GrowthDifferentiation Factors in Organogenesis

器官发生中的生长分化因素

• 批准号: 7964999
• 负责人: ALAN PERANTONI
• 金额: $ 116.8万
• 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/7964999
• 关键词: Adherens Junction; Affect; Alleles; Animal Model; Animals; Area; Binding; Biochemical; Biological; Cadherins; Cell Count; Cell Line; Cells; Characteristics; Chemicals; Commit; Communities; Complex; Congenital Mesoblastic Nephroma; Cultured Cells; Data; Development; Duct (organ) structure; Elements; Embryo; Epithelial; Epithelium; Family member; Genes; Genetic Models; Genetic Transcription; Glycogen Synthase Kinases; Goals; Harvest; Histology; Human; Inbred F344 Rats; Incidence; Interferon Type II; Invaded; Kidney; LacZ Genes; Lead; Ligands; MG132; Malignant Neoplasms; Mesenchymal; Mesenchyme; Metanephric Diverticulum; Metanephric structure; Molecular Target; Morphogenesis; Mus; Mutate; Mutation; N Domain; Neoplasms; Nephroblastoma; Nephrons; Nuclear; Organogenesis; Oximes; Pathway interactions; Pediatric Neoplasm; Peptides; Pharmaceutical Preparations; Phosphorylation; Play; Population; Predisposition; Process; Proteasome Inhibitor; Publications; Rattus; Reporter; Reporting; Research; Resources; Role; STAT1 gene; STAT3 gene; STAT5A gene; Series; Serine; Signal Transduction; Site; Source; System; Terminator Codon; Testing; Tight Junctions; Time; Transactivation; Transcriptional Activation; Transgenic Mice; Tumor Tissue; Tumorigenicity; Tyrosine; Voice; Work; Xenograft Model; Xenograft procedure; antitumor agent; beta catenin; cytokine; design; drug development; effective therapy; embryo tissue; established cell line; improved; in vivo; inhibitor/antagonist; leukemia inhibitory factor; medulloblastoma; mutant; nephrogenesis; neutralizing antibody; novel; prevent; progenitor; prototype; small molecule; tissue culture; tumor; tumorigenesis

In our previous studies, we reported the constitutive activation of STAT1 in Wilms tumors. Activation of a STAT family member occurs in a variety of human neoplasms but generally involves Y705 phosphorylation of STAT3. While investigating the possible dysregulation of STATs in Wilms tumors, we discovered the constitutive phosphorylation of S727 in STAT1 and demonstrated its role in Wilms tumorigenesis. Since this neoplasm is a prototype for arrested cellular differentiation in cancer, we hypothesized that STAT1 signaling might also play an important role in the developing kidney. In order to determine if STAT signaling is active during metanephric development, we analyzed a temporal series of embryonic kidneys and discovered that both STAT1 and STAT3 are activated by tyrosine and serine phosphorylation during the peak of nephronic development. STAT5 is also expressed and may function in this process. To examine their roles in nephrogenesis, we utilized explant cultures of metanephric mesenchyme (MM), the progenitor of nephronic epithelia and putative origin of Wilms tumor. Interferon-gamma (IFNg), unlike differentiation-inducing leukemia inhibitory factor (LIF), caused STAT1, but not STAT3, activation in MM. In functional opposition to LIF, which induces tubulogenesis in MM, IFNg stimulated proliferation in MMs and inhibited nephron formation following induction. Furthermore, a peptide inhibitor was designed by our NCI-Frederick collaborator Nadya Tarasova, to target the STAT1 N-domain, and it specifically disrupts STAT1-dependent transcription and induces tubule formation in explanted MMs. These findings indicate that STAT1 activation provides a critical regulatory signal in MM specification, consistent with its role in Wilms tumorigenesis, and further suggest that disruption of STAT1 activation may provide a novel target for Wilms tumor therapy. We are currently evaluating the efficacy of using this novel STAT1 peptide as an anti-tumor agent in vivo. To directly examine the role of STAT signaling in renal development, we are also developing mouse genetic models for mutant forms of STAT1 that conditionally target S727 activity (floxed stop codons upstream of the mutant Stat form). These include a S727-to-E727 mutation, which is constitutively active, and a S727-to-A727, which is inactive for phosphorylation at this site. We also have generated a floxed-stop codon form of IFNg, which causes increased expression of this cytokine conditionally with Cre activation. We speculate that this will increase the progenitor population in the developing kidney or at least disrupt normal development. A similar strategy has been used by others to induce medulloblastomas which are STAT1 dependent in mice. Finally, we have also obtained mouse lines with floxed Stat1 or Stat3 alleles to assess the impact of the loss of each of these genes either separately or in combination. In addition to these mechanistic studies, we are attempting to leverage our findings with LIF and IFNg in tissue culture in order to massively expand populations of MM for biochemical analyses. A longstanding problem in working with embryonic tissues is the small numbers of cells that can be harvested for study. We estimate that the average MM contains about 4000 cells. By culturing these cells on a matrix using LIF and IFNg, we have now been able to expand 30 MMs (120,000 cells) into over 20 million cells in a weeks time without sacrificing their ability to be induced to differentiate. We are currently evaluating subsequent passages to determine if they retain this capacity. This would provide an invaluable resource for the research community if we are successful in achieving this. In addition, we have applied similar conditions to Wilms tumor tissues in order to establish cell lines from these as well. There is currently only one cell line that has been successfully established from a Wilms tumor, and clinicians have voiced a need for more appropriate lines for drug trial xenograft studies. With our conditions, we have established an early passage cell line from a Wilms tumor that expresses characteristics of Wilms including NCam, cadherin11 and Osr1. We are in the process of acquiring more tumors for culturing and will eventually use them in xenograft studies to demonstrate tumorigenicity and evaluate histology. There has been considerable effort and resource committed by others in the pursuit of this goal, so it would be a major step forward for this research area to have a replenishable biological source for study and an improved model for xenograft drug trials. Another feature of Wilms tumor is the frequent activation of the Wnt pathway, as demonstrated in our recent publication of nephroblastomas, which show nuclear localization of beta-catenin in most tumors. This pathway is also critical to nephronic development; however, the exact mechanism involved is not clear. Studies have shown an essential role for Wnt/beta-catenin in mesenchymal-epithelial transition (MET) during nephron formation. Intriguingly, analysis of BATlacZ transgenic mice expressing a beta-catenin-transactivated lacZ reporter revealed that the reporter is strongly expressed in the ureteric bud but not in surrounding metanephric mesenchyme (MM) or newly formed nephronic tubules, raising the possibility that beta-catenin may act through a TCF/LEF-independent mechanism to promote MET and tubule formation. To explore the mechanism, isolated MMs were treated with a variety of small molecules that affect the Wnt/beta-catenin pathway. An activator of Wnt signaling (WA), which stimulates TCF/LEF-dependent transactivation without altering beta-catenin levels, could not induce MET. Three glycogen synthase kinase-3beta inhibitors, including bromoindirubin-3-oxime (BIO), were able to increase beta-catenin levels and induce MET. Proteasome inhibitor MG132 also increased beta-catenin levels, but did not induce MET. Using our newly developed approach for expanding the population of MM cells, we are now able to perform GST pull-down studies of beta-catenin to determine how the various chemical treatments affect its binding form. We have found that BIO treatment increased both TCF- and cadherin-binding forms of beta-catenin, while MG132 elevated only the TCF-binding form. Expression of a mutated cadherin that disrupts normal beta-catenin/cadherin complex formation prevented BIO-induced tight junction formation. Consistent with these observations, neutralizing antibody against cadherin abrogated BIO-induced tubulogenesis in cultured MMs. These data indicate that a beta-catenin interaction with cadherin plays an essential role in MET and tubule formation during renal development. We are currently testing peptides generated by Dr. Tarasova that target beta-catenin and its interaction with TCF/LEF1 in efforts to specifically block canonical activation. Such peptides may have the ability to biochemically dissect Wnt signaling, separating its role in transcriptional activation through TCF from its function in adherens junctions. Since many different types of tumors show nuclear localization of beta-catenin and aberrant activation of Wnt signaling, such an approach could impact the development of effective therapies directed at this target. We have also investigated the susceptibility of rat to develop nephroblastomas. The Noble rat is exquisitely sensitive to chemical induction of these tumors, while the F344 rat is insensitive. When crossed, animals develop a tumor incidence suggestive of the involvemen [summary truncated at 7800 characters]

在我们之前的研究中，我们报道了肾母细胞瘤中 STAT1 的组成型激活。 STAT 家族成员的激活发生在多种人类肿瘤中，但通常涉及 STAT3 的 Y705 磷酸化。在研究肾母细胞瘤中 STAT 可能的失调时，我们发现了 STAT1 中 S727 的组成型磷酸化，并证明了其在肾母细胞瘤发生中的作用。由于这种肿瘤是癌症中细胞分化停滞的原型，我们假设 STAT1 信号传导也可能在发育中的肾脏中发挥重要作用。为了确定 STAT 信号在后肾发育过程中是否活跃，我们分析了一系列胚胎肾脏的时间序列，发现 STAT1 和 STAT3 在肾发育高峰期间均被酪氨酸和丝氨酸磷酸化激活。 STAT5 也在此过程中表达并可能发挥作用。为了检查它们在肾发生中的作用，我们利用了后肾间充质（MM）的外植体培养物，MM是肾单位上皮细胞的祖细胞，也是肾母细胞瘤的假定起源。 与分化诱导白血病抑制因子 (LIF) 不同，干扰素-γ (IFNg) 会导致 MM 中 STAT1 激活，但不会激活 STAT3。与 LIF 诱导 MM 肾小管形成的功能相反，IFNg 刺激 MM 增殖并抑制诱导后的肾单位形成。此外，我们的 NCI-Frederick 合作者 Nadya Tarasova 设计了一种肽抑制剂，针对 STAT1 N 结构域，它特异性破坏 STAT1 依赖性转录并诱导外植 MM 中的小管形成。这些发现表明 STAT1 激活在 MM 规范中提供了关键的调节信号，与其在肾母细胞瘤发生中的作用一致，并进一步表明 STAT1 激活的破坏可能为肾母细胞瘤治疗提供新的靶点。我们目前正在评估使用这种新型 STAT1 肽作为体内抗肿瘤剂的功效。为了直接检查 STAT 信号传导在肾脏发育中的作用，我们还在开发 STAT1 突变形式的小鼠遗传模型，该模型有条件地靶向 S727 活性（突变 Stat 形式上游的 floxed 终止密码子）。其中包括 S727-to-E727 突变（具有组成型活性）和 S727-to-A727（对该位点的磷酸化无活性）。我们还生成了 IFNg 的 floxed 终止密码子形式，它会随着 Cre 激活而有条件地导致该细胞因子的表达增加。我们推测这将增加发育中肾脏中的祖细胞数量，或者至少破坏正常发育。其他人也使用类似的策略在小鼠中诱导 STAT1 依赖性髓母细胞瘤。最后，我们还获得了具有 floxed Stat1 或 Stat3 等位基因的小鼠品系，以评估这些基因单独或组合缺失的影响。除了这些机制研究之外，我们还尝试利用组织培养中 LIF 和 IFNg 的研究结果，大规模扩大 MM 群体进行生化分析。胚胎组织研究中的一个长期存在的问题是可以收获用于研究的细胞数量很少。我们估计平均 MM 包含约 4000 个细胞。通过使用 LIF 和 IFNg 在基质上培养这些细胞，我们现在能够在一周内将 30 个 MM（120,000 个细胞）扩增为超过 2000 万个细胞，而不会牺牲它们诱导分化的能力。我们目前正在评估后续段落，以确定它们是否保留了这种能力。如果我们成功实现这一目标，这将为研究界提供宝贵的资源。此外，我们对肾母细胞瘤组织应用了类似的条件，以便也从这些组织中建立细胞系。目前仅从肾母细胞瘤中成功建立了一种细胞系，临床医生表示需要更合适的细胞系用于药物试验异种移植研究。 根据我们的条件，我们建立了来自肾母细胞瘤的早期传代细胞系，该细胞系表达肾母细胞瘤的特征，包括NCam、钙粘蛋白11和Osr1。我们正在获取更多的肿瘤进行培养，并最终将它们用于异种移植研究，以证明致瘤性并评估组织学。为了实现这一目标，其他人已经付出了相当大的努力和资源，因此，拥有可补充的研究生物来源和改进的异种移植药物试验模型将是该研究领域向前迈出的重要一步。肾母细胞瘤的另一个特征是 Wnt 通路的频繁激活，正如我们最近发表的肾母细胞瘤中所证明的那样，该肿瘤显示大多数肿瘤中 β-连环蛋白的核定位。该途径对于肾单位的发育也至关重要。然而，具体的机制尚不清楚。研究表明 Wnt/β-连环蛋白在肾单位形成过程中的间质-上皮转化 (MET) 中发挥重要作用。有趣的是，对表达β-连环蛋白反式激活的lacZ报告基因的BATlacZ转基因小鼠的分析表明，该报告基因在输尿管芽中强烈表达，但在周围的后肾间充质（MM）或新形成的肾小管中不表达，这提高了β-连环蛋白可能通过TCF/LEF独立机制发挥作用以促进MET和 肾小管的形成。为了探索其机制，用多种影响 Wnt/β-catenin 通路的小分子处理分离的 MM。 Wnt 信号传导 (WA) 激活剂可刺激 TCF/LEF 依赖性反式激活而不改变 β-连环蛋白水平，但不能诱导 MET。三种糖原合酶激酶激酶 3β 抑制剂，包括溴靛红宾-3-肟 (BIO)，能够增加 β-连环蛋白水平并诱导 MET。蛋白酶体抑制剂 MG132 也会增加 β-连环蛋白水平，但不会诱导 MET。使用我们新开发的方法来扩大 MM 细胞群，我们现在能够对 β-连环蛋白进行 GST 下拉研究，以确定各种化学处理如何影响其结合形式。我们发现 BIO 处理增加了 β-连环蛋白的 TCF 和钙粘蛋白结合形式，而 MG132 仅增加了 TCF 结合形式。突变的钙粘蛋白的表达破坏了正常的β-连环蛋白/钙粘蛋白复合物的形成，从而阻止了 BIO 诱导的紧密连接的形成。与这些观察结果一致，针对钙粘蛋白的中和抗体消除了培养的 MM 中 BIO 诱导的肾小管发生。 这些数据表明，β-连环蛋白与钙粘蛋白的相互作用在肾脏发育过程中的 MET 和肾小管形成中起着重要作用。我们目前正在测试 Tarasova 博士生成的肽，这些肽以 β-连环蛋白及其与 TCF/LEF1 的相互作用为目标，以特异性阻断经典激活。此类肽可能具有生化剖析 Wnt 信号传导的能力，将其通过 TCF 进行转录激活的作用与其在粘附连接中的功能分开。由于许多不同类型的肿瘤表现出 β-连环蛋白的核定位和 Wnt 信号传导的异常激活，因此这种方法可能会影响针对该靶点的有效疗法的开发。我们还研究了大鼠患肾母细胞瘤的易感性。 Noble 大鼠对这些肿瘤的化学诱导极其敏感，而 F344 大鼠则不敏感。当杂交时，动物会出现肿瘤发病率，暗示涉及者[摘要被截断为 7800 个字符]