Robinson Shaiya C, Klobucar Kristina, Pierre Christina C, Ansari Amna, Zhenilo Svetlana, Prokhortchouk Egor, Daniel Juliet M
Department of Biology, McMaster University, Hamilton, L8S 4K1, ON, Canada.
Current address: Department of Biochemistry and Biomedical Sciences, Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, L8N 3Z5, ON, Canada.
Cell Commun Signal. 2017 Jun 21;15(1):24. doi: 10.1186/s12964-017-0178-x.
In mammalian intestines, Notch signaling plays a critical role in mediating cell fate decisions; it promotes the absorptive (or enterocyte) cell fate, while concomitantly inhibiting the secretory cell fate (i.e. goblet, Paneth and enteroendocrine cells). We recently reported that intestinal-specific Kaiso overexpressing mice (Kaiso ) exhibited chronic intestinal inflammation and had increased numbers of all three secretory cell types, hinting that Kaiso might regulate Notch signaling in the gut. However, Kaiso's precise role in Notch signaling and whether the Kaiso secretory cell fate phenotype was linked to Kaiso-induced inflammation had yet to be elucidated.
Intestines from 3-month old Non-transgenic and Kaiso mice were "Swiss" rolled and analysed for the expression of Notch1, Dll-1, Jagged-1, and secretory cell markers by immunohistochemistry and immunofluorescence. To evaluate inflammation, morphological analyses and myeloperoxidase assays were performed on intestines from 3-month old Kaiso and control mice. Notch1, Dll-1 and Jagged-1 expression were also assessed in stable Kaiso-depleted colon cancer cells and isolated intestinal epithelial cells using real time PCR and western blotting. To assess Kaiso binding to the DLL1, JAG1 and NOTCH1 promoter regions, chromatin immunoprecipitation was performed on three colon cancer cell lines.
Here we demonstrate that Kaiso promotes secretory cell hyperplasia independently of Kaiso-induced inflammation. Moreover, Kaiso regulates several components of the Notch signaling pathway in intestinal cells, namely, Dll-1, Jagged-1 and Notch1. Notably, we found that in Kaiso mice intestines, Notch1 and Dll-1 expression are significantly reduced while Jagged-1 expression is increased. Chromatin immunoprecipitation experiments revealed that Kaiso associates with the DLL1 and JAG1 promoter regions in a methylation-dependent manner in colon carcinoma cell lines, suggesting that these Notch ligands are putative Kaiso target genes.
Here, we provide evidence that Kaiso's effects on intestinal secretory cell fates precede the development of intestinal inflammation in Kaiso mice. We also demonstrate that Kaiso inhibits the expression of Dll-1, which likely contributes to the secretory cell phenotype observed in our transgenic mice. In contrast, Kaiso promotes Jagged-1 expression, which may have implications in Notch-mediated colon cancer progression.
在哺乳动物肠道中，Notch信号通路在介导细胞命运决定中起关键作用；它促进吸收性（或肠上皮细胞）细胞命运，同时抑制分泌性细胞命运（即杯状细胞、潘氏细胞和肠内分泌细胞）。我们最近报道，肠道特异性过表达Kaiso的小鼠（Kaiso ）表现出慢性肠道炎症，并且所有三种分泌性细胞类型的数量均增加，这暗示Kaiso可能调节肠道中的Notch信号通路。然而，Kaiso在Notch信号通路中的精确作用以及Kaiso 分泌性细胞命运表型是否与Kaiso诱导的炎症有关尚待阐明。
对3个月大的非转基因和Kaiso 小鼠的肠道进行“瑞士卷”处理，并通过免疫组织化学和免疫荧光分析Notch1、Dll-1、Jagged-1和分泌性细胞标志物的表达。为了评估炎症，对3个月大的Kaiso 和对照小鼠的肠道进行形态学分析和髓过氧化物酶测定。还使用实时PCR和蛋白质印迹法在稳定的Kaiso缺失的结肠癌细胞和分离的肠上皮细胞中评估Notch1、Dll-1和Jagged-1的表达。为了评估Kaiso与DLL1、JAG1和NOTCH1启动子区域的结合，对三种结肠癌细胞系进行染色质免疫沉淀。
在此我们证明，Kaiso促进分泌性细胞增生，且独立于Kaiso诱导的炎症。此外，Kaiso调节肠道细胞中Notch信号通路的几个组分，即Dll-1、Jagged-1和Notch1。值得注意的是，我们发现在Kaiso 小鼠的肠道中，Notch1和Dll-1的表达显著降低，而Jagged-1的表达增加。染色质免疫沉淀实验表明，在结肠癌细胞系中，Kaiso以甲基化依赖的方式与DLL1和JAG1启动子区域结合，表明这些Notch配体是假定的Kaiso靶基因。
在此，我们提供证据表明，在Kaiso 小鼠中，Kaiso对肠道分泌性细胞命运的影响先于肠道炎症的发展。我们还证明，Kaiso抑制Dll-1的表达，这可能导致我们在转基因小鼠中观察到的分泌性细胞表型。相反，Kaiso促进Jagged-1的表达，这可能对Notch介导的结肠癌进展有影响。
