Background & Aims

Diarrhea associated with inflammatory bowel diseases has been associated with increased levels of inflammatory cytokines, including tumor necrosis factor (TNF). The intestinal mucosa of patients with inflammatory bowel diseases has reduced expression of solute carrier family 26 member 3 (SLC26A3, also called DRA). We investigated whether TNF directly affects expression of DRA in human intestinal epithelial cells (IECs) and in the intestines of mice, and studied the mechanisms of these effects.

Methods

We performed quantitative reverse transcription polymerase chain reaction, immunofluorescence, and immunoblot analyses in Caco-2, HT-29, and T-84 cells human IECs cultured in 2 or 3 dimensions with or without TNF (50 ng/mL for 6–24 hours). We purified nuclear extracts and quantified nuclear factor−κB (NF-κB) activation and DNA binding. We isolated intestinal crypts from C57BL/6 mice, cultured enteroids, incubated these with TNF (50 ng/mL, 24 hours), and quantified messenger RNAs. DRA-mediated exchange of Cl⁻ for HCO₃⁻ was measured by uptake of ¹²⁵I. Expression of the NF-κB inhibitor α (IkBa) was knocked down in Caco-2 cells with small interfering RNAs. Activation of NF-κB in response to TNF was measured by luciferase reporter assays; binding of the NF-κB subunit p65 in cells was analyzed in chromatin immunoprecipitation assays. DRA promoter activity was measured in a luciferase reporter assay. C57BL/6 mice were injected with TNF (5 μg/mouse for 3–6 hours) or vehicle (control); intestines were collected and analyzed by immunofluorescence, or RNA and protein were collected from the mucosa.

Results

Incubation of IECs with TNF reduced expression of DRA. Knockdown of NF-κB inhibitor α in IECs led to nuclear translocation of the NF-κB subunit p65 and reduced levels of DRA messenger RNA and protein. Expression of a transgene encoding p65 or p50 in IECs led to significant reductions in the promoter activity of DRA and its expression. In chromatin immunoprecipitation assays, p65 bound directly to the promoter of DRA, at the regions of −935 to −629 and −375 to −84. Injection of mice with TNF or incubation of crypt-derived enteroids with TNF reduced their expression of DRA messenger RNA and protein.

Conclusions

In human IECs and intestinal tissues from mice, we found TNF to activate NF-κB, which reduced expression of the Cl⁻ / HCO₃⁻ exchanger DRA (SLC26A3), via direct binding to the promoter of DRA. This pathway is an important therapeutic target for inflammatory bowel disease−associated diarrhea.

中文翻译：

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肠上皮细胞和小鼠肠上皮中肿瘤坏死因子激活核因子−κB 降低氯离子转运蛋白 SLC26A3 的表达

 背景与目标

与炎症性肠病相关的腹泻与炎症细胞因子水平升高有关，包括肿瘤坏死因子（TNF）。炎症性肠病患者的肠粘膜溶质载体家族26成员3（SLC26A3，也称为DRA）的表达减少。我们研究了 TNF 是否直接影响人肠上皮细胞 (IEC) 和小鼠肠道中 DRA 的表达，并研究了这些影响的机制。

 方法

我们对 Caco-2、HT-29 和 T-84 细胞人类 IEC 进行定量逆转录聚合酶链反应、免疫荧光和免疫印迹分析，这些细胞在 2 或 3 维中培养，有或没有 TNF（50 ng/mL，6-24 小时） ）。我们纯化了核提取物并量化了核因子-κB (NF-κB) 激活和 DNA 结合。我们从 C57BL/6 小鼠中分离出肠隐窝，培养肠类，将其与 TNF（50 ng/mL，24 小时）一起孵育，并对信使 RNA 进行定量。 DRA 介导的 Cl ^-与 HCO ₃ ^-的交换通过¹²⁵ I 的摄取来测量。Caco-2 细胞中 NF-κB 抑制剂 α (IkBa) 的表达被小干扰 RNA 敲低。通过荧光素酶报告基因测定来测量 NF-κB 响应 TNF 的激活；通过染色质免疫沉淀试验分析细胞中 NF-κB 亚基 p65 的结合。 DRA 启动子活性通过荧光素酶报告基因测定进行测量。 C57BL/6 小鼠注射 TNF（5 μg/只，持续 3-6 小时）或载体（对照）；收集肠并通过免疫荧光分析，或从粘膜收集RNA和蛋白质。

 结果

IECs 与 TNF 一起孵育可降低 DRA 的表达。 IEC 中 NF-κB 抑制剂 α 的敲除导致 NF-κB 亚基 p65 的核转位，并降低 DRA 信使 RNA 和蛋白质的水平。编码 p65 或 p50 的转基因在 IEC 中的表达导致 DRA 启动子活性及其表达显着降低。在染色质免疫沉淀测定中，p65 直接与DRA启动子结合，位于 -935 至 -629 和 -375 至 -84 区域。给小鼠注射 TNF 或将隐窝来源的肠类与 TNF 一起孵育，会降低小鼠 DRA 信使 RNA 和蛋白质的表达。

 结论

在人类 IEC 和小鼠肠道组织中，我们发现 TNF 可以激活 NF-κB，从而通过直接结合到 DRA 启动子来减少 Cl ^- / HCO ₃ ^-交换剂 DRA (SLC26A3) 的表达。该途径是炎症性肠病相关腹泻的重要治疗靶点。