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.

分化和肿瘤部门研究了组织发育/形态发生中的电感信号传导，并同时在肿瘤发生中其失调，重点是介导正常组织相互作用的配体以及响应信号响应的途径和靶标。我们的重点是泌尿生殖道的发展，泌尿生殖道具有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，间质 - 上皮 - 上皮过渡，来自不同谱系的结构的整合，发育的重复性循环以及繁殖肾病的肿瘤。更具体地说，我们对直接延髓间充质（MM）转换为肾单位上皮的信号传导机制感兴趣。威尔姆斯肿瘤（WT）的特征是胚胎/祖先种群扩大，上皮转化的能力受限。我们的长期目标是确定WT细胞依赖生存或失调信号传导的目标，可以重新编程以允许细胞区分到更良性的表型。 我们和其他人已经暗示了细胞因子白血病抑制因子（LIF）在MM中诱导肾上腺素上皮。 LIF刺激MM祖细胞中Stat3和Stat1的酪氨酸和丝氨酸磷酸化。尽管有关于统计信号传导的巨大文献，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细胞的研究外，在开发过程中的数据参与很大程度上没有探索。为了更好地了解这些因素在分化中的作用，我们研究了每个Stat家族成员的表达，并在可能的情况下它们在肾脏发生过程中的磷酸化。统计1、3、5和6在整个跨肾脏发育和成年肾脏中都高度表达，而数据1、3和5仅在发育过程中才磷酸化。这些发现使我们使用条件功能丧失（LOF）小鼠模型研究了这些分子的生物学功能。尽管已经报道了单个统计敲除的非肾脏表型，但单个细胞因子激活多个统计局家族成员的能力已得到充分确立，并表明可能有功能冗余。对STAT3的LOF突变体的初步评估显示了骨骼系统中的广泛缺陷，但除尺寸差异外，肾脏没有明显的改变。因此，我们正在为Stats 1和3以及Stats 1和5的条件双突变体生成条件双突变体，以规避冗余并发症。 已显示Wnt4在体内MM中介导间充质 - 上皮过渡（MET），LIF或GSK3抑制剂在Explant培养物中类似地诱导MET。为了理解负责此签名事件的肾单位形成的信号传导机制，我们研究了三个分子中每个分子激活钙依赖性NFAT信号传导的能力，这证明了MET过程独立于β-catenin/canonical Wnt信号传导。现在，我们已经确定NFAT被每个电感因子激活，并且NFAT的本构激活足以在MM中诱导上皮标记。此外，抑制NFAT信号传导阻碍了这些标记的表达。通过1。细胞培养条件的发展促进了这些研究，这些细胞培养条件促进了肾原生祖细胞的扩展，以进行生化分析，而不会损害其形成小管和2的能力。设计，合成和分析有效的肽抑制剂对β-catenin的有效肽抑制剂，以靶向Canonical Wnt WNT信号。该抑制剂已由NCI批准为专利。 在MM的MET中，我们还询问WTS中是否发生了统计激活。在许多类型的固体和血液学癌症中，已经观察到了STAT家族成员的组成型磷酸化。对WTS的评估表明，大多数在STAT1中包含磷酸化的S727。当肿瘤细胞表达了无法磷酸化的STAT1突变形式（S727a）时，它们失去了独立于锚定生长的能力。此外，STAT1的下调诱导这些细胞的凋亡。微阵列分析将MCL1，HSP27和CUX1识别为STAT1信号的下游靶标，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，我们报道说，与干扰素γ（IFN-GAMMA）正常MM激活STAT1刺激了细胞的增殖，抑制了小管的形成，并诱导了MCL1和CUX1的表达，这是促进细胞存活和增殖的因素。缺乏良好的动物模型或肿瘤细胞系，WT的研究受到了阻碍。我们采用了三种独立的方法来解决这个问题。首先，我们已经生成了一条携带可活化的STAT1-S727E转基因的小鼠线，该系应该模仿分子的磷酸化形式。此外，我们还有条件地表达了小鼠MM中的IFN-gamma转基因，以组成性激活STAT1并确认其在WT发病机理中的作用。类似的方法在神经组织中产生髓母细胞瘤。其次，我们正在绘制易感性NB菌株大鼠的后代与抗耐药性F344菌株大鼠的后代相关的遗传基因座，因为遗传因常染色体不完全的主导性特征传播。第三，我们使用针对MM培养的条件开发了新的WT细胞系，用于生化和分子研究。这些条件促进了细胞从组织学不同的Wilms肿瘤中的生长和传播，目前正在研究其在软琼脂和异种移植中形成肿瘤的能力。