R-spondins, Secreted Frizzled-Related Proteins and the Regulation of Wnt Signali

R-spondins、分泌型卷曲相关蛋白和 Wnt 信号的调节

• 批准号: 7965209
• 负责人: Jeffrey Rubin
• 金额: $ 56.88万
• 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/7965209
• 关键词: Adult; Aftercare; Amino Acid Substitution; Biological; Biological Assay; Bone Development; Brain; Breast; CXCR6 gene; Cell surface; Cells; Colorectal; Development; Diagnosis; Disease; Embryonic Development; Epigenetic Process; Epithelial Cells; Gene Expression; Gene Targeting; Genetic; Glaucoma; Histologic; Homeostasis; Immunoblotting; Individual; Kidney; Knockout Mice; Knowledge; Lead; Link; MAPK8 gene; Malignant Neoplasms; Mammary gland; Mediating; Microarray Analysis; Mus; Neoplasm Metastasis; Nude Mice; Oncogenic; Ovary; Pathway interactions; Pattern; Phenotype; Phosphorylation; Physiologic Intraocular Pressure; Physiological; Process; Property; Prostate; Publications; Recombinant Proteins; Regulation; Relative (related person); Reverse Transcriptase Polymerase Chain Reaction; Role; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Stem cells; Structure-Activity Relationship; Subcutaneous Injections; Testing; Tissues; Transfection; Wnt proteins; Work; beta catenin; cancer type; chemokine receptor; embryonic stem cell; expression vector; frizzled related protein-1; human SFRP4 protein; interest; lipoprotein receptor-related protein 6; malignant breast neoplasm; matrigel; mouse model; mutant; novel; tumor

R-spondins (Rspos) are known to potentiate Wnt/beta-catenin signaling in various settings. Activation of this pathway is critical for many processes during embryonic development and tissue homeostasis in the adult. It is one of the mechanisms that supports the proliferation of embryonic stem cells and multi-potential progenitor cells. Constitutive activation of the beta-catenin pathway is common in many types of cancers, including colorectal, hepatocellular, mammary, prostate, renal, ovary and others. Understanding the properties of factors such as the Rspos that enhance signaling through this pathway and factors like sFRP-1 that block could lead to better ways to diagnose and treat cancer. In addition, because Wnt signaling has many functions during embryonic development and in the adult increased knowledge of its regulation by Rspos and sFRPs could have applications in the treatment of other diseases. Our site-directed mutagenesis of R-spondin2 demonstrated that specific amino acid substitutions dramatically reduced R-spondin2 activity in the beta-catenin pathway. Rspo2 activation of this pathway was associated with an unusually prolonged stimulation of Wnt co-receptor LRP6 phosphorylation and an accumulation of LRP6 at the cell surface. R-spondin2 co-immunoprecipitated with LRP6 and Kremens, but the lack of major differences between wild-type and mutant Rspo2 implied that additional interactions might be important for biological activity. Microarray analysis of C57MG mouse mammary epithelial cells treated with recombinant proteins showed that Rspo2 potentiated a large percentage of Wnt-3a-dependent changes in gene expression. Many novel Wnt target genes were identified and validated by quantitative RT-PCR and immunoblotting, including CXCR6, a chemokine receptor recently implicated in breast cancer metastasis. Several changes in gene expression induced by the combination of Rspo2 and Wnt-3a did not appear to be mediated by the canonical Wnt/beta-catenin/TCF pathway, suggesting that other mechanisms were associated with Rspo activity. Double transfection of C57MG cells with Rspo2 and Wnt-1 stimulated cell invasiveness in three-dimensional Matrigel cultures as well as transwell assays. Invasive properties were linked to JNK activation rather than beta-catenin signaling. Subcutaneous injection of mammary cells transfected either with Rspo2 or Wnt-1 expression vectors produced tumors in nude mice. Co-expression of Rspo2 and Wnt-1 altered the histologic phenotype of tumors relative to the pattern seen with the Wnt-1 transfectant. During the previous fiscal year, two collaborative studies concerning the physiological or pathophysiological activities of sFRP-1 culminated in publications. In one study, we showed that increases in sFRP-1 expression and concomitant inhibition of Wnt/beta-catenin signaling could elevant intraocular pressure, predisposing individuals to glaucoma. We are continuing to work with Dr. Clark to identify genetic and/or epigenetic mechanisms that would predispose individuals to glaucoma by inhibiting the Wnt/beta-catenin pathway. The other article demonstrated that sFRP-1 regulates development of the prostate in mouse, using an Sfrp1 null mouse model created in my lab. We have provided these mice to several other labs interested in exploring the role of sFRP-1 in normal brain, breast and bone development as well as corresponding pathological conditions.

已知R-spondin（Rspos）在多种细胞中增强Wnt/β-连环蛋白信号传导。 设置.该途径的激活对于胚胎发育过程中的许多过程至关重要。 发育和组织稳态。它是支持 胚胎干细胞和多潜能祖细胞的增殖。本构 β-连环蛋白途径的激活在许多类型的癌症中是常见的，包括 结直肠、肝细胞、乳腺、前列腺、肾、卵巢和其他。了解 诸如Rspos的因子的性质，其增强通过该途径的信号传导， 像sFRP-1这样的阻断因子可能会导致更好的诊断和治疗癌症的方法。在 此外，由于Wnt信号传导在胚胎发育过程中以及在胚胎发育过程中具有多种功能， 成年人对Rspos和sFRPs调控的认识增加，可能会应用于 其他疾病的治疗。我们对R-spondin2的定点突变表明， 特定的氨基酸取代显著降低了β-连环蛋白中的R-spondin2活性 通路该通路的Rspo 2激活与异常延长的 刺激Wnt共受体LRP6磷酸化和LRP6在细胞中的积累 面R-spondin2与LRP 6和Kremens共免疫沉淀，但缺乏主要的 野生型和突变型Rspo 2之间的差异暗示， 对生物活性很重要。C57 MG小鼠乳腺上皮细胞基因芯片分析 用重组蛋白处理显示，Rspo2增强了大百分比的 Wnt-3a依赖的基因表达变化。许多新的Wnt靶基因被鉴定， 通过定量RT-PCR和免疫印迹验证，包括趋化因子受体CXCR6 最近与乳腺癌转移有关诱导的基因表达的几种变化 Rspo 2和Wnt-3a的结合似乎不是由经典的 Wnt/β-catenin/TCF通路，提示其他机制与Rspo相关 活动Rspo2和Wnt-1双转染C57 MG细胞增强细胞侵袭力 在三维基质胶培养以及transwell测定中。侵入性特征是 与JNK激活有关，而不是β-连环蛋白信号。乳房皮下注射 用Rspo2或Wnt-1表达载体转染的细胞在裸鼠中产生肿瘤。 与对照组相比，Rspo 2和Wnt-1的共表达改变了肿瘤的组织学表型。 用Wnt-1转染子观察到的模式。在上一个财政年度，两个合作 关于sFRP-1的生理学或病理生理学活性的研究最终导致 出版物。在一项研究中，我们发现sFRP-1表达的增加和伴随的 抑制Wnt/β-catenin信号通路可升高眼内压， 青光眼的危害我们正在继续与克拉克博士合作，以确定遗传和/或 表观遗传机制，通过抑制 Wnt/β-连环蛋白途径。另一篇文章表明sFRP-1调节了 前列腺在小鼠中，使用Sfrp1 null小鼠模型在我的实验室创建。我们提供了 将这些小鼠送到其他几个对探索sFRP-1在正常大脑中的作用感兴趣的实验室， 乳房和骨骼发育以及相应的病理状况。