Secreted Frizzled-Related Proteins and Wnt Signaling

我们建立了尤文氏肉瘤肿瘤家族(ESFT)细胞对WNT-3a的反应形成神经突起，并已开始确定解释这种细胞反应的机制。Fzd3被认为是介导这一过程的主要Wnt受体，它还涉及先前发现的Wnt效应分子，Disheveled-2和Disheveled-3，以及氨基末端c-Jun激酶(JNK)。与我们的一个总体目标一致(见上文)，我们观察到Dickkopf-1也促进了ESFT细胞中的轴突生长，显然是通过改变内源性Wnts的活性来刺激类似的Wnt信号通路。进一步的分析表明，Wnt-3a诱导的轴突生长需要酪蛋白激酶I增量(CKI Delta)紊乱的磷酸化。此外，初步实验表明，CKI Delta和Dishevelleds可能在初级纤毛的形成中起关键作用，这意味着这些蛋白质有助于形成需要中心体功能的多种细胞结构。在过去的两年里，我们一直在研究R-Spinins在刺激Wnt/β-catenin信号和肿瘤发生中的作用。在与Robert Callahan博士的合作中，我们证明了Rsp2增强了乳腺上皮细胞中的Wnt/β-catenin信号，并有助于它们的致癌特性。然而，一些细胞反应似乎依赖于其他尚未确定的信号通路。定点突变表明，特定的点突变显著降低了β-连环蛋白途径中R-Spindin2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。在本财政年度，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。另一篇报道包含SFRP-1调节小鼠前列腺发育的证据。在一项正在进行的Wnt/FrizzledFZD/SFRP相互作用的研究中，我的实验室成员使用了全套10个表位标记的FZD来定义SFRP-1/FZD结合的特异性，并证实这种相互作用是由SFRP-1的富含半胱氨酸的结构域介导的。到目前为止，我们证明SFRP-1/FZD结合的功能意义的努力还没有定论。

We established that cells from Ewing's sarcoma family of tumors (ESFT) form neurites in response to Wnt-3a and have begun to define the mechanisms that account for this cellular response. Fzd3 was identified as the primary Wnt receptor that mediates this process, which also involves the previously identified Wnt effector molecules, Dishevelled-2 and Dishevelled-3, and amino-terminal c-Jun kinase (JNK). Consistent with one of our general objectives (see above), we observed that Dickkopf-1 also promoted neurite outgrowth in ESFT cells, apparently by shifting the activity of endogenous Wnts to stimulate similar Wnt signaling pathways. Further analysis suggests that neurite outgrowth induced by Wnt-3a requires Dishevelled phosphorylation by casein kinase I delta (CKI delta). Moreover, preliminary experiments indicate that CKI delta and Dishevelleds may have critical roles in the formation of the primary cilium, implying that these proteins contribute to the formation of multiple cellular structures that require centrosomal function. For the past two years we have been investigating the role of R-spondins in the stimulation of Wnt/beta-catenin signaling and tumorigenesis. In collaboration with Dr. Robert Callahan's group, we demonstrated that Rspo2 potentiates Wnt/beta-catenin signaling in mammary epithelial cells and contributes to their oncogenic properties. However, some cellular responses appear to depend on other, yet to be determined, signaling pathways. Site-directed mutagenesis showed that specific point mutations 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. During the current 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. The other report contained evidence that sFRP-1 regulates development of the prostate in mouse. In an ongoing examination of Wnt/Frizzled(Fzd)/sFRP interactions, members of my lab used a full set of ten epitope-tagged Fzds to define the specificity of sFRP-1/Fzd binding and confirmed that such interactions are mediated by the cysteine-rich domain of sFRP-1. Thus far, our efforts to demonstrate the functional significance of sFRP-1/Fzd binding are inconclusive.