Laboratory of Biochemistry, Department of Biopharmaceutical Sciences, Gifu Pharmaceutical University, Gifu 501-1196, Japan.
School of Food and Nutritional Sciences, University of Shizuoka, Shizuoka 422-8526, Japan.
Int J Mol Sci. 2020 Feb 20;21(4):1442. doi: 10.3390/ijms21041442.
Dietary NaCl depletion increases Na and Cl absorption in the colon, but the mechanisms are not fully understood. So far, we reported that the expression of claudin-7 (CLDN7), a tight junction (TJ) protein, was upregulated in the mice fed with NaCl-depleted diets, but the regulatory mechanism has not been clarified. Here, we found that angiotensin II (ANGII) increases the mRNA level of CLDN7, which was inhibited by losartan, a type 1 ANGII (AT1) receptor antagonist. Immunofluorescence measurement showed that CLDN7 is colocalized with zonula occludens-1 at the TJ in untreated and ANGII-treated cells. ANGII decreased transepithelial electrical resistance (TER) and increased permeability to C1 without affecting permeability to lucifer yellow, a paracellular flux marker. In contrast, TER was increased by CLDN7 knockdown in the absence and presence of ANGII. ANGII increased the nuclear distribution of phosphorylated p65 subunit of NF-κB, which was inhibited by losartan. The ANGII-induced elevation of CLDN7 expression was blocked by BAY 11-7082 (BAY), an NF-κB inhibitor. Luciferase reporter assay showed that ANGII increases promoter activity of CLDN7, which was inhibited by the treatment with losartan or BAY, and introduction of mutations in κB-binding motifs in the promoter. The binding of p65 on the promoter region of CLDN7 was increased by ANGII, which was inhibited by losartan and BAY in chromatin immunoprecipitation assay. Our data suggest that ANGII acts on AT1 receptor and increases paracellular permeability to Cl mediated by the elevation of CLDN7 expression in the colon.
饮食中 NaCl 缺乏会增加结肠对 Na 和 Cl 的吸收，但具体机制尚不完全清楚。到目前为止，我们报道了在给予 NaCl 缺乏饮食的小鼠中，紧密连接（TJ）蛋白 Claudin-7（CLDN7）的表达上调，但调节机制尚不清楚。在这里，我们发现血管紧张素 II（ANGII）增加了 CLDN7 的 mRNA 水平，该水平被血管紧张素 II 型 1（AT1）受体拮抗剂 losartan 抑制。免疫荧光测量显示，CLDN7 在未经处理和 ANGII 处理的细胞中与 zonula occludens-1 共定位在 TJ 处。ANGII 降低了跨上皮电阻（TER）并增加了对 C1 的通透性，而对细胞旁通量标记物荧光素黄的通透性没有影响。相比之下，CLDN7 敲低在不存在和存在 ANGII 的情况下增加了 TER。ANGII 增加了 NF-κB 磷酸化 p65 亚单位的核分布，该分布被 losartan 抑制。ANGII 诱导的 CLDN7 表达升高被 NF-κB 抑制剂 BAY 11-7082（BAY）阻断。荧光素酶报告基因检测显示，ANGII 增加了 CLDN7 启动子活性，该活性被 losartan 和 BAY 处理以及在启动子中引入 κB 结合基序的突变所抑制。ANGII 增加了 CLDN7 启动子区域 p65 的结合，该结合在染色质免疫沉淀检测中被 losartan 和 BAY 抑制。我们的数据表明，ANGII 作用于 AT1 受体并通过增加结肠中 CLDN7 表达来增加 Cl 的细胞旁通透性。
