Growth/Differentiation Factors in Organogenesis

器官发生中的生长/分化因素

• 批准号: 7038174
• 负责人: ALAN PERANTONI
• 金额: --
• 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/7038174
• 关键词: Wilms' tumor; biological signal transduction; carcinogenesis; cell differentiation; cell growth regulation; cell line; chemokine receptor; fibroblast growth factor; growth factor; growth factor receptors; histogenesis; human tissue; kidney; kidney neoplasms; leukemia inhibitory factor; mesenchyme; microarray technology; receptor binding; tissue /cell culture; transforming growth factors

Morphogenesis in the differentiating metanephros is regulated by reciprocal interactions between ureteric bud (UB) epithelia and the metanephric mesenchyme (MM). The UB invades the overlying MM and induces conversion of the mesenchyme into stromal and epithelial elements, which form the nephron. In turn, the MM stimulates the UB to grow and branch, forming the collecting duct system. The Differentiation and Neoplasia Section has focused on the elucidation of mechanisms of inductive signaling in metanephric development, seeking (1) the ligands responsible for nephronic differentiation, (2) other non-inductive regulatory factors of nephrogenesis, (3) the molecular targets of induction, and (4) alterations in inductive signaling during tumorigenesis. Thus far, we have identified three families of secreted soluble growth factors/cytokines produced by the UB that cooperate in inductive signaling to generate nephronic tubules. These include the fibroblast growth factors (notably FGF2 and 9), gp130-binding proteins (leukemia inhibitory factor; LIF), and transforming growth factor-beta (Tgf-beta2, activin A and B, and gdf11). In a recent collaborative effort, we have additionally identified, through gene targeting studies, Fgf8, a factor expressed by MM, as a critical player in nephrogenesis through upregulation of secreted patterning molecule Wnt4 and homeodomain transcription factor Lim1. We now plan to verify the roles of these factors in experiments designed to rescue kidney development through their replacement and overexpression. In the genetic studies, we have also discovered a role for Fgf8 in the development of male reproductive tract accessory tissues. To understand the targets of individual signaling pathways and their cooperative output, we have begun a systematic assessment of activated signaling in the MM. As part of this effort, we have generated an inducible immortalized cell line of MM that, under inductive conditions, differentiates to tubular epithelia and responds to the three described families of inductive factors. Furthermore, microarray analysis has provided a comprehensive characterization of transcriptional targets associated with induction. This line is now also being applied to the elucidation of proteomic changes that mediate or regulate differentiation of the MM progenitor. For this, we are specifically evaluating canonical and noncanonical pathways of Wnt signaling in order to clarify the mechanism invoked in tubule formation.Microarray analysis of a line we derived from the UB has identified high expression levels of multiple CXC chemokines. Primarily studied for their involvement in inflammatory responses, we find that they also function in normal kidney differentiation. CXCL7 is the principal ligand from this family expressed during development of the metanephros prior to induction, while CXCL1 is upregulated in induced MM. Furthermore, inhibition of signaling through their receptor CXCR2 dramatically inhibits development of the metanephros and promotes apoptosis of the MM, suggesting that it plays a critical role in the growth and maintenance of metanephric tissues. Adult tissues in general lack CXCR2, but a variety of human tumors express the receptors, suggesting that they function in tumorigenesis. Indeed, examination of several Wilms tumors reveals the expression of the CXCR2 receptor, and treatment of a Wilms tumor cell line with a CXCR2 inhibitor induces apoptosis in these cells. We are currently acquiring other chemical inhibitors described as more specific as well as evaluating the action of a soluble truncated CXCR2 receptor with possible inhibitory activity for CXCR2 signaling, which we have produced, that may target Wilms tumor cells by sequestering CXCR2 ligands and thus promote apoptosis in blastemal tumor cell populations. The ultimate goal of the DNS is to define subverted signaling pathways in tumorigenesis based upon normal growth and differentiation mechanisms required by tissue progenitors in development and tissue renewal. Accordingly, having identified several pathways responsible for normal cell maintenance and differentiation in the kidney, we have shifted efforts to the Wilms tumor, a caricature of metanephric development characterized by an accumulation of blastemal progenitors. Beginning with proteomic studies of Stat phosphorylation, the mediator of LIF induction, we have identified patterns of tyrosine and serine phosphorylation of Stats 1 and 3 that contribute to tumor behaviors. Thus far, we have evaluated several cell lines, including those from a Wilms tumor and the NCI-60 panel. We also expanded our findings to include 16 primary Wilms tumors and 4 normal tissues from tumor-bearing patients. In these studies, we have observed a consistent pattern of constitutive phosphorylation of Stat1 serine 727, but not tyrosine 701, in tumors - a pattern which does not occur in normal tissues. Furthermore, abrogation of altered Stat signaling, using a mutant form that prevents serine 727 phosphorylation, inhibits tumorigenesis as measured by Wilms tumor cell growth in soft agar and tumor cell invasion, and enhances cell survival under hypoxic conditions, suggesting that the altered patterns contribute to the neoplastic process. We have also implicated protein kinase CK2 in the aberrant phosphorylation and find that inhibition of CK2 also blocks tumorigenic behavior. Remarkably, gene expression profiles determined by microarray analyses of cells transfected with a mutant inactive or constitutively active Stat1 demonstrate regulation of many of the same genes profiled by others in Wilms tumor, suggesting that expression of this molecule is critical to the Wilms tumor phenotype. In addition, these profiles reveal the induction of invasive factors, e.g., fibulin 5 and MMP3, and downregulation of differentiation properties, e.g. K-cadherin and alpha8 integrin, with serine 727 phosphorylation. A test of a peptide inhibitor for Stat1 developed by the SBL proved unsuccessful, so they are currently designing another to target a different portion of the molecule. These studies provide compelling support for the principle that tumorigenesis is a disease of differentiation and that by dissecting inductive signaling, we may indeed gain insight into the mechanisms of tumor formation.

后肾分化过程中的形态发生受输尿管芽上皮（UB）和后肾间质（MM）的相互作用调控。UB侵入覆盖的MM并诱导间质转化为间质和上皮成分，形成肾元。反过来，MM刺激UB生长和分支，形成收集管系统。分化和瘤变部分侧重于阐明后肾发育中诱导信号的机制，寻求(1)负责肾分化的配体，(2)肾形成的其他非诱导调节因子，(3)诱导的分子靶点，(4)肿瘤发生过程中诱导信号的改变。到目前为止，我们已经确定了三个由UB产生的可分泌的可溶性生长因子/细胞因子家族，它们在诱导信号传导中合作产生肾小管。这些包括成纤维细胞生长因子（特别是FGF2和9），gp130结合蛋白（白血病抑制因子；LIF）和转化生长因子- β （tgf - β 2，激活素A和B， gdf11）。在最近的合作研究中，我们还通过基因靶向研究发现，MM表达的因子Fgf8通过上调分泌的模式分子Wnt4和同源结构域转录因子Lim1在肾形成中起关键作用。我们现在计划在实验中验证这些因子的作用，通过它们的替代和过表达来挽救肾脏的发育。在遗传学研究中，我们也发现了Fgf8在男性生殖道副组织发育中的作用。为了了解单个信号通路的目标及其协同输出，我们已经开始系统地评估MM中的激活信号。作为这项工作的一部分，我们已经产生了MM的可诱导永生化细胞系，在诱导条件下，分化为小管上皮，并对所描述的三个诱导因子家族做出反应。此外，微阵列分析提供了与诱导相关的转录靶点的全面表征。这条线现在也被应用于阐明介导或调节MM祖细胞分化的蛋白质组学变化。为此，我们特别评估了Wnt信号的典型和非典型途径，以阐明小管形成的机制。我们从UB获得的细胞系进行微阵列分析，发现多种CXC趋化因子的高表达水平。主要研究它们参与炎症反应，我们发现它们也在正常肾脏分化中起作用。CXCL7是该家族在诱导前后肾发育过程中表达的主要配体，而CXCL1在诱导MM中表达上调。此外，通过其受体CXCR2抑制信号传导可显著抑制后肾的发育并促进MM的凋亡，提示其在后肾组织的生长和维持中起关键作用。成人组织一般缺乏CXCR2，但多种人类肿瘤表达该受体，表明它们在肿瘤发生中起作用。事实上，对几种Wilms肿瘤的检查揭示了CXCR2受体的表达，并且用CXCR2抑制剂治疗Wilms肿瘤细胞系可诱导这些细胞凋亡。我们目前正在获得其他被描述为更具特异性的化学抑制剂，并评估一种可溶性截断的CXCR2受体的作用，该受体可能对CXCR2信号传导具有抑制活性，我们已经生产的这种受体可能通过隔离CXCR2配体来靶向Wilms肿瘤细胞，从而促进胚质肿瘤细胞群的凋亡。DNS的最终目标是根据组织祖细胞在发育和组织更新过程中所需的正常生长和分化机制，定义肿瘤发生过程中被破坏的信号通路。因此，在确定了肾脏中负责正常细胞维持和分化的几种途径后，我们将研究重点转向肾母细胞瘤，这是一种以胚祖细胞积累为特征的后肾发育的漫画。从Stat磷酸化（LIF诱导的中介）的蛋白质组学研究开始，我们已经确定了Stat 1和3的酪氨酸和丝氨酸磷酸化模式，这些模式有助于肿瘤行为。到目前为止，我们已经评估了几种细胞系，包括来自Wilms肿瘤和NCI-60组的细胞系。我们还扩大了我们的研究结果，包括16例原发Wilms肿瘤和4例来自荷瘤患者的正常组织。在这些研究中，我们观察到肿瘤中Stat1丝氨酸727的一致组成磷酸化模式，而不是酪氨酸701 -这种模式在正常组织中不会发生。此外，使用一种阻止丝氨酸727磷酸化的突变形式来消除改变的Stat信号，通过软琼脂中Wilms肿瘤细胞生长和肿瘤细胞侵袭来抑制肿瘤发生，并增强缺氧条件下的细胞存活，这表明改变的模式有助于肿瘤过程。我们还发现蛋白激酶CK2与异常磷酸化有关，并发现抑制CK2也能阻断致瘤行为。值得注意的是，通过微阵列分析转染突变型失活或构成活性Stat1的细胞确定的基因表达谱表明，在Wilms肿瘤中，许多与其他基因相同的基因都受到调控，这表明该分子的表达对Wilms肿瘤表型至关重要。此外，这些谱还揭示了通过丝氨酸727磷酸化诱导侵袭因子（如fibulin 5和MMP3）和分化特性（如K-cadherin和alpha8整合素）的下调。由SBL开发的Stat1肽抑制剂的测试被证明是不成功的，因此他们目前正在设计另一种靶向分子的不同部分。这些研究为肿瘤发生是一种分化疾病的原理提供了强有力的支持，并且通过剖析诱导信号，我们确实可以深入了解肿瘤形成的机制。