GrowthDifferentiation Factors in Organogenesis

器官发生中的生长分化因素

• 批准号: 8157163
• 负责人: ALAN PERANTONI
• 金额: $ 119.2万
• 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/8157163
• 关键词: AXIN2 protein; Affect; Alkylating Agents; Alleles; Apoptosis; Biological; Chemicals; Congenital Mesoblastic Nephroma; Defect; Development; Dose; Ectopic Expression; Epithelial; Epithelium; Ethylnitrosourea; Etiology; Exhibits; Histology; Human; Incidence; Individual; Interferon Type II; Intermediate Mesoderm; Invaded; Ionomycin; Kidney; LacZ Genes; Lead; Lesion; Ligands; Mesenchymal; Mesenchyme; Metanephric Diverticulum; Metanephric structure; Molecular Target; N Domain; Nephrogenic Cord; Organogenesis; Pathway interactions; Pediatric Neoplasm; Predisposition; Recombinants; STAT1 gene; STAT3 gene; STAT5A gene; Site; Transcriptional Activation; beta catenin; in utero; leukemia inhibitory factor; nephrogenesis; tumorigenesis

In 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. An analysis of early rat metanephric development revealed both tyrosine and serine phosphorylation of STAT1 and STAT3 from the beginning of rudiment formation. For STAT3, pY705 was detected throughout the nephrogenic zone including the cap, which is populated with renal progenitors. Levels were maximal at the initiation of metanephric development (E13.5). For STAT1, pY701 was also observed in the nephrogenic zone but was additionally detected in newly formed nephronic epithelia. Levels were maximal at E15.5 and diminished postnataly with maturation. STAT5 pY694 was localized primarily to epithelial structures but was maximal at E18.5. These results suggest that these transcription factors function at different stages of metanephric development. To better understand the relationship among the various STATs during nephrogenesis, we utilized explant cultures of metanephric mesenchyme (MM), the progenitor of nephronic epithelia and putative origin of Wilms tumor. Interferon-gamma (IFNg), unlike 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 designed by our NCI-Frederick collaborator Nadya Tarasova to target the STAT1 N-domain specifically disrupted STAT1-dependent transcription and induced 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 role of individual STATs during metanephric development by conditional loss-of-function alleles, targeting the loss to the mesoderm, the metanephric mesenchyme, or the ureteric bud. We are also examining conditional gain-of-function alleles for biological activity. The ectopic expression of interferon-gamma in MM resulted in renal hypoplasia. The kidneys exhibited one of two phenotypes: either a 50% size reduction or complete loss of the kidney. Mutants with reduced kidney size had comparable rates of proliferation and apoptosis with normal kidneys. However, the mutants also display a looped-tail phenotype typical of a defect in the planar cell polarity (PCP) pathway. We are now assessing this possibility in the kidney. Additionally, we have generated constructs with 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. Mouse lines have now been produced bearing these constructs. In addition to our work on STATs, we are actively collaborating with CDBL PI Terry Yamaguchi on a characterization of the renal phenotype of Wnt5a conditional loss-of-function mutants. Wnt5a has previously been associated with noncanonical Wnt mechanisms, notably the PCP pathway. It is expressed normally in the ureteric bud and surrounding interstitial stroma. Inactivation of Wnt5a in mesoderm with TCre yields mutants with duplexed kidneys, shortened ureters, and aberrant branching morphogenesis in the metanephros. The presence of hypercellular thickened collecting ducts suggests PCP involvement, so we are exploring this possibility. Another feature of Wilms tumor is the frequent activation of the Wnt pathway, as demonstrated in our 2010 publication on rat 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 transgenic mice expressing a beta-catenin-transactivated lacZ reporter revealed that the reporter is not expressed in metanephric mesenchyme (MM) or nephronic tubules, raising the possibility that beta-catenin acts through a TCF/LEF-independent mechanism to promote MET and tubule formation. To better define the role of Wnt signaling, we treated rat metanephric mesenchymal progenitors directly with recombinant Wnt proteins. These studies revealed that Wnt4 protein, which is required for nephron formation, induces tubule formation and differentiation markers Lim1 and E-cadherin in MM cells, but does not activate a TCF reporter or up regulate expression of canonical Wnt target gene Axin-2 and has little effect on the stabilization of beta-catenin or phosphorylation of disheveled-2. Furthermore, Wnt4 causes membrane localization of ZO-1 and occludin in tight junctions. To directly examine the role of beta-catenin/TCF-dependent transcription, we developed synthetic cell-permeable analogs of beta-catenins helix C, which is required for transcriptional activation. One inhibitor blocked TCF-dependent transcription and induced degradation of beta-catenin but did not affect tubule formation and stimulated the expression of Lim1 and E-cadherin. Since a canonical mechanism appears not to be operative in tubule formation, we assessed the involvement of the non-canonical Ca2+-dependent pathway. Treatment of MM cells with Wnt4 induced an influx of Ca2+ and caused phosphorylation of CaMKII. Moreover, treatment with Ionomycin, a Ca2+-dependent pathway activator, stimulated tubule formation. These results demonstrate that the canonical Wnt pathway is not responsible for mesenchymal-epithelial transition (MET) in nephron formation and suggest that the non-canonical calcium/Wnt pathway mediates Wnt4-induced tubulogenesis in the kidney. Furthermore, they demonstrate that canonical Wnt activation can be chemically dissected in cells without interfering with mechanisms of morphogenesis or differentiation. Thus it may be possible to selectively block canonical Wnt activation in tumors that depend upon signaling by this mechanism for survival without affecting their ability to differentiate. We are currently testing the efficacy of these peptides on tumors with constitutively active beta-catenin. Finally, we also continue to investigate 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 involvement of a single autosomal locus with incomplete dominance. Since the etiology of Wilms tumor remains largely undefined and since we have ruled out known genetic lesions, it is possible that we are targeting an important novel event in this model. Accordingly, we are currently mapping that locus. Thus far, we have completed the animal portion of the study, generating F1s which were then backcrossed with susceptible Noble rats. The F2s were exposed in utero to a single dose of the direct alkylating agent ethylnitrosourea, which induces a high incidence of tumors in susceptible rats. We have also completed the histology and are currently concluding the SNP analyses for this genome-wide association study.

在之前的研究中，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。对早期大鼠后肾发育的分析表明，从雏形形成开始，STAT1 和 STAT3 的酪氨酸和丝氨酸就被磷酸化。对于 STAT3，在整个肾源区（包括充满肾祖细胞的帽）中检测到 pY705。后肾发育开始时水平最高（E13.5）。对于 STAT1，pY701 也在肾源区中观察到，但在新形成的肾上皮中也检测到。水平在 E15.5 时达到最高，并在出生后随着成熟而降低。 STAT5 pY694 主要定位于上皮结构，但在 E18.5 时达到最大值。这些结果表明这些转录因子在后肾发育的不同阶段发挥作用。为了更好地了解肾发生过程中各种 STAT 之间的关系，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 设计的一种肽抑制剂，以 STAT1 N 结构域为靶点，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。我们目前正在通过条件性功能丧失等位基因评估个体 STAT 在后肾发育过程中的作用，针对中胚层、后肾间质或输尿管芽的损失。我们还在检查条件功能获得等位基因的生物活性。 MM中干扰素-γ的异位表达导致肾脏发育不全。肾脏表现出两种表型之一：尺寸缩小 50% 或肾脏完全丧失。肾脏尺寸减小的突变体的增殖和凋亡率与正常肾脏相当。然而，突变体还表现出典型的平面细胞极性（PCP）途径缺陷的环尾表型。我们现在正在评估肾脏中的这种可能性。此外，我们还生成了具有 STAT1 突变形式的构建体，该结构有条件地靶向 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）。现在已经生产出带有这些构建体的小鼠品系。 除了 STAT 方面的工作外，我们还与 CDBL PI Terry Yamaguchi 积极合作，对 Wnt5a 条件性功能丧失突变体的肾表型进行表征。 Wnt5a 此前曾与非经典 Wnt 机制相关，尤其是 PCP 通路。它通常在输尿管芽和周围间质中表达。用 TCre 灭活中胚层中的 Wnt5a 会产生具有双肾、缩短的输尿管和后肾分支形态发生异常的突变体。细胞增多、增厚的集合管的存在表明 PCP 参与，因此我们正在探索这种可能性。 肾母细胞瘤的另一个特征是 Wnt 通路的频繁激活，正如我们在 2010 年发表的关于大鼠肾母细胞瘤的出版物中所证明的那样，该出版物显示大多数肿瘤中 β-连环蛋白位于核内。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报告基因的转基因小鼠的分析表明，该报告基因在后肾间充质（MM）或肾小管中不表达，这提高了β-连环蛋白通过TCF/LEF独立机制促进MET和肾小管形成的可能性。为了更好地确定 Wnt 信号传导的作用，我们直接用重组 Wnt 蛋白处理大鼠后肾间质祖细胞。这些研究表明，肾单位形成所需的Wnt4蛋白可诱导MM细胞中的肾小管形成和分化标记物Lim1和E-cadherin，但不会激活TCF报告基因或上调经典Wnt靶基因Axin-2的表达，并且对β-catenin的稳定或disheveled-2的磷酸化影响不大。此外，Wnt4 导致 ZO-1 和 occludin 在紧密连接中的膜定位。为了直接检查β-连环蛋白/TCF依赖性转录的作用，我们开发了转录激活所需的β-连环蛋白螺旋C的合成细胞可渗透类似物。一种抑制剂阻断 TCF 依赖性转录并诱导 β-连环蛋白降解，但不影响肾小管形成并刺激 Lim1 和 E-钙粘蛋白的表达。由于规范机制似乎在肾小管形成中不起作用，因此我们评估了非规范 Ca2+ 依赖性途径的参与。用 Wnt4 处理 MM 细胞诱导 Ca2+ 流入并引起 CaMKII 磷酸化。此外，离子霉素（一种 Ca2+ 依赖性途径激活剂）治疗可刺激肾小管形成。这些结果表明，经典 Wnt 通路不负责肾单位形成中的间质-上皮转化 (MET)，并表明非经典钙/Wnt 通路介导 Wnt4 诱导的肾脏肾小管生成。此外，他们证明可以在细胞中对经典 Wnt 激活进行化学剖析，而不会干扰形态发生或分化机制。因此，有可能选择性地阻断肿瘤中典型的 Wnt 激活，这些肿瘤依赖于这种机制的信号传导而生存，而不影响其分化能力。我们目前正在测试这些肽对具有组成型活性β-连环蛋白的肿瘤的功效。 最后，我们还继续研究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，提示涉及不完全显性的单个常染色体基因座。由于维尔姆斯瘤的病因在很大程度上仍未确定，而且我们已经排除了已知的遗传病变，因此我们可能正在针对该模型中的一个重要的新事件。因此，我们目前正在绘制该基因座的图谱。到目前为止，我们已经完成了研究的动物部分，产生了 F1，然后与易感的 Noble 大鼠回交。 F2在子宫内暴露于单剂量的直接烷化剂乙基亚硝基脲，这会在易感大鼠中诱导高肿瘤发生率。我们还完成了组织学研究，目前正在总结这项全基因组关联研究的 SNP 分析。