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Wilms肿瘤中STAT1的组成性激活。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Wilms肿瘤中stat可能的失调时，我们发现STAT1中S727的组成性磷酸化，并证明其在Wilms肿瘤发生中的作用。由于这种肿瘤是癌症中细胞分化受阻的原型，我们假设STAT1信号也可能在肾脏发育中发挥重要作用。为了确定STAT信号在肾后发育过程中是否活跃，我们分析了胚胎肾脏的时间序列，发现STAT1和STAT3在肾后发育的高峰期都被酪氨酸和丝氨酸磷酸化激活。STAT5也表达，并可能在此过程中发挥作用。为了研究它们在肾形成中的作用，我们利用肾后肾间质（MM）的外植体培养，肾后肾间质是肾上皮的祖细胞，也是肾母细胞瘤的推定来源。干扰素- γ (IFNg)与分化诱导白血病抑制因子（LIF）不同，能在MM中激活STAT1，而不是STAT3。与LIF在MM中诱导小管形成的功能相反，IFNg刺激MM中的增殖，并在诱导后抑制肾元的形成。此外，我们的NCI-Frederick合作者Nadya Tarasova设计了一种肽抑制剂，用于靶向STAT1 n结构域，它特异性地破坏STAT1依赖的转录并诱导外体mm中的小管形成。这些发现表明STAT1激活在MM规范中提供了一个关键的调控信号，与其在Wilms肿瘤发生中的作用一致，并进一步表明STAT1激活的破坏可能为Wilms肿瘤治疗提供新的靶点。我们目前正在评估使用这种新型STAT1肽作为体内抗肿瘤药物的疗效。为了直接研究STAT信号在肾脏发育中的作用，我们还开发了STAT1突变形式的小鼠遗传模型，该突变形式有条件地靶向S727活性（位于突变STAT形式上游的封闭终止密码子）。这些突变包括s727 - e727突变，它具有组成性活性，以及s727 - a727突变，它对该位点的磷酸化无活性。我们还生成了IFNg的一种柔停密码子形式，它可以在Cre激活的情况下有条件地增加该细胞因子的表达。我们推测这将增加发育中的肾脏中的祖细胞数量，或者至少破坏正常的发育。类似的策略已被其他人用于诱导小鼠中依赖STAT1的成神经管细胞瘤。最后，我们还获得了带有固定Stat1或Stat3等位基因的小鼠系，以评估这些基因单独或组合丢失的影响。除了这些机制研究，我们正试图利用我们的发现与组织培养中的LIF和IFNg，以大规模扩大MM种群进行生化分析。研究胚胎组织的一个长期存在的问题是，可以采集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，我们现在已经能够在一周的时间内将30 mm（12万个细胞）扩增成超过2000万个细胞，而不会牺牲它们被诱导分化的能力。我们目前正在评估后续段落，以确定它们是否保留这种能力。如果我们成功地实现了这一点，这将为研究界提供宝贵的资源。此外，我们已经将类似的条件应用于Wilms肿瘤组织，以便从这些组织中建立细胞系。目前只有一种细胞系成功地从Wilms肿瘤中建立，临床医生已经表示需要更多适合药物试验异种移植研究的细胞系。在我们的条件下，我们从Wilms肿瘤中建立了一个表达Wilms特征的早期传代细胞系，包括NCam， cadherin11和Osr1。我们正在获取更多的肿瘤进行培养，并最终将其用于异种移植研究，以证明致瘤性和评估组织学。在追求这一目标的过程中，其他人已经付出了相当大的努力和资源，因此，拥有可补充的生物来源和改进的异种移植药物试验模型将是这一研究领域向前迈出的重要一步。Wilms肿瘤的另一个特征是Wnt通路的频繁激活，正如我们最近发表的肾母细胞瘤所证明的那样，在大多数肿瘤中显示β -连环蛋白的核定位。这一途径对肾脏发育也至关重要；然而，其中的确切机制尚不清楚。研究表明，在肾元形成过程中，Wnt/ β -连环蛋白在间充质-上皮转化（MET）中起重要作用。有趣的是，对表达β -catenin-transactivated lacZ报告基因的BATlacZ转基因小鼠的分析显示，该报告基因在输尿管芽中强烈表达，而不在周围的后肾间质（MM）或新形成的肾小管中表达，这提出了β -catenin可能通过TCF/ lef不依赖的机制促进MET和小管形成的可能性。为了探索其机制，用多种影响Wnt/ β -连环蛋白途径的小分子处理分离的mm。Wnt信号（WA）的激活剂，在不改变β -连环蛋白水平的情况下刺激TCF/ lef依赖性的交易激活，不能诱导MET。三种糖原合成酶激酶-3 β抑制剂，包括溴茚红素-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也增加β -连环蛋白水平，但不诱导MET。利用我们新开发的扩展MM细胞群的方法，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TCF-和cadherin-结合形式的β -catenin，而MG132只提高了TCF-结合形式。表达一种突变的钙粘蛋白，破坏正常的β -连环蛋白/钙粘蛋白复合物的形成，阻止生物诱导的紧密连接的形成。与这些观察结果一致，抗钙粘蛋白的中和抗体消除了培养的mm细胞中生物诱导的小管形成。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MET和小管形成中起重要作用。我们目前正在测试Tarasova博士制造的肽，该肽靶向β -连环蛋白及其与TCF/LEF1的相互作用，以特异性阻断规范激活。这些肽可能具有生化剖析Wnt信号的能力，将其通过TCF在转录激活中的作用与其在粘附连接中的功能区分开来。由于许多不同类型的肿瘤显示β -连环蛋白的核定位和Wnt信号的异常激活，这种方法可能会影响针对该靶点的有效治疗方法的发展。我们还研究了大鼠对肾母细胞瘤的易感性。Noble大鼠对这些肿瘤的化学诱导非常敏感，而F344大鼠则不敏感。当杂交时，动物的肿瘤发病率表明了这种累及[摘要截短为7800个字符]