NOTCH1 和 NOTCH3 通过一个 CSL 依赖性转录网络协调食管鳞状细胞分化。
NOTCH1 and NOTCH3 coordinate esophageal squamous differentiation through a CSL-dependent transcriptional network.
机构信息
Gastroenterology Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
出版信息
Gastroenterology. 2010 Dec;139(6):2113-23. doi: 10.1053/j.gastro.2010.08.040. Epub 2010 Aug 27.
BACKGROUND & AIMS: The Notch receptor family regulates cell fate through cell-cell communication. CSL (CBF-1/RBP-jκ, Su(H), Lag-1) drives canonical Notch-mediated gene transcription during cell lineage specification, differentiation, and proliferation in the hematopoietic system, the intestine, the pancreas, and the skin. However, the functional roles of Notch in esophageal squamous epithelial biology are unknown.
METHODS
Normal esophageal keratinocytes were stimulated with calcium chloride to induce terminal differentiation. The squamous epithelia were reconstituted in organotypic 3-dimensional culture, a form of human tissue engineering. Notch was inhibited in culture with a γ-secretase inhibitor or dominant negative mastermind-like 1 (DNMAML1). The roles of Notch receptors were evaluated by in vitro gain-of-function and loss-of-function experiments. Additionally, DNMAML1 was targeted to the mouse esophagus by cytokeratin K14 promoter-driven Cre (K14Cre) recombination of Lox-STOP-Lox-DNMAML1. Notch-regulated gene expression was determined by reporter transfection, chromatin immunoprecipitation assays, quantitative reverse-transcription polymerase chain reaction, Western blotting, immunofluorescence, and immunohistochemistry.
RESULTS
NOTCH1 (N1) was activated at the onset of squamous differentiation in the esophagus. Intracellular domain of N1 (ICN1) directly activated NOTCH3 (N3) transcription, inducing HES5 and early differentiation markers such as involucrin (IVL) and cytokeratin CK13 in a CSL-dependent fashion. N3 enhanced ICN1 activity and was required for squamous differentiation. Loss of Notch signaling in K14Cre;DNMAML1 mice perturbed esophageal squamous differentiation and resulted in N3 loss and basal cell hyperplasia.
CONCLUSIONS
Notch signaling is important for esophageal epithelial homeostasis. In particular, the cross talk of N3 with N1 during differentiation provides novel, mechanistic insights into Notch signaling and squamous epithelial biology.
背景与目的
Notch 受体家族通过细胞间通讯调节细胞命运。CSL（CBF-1/RBP-jκ、Su(H)、Lag-1）在造血系统、肠道、胰腺和皮肤的细胞谱系特化、分化和增殖过程中驱动经典的 Notch 介导的基因转录。然而，Notch 在食管鳞状上皮生物学中的功能作用尚不清楚。
方法
用氯化钙刺激正常食管角质形成细胞诱导终末分化。将鳞状上皮在器官型 3 维培养中重建，这是一种人体组织工程形式。用 γ-分泌酶抑制剂或显性负性主样蛋白 1（DNMAML1）抑制培养中的 Notch。通过体外获得功能和丧失功能实验评估 Notch 受体的作用。此外，通过细胞角蛋白 K14 启动子驱动 Cre（K14Cre）重组的 Lox-STOP-Lox-DNMAML1 将 DNMAML1 靶向到小鼠食管。通过报告基因转染、染色质免疫沉淀分析、定量逆转录聚合酶链反应、Western 印迹、免疫荧光和免疫组织化学确定 Notch 调节的基因表达。
结果
NOTCH1（N1）在食管鳞状分化开始时被激活。N1 的细胞内结构域（ICN1）直接激活 NOTCH3（N3）转录，以 CSL 依赖的方式诱导 HES5 和早期分化标志物，如 involucrin（IVL）和细胞角蛋白 CK13。N3 增强 ICN1 活性，是鳞状分化所必需的。K14Cre；DNMAML1 小鼠中 Notch 信号的缺失扰乱了食管鳞状分化，导致 N3 丢失和基底细胞增生。
结论
Notch 信号对食管上皮稳态很重要。特别是，分化过程中 N3 与 N1 的相互作用为 Notch 信号和鳞状上皮生物学提供了新的、机制上的见解。
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