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: 8941529
• 负责人: Yun-Bo Shi
• 金额: $ 129.19万
• 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/8941529
• 关键词: Adipose tissue; Adult; African; Amino Acids; Animals; Antigen Receptors; Apoptosis; Basal lamina; Biological Metamorphosis; Catalytic Domain; Cell Communication; Cell Culture Techniques; Cell Differentiation process; Cell physiology; Cells; Cessation of life; Collaborations; Complement; Connective Tissue; Control Animal; Development; Diet; Dietary Proteins; Diploidy; Embryonic Development; Enzymes; Epithelial; Epithelial Cells; Epithelium; Exhibits; Extracellular Matrix; Failure; Food Processing; Gastrocnemius Muscle; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Glucose; Goals; Growth; Histidine Ammonia-Lyase; Injection of therapeutic agent; Insulin Resistance; Intestines; Knock-out; Knockout Mice; Leucine; Life; Light; Lyase Gene; Maintenance; Mediating; Metabolic; Molecular; Mus; Muscle; Neonatal; Neutral Amino Acids; Nutrient; Organ; Organ Culture Techniques; Pathway interactions; Phenotype; Phosphorylation; Physiological; Play; Primary Cell Cultures; Process; Proliferating; Protein Biosynthesis; Proteins; Rana; Regulation; Relative (related person); Role; Signal Pathway; Signal Transduction; Signaling Pathway Gene; Skeletal Muscle; Stem Cell Development; Stem cells; Structure; System; T cell differentiation; Tadpoles; Thyroid Hormone Receptor; Thyroid Hormones; Tissues; Xenopus; Xenopus laevis; absorption; adult stem cell; cell growth; human FRAP1 protein; intestinal epithelium; intraperitoneal; mRNA Expression; muscle form; neurodevelopment; progenitor; programs; promoter; recombinase; response; stem cell division; tissue regeneration; tissue repair; toad; transcription factor; xenopus development

DIFFERENTIAL REGULATION OF TWO HISTIDINE AMMONIA-LYASE GENES DURING XENOPUS DEVELOPMENT IMPLICATES DISTINCT FUNCTIONS DURING THYROID HORMONE-INDUCED FORMATION OF ADULT STEM CELLS. We have shown earlier that the de novo formation of adult intestinal stem cells that requires TH signaling in both the larval epithelium (Ep) and non-epithelial tissues (non-Ep). To understand the underlying molecular mechanisms, it is critical to determine the genes and signaling pathways involved in this process. Thus, we have profiled the gene expression programs in the epithelial and non-epithelial tissues in X. laevis intestine to systematically determine changes underlying the cell autonomous and cell-cell interaction-dependent processes that are required for stem cell formation (1). One of the genes thus identified is the histidine ammonia-lyase (HAL) gene, which encodes an enzyme known as histidase or histidinase (2). We showed that there are two duplicated HAL genes, HAL1 and HAL2, in both Xenopus laevis and Xenopus tropicalis, a highly related but diploid species. Interestingly, only HAL2 is highly upregulated by TH and appears to be specifically expressed in the adult intestinal progenitor/stem cells while HAL1 is not expressed in the intestine during metamorphosis. Furthermore, when analyzed in whole animals, HAL1 appears to be expressed only during embryogenesis but not metamorphosis while the opposite appears to be true for HAL2. Our results suggest that the duplicated HAL genes have distinct functions with HAL2 likely involved in the formation and/or proliferation of the adult stem cells during metamorphosis. DIRECT ACTIVATION OF SOX3 GENE TRANSCRIPTION BY THYROID HORMONE IN THE DEVELOPING ADULT INTESTINAL STEM CELL DURING XENOPUS METAMORPHOSIS. In addition, we also discovered from our microarray studies that the transcription factor Sox3, well known for its involvement in neural development, was upregulated in the intestinal epithelium during metamorphosis. Here, we have now shown that Sox3 is highly and specifically expressed in the developing adult intestinal progenitor/stem cells. We further demonstrated that Sox3 is a direct target gene of TH as its induction by TH is independent of new protein synthesis, suggesting that TH activate the promoter directly via TH receptor. Thus, TH activates Sox3 as one of the earliest changes in the epithelium and that Sox3 in turn may facilitate the dedifferentiation of the larval epithelial cells into adult stem cells. THE CATALYTIC SUBUNIT OF THE SYSTEM L1 AMINO ACID/THYROID HORMONE TRANSPORTER (LAT1 OR SLC7A5) FACILITATES NUTRIENT SIGNALING IN MOUSE SKELETAL MUSCLE. To regulate cellular processes, TH has to be actively transported into cells and this process is mediated by several different types of transporters. One of our previously identified TH-response genes in the intestine, LAT1, encodes the light chain of a heterodimeric system L type of TH transporter, which also transports several amino acids. Interestingly, LAT1 is highly upregulated at the climax of metamorphosis in the tadpole intestine, coinciding with the formation and rapid proliferation of the adult intestinal stem cells. We recently found out that LAT1 was also highly expressed in the mouse intestine during the neonatal period when the mouse intestine matured into the adult form, a process that appears also involves TH-dependent formation and/proliferation of the adult intestinal stem cells. Through a collaborative study, we generated a mouse line with the LAT1 gene floxed, which allows conditional knockout of the LAT1 upon expression of the Cre recombinase (3). When we crossed floxed LAT1 mice with mice expressing Cre driven by a global promoter, we obtained global LAT1 heterozygous knockout (Slc7a5+/-) animals, although failure to produce homozygous Slc7a5 animals indicate that the global knockout of Slc7a5 is embryonically lethal. Slc7a5 mRNA expression and functional System L1-type transport activity were reduced in the Slc7a5+/- animals, although no growth phenotype was detected. Muscle-specific (MCK Cre-mediated) Slc7a5 knockout mice were established and used to study the role of SLC7A5 in the intracellular delivery large neutral amino acids (LNAA) such as leucine, which are required for full activation of the mTOR-S6K signaling pathway promoting protein synthesis and cell growth. Activation of muscle mTOR-S6K (Thr389 phosphorylation) by intraperitoneal leucine injection was blunted in homozygous MCK Cre-Slc7a5-Flox mice relative to wild-type animals. Leu and Ile concentrations in gastrocnemius muscle were reduced by 40% as dietary protein content was reduced from 30 to 10%. These changes were associated with >50% decrease in S6K Thr389 phosphorylation in Slc7a5-knockout muscle, indicating reduced mTOR-S6K pathway activation, despite no significant differences in lean tissue mass between groups on the same diet. MCK Cre-Slc7a5-Flox mice on 30% protein diet exhibited mild insulin resistance (e.g. reduced glucose clearance, larger gonadal adipose depots) relative to control animals. Thus, SLC7A5 modulates LNAA-dependent muscle mTOR-S6K signaling in mice, although it appears non-essential for maintenance of normal muscle mass. In addition, we had earlier shown through another collaboration that LAT1 is critical for antigen receptor-mediated the metabolic re-programming (4), a process essential for T cell differentiation, suggesting LAT1 plays diverse roles in multiple tissues/organs.

非洲爪蟾发育过程中两个组氨酸解氨酶基因的差异调节意味着甲状腺激素诱导的成体干细胞形成过程中的不同功能。我们之前已经证明，成体肠道干细胞的从头形成需要幼虫上皮 (Ep) 和非上皮组织 (non-Ep) 中的 TH 信号传导。为了了解潜在的分子机制，确定参与该过程的基因和信号通路至关重要。因此，我们对 X. laevis 肠道上皮和非上皮组织中的基因表达程序进行了分析，以系统地确定干细胞形成所需的细胞自主和细胞间相互作用依赖性过程的变化 (1)。由此鉴定出的基因之一是组氨酸解氨酶 (HAL) 基因，它编码一种称为组氨酸酶或组氨酸酶的酶 (2)。我们发现，非洲爪蟾和热带爪蟾（一种高度相关的二倍体物种）中都有两个重复的 HAL 基因：HAL1 和 HAL2。有趣的是，只有 HAL2 被 TH 高度上调，并且似乎在成体肠道祖细胞/干细胞中特异性表达，而 HAL1 在变态过程中不在肠道中表达。此外，在整个动物中进行分析时，HAL1 似乎仅在胚胎发生过程中表达，而在变态过程中不表达，而 HAL2 的情况则相反。我们的结果表明，复制的 HAL 基因具有不同的功能，其中 HAL2 可能参与变态过程中成体干细胞的形成和/或增殖。 爪蟾变态过程中发育中的成年肠干细胞中甲状腺激素直接激活 SOX3 基因转录。此外，我们还从微阵列研究中发现，众所周知参与神经发育的转录因子Sox3在变态过程中肠上皮细胞中的表达上调。 在这里，我们现已证明 Sox3 在发育中的成体肠道祖细胞/干细胞中高度特异性表达。我们进一步证明 Sox3 是 TH 的直接靶基因，因为 TH 的诱导不依赖于新蛋白质的合成，这表明 TH 直接通过 TH 受体激活启动子。因此，TH 激活 Sox3 作为上皮中最早的变化之一，而 Sox3 反过来可能促进幼虫上皮细胞去分化为成体干细胞。 L1 氨基酸/甲状腺激素转运蛋白系统（LAT1 或 SLC7A5）的催化亚基促进小鼠骨骼肌中的营养信号传导。为了调节细胞过程，TH 必须主动转运到细胞中，并且该过程由几种不同类型的转运蛋白介导。我们之前在肠道中发现的 TH 反应基因之一 LAT1，编码 TH 转运蛋白异二聚体系统 L 型的轻链，该转运蛋白还转运多种氨基酸。有趣的是，LAT1在蝌蚪肠道变态高峰期高度上调，与成体肠道干细胞的形成和快速增殖同时发生。我们最近发现，当小鼠肠道成熟为成年形式时，LAT1 在小鼠肠道中也在新生儿期高度表达，这一过程似乎也涉及成体肠道干细胞的 TH 依赖性形成和/增殖。 通过一项合作研究，我们培育了 LAT1 基因 floxed 的小鼠品系，该品系允许在 Cre 重组酶表达时条件性敲除 LAT1 (3)。当我们将 floxed LAT1 小鼠与表达由全局启动子驱动的 Cre 的小鼠杂交时，我们获得了全局 LAT1 杂合敲除 (Slc7a5+/-) 动物，尽管未能产生纯合 Slc7a5 动物表明 Slc7a5 全局敲除是胚胎致死的。尽管没有检测到生长表型，但 Slc7a5+/- 动物中的 Slc7a5 mRNA 表达和功能性系统 L1 型转运活性降低。建立了肌肉特异性（MCK Cre 介导的）Slc7a5 敲除小鼠，并用于研究 SLC7A5 在细胞内递送大中性氨基酸 (LNAA)（如亮氨酸）中的作用，这是完全激活 mTOR-S6K 信号通路所必需的促进蛋白质合成和细胞生长。与野生型动物相比，纯合 MCK Cre-Slc7a5-Flox 小鼠腹腔注射亮氨酸对肌肉 mTOR-S6K（Thr389 磷酸化）的激活减弱。随着膳食蛋白质含量从 30% 降低至 10%，腓肠肌中的 Leu 和 Ile 浓度降低了 40%。这些变化与 Slc7a5 敲除肌肉中 S6K Thr389 磷酸化减少 > 50% 相关，表明 mTOR-S6K 通路激活减少，尽管相同饮食组之间的瘦组织质量没有显着差异。相对于对照动物，接受 30% 蛋白质饮食的 MCK Cre-Slc7a5-Flox 小鼠表现出轻度胰岛素抵抗（例如葡萄糖清除率降低、性腺脂肪库增大）。因此，SLC7A5 调节小鼠中 LNAA 依赖性肌肉 mTOR-S6K 信号传导，尽管它对于维持正常肌肉质量似乎不是必需的。此外，我们之前通过另一项合作表明，LAT1 对于抗原受体介导的代谢重编程至关重要 (4)，这是 T 细胞分化所必需的过程，表明 LAT1 在多个组织/器官中发挥着不同的作用。