Trefoil factor 2 activation of CXCR4 requires calcium mobilization to drive epithelial repair in gastric organoids
Trefoil factor 2 activation of CXCR4 requires calcium mobilization to drive epithelial repair in gastric organoids
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DOI：
10.1113/jp277259
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发表时间：
2019-05-15
影响因子：
5.5
通讯作者：
Montrose, Marshall H.
中科院分区：
文献类型：
--
作者：
Engevik, Kristen A.;Hanyu, Hikaru;Montrose, Marshall H.
要点使用小鼠胃类器官确定上皮修复的信号级联，可以定义上皮细胞固有的调节过程，同时比体内更精确地验证和剖析信号级联。单细胞损伤后，细胞内钙在损伤部位附近的细胞内选择性增加，这对于促进修复至关重要。三叶因子 2 (TFF2) 通过趋化因子 C-X-C 受体 4 和表皮生长因子受体信号传导（包括细胞外信号调节激酶激活）发挥作用，驱动钙动员并促进胃修复。钠氢交换器 2 虽然对于修复至关重要，但它在 TFF2 和钙动员的下游发挥作用。胃的胃粘膜不断暴露于环境和生理应激因素下，可引起局部上皮损伤。尽管人们对胃伤口修复的复杂性了解很多，但表征上皮恢复的逐步过程仍然不明确。本研究旨在使用还原培养模型确定驱动胃上皮修复的效应器。为了确定三叶因子 2 (TFF2) 和细胞内钙 (Ca2+) 动员在胃恢复中的作用，分别从 TFF2 敲除 (KO) 小鼠和黄色 Cameleon-Nano15（荧光钙报告基因）转基因小鼠中获得胃类器官。抑制剂和重组蛋白用于确定胃类器官内单细胞光损伤（PD）后胃恢复的上游和下游效应子。单细胞PD导致死细胞脱落和完整相邻细胞迁移以恢复损伤部位的连续上皮的并行事件。在正常情况下，PD 后，相邻迁移细胞内的 Ca2+ 水平增加，在大约 1 分钟时达到峰值，表明细胞突出/迁移部位存在局部 Ca2+ 动员。 TFF2 KO 类器官表现出修复延迟；然而，这种延迟可以通过添加外源 TFF2 来挽救。抑制表皮生长因子受体 (EGFR)、细胞外信号调节激酶 (ERK)1/2 或 TFF2 受体、趋化因子 C-X-C 受体 4 (CXCR4)，导致 Ca2+ 动员显着延迟和减弱。钠氢交换器 2 (NHE2) 的抑制导致显着延迟，但不影响 Ca2+ 动员。在 NHE2 KO 类器官中也观察到类似的延迟。在 TFF2 KO 胃类器官中，在 EGFR 或 CXCR4 抑制存在的情况下添加外源 TFF2 无法挽救修复。本研究表明，细胞内 Ca2+ 动员发生在损伤部位附近的胃上皮细胞内，通过涉及 CXCR4 的 TFF2 信号传导以及 EGFR 和 ERK1/2 激活的机制促进修复。此外，NHE2 对有效修复很重要，并且通过下游或独立于钙动员的机制进行操作。
Key pointsDetermining the signalling cascade of epithelial repair, using murine gastric organoids, allows definition of regulatory processes intrinsic to epithelial cells, at the same time as validating and dissecting the signalling cascade with more precision than is possible in vivo Following single cell damage, intracellular calcium selectively increases within cells adjacent to the damage site and is essential for promoting repair. Trefoil factor 2 (TFF2) acts via chemokine C-X-C receptor 4 and epidermal growth factor receptor signalling, including extracellular signal-regulated kinase activation, to drive calcium mobilization and promote gastric repair. Sodium hydrogen exchanger 2, although essential for repair, acts downstream of TFF2 and calcium mobilization. The gastric mucosa of the stomach is continually exposed to environmental and physiological stress factors that can cause local epithelial damage. Although much is known about the complex nature of gastric wound repair, the stepwise process that characterizes epithelial restitution remains poorly defined. The present study aimed to determine the effectors that drive gastric epithelial repair using a reductionist culture model. To determine the role of trefoil factor 2 (TFF2) and intracellular calcium (Ca2+) mobilization in gastric restitution, gastric organoids were derived from TFF2 knockout (KO) mice and yellow Cameleon-Nano15 (fluorescent calcium reporter) transgenic mice, respectively. Inhibitors and recombinant protein were used to determine the upstream and downstream effectors of gastric restitution following photodamage (PD) to single cells within the gastric organoids. Single cell PD resulted in parallel events of dead cell exfoliation and migration of intact neighbouring cells to restore a continuous epithelium in the damage site. Under normal conditions following PD, Ca2+ levels increased within neighbour migrating cells, peaking at similar to 1 min, suggesting localized Ca2+ mobilization at the site of cell protrusion/migration. TFF2 KO organoids exhibit delayed repair; however, this delay can be rescued by the addition of exogenous TFF2. Inhibition of epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK)1/2 or a TFF2 receptor, chemokine C-X-C receptor 4 (CXCR4), resulted in significant delay and dampened Ca2+ mobilization. Inhibition of sodium hydrogen exchanger 2 (NHE2) caused significant delay but did not affect Ca2+ mobilization. A similar delay was observed in NHE2 KO organoids. In TFF2 KO gastric organoids, the addition of exogenous TFF2 in the presence of EGFR or CXCR4 inhibition was unable to rescue repair. The present study demonstrates that intracellular Ca2+ mobilization occurs within gastric epithelial cells adjacent to the damage site to promote repair by mechanisms that involve TFF2 signalling via CXCR4, as well as activation of EGFR and ERK1/2. Furthermore NHE2 is shown to be important for efficient repair and to operate via a mechanism either downstream or independent of calcium mobilization.