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GrowthDifferentiation Factors in Organogenesis

器官发生中的生长分化因素

基本信息

批准号：
7732862
负责人：
ALAN PERANTONI
金额：
$ 104.1万
依托单位：
DIVISION OF BASIC SCIENCES - NCI
依托单位国家：
美国
项目类别：
财政年份：
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/7732862
关键词：
Address Affect Animal Model Animals Backcrossings Binding Breeding Cadherins Cell Nucleus Cells Chemicals Childhood Complex Congenital Mesoblastic Nephroma Cycloheximide Data Decision Making Development Disruption Duct (organ) structure E-Cadherin Elements Embryo Epithelial Epithelium Etiology Event Exposure to Frequencies Gatekeeping Gene Expression Genes Genetic Genetic Transcription Glutathione S-Transferase Glycogen Synthase Kinases Human Inbred F344 Rats Incidence Interferon Type II Invaded Investigation Kidney LacZ Genes Lead Ligands MG132 Malignant Neoplasms Mediating Mesenchymal Mesenchyme Metanephric Diverticulum Metanephric structure Modeling Molecular Target Morphogenesis Mutate Mutation N Domain Neoplasms Nephroblastoma Nephrons Nuclear Organogenesis Oximes Pathogenesis Pathway interactions Pediatric Neoplasm Peptides Phosphorylation Play Predisposition Primary Neoplasm Process Proteasome Inhibitor Protein Synthesis Inhibitors Rat Strains Rattus Reporter Reporting Rodent Role STAT1 gene STAT3 gene Signal Pathway Signal Transduction System Tight Junctions Transactivation Transcriptional Activation Transgenic Mice Tumor Suppressor Proteins Tumor Tissue antitumor agent beta catenin casein kinase II drug development in vivo inhibitor/antagonist leukemia inhibitory factor nephrogenesis neutralizing antibody novel peptide A prevent progenitor prototype small molecule tumor tumorigenesis

项目摘要

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. Furthermore, short exposures to protein synthesis inhibitor cycloheximide did not prevent BIO-induced tubule formation despite inhibition of TCF activation. In human embryonic kidney-derived HEK293 cells, treatment with BIO, but not WA, promoted tight junction formation, a beta-catenin-involved process often regarded as a hallmark of epithelial transformation. GST pull-down studies 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. Human Wilms tumor is formed in the embryonic progenitor metanephric mesenchyme, so a possible role for beta-catenin in its pathogenesis would seem reasonable. Collaborative investigations of beta-catenin in rat nephroblastomas, the rodent equivalent of the pediatric Wilms tumor, have revealed its nuclear localization in the majority of tumors and at a similar frequency as reported for Wilms tumors. This suggests that beta-catenin-dependent transcriptional activation is important in nephroblastoma pathogenesis. Since nephroblastoma cells are inhibited in their ability to undergo MET, it is possible that beta-catenin may function as the gatekeeper for proliferation or differentiation in metanephric mesenchymal progenitors. If directed toward E-cadherin binding, beta-catenin may mediate MET; whereas, it may induce proliferation if translocated to the nucleus to activate Wnt target gene expression, which appears to be required for tumorigenesis. We plan to further analyze the decision making that occurs in generating these different binding forms of beta-catenin. We have also investigated the susceptibility of rat to develop nephroblastomas. The Noble rat is exquistely sensitive to chemical induction of these tumors, while the F344 rat is insensitive. When these animals are cross bred, they develop a tumor incidence that is intermediate between the two and depending upon the backcross the tumor incidence is changed in such a manner as to suggest the involvement of a single autosomal locus with incomplete dominance. We are now beginning studies to identify that genetic locus. Previous studies with another rat strain implicated the Wilms tumor suppressor-1 gene (Wt1) in nephroblastoma pathogenesis. However, an examination of this gene along with Wtx, another Wilms tumor associated gene, has revealed no mutations in these tumors, suggesting the involvement of a novel locus in the Noble rat. Since the etiology of Wilms tumor remains largely undefined, it is quite possible that we may be targeting an important novel genetic event in this model. In addition to the involvement of the Wnt signaling pathway in Wilms tumor development, we previously reported the constitutive activation of STAT1 in human tumors. Constitutive STAT activation occurs in a variety of human neoplasms. While investigating the possible dysregulation of STATs in Wilms tumors, we discovered constitutive S727 phosphorylation of STAT1 in primary tumor tissues and demonstrated its role in Wilms tumorigenesis. Since this neoplasm is a prototype for arrested cellular differentiation in cancer, STAT1 signaling might also play an important role in the developing kidney. To address this, we utilized explant cultures of metanephric mesenchyme (MM), the progenitor of nephronic epithelia and putative tumor origin. 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. Moreover, a protein kinase CK2 inhibitor, which blocks activation of STAT1, promotes tubulogenesis in MMs. A peptide inhibitor that targets the STAT1 N-domain and specifically disrupts STAT1-dependent transcription induces massive 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.