Secreted Frizzled-Related Proteins and Wnt Signaling

我们确定来自尤文氏肉瘤家族肿瘤（ESFT）的细胞在肿瘤中形成神经突。 对Wnt-3a的反应，并已开始定义解释这种细胞反应的机制。 反应Fzd 3被鉴定为介导该过程的主要Wnt受体， 还涉及先前鉴定的Wnt效应分子Dishevelled-2和 Dishevelled-3和氨基末端c-Jun激酶（JNK）。与我们的一位将军吻合 目的（见上文），我们观察到Dickkopf-1也促进ESFT中的神经突生长， 细胞，显然是通过改变内源性Wnt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 δ（CKI δ）的Dishevelled磷酸化。此外，委员会认为， 初步实验表明，CKI delta和Dishevelleds可能在 初级纤毛的形成，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， 一直在研究R-spondins在Wnt/β-连环蛋白刺激中的作用 信号传导和肿瘤发生。与罗伯特卡拉汉博士的小组合作，我们 证明Rspo 2增强乳腺上皮细胞中的Wnt/β-连环蛋白信号传导， 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-连环蛋白途径中的R-spondin 2活性。 Rspo 2激活该通路与Wnt异常延长的刺激有关 辅助受体LRP 6磷酸化和LRP 6在细胞表面的积累。期间 本财政年度，两项关于生理或 sFRP-1的病理生理学活性在出版物中达到顶峰。在一项研究中， 增加sFRP-1表达并伴随抑制Wnt/β-连环蛋白信号传导 可能导致眼内压升高，使个体易患青光眼。另一份报告 包含sFRP-1调节小鼠前列腺发育的证据。以不间断 为了检查Wnt/Frizzled（Fzd）/sFRP相互作用，我实验室的成员使用了一整套10个 表位标记的Fzd来定义sFRP-1/Fzd结合的特异性，并证实这种结合是有效的。 相互作用由sFRP-1的富含半胱氨酸的结构域介导。到目前为止，我们努力 证明sFRP-1/Fzd结合的功能意义是不确定的。

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.