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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肿瘤坏死因子激活肠上皮细胞和小鼠肠上皮中的核因子-κB减少氯化物转运蛋白SLC26A3的表达。

背景与目标

与炎性肠病有关的腹泻与包括肿瘤坏死因子（TNF）在内的炎性细胞因子水平升高有关。炎性肠病患者的肠粘膜具有降低的溶质载体家族26成员3（SLC26A3，也称为DRA）的表达。我们调查了TNF是否直接影响人肠道上皮细胞（IEC）和小鼠肠道中DRA的表达，并研究了这些作用的机制。

方法

我们在Caco-2，HT-29和T-84细胞中以2或3个维度在有或没有TNF（50 ng / mL）的条件下培养6-24小时的人类IEC，进行了定量逆转录聚合酶链反应，免疫荧光和免疫印迹分析。 ）。我们纯化了核提取物，并定量了核因子-κB（NF-κB）的活化和DNA结合。我们从C57BL / 6小鼠中分离出肠道隐窝，培养肠溶蛋白，将它们与TNF（50 ng / mL，24小时）孵育，并定量信使RNA。DRA介导的Cl交换^-为HCO ₃ ^-通过摄取测量¹²⁵I.NF-κB抑制剂α（IkBa）的表达在带有小干扰RNA的Caco-2细胞中被敲低。通过萤光素酶报告基因测定法测定了对TNF应答的NF-κB活化。用染色质免疫沉淀分析法分析细胞中NF-κB亚基p65的结合。在荧光素酶报告基因测定中测量DRA启动子活性。给C57BL / 6小鼠注射TNF（5μg/小鼠，持续3-6小时）或溶媒（对照组）。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

将IEC与TNF一起孵育会降低DRA的表达。在IECs中敲低NF-κB抑制剂α会导致NF-κB亚基p65的核易位，并降低DRA信使RNA和蛋白质的水平。IEC中编码p65或p50的转基因表达导致DRA启动子活性及其表达显着降低。在染色质免疫沉淀试验中，p65在-935至-629和-375至-84区域直接结合DRA启动子。给小鼠注射TNF或与TNF一起孵育隐窝衍生的类固醇会降低其DRA信使RNA和蛋白质的表达。

结论

在人类的IEC和来自小鼠的肠组织中，我们发现TNF激活NF-κB，从而减少所述的Cl表达^- / HCO ₃ ^-交换DRA（SLC26A3），通过直接结合至DRA的启动子。该途径是与炎症性肠病相关的腹泻的重要治疗靶标。