Regulation of stem cell development during tissue remodeling

组织重塑过程中干细胞发育的调节

• 批准号: 8553964
• 负责人: Yun-Bo Shi
• 金额: $ 97.73万
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553964
• 关键词: Adult; African; Apoptosis; Basal lamina; Binding; Biological Metamorphosis; Caco-2 Cells; Cell Culture Techniques; Cell Differentiation process; Cells; Cessation of life; Characteristics; Complement; Connective Tissue; Development; Epithelial Cells; Epithelium; Erinaceidae; Event; Extracellular Matrix; Food Processing; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Human Cell Line; Intestines; Introns; Knock-out; Life; Mesenchymal; Molecular; Mus; Muscle; Nutrient; Organ; Organ Culture Techniques; Pathway interactions; Physiological; Play; Primary Cell Cultures; Process; Proliferating; Protein Synthesis Inhibitors; Protein-Arginine N-Methyltransferase; Proto-Oncogene Proteins c-myc; Rana; Regulation; Role; Series; Signal Pathway; Site; Stem Cell Development; Stem cells; Structure; Tadpoles; Tail; Thyroid Hormone Receptor; Thyroid Hormones; Tissues; Up-Regulation; Vertebrates; Xenopus; Xenopus Proteins; Xenopus laevis; Zebrafish; absorption; adult stem cell; c-myc Genes; human SMO protein; intestinal epithelium; overexpression; paracrine; promoter; research study; response; smoothened signaling pathway; sonic hedgehog receptor; spatiotemporal; stem cell division; tissue regeneration; tissue repair; toad; transcription factor

THYROID HORMONE-INDUCED SONIC HEDGEHOG SIGNAL UP-REGULATES ITS OWN PATHWAY IN A PARACRINE MANNER IN THE XENOPUS LAEVIS INTESTINE DURING METAMORPHOSIS. We have shown previously that during Xenopus laevis metamorphosis, Sonic hedgehog (Shh) is directly induced by thyroid hormone (TH) at the transcription level as one of the earliest events in intestinal remodeling. However, the regulation of other components of this signaling pathway remains to be analyzed. We have now analyzed the spatiotemporal expression of Shh receptors Patched (Ptc)-1 and Smoothened (Smo), and its downstream transcription factors Gli1, Gli2 and Gli3 during natural and TH-induced intestinal remodeling. We showed that all of the genes examined were transiently up-regulated in the mesenchymal tissues during intestinal metamorphosis. Interestingly, in the presence of protein synthesis inhibitors, Gli2 but not the others was induced by TH, suggesting that Gli2 is a direct TH response gene, while the others are likely indirect ones. Furthermore, we demonstrate by the organ culture experiment that overexpression of Shh enhances the expression of Ptc-1, Smo and Glis even in the absence of TH, indicating that Shh regulates its own pathway components during intestinal remodeling. These findings indicate that Shh expressed in the epithelium regulates mesenchymal tissue development, which in turn facilitate the development of the adult intestinal stem cells during metamorphosis. THYROID HORMONE ACTIVATES PROTEIN ARGININE METHYLTRANSFERASE 1 EXPRESSION BY DIRECTLY INDUCING C-MYC TRANSCRIPTION DURING XENOPUS INTESTINAL STEM CELL DEVELOPMENT. We have previously shown that protein arginine methyltransferase 1 (PRMT1) is upregulated in and required for adult intestinal stem cells during metamorphosis and that this role is evolutionally conserved in vertebrates. PRMT1 upregulation is the earliest known molecular event for the developing stem cells and is also conserved during zebrafish and mouse adult intestinal stem cell development. To analyze how PRMT1 is specifically upregulated during the formation of the adult intestinal stem cells, we cloned Xenopus PRMT1 promoter and characterized it in CaCo-2 cells, a human cell line with intestinal stem cell characteristics. Through a series deletion and mutational analyses, we showed that the stem cell associated transcription factor c-Myc could bind to an evolutionally conserved site in the first intron to activate the promoter. Furthermore, we demonstrated that during metamorphosis, both c-Myc and PRMT1 were highly upregulated specifically in the remodeling intestine but not the resorbing tail and that c-Myc was induced by TH prior to PRMT1 upregulation. In addition, we showed that TH directly activated c-Myc gene during metamorphosis in the intestine via binding of TH receptor to the c-Myc promoter. These results suggest that TH induces c-Myc transcription directly in the intestine and c-Myc in turn activates PRMT1 expression and that this is an important gene regulation cascade controlling intestinal stem cell development.

甲状腺激素诱导的声波刺猬信号在非洲爪蟾的变态过程中以旁分泌方式上调其自身的通路。我们之前已经证明，在非洲爪蟾变态过程中，甲状腺激素（TH）在转录水平上直接诱导音刺猬（Shh），这是肠道重塑中最早的事件之一。然而，该信号通路其他成分的调节仍有待分析。我们现在分析了在自然和 TH 诱导的肠道重塑过程中 Shh 受体 Patched (Ptc)-1 和 Smoothened (Smo) 及其下游转录因子 Gli1、Gli2 和 Gli3 的时空表达。我们发现，在肠变态过程中，所有检查的基因在间充质组织中都短暂上调。有趣的是，在蛋白质合成抑制剂存在的情况下，TH 诱导 Gli2 而不是其他基因，这表明 Gli2 是直接 TH 反应基因，而其他基因可能是间接反应基因。此外，我们通过器官培养实验证明，即使在TH不存在的情况下，Shh的过度表达也会增强Ptc-1、Smo和Glis的表达，表明Shh在肠道重塑过程中调节其自身的通路成分。 这些发现表明，上皮细胞中表达的 Shh 调节间充质组织的发育，进而促进成体肠道干细胞在变态过程中的发育。 甲状腺激素通过在爪蟾肠干细胞发育过程中直接诱导 C-MYC 转录来激活蛋白质精氨酸甲基转移酶 1 的表达。我们之前已经证明，蛋白质精氨酸甲基转移酶 1 (PRMT1) 在成体肠道干细胞变态过程中上调，并且是成体肠道干细胞所必需的，并且这种作用在脊椎动物中在进化上是保守的。 PRMT1 上调是发育干细胞中最早已知的分子事件，并且在斑马鱼和小鼠成体肠道干细胞发育过程中也是保守的。为了分析 PRMT1 在成体肠道干细胞形成过程中如何特异性上调，我们克隆了非洲爪蟾 PRMT1 启动子，并在 CaCo-2 细胞（一种具有肠道干细胞特征的人类细胞系）中对其进行了表征。通过一系列缺失和突变分析，我们发现干细胞相关转录因子c-Myc可以与第一个内含子中的进化保守位点结合以激活启动子。此外，我们证明，在变态过程中，c-Myc 和 PRMT1 均在重塑肠中高度上调，但在吸收尾部则不然，并且 c-Myc 在 PRMT1 上调之前由 TH 诱导。 此外，我们还发现，TH 在肠道变态过程中通过 TH 受体与 c-Myc 启动子结合直接激活 c-Myc 基因。这些结果表明，TH 直接在肠道中诱导 c-Myc 转录，而 c-Myc 反过来又激活 PRMT1 表达，这是控制肠道干细胞发育的重要基因调控级联。