GrowthDifferentiation Factors in Organogenesis

器官发生中的生长分化因素

• 批准号: 7592511
• 负责人: ALAN PERANTONI
• 金额: $ 84.22万
• 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/7592511
• 关键词: Adhesions; Agonist; Animal Model; Animals; Apoptosis; Biophysics; Blocking Antibodies; CXC Chemokines; Cadherins; Cell Adhesion; Cell Proliferation; Cells; Cephalic; Collaborations; Competence; Complex; Congenital Mesoblastic Nephroma; Cultured Cells; Development; Dominant-Negative Mutation; Duct (organ) structure; Ectopic Expression; Elements; Embryo; Epithelial; Epithelium; Evaluation; Family member; Female; Gatekeeping; Gene Targeting; Genetic Transcription; Goals; Growth; HSPB1 gene; Head; Interferon Type II; Interleukin 8A Receptor; Invaded; Kidney; Knock-out; Laboratories; Lead; Ligands; Mammalian Oviducts; Mediating; Mediator of activation protein; Mesenchymal; Mesenchyme; Mesonephric structure; Metanephric Diverticulum; Metanephric structure; Modeling; Molecular Target; Morphogenesis; Mus; N-terminal; Neoplasms; Nephroblastoma; Nephrons; Normal tissue morphology; Nuclear; Oncogenic; Organogenesis; Ovary; Pathogenesis; Pathway interactions; Pattern; Pediatric Neoplasm; Peptides; Phenotype; Phosphorylation; Play; Population; Process; Proteins; Purpose; RNA Interference; Rattus; Reporter; Reporting; Role; STAT1 gene; STAT3 gene; Serine; Signal Transduction; Stress; Structure of efferent ductule of testis; Structure of mesonephric duct; Structure of paramesonephric duct; System; Time; Tissues; Toxic effect; Transcription Repressor/Corepressor; Transcriptional Activation; Transformed Cell Line; Transgenic Organisms; Tumor Suppressor Proteins; Universities; Vas deferens structure; Xenograft procedure; base; beta catenin; cell growth; design; drug development; epididymis body; gene function; glycogen synthase kinase 3 beta inhibitor; homeodomain; inhibitor/antagonist; leukemia inhibitory factor; male; medical schools; mutant; neoplastic cell; nephrogenesis; neutralizing antibody; novel; penetratin; prevent; progenitor; receptor; receptor expression; reproductive; sex; small molecule; therapeutic target; transcription factor; tumor; tumor growth; tumorigenesis; tumorigenic; vasculogenesis

Through mouse gene targeting studies, we have identified Fgf8, a factor expressed by metanephric mesenchyme (MM), to be essential for nephrogenesis through its upstream effects on expression of the secreted patterning molecule Wnt4 and homeodomain transcription factor Lim1. We have also discovered an epistatic relationship between Fgf8 and Wnt signaling in nephron formation. Moreover, we have now also established a role for Fgf8 in the development of male sex accessory tissues. During development of the mesonephros, the rostral aspect of the Wolffian duct (WD) (or cranial tubules) is absent in mutant animals, resulting in the loss of the head and body of the epididymis, efferent ductules, and much of the vas deferens. In the female, the epoophoron is missing but development of the mature female reproductive tract, including the ovaries and oviducts, is normal, since the epoophoron does not contribute to these tissues. Surprisingly, the Mullerian duct appears unaffected despite its putative growth requirement for interaction with the WD, suggesting that this relationship is mediated by caudal elements of the WD. These findings demonstrate for the first time the critical role of Fgf8 signaling in formation of the male reproductive tract tissues. Moreover, they suggest that formation of the WD is a discontinuous process. Since the loss of Lim1 expression in the WD also results in a similar phenotype in the male reproductive tract, Lim1 expression in the WD may occur downstream of Fgf8 signaling as it does in nephronic differentiation and may indicate a common mechanism for Fgf8 activity. In addition to the critical requirement for Fgf signaling during nephronic differentiation, Wnt signaling appears to play a complementary role in this process as demonstrated in studies of our conditional knockout of Fgf8. Since Wnt4 and Wnt9 are essential for the epithelial conversion of MM, but Wnt-mediated transcriptional activation is not evident using a transgenic reporter, we have assessed the role of the canonical Wnt mediator beta-catenin in cells from MM. A GSK-3beta inhibitor BIO proved to be a potent inducer of MM progenitor survival, proliferation, and tubule formation. We have found that BIO stabilizes beta-catenin and activates Wnt-dependent signaling in cultured cells from MM. It also induces adhesion complex formation necessary for mesenchymal- epithelial transition (MET). A small molecule called Wnt agonist, which activates TCF-dependent signaling independent of beta-catenin stabilization, fails to induce tubule formation. Furthermore, interference with adhesion complex formation using neutralizing antibodies blocks MET. These findings are consistent with MET being a TCF-independent process. In order to better understand the role of canonical Wnt signaling (beta-catenin/TCF-dependent transcriptional activation) in MET, we have developed a model using the transformed cell line HEK293, which was derived from MM. These cells rapidly form adhesion complexes when exposed to BIO. This process is not inhibited when cells are transfected with dominant-negative TCF constructs or beta-catenin that is incapable of interaction with TCF; however, MET is disrupted in cells expressing a dominant-negative cadherin construct, suggesting that MET is beta-catenin-dependent, but TCF-independent and therefore not the result of canonical signaling. Since Wilms tumor cells show significant activity through canonical signaling, i.e., beta-catenin-mediated TCF-dependent transcription, these studies suggest that if we block TCF activity and promote instead beta-catenin-dependent cell adhesion complex formation, we may be able to re-regulate tumor cells and inhibit tumor formation. The ultimate goal of the DNS is to define inappropriate signaling in tumorigenesis based upon mechanisms required for the differentiation of normal tissue progenitors during development and tissue renewal. In evaluations of one implicated pathway, i.e., STAT signaling, we delineated the role of STAT1 serine 727 phosphorylation in Wilms tumor pathogenesis and identified at least three genes that function downstream of STAT1 signaling, and these are involved in either blocking apoptosis or stimulating proliferation in Wilms tumor cells. These include the BCL-2 family member MCL-1, the stress-related protein HSP-27, and transcriptional repressor and proliferation promoting factor CDP or CUX-1. Targeting of any of these factors with RNAi inhibits tumor cell growth and for MCL-1 and HSP27 induces apoptosis, suggesting that they are required to sustain the tumorigenic phenotype. We are now evaluating the role of STAT1 signaling in the differentiation of normal MM from which Wilms tumor is derived. S727 phosphorylation of STAT1 is demonstrable in embryonic metanephroi, beginning prior to induction at E13.5 in rat and is lost with tissue maturation. Furthermore, induction of STAT1 activation (both Y701 and S727) with gamma-interferon treatment stimulates cell proliferation and inhibits apoptosis in primary MM but also blocks tubule formation. In a collaboration with the Structural Biophysics Laboratory, we have helped in the characterization and development of a penetratin-fusion peptide designed to interact specifically with the N-terminal portion of STAT1, preventing oligomerization. This peptide shows a potent and selective toxicity for tumor cells which express STAT1 and interferes with STAT1 transcriptional activation. When applied to MMs treated with gamma-interferon, this inhibitor blocks STAT1 activity and induces tubule formation. These studies provide compelling evidence that STAT1 signaling in normal renal progenitors inhibits differentiation in opposition to our observed induction of differentiation with STAT3 activation following treatment of MM with leukemia inhibitory factor. Thus, in this particular embryonic progenitor population, STAT1 is oncogenic, which contrasts with its putative role as a tumor suppressor in other tissues. In collaboration with Dr. Mark de Caestecker, Vanderbilt University Medical School, we have continued studies on transcriptional co-activator CITED1, which is highly expressed in condensed cap cell MM that overlays the ureteric bud. We previously proposed a role for CITED1 as a gatekeeper in its differentiation to the epithelia of the nephron. Now we have characterized the expression of this factor in Wilms tumor and have determined that it is an important marker for blastemal populations in these tumors and that its expression and nuclear localization correlates with the more aggressive tumors. We also demonstrate that ectopic expression of CITED1 in Wilms tumor cells stimulates proliferation and tumor growth in xenografts while a dominant-negative form inhibits proliferation in and tumor growth by these cells. These findings indicate the CITED1 functions in tumorigenesis. Finally, we reported the surprising finding that ELR+-CXC chemokine, CXCL-7, and its receptors CXCR1 and 2 play a fundamental role in nephrogenesis by allowing competence to respond to a variety of signaling ligands through induction of receptor expression as well as inducing vasculogenesis in metanephric blastemal cells. Furthermore, we established that Wilms tumors express these receptors and that Wilms tumor cells depend upon signaling through these receptors for survival. This may provide a possible target for therapeutic purposes

通过小鼠基因靶向研究，我们已经确定了通过元素间充质（MM）表达的FGF8，这对于肾脏的发生是通过其上游对分泌构图分子Wnt4和同源域转录因子LIM1表达的上游作用而至关重要的。我们还发现了肾单位形成中FGF8和Wnt信号传导之间的同志关系。此外，我们现在还确立了FGF8在男性性辅助组织发展中的作用。在中源的发育过程中，突变动物中不存在沃尔夫管道（WD）（或颅管）的延髓方面，从而导致附加膜的头部和身体丧失，衰弱的导管和大部分VAS deferens。在雌性中，缺失了卵圆龙，但是包括卵巢和卵形在内的成熟雌性生殖道的发展是正常的，因为Epoophoron不影响这些组织。令人惊讶的是，尽管穆勒（Mullerian）的管道与WD相互作用，但穆勒（Mullerian）管道似乎不受影响，这表明这种关系是由WD的尾部元素介导的。这些发现首次证明了FGF8信号在形成男性生殖道组织中的关键作用。此外，他们认为WD的形成是一个不连续的过程。由于WD中LIM1表达的丧失也导致男性生殖道中的表型相似，因此WD中的LIM1表达可能像FGF8信号的下游发生，因为它在肾脏分化中的表达可能会发生，并且可能表明FGF8活性的常见机制。除了在肾脏分化过程中FGF信号传导的关键要求外，Wnt信号在此过程中似乎在此过程中起着互补作用，如我们对FGF8的条件敲除的研究所证明。由于Wnt4和Wnt9对于MM的上皮转化至关重要，但是使用转基因报告基因WNT介导的转录激活并不明显，因此我们评估了从MM的细胞中规范Wnt Wnt介质β-catenin在细胞中的作用。 GSK-3BETA抑制剂生物被证明是MM祖细胞存活，增殖和肾小管形成的有效诱导剂。我们发现，生物可以稳定β-catenin并激活来自MM培养的细胞中Wnt依赖性信号传导。它还诱导间质上皮转变所需的粘附复合物的形成（MET）。一种称为Wnt激动剂的小分子，它激活与β-蛋白稳定无关的TCF依赖性信号传导，无法诱导小管形成。此外，使用中和抗体阻断的干扰对粘附复合物的形成遇到。这些发现与MET是独立于TCF的过程一致。为了更好地了解MET中规范Wnt信号传导（β-catenin/TCF依赖性转录激活）的作用，我们使用了转换的细胞系HEK293开发了一个模型，该模型源自MM。这些细胞暴露于生物时会迅速形成粘附复合物。当细胞用无法与TCF相互作用的显性阴性TCF构建体或β-catenin转染时，不会抑制此过程。然而，在表达显性阴性的钙粘蛋白构建体的细胞中，MET被破坏，表明Met是β-catenin依赖性的，但不依赖于TCF，因此不是规范信号传导的结果。由于Wilms肿瘤细胞通过规范信号传导（即β-catenin介导的TCF依赖性转录）表现出显着的活性，因此这些研究表明，如果我们阻断TCF活性并促进β-蛋白依赖性细胞粘附络合物形成，那么我们可能能够重新调节肿瘤细胞并抑制肿瘤的形成。 DNS的最终目标是根据在发育和组织更新过程中正常组织祖细胞分化所需的机制来定义肿瘤发生中的不适当信号传导。在评估一个涉及的途径（即STAT信号传导）中，我们描述了STAT1丝氨酸727在Wilms肿瘤发病机理中的作用，并鉴定了至少三个STAT1信号传导起作用的基因，并且这些基因在Wilms thmor thmor thmor thmor thmor thmor thmor thmor thmor thmor thmofer的下游作用。其中包括Bcl-2家族成员MCL-1，与应力相关的蛋白HSP-27，以及转录阻遏物和增殖促进因子CDP或CUX-1。用RNAi靶向任何这些因素都会抑制肿瘤细胞的生长，而Mcl-1和Hsp27诱导凋亡，这表明它们是维持肿瘤性表型所必需的。现在，我们正在评估STAT1信号在得出Wilms肿瘤的正常MM分化中的作用。 S727 STAT1的S727磷酸化在胚胎元素中是可以证明的，在大鼠诱导之前，在E13.5诱导之前，随着组织成熟而丢失。此外，使用伽马 - 互化处理的STAT1激活（Y701和S727）诱导刺激细胞增殖并抑制原发时MM的凋亡，但也阻塞了小管的形成。在与结构生物物理学实验室的合作中，我们帮助了渗透蛋白融合肽的表征和开发，旨在与STAT1的N末端部分相互作用，从而防止低聚。该肽显示出对肿瘤细胞的有效和选择性毒性，该肿瘤细胞表达STAT1并干扰STAT1转录激活。当应用于用伽马间膜处理的MMS时，该抑制剂会阻止STAT1活性并诱导小管形成。这些研究提供了令人信服的证据，表明正常肾脏祖细胞中的STAT1信号传导抑制了与我们观察到的与白血病抑制因子MM治疗后STAT3激活诱导分化的分化。因此，在这种特定的胚胎祖细胞中，STAT1具有致癌性，与其在其他组织中作为肿瘤抑制剂的推定作用形成鲜明对比。与范德比尔特大学医学院的马克·德·卡斯特克（Mark de Caestecker）博士合作，我们继续研究转录共同激活剂CITED1，它在凝结的凝盖帽室中高度表达，覆盖了输尿管芽。我们以前提出了引用1作为守门人在与肾单位上皮的区分中的作用。现在，我们已经表征了该因子在Wilms肿瘤中的表达，并确定它是这些肿瘤中胚胎种群的重要标记，其表达和核定位与更具侵略性的肿瘤相关。我们还证明，在Wilms肿瘤细胞中引用1的异位表达刺激异种移植物中的增殖和肿瘤生长，而显性阴性形式抑制了这些细胞的增殖和肿瘤生长。这些发现表明肿瘤发生中引用的1功能。最后，我们报道了一个令人惊讶的发现，即ELR+-CXC趋化因子，CXCL-7及其受体CXCR1和2通过允许能力通过诱导受体表达诱导的多种信号配体反应，并在肾上腺表达以及诱导Metanaphric甲基胚胚细胞中的血管生成来响应各种信号配体，从而在肾病中起基本作用。此外，我们确定Wilms肿瘤表达了这些受体，并且Wilms肿瘤细胞依赖于通过这些受体的信号传导以生存。这可能为治疗目的提供可能的目标