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

已知R-Spinins(RSPO)在各种环境中增强Wnt/β-catenin信号转导。这一途径的激活对胚胎发育和成人组织动态平衡的许多过程都是至关重要的。它是支持胚胎干细胞和多潜能祖细胞增殖的机制之一。β-连环蛋白途径的结构性激活在许多类型的癌症中都很常见，包括结直肠癌、肝细胞癌、乳腺癌、前列腺癌、肾癌、卵巢癌等。了解通过这一途径增强信号的Rspos等因子和阻断SFRP-1等因子的特性可能会导致更好的癌症诊断和治疗方法。此外，由于Wnt信号在胚胎发育和成人中具有许多功能，因此Rspos和sFRPs对其调控的了解增加可能会应用于其他疾病的治疗。我们对R-Spin2进行的定点突变表明，特定的氨基酸取代显著降低了R-Spin2在β-连环蛋白途径中的活性。Rsp2的激活与Wnt共同受体LRP6磷酸化异常长时间的刺激和LRP6在细胞表面的积聚有关。R-pondin2与LRP6和Kremens共沉淀，但野生型和突变型Rspo2之间没有重大差异，这意味着额外的相互作用可能对生物活性很重要。对重组蛋白处理的C57 MG小鼠乳腺上皮细胞的微阵列分析表明，Rsp2增强了Wnt-3a依赖的基因表达的很大比例的变化。许多新的Wnt靶基因通过定量RT-PCR和免疫印迹被鉴定和验证，包括CXCR6，一个最近与乳腺癌转移有关的趋化因子受体。Rsp2和Wnt-3a联合诱导的基因表达的一些变化似乎不是由典型的Wnt/β-catenin/Tcf途径介导的，这表明其他机制与RSPO活性有关。用Rspo2和Wnt-1双转染C57 MG细胞，在三维Matrigel培养和Transwell实验中刺激细胞侵袭。侵袭性特性与JNK激活有关，而不是与β-连环蛋白信号有关。皮下注射转Rspo2或Wnt-1表达载体的乳腺细胞在裸鼠体内产生肿瘤。Rspo2和Wnt-1的共同表达改变了肿瘤的组织学表型，相对于Wnt-1转染体所见的模式。在上一财政年度，关于SFRP-1的生理或病理生理活性的两项合作研究发表了论文。在一项研究中，我们发现，SFRP-1表达的增加和Wnt/β-catenin信号的抑制可以提高眼压，使个人更容易患上青光眼。我们正在继续与Clark博士合作，以确定通过抑制Wnt/β-catenin途径而使个人易患青光眼的遗传和/或表观遗传机制。另一篇文章使用我实验室建立的SFRP1缺失的小鼠模型，证明了SFRP-1调节小鼠前列腺的发育。我们已经将这些小鼠提供给了其他几个实验室，这些实验室有兴趣探索SFRP-1在正常大脑、乳房和骨骼发育以及相应的病理条件中的作用。

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.