GrowthDifferentiation Factors in Organogenesis

器官发生中的生长分化因素

• 批准号: 8348866
• 负责人: ALAN PERANTONI
• 金额: $ 124.69万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/8348866
• 关键词: Address; Adult; Agar; Anchorage-Independent Growth; Animal Model; Apoptosis; Benign; Biochemical; Biological Process; Calcium; Caricatures; Cell Culture Techniques; Cell Proliferation; Cell Survival; Cells; Congenital Mesoblastic Nephroma; Defect; Development; Dominant Genetic Conditions; Down-Regulation; Drosophila genus; Duct (organ) structure; Epithelial; Epithelium; Evaluation; Event; Family member; Genes; Genetic; Genitourinary system; Goals; Hematologic Neoplasms; Histologic; Individual; Inflammatory; Interferon Type II; Intermediate Mesoderm; Invaded; Kidney; Knock-out; Lead; Legal patent; Ligands; Link; Literature; MCL1 gene; Maps; Mediating; Mesenchymal; Mesenchyme; Metanephric Diverticulum; Metanephric structure; Microarray Analysis; Molecular; Molecular Target; Morphogenesis; Movement; Mus; Neoplasms; Nephroblastoma; Nephrogenic Cord; Nephrons; Normal tissue morphology; Organogenesis; Pathogenesis; Pathway interactions; Pediatric Neoplasm; Peptides; Phenotype; Phosphorylation; Population; Predisposition; Process; Rat Strain F344; Rat Strains; Reporting; Resistance; Role; STAT1 gene; STAT3 gene; Serine; Signal Transduction; Skeletal system; Solid; Structure; Structure of mesonephric duct; Study Section; System; Tissues; Transgenes; Tumor Cell Line; Tyrosine; WNT4 gene; Xenograft procedure; beta catenin; cytokine; design; drug development; embryonic stem cell; immune function; in vivo; inhibitor/antagonist; interest; leukemia inhibitory factor; loss of function; medulloblastoma; mouse model; mutant; neoplastic cell; nephrogenesis; novel; offspring; progenitor; relating to nervous system; reproductive; response; tumor; tumorigenesis

The Differentiation and Neoplasia Section studies inductive signaling in tissue development/morphogenesis and, in parallel, its dysregulation in tumorigenesis with emphasis on the ligands that mediate normal tissue interactions and the pathways and targets that are activated in response to signaling. Our focus has been on development of the urogenital tract, which features reciprocal interactions between two distinct mesodermal progenitors, highly coordinated tissue movements, mesenchymal-epithelial transition, integration of structures from different lineages, reiterative cycles of development, and a tumor that caricatures nephrogenesis. More specifically we are interested in the signaling mechanisms that direct metanephric mesenchyme (MM) to convert to the epithelia of the nephron. Wilms tumor (WT) is characterized by an expanded blastemal/progenitor population with a restricted capacity for epithelial conversion. It is our long-term goal to identify targets on which WT cells depend for survival or dysregulated signaling that can be reprogrammed to allow cells to differentiate to a more benign phenotype. We and others have implicated the cytokine leukemia inhibitory factor (LIF) in the induction of nephronic epithelia from MM. LIF stimulates tyrosine and serine phosphorylation of STAT3 and STAT1 in MM progenitors. Despite the enormous literature on STAT signaling, the vast majority of studies have focused on its role in immune function or the inflammatory process. The involvement of STATs during development, with the exception of studies in Drosophila or ES cells, has largely gone unexplored. In efforts to better understand the role of these factors in differentiation, we have examined the expression of each STAT family member and, where possible, their phosphorylation during nephrogenesis. STATs 1, 3, 5, and 6 are all highly expressed throughout metanephric development and in the adult kidney, and STATs 1, 3, and 5 are phosphorylated only during development. These findings have led us to investigate the biological function of these molecules using conditional loss-of-function (LOF) mouse models. Although non-renal phenotypes have been reported for individual Stat knockouts, the ability of a single cytokine to activate more than one STAT family member is well established and suggests that functional redundancy is likely. A preliminary assessment of a LOF mutant for Stat3 has revealed extensive defects in the skeletal system but no obvious alterations in the kidney other than size differences. We are therefore generating conditional double mutants for Stats 1 and 3 and Stats 1 and 5 in order to circumvent the complications of redundancy. WNT4 has been shown to mediate mesenchymal-epithelial transition (MET) in MM in vivo, and LIF or GSK3 inhibitors similarly induce MET in explant culture. In efforts to understand the signaling mechanism(s) responsible for this signature event in nephron formation,we have examined the ability of each of the three molecules to activate calcium-dependent NFAT signaling, having demonstrated that the MET process occurs independently of beta-catenin/canonical Wnt signaling. We have now determined that NFAT is activated by each inductive factor and that constitutive activation of NFAT is sufficient to induce epithelial markers in MM. Moreover, inhibition of NFAT signaling blocks the expression of these markers. These studies were facilitated by 1. development of cell culture conditions that promote the expansion of nephrogenic progenitors for biochemical analyses without compromising their capacity to form tubules and 2. the design, synthesis, and analysis of a potent peptide inhibitor for beta-catenin that targets canonical Wnt signaling. This inhibitor was approved for patenting by NCI. Having implicated LIF signaling in MET of MM, we also asked whether STAT activation occurs in WTs. Constitutive phosphorylation of a STAT family member has been observed in many types of solid and hematologic cancers. An evaluation of WTs, revealed that most contain phosphorylated S727 in STAT1. When tumor cells expressed a mutant form (S727A) of STAT1, which cannot be phosphorylated, they lost their ability for anchorage-independent growth. Furthermore, downregulation of STAT1 induced apoptosis in these cells. Microarray analysis identified Mcl1, Hsp27, and Cux1 as downstream targets of STAT1 signaling, and these same genes were found to be highly expressed in WTs. Consistent with these observations, we reported that activation of STAT1 in normal MM with interferon-gamma (IFN-gamma) stimulated cell proliferation, inhibited tubule formation, and also induced the expression of MCL1 and CUX1, factors that promote cell survival and proliferation. Studies of WTs have been hampered by the absence of good animal models or tumor cell lines. We have taken three independent approaches to address this problem. Firstly, we have generated a mouse line carrying an activatable Stat1-S727E transgene that should mimic the phosphorylated form of the molecule. Additionally,we have conditionally expressed an Ifn-gamma transgene in mouse MM in order to constitutively activate STAT1 and confirm its role in WT pathogenesis. A similar approach yielded medulloblastomas in neural tissues. Secondly, we are mapping a genetic locus linked to nephroblastoma susceptibility/resistance in offspring of susceptible Nb strain rats versus resistant F344 strain rats, since inheritance was transmitted as an autosomal incomplete dominant trait. Thirdly, we are developing new WT cell lines for biochemical and molecular studies using conditions optimized for the culture of MM. These conditions promote the outgrowth and passage of cells from histologically distinct Wilms tumors and are currently examining their ability to form tumors in soft agar and in xenotransplantation.

分化和肿瘤部分研究组织发育/形态发生中的诱导信号传导，同时研究其在肿瘤发生中的失调，重点关注介导正常组织相互作用的配体以及响应信号传导而激活的途径和靶点。我们的重点是泌尿生殖道的发育，其特征是两个不同的中胚层祖细胞之间的相互相互作用、高度协调的组织运动、间充质-上皮转化、不同谱系结构的整合、反复的发育周期以及讽刺肾发生的肿瘤。更具体地说，我们对引导后肾间充质（MM）转化为肾单位上皮的信号传导机制感兴趣。肾母细胞瘤（WT）的特点是胚细胞/祖细胞群扩大，上皮转化能力有限。我们的长期目标是确定 WT 细胞生存或失调信号传导所依赖的靶标，这些靶标可以被重新编程以允许细胞分化为更良性的表型。 我们和其他人发现细胞因子白血病抑制因子 (LIF) 与 MM 肾病上皮的诱导有关。 LIF 刺激 MM 祖细胞中 STAT3 和 STAT1 的酪氨酸和丝氨酸磷酸化。尽管关于 STAT 信号传导的文献数量巨大，但绝大多数研究都集中在其在免疫功能或炎症过程中的作用。除了果蝇或 ES 细胞的研究之外，STAT 在发育过程中的参与在很大程度上尚未得到探索。为了更好地了解这些因素在分化中的作用，我们检查了每个 STAT 家族成员的表达，并在可能的情况下检查了它们在肾发生过程中的磷酸化。 STAT 1、3、5 和 6 在整个后肾发育过程和成人肾脏中均高度表达，STAT 1、3 和 5 仅在发育过程中被磷酸化。这些发现促使我们使用条件性功能丧失（LOF）小鼠模型来研究这些分子的生物学功能。尽管已经报道了个体 Stat 敲除的非肾脏表型，但单一细胞因子激活多个 STAT 家族成员的能力已得到充分证实，并表明可能存在功能冗余。对 Stat3 LOF 突变体的初步评估显示，骨骼系统存在广泛缺陷，但肾脏除了大小差异外没有明显变化。因此，我们为统计 1 和 3 以及统计 1 和 5 生成条件双突变体，以避免冗余的复杂性。 WNT4 已被证明可介导体内 MM 的间充质-上皮转化 (MET)，而 LIF 或 GS​​K3 抑制剂同样可在外植体培养中诱导 MET。为了了解肾单位形成中这一标志性事件的信号传导机制，我们检查了这三种分子激活钙依赖性 NFAT 信号传导的能力，并证明 MET 过程的发生独立于 β-连环蛋白/经典 Wnt 信号传导。我们现在已经确定，NFAT 被每种诱导因子激活，并且 NFAT 的组成型激活足以诱导 MM 中的上皮标记。此外，抑制 NFAT 信号传导会阻止这些标记物的表达。这些研究得到了促进：1. 开发细胞培养条件，促进肾源性祖细胞的扩增以进行生化分析，而不损害其形成肾小管的能力；2. 设计、合成和分析针对经典 Wnt 信号传导的有效 β-连环蛋白肽抑制剂。该抑制剂已获得 NCI 批准申请专利。 在 MM 的 MET 中涉及 LIF 信号传导后，我们还询问 WT 中是否发生 STAT 激活。在许多类型的实体癌和血液癌中观察到 STAT 家族成员的组成型磷酸化。对 WT 的评估表明，大多数 WT 的 STAT1 中含有磷酸化的 S727。当肿瘤细胞表达不能被磷酸化的 STAT1 突变体 (S727A) 时，它们就失去了不依赖锚定的生长能力。此外，STAT1 的下调诱导这些细胞凋亡。微阵列分析确定 Mcl1、Hsp27 和 Cux1 是 STAT1 信号传导的下游靶标，并且发现这些相同的基因在 WT 中高度表达。与这些观察结果一致，我们报道用干扰素-γ (IFN-γ) 激活正常 MM 中的 STAT1 刺激细胞增殖，抑制肾小管形成，并诱导 MCL1 和 CUX1 的表达，这些因子促进细胞存活和增殖。由于缺乏良好的动物模型或肿瘤细胞系，WT 的研究受到了阻碍。我们采取了三种独立的方法来解决这个问题。首先，我们生成了携带可激活 Stat1-S727E 转基因的小鼠品系，该转基因应模仿该分子的磷酸化形式。此外，我们在小鼠 MM 中条件性表达了 Ifn-gamma 转基因，以组成型激活 STAT1 并确认其在 WT 发病机制中的作用。类似的方法在神经组织中产生了髓母细胞瘤。其次，我们绘制了易感 Nb 品系大鼠与耐药 F344 品系大鼠后代中与肾母细胞瘤易感性/耐药性相关的遗传位点，因为遗传是作为常染色体不完全显性性状传递的。第三，我们正在开发新的 WT 细胞系，用于使用针对 MM 培养优化的条件进行生化和分子研究。这些条件促进组织学上不同的肾母细胞瘤细胞的生长和传代，目前正在检查它们在软琼脂和异种移植中形成肿瘤的能力。