis of the sequence showed high homology to the ETS domain-containing protein-1 (ELK1)-binding sequence which is involved in cell growth, differentiation, angiogenesis, and cancer. Transcriptional ELK1 activation is associated with the mitogen-activated protein kinase (MAPK) signaling cascade originating from the epidermal growth factor receptor (EGFR). Here, we evaluated the effect of gefitinib, an EGFR tyrosine kinase inhibitor, on the suppression of CLDND1 expression using ELK1 overexpression in luciferase reporter and chromatin immunoprecipitation assays. ELK1 was found to be an activator of the enhancer region, and its transient expression increased that of CLDND1 at the mRNA and protein levels. CLDND1 expression was increased following EGF-induced ELK1 phosphorylation. Furthermore, this increase in CLDND1 was significantly suppressed by gefitinib. Therefore, EGF-dependent activation of ELK1 contributes to the induction of CLDND1 expression. These findings open avenues for the development of new anticancer agents targeting CLDND1.

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