Section of Surgical Sciences, Department of Surgery, Vanderbilt University Medical Center, Nashville, Tennessee.
Graduate Program in Cancer Biology, Vanderbilt University School of Medicine, Nashville, Tennessee.
Am J Physiol Gastrointest Liver Physiol. 2021 Jun 1;320(6):G936-G957. doi: 10.1152/ajpgi.00053.2021. Epub 2021 Mar 24.
Defective barrier function is a predisposing factor in inflammatory bowel disease (IBD) and colitis-associated cancer (CAC). Although TGFβ signaling defects have been associated with IBD and CAC, few studies have examined the relationship between TGFβ and intestinal barrier function. Here, we examine the role of TGFβ signaling via SMAD4 in modulation of colon barrier function. The gene was conditionally deleted in the intestines of adult mice and intestinal permeability assessed using an in vivo 4 kDa FITC-Dextran (FD4) permeability assay. Mouse colon was isolated for gene expression (RNA-sequencing), Western blot, and immunofluorescence analysis. In vitro colon organoid culture was utilized to assess junction-related gene expression by qPCR and transepithelial resistance (TER). In silico analyses of human IBD and colon cancer databases were performed. Mice lacking intestinal expression of demonstrate increased colonic permeability to FD4 without gross mucosal damage. mRNA/protein expression analyses demonstrate significant increases in /Claudin 2 and /Claudin 8, and decreases in , , and /Claudin 7 with intestinal SMAD4 loss in vivo without changes in Claudin protein localization. TGFβ1/BMP2 treatment of polarized SMAD4+ colonoids increases TER. and are regulated by canonical TGFβ signaling, and TGFβ-dependent regulation of these genes is dependent on nascent RNA transcription () but not nascent protein translation (, ). Human IBD/colon cancer specimens demonstrate decreased and and increased compared with healthy controls. Canonical TGFβ signaling modulates the expression of tight junction proteins and barrier function in mouse colon. We demonstrate that canonical TGFβ family signaling modulates the expression of critical tight junction proteins in colon epithelial cells, and that expression of these tight junction proteins is associated with maintenance of colon epithelial barrier function in mice.
屏障功能缺陷是炎症性肠病（IBD）和结肠炎相关癌症（CAC）的一个诱发因素。尽管 TGFβ 信号缺陷与 IBD 和 CAC 有关，但很少有研究检查 TGFβ 与肠道屏障功能之间的关系。在这里，我们研究了通过 SMAD4 进行的 TGFβ 信号在调节结肠屏障功能中的作用。该基因在成年小鼠的肠道中条件性缺失，并使用体内 4 kDa FITC-右旋糖酐（FD4）通透性测定法评估肠道通透性。分离小鼠结肠进行基因表达（RNA 测序）、Western blot 和免疫荧光分析。利用体外结肠类器官培养物通过 qPCR 和跨上皮电阻（TER）评估连接相关基因的表达。对人类 IBD 和结肠癌数据库进行了计算机分析。缺乏肠道表达 的小鼠对 FD4 的结肠通透性增加，而无明显的粘膜损伤。体内肠道 SMAD4 缺失后，mRNA/蛋白表达分析表明 /Claudin 2 和 /Claudin 8 的表达显著增加，而 、 和 /Claudin 7 的表达减少，但 Claudin 蛋白定位没有变化。极化 SMAD4+类器官中 TGFβ1/BMP2 的处理增加了 TER。 和 受经典 TGFβ 信号调节，TGFβ 对这些基因的依赖性调节依赖于新生 RNA 转录（）而不是新生蛋白翻译（，）。与健康对照相比，人类 IBD/结肠癌标本中的 和 减少，而 增加。经典 TGFβ 信号调节小鼠结肠中紧密连接蛋白的表达和屏障功能。我们证明，经典 TGFβ 家族信号调节结肠上皮细胞中关键紧密连接蛋白的表达，并且这些紧密连接蛋白的表达与维持小鼠结肠上皮屏障功能有关。
