Intrinsic and extrinsic regulation of epidermal stem cells

表皮干细胞的内在和外在调节

• 批准号: 8765810
• 负责人: Makoto Senoo
• 金额: $ 35.2万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-07-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8765810
• 关键词: Address; Alternative Splicing; Animal Model; Bass; Binding; Binding Proteins; Burn injury; Cell Cycle Progression; Cell physiology; Cells; Characteristics; Chromatin; Coupled; DNA; Data; Defect; Degenerative Disorder; Deletion Mutation; Development; Disease; Dominant-Negative Mutation; Ectodermal Dysplasia; Exhibits; Genes; Genetic; Genetic Transcription; Goals; Growth; Growth Factor; Health; Hepatocyte Growth Factor; Homeostasis; Homologous Gene; Human; In Vitro; Individual; Insulin-Like Growth Factor II; Intrinsic factor; Knockout Mice; Link; Lip structure; Maintenance; Malignant Neoplasms; Mediator of activation protein; Molecular; Molecular Analysis; Mus; Mutate; Mutation; Natural regeneration; Oncogenic; Palate; Pathogenesis; Play; Property; Protein Binding Domain; Protein Isoforms; Protein p53; Proteins; RNA; Regenerative Medicine; Regulation; Reporter; Role; Running; Signal Pathway; Signal Transduction; Skin Cancer; Stem cell transplant; Stem cells; Sterility; Stimulus; Syndrome; Therapeutic; Tissues; Variant; adult stem cell; base; cleft lip and palate; gene therapy; genome-wide; improved; in vivo; insight; mouse model; mutant; mutant mouse model; novel; novel strategies; premature; promoter; reconstitution; self-renewal; skin disorder; stem cell niche; tissue regeneration; transcription factor; wound

DESCRIPTION (provided by applicant): The long-term goal of this project is to improve the quality of epidermal stem cells (EpiSCs) for therapeutic transplantation, stem cell (SC)-directed gene therapy and for the treatment of degenerative skin disorders by elucidating both intrinsic and extrinsic mechanisms regulating EpiSC proliferative potential. We previously demonstrated that the transcription factor p63 is essential for the proliferative potential of EpiSCs. However, dual promoters in the p63 gene drive expression of TA isoforms with growth suppressive functions as well as ΔN dominant negative isoforms with oncogenic properties. Both isoforms undergo alternative splicing to generate multiple C-terminus variants, complicating molecular analysis of p63 function. Notably, α-type C- terminus (Cα) uniquely harbors a sterile α-motif (SAM) protein-protein interaction domain and a transcription inhibitory (TI) domain. Their mutations and deletions in ankyloblepharon-ectodermal defects-cleft lip/palate (AEC) syndrome, a unique form of p63-associated ectodermal dysplasia, highlight their functional importance. To further dissect the role of the Cα domains, we have generated mice deficient in Cv (p63C-/- mice). Notably, p63C-/- mice exhibit severe epidermal hypoplasia and fragility due to premature run down of the proliferative potential of EpiSCs. As we hypothesized that p63SAM interacting proteins were at least partially responsible for these effects, we screened for p63SAM interacting proteins and found that many of the identified proteins were involved in transcriptional and posttranscriptional activities. In Aim 1, we will further characterize p63SAM binding proteins by assessing their role in controlling the proliferative potential of EpiSCs and determine how deletion and AEC mutations in the individual Cα domains impact their interactions. We will also interrogate the role of each Cα domain in TA- and ΔNp63α isoforms by reconstituting p63C-/- epidermal cells with wild type or Cα domain mutant p63 isoforms. Finally, our preliminary data utilizing genome-wide array analysis coupled with clonogenic cultures suggest that the synergistic effects of Hgf and Igf2, regulated by the Wnt signaling mediator Dact1, are directly involved in EpiSC maintenance. In Aim 2, we will further assess the synergistic role of Hgf/Igf2 in regulating EpiSC self-renewal, proliferation and differentiation boh in vitro and in animal models and will determine if these growth factors regulate p63 levels to maintain the proliferative potential of EpiSCs. To further characterize the EpiSC niche, we have generated novel reporter mice to identify cells in which the Dact1 gene promoter is active, thereby allowing us to determine if they contribute to niche function in vivo. Together, these studies will provide unique insight into the intrinsic and extrinsic control of EpiSCs and will sere as a basis for development of novel strategies to improve the quality of EpiSCs for therapeutic transplantation and SC-directed gene therapy and for the treatment of wounds, burns and other degenerative epidermal disorders including cancer.

描述（由申请人提供）：该项目的长期目标是通过阐明调节EpiSC增殖潜力的内在和外在机制，提高用于治疗性移植、干细胞（SC）定向基因治疗和治疗退行性皮肤病的表皮干细胞（EpiSC）的质量。我们以前证明，转录因子p63是必不可少的增殖潜能的上皮干细胞。然而，p63基因中的双启动子驱动具有生长抑制功能的TA同种型以及具有致癌特性的ΔN显性负性同种型的表达。这两种异构体都经过选择性剪接产生多个C-末端变体，使p63功能的分子分析复杂化。值得注意的是，α-型C-末端（Cα）独特地具有无菌α-基序（SAM）蛋白质-蛋白质相互作用结构域和转录抑制（TI）结构域。他们的突变和缺失，在ankyloblepharon-ectodermal defects-cleft lip/palate（AEC）综合征，一种独特的形式p63相关的外胚层发育不良，突出了他们的功能的重要性。为了进一步剖析Cα结构域的作用，我们产生了Cv缺陷小鼠（p63 C-/-小鼠）。值得注意的是，p63 C-/-小鼠表现出严重的表皮发育不全和脆性，这是由于EpiSC的增殖潜力过早下降。由于我们假设p63 SAM相互作用蛋白至少部分负责这些影响，我们筛选p63 SAM相互作用蛋白，发现许多已鉴定的蛋白质参与转录和转录后活动。在目标1中，我们将通过评估p63 SAM结合蛋白在控制EpiSC增殖潜力中的作用来进一步表征它们，并确定单个Cα结构域中的缺失和AEC突变如何影响它们的相互作用。我们还将通过用野生型或Cα结构域突变型p63同种型重建p63 C-/-表皮细胞来探究每个Cα结构域在TA-和Δ Np 63 α同种型中的作用。最后，我们利用全基因组阵列分析结合克隆培养的初步数据表明，由Wnt信号传导介质Dact 1调节的Hgf和Igf 2的协同作用直接参与EpiSC的维持。在目的2中，我们将进一步评估Hgf/Igf 2在体外和动物模型中调节EpiSC自我更新、增殖和分化的协同作用，并将确定这些生长因子是否调节p63水平以维持EpiSC的增殖潜力。为了进一步表征EpiSC生态位，我们已经产生了新的报告小鼠，以识别其中Dact 1基因启动子活跃的细胞，从而使我们能够确定它们是否有助于体内生态位功能。总之，这些研究将为EpiSC的内在和外在控制提供独特的见解，并将作为开发新策略的基础，以提高EpiSC的质量，用于治疗性移植和SC定向基因治疗以及治疗伤口，烧伤和其他退行性表皮疾病，包括癌症。