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，我们发现Fgf8是一个由后肾间质（MM）表达的因子，通过其对分泌的模式分子Wnt4和同源结构域转录因子Lim1表达的上游作用，对肾形成至关重要。我们还发现了Fgf8和Wnt信号在肾元形成过程中的上位性关系。此外，我们现在也确定了Fgf8在男性性附属组织发育中的作用。在中肾发育过程中，突变动物的沃尔夫管（WD）或颅小管的吻侧缺失，导致附睾的头部和体部、输出小管和大部分输精管的缺失。在女性中，卵泡囊是缺失的，但成熟的女性生殖道，包括卵巢和输卵管的发育是正常的，因为卵泡囊对这些组织没有贡献。令人惊讶的是，尽管假定缪勒管的生长需要与WD相互作用，但它并未受到影响，这表明这种关系是由WD的尾端元件介导的。这些发现首次证明了Fgf8信号在男性生殖道组织形成中的关键作用。此外，他们认为WD的形成是一个不连续的过程。由于Lim1在WD中的表达缺失也会导致男性生殖道中出现类似的表型，因此Lim1在WD中的表达可能发生在Fgf8信号传导的下游，就像在肾分化中一样，这可能表明Fgf8活性的共同机制。除了在肾细胞分化过程中对Fgf信号的关键要求外，Wnt信号似乎在这一过程中起着互补作用，正如我们的条件敲除Fgf8的研究所证明的那样。由于Wnt4和Wnt9对MM的上皮转化至关重要，但Wnt介导的转录激活在转基因报告基因中并不明显，因此我们评估了典型Wnt介质β -连环蛋白在MM细胞中的作用。gsk -3 β抑制剂BIO被证明是MM祖细胞存活、增殖和小管形成的有效诱导剂。我们发现BIO稳定β -连环蛋白并激活MM培养细胞中wnt依赖的信号传导。它还诱导间充质-上皮转化（MET）所需的粘附复合物的形成。一种名为Wnt激动剂的小分子，可以激活tcf依赖性信号，不依赖于β -连环蛋白的稳定，但不能诱导小管的形成。此外，使用中和抗体干扰粘附复合物的形成会阻断MET。这些发现与MET是一个与tcf无关的过程是一致的。为了更好地理解典型Wnt信号（β -catenin/ tcf依赖性转录激活）在MET中的作用，我们利用转化细胞系HEK293建立了一个模型，该细胞系来源于MM。这些细胞在暴露于BIO时迅速形成粘附复合物。当细胞转染显性阴性TCF构建体或不能与TCF相互作用的β -连环蛋白时，这一过程不受抑制；然而，MET在表达显性阴性钙粘蛋白结构的细胞中被破坏，这表明MET依赖于β -连环蛋白，但不依赖于tcf，因此不是典型信号传导的结果。由于Wilms肿瘤细胞通过典型信号，即β -catenin介导的TCF依赖性转录表现出显著的活性，这些研究表明，如果我们阻断TCF活性，转而促进β -catenin依赖性细胞粘附复合物的形成，我们可能能够重新调节肿瘤细胞并抑制肿瘤的形成。DNS的最终目标是根据正常组织祖细胞在发育和组织更新过程中分化所需的机制来定义肿瘤发生中的不适当信号。在评估一条相关通路，即STAT信号通路时，我们描述了STAT1丝氨酸727磷酸化在Wilms肿瘤发病机制中的作用，并确定了至少三个在STAT1信号通路下游起作用的基因，这些基因参与了阻断Wilms肿瘤细胞凋亡或刺激细胞增殖。这些包括BCL-2家族成员MCL-1，应激相关蛋白HSP-27，转录抑制因子和增殖促进因子CDP或CUX-1。用RNAi靶向这些因子中的任何一个都会抑制肿瘤细胞生长，MCL-1和HSP27会诱导细胞凋亡，这表明它们是维持致瘤表型所必需的。我们现在正在评估STAT1信号在正常MM （Wilms肿瘤的来源）分化中的作用。S727 STAT1的磷酸化在胚胎后肾中被证实，在大鼠E13.5诱导之前开始，并随着组织成熟而消失。此外，γ -干扰素治疗诱导STAT1激活（Y701和S727）刺激细胞增殖，抑制原发性MM细胞凋亡，但也阻断小管形成。在与结构生物物理实验室的合作中，我们帮助表征和开发了一种穿透融合肽，该肽旨在与STAT1的n端特异性相互作用，防止寡聚化。该肽对表达STAT1并干扰STAT1转录激活的肿瘤细胞具有强效和选择性毒性。当应用于经γ -干扰素处理的mm时，这种抑制剂阻断STAT1活性并诱导小管形成。这些研究提供了令人信服的证据，表明正常肾祖细胞中的STAT1信号抑制分化，这与我们观察到的用白血病抑制因子治疗MM后STAT3激活诱导分化相反。因此，在这个特殊的胚胎祖细胞群体中，STAT1是致癌的，这与它在其他组织中作为肿瘤抑制因子的假定作用形成对比。我们与范德比尔特大学医学院的Mark de Caestecker博士合作，继续研究转录共激活因子CITED1，它在覆盖输尿管芽体的浓缩帽细胞MM中高度表达。我们之前提出了CITED1在其向肾细胞上皮分化的过程中作为看门人的作用。现在我们已经确定了该因子在Wilms肿瘤中的表达特征，并确定它是这些肿瘤中囊胚群的重要标记物，其表达和核定位与更具侵袭性的肿瘤相关。我们还证明，CITED1在Wilms肿瘤细胞中的异位表达刺激异种移植物的增殖和肿瘤生长，而显性阴性形式抑制这些细胞的增殖和肿瘤生长。这些发现提示了CITED1在肿瘤发生中的作用。最后，我们报告了令人惊讶的发现，ELR+-CXC趋化因子CXCL-7及其受体CXCR1和2在肾后胚细胞中通过诱导受体表达和诱导血管生成，使能力响应各种信号配体，从而在肾形成中发挥重要作用。此外，我们确定了Wilms肿瘤表达这些受体，并且Wilms肿瘤细胞依赖于通过这些受体的信号传导来生存。这可能为治疗目的提供一个可能的靶点