Secreted Frizzled-Related Proteins and Wnt Signaling

我们确定来自尤文氏肉瘤家族（ESFT）的细胞在Wnt-3a的反应下形成神经突，并开始确定这种细胞反应的机制。Fzd3被鉴定为介导这一过程的主要Wnt受体，该过程还涉及先前鉴定的Wnt效应分子disheveled -2和disheveled -3以及氨基末端c-Jun激酶（JNK）。与我们的总体目标一致（见上文），我们观察到Dickkopf-1也促进ESFT细胞的神经突生长，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。进一步分析表明，Wnt-3a诱导的神经突生长需要酪蛋白激酶I δ (CKI δ)的无序磷酸化。此外，初步实验表明CKI δ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。在过去的两年中，我们一直在研究R-spondins在刺激Wnt/ β -连环蛋白信号传导和肿瘤发生中的作用。在与Robert Callahan博士的团队合作中，我们证明了Rspo2增强了乳腺上皮细胞中的Wnt/ β -连环蛋白信号，并有助于其致癌特性。然而，一些细胞反应似乎依赖于其他尚未确定的信号通路。位点定向突变表明，特定的点突变显著降低了β -catenin途径中R-spondin2的活性。该通路的Rspo2激活与Wnt共受体LRP6磷酸化的异常长时间刺激和LRP6在细胞表面的积累有关。在本财政年度，两项关于sFRP-1生理或病理生理活动的合作研究最终发表。在一项研究中，我们发现sFRP-1表达的增加和伴随的Wnt/ β -连环蛋白信号的抑制可能导致眼压升高，使个体易患青光眼。另一篇报道包含了sFRP-1调节小鼠前列腺发育的证据。在对Wnt/ frizzed (Fzd)/sFRP相互作用的持续研究中，我的实验室成员使用了全套10个表位标记的Fzds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.