POLYCYSTIN-1 MEDIATED CALCIUM AND cAMP SIGNALING

一些观察结果表明，cAMP是PKD发病机制的核心，通过激活Ras/MAPK途径导致囊肿形成和囊肿生长所需的细胞增殖，并通过激活CFTR刺激液体分泌以填充囊肿。cAMP的重要性最近通过在四种动物模型中成功治疗PKD而得到进一步证实，使用加压素V2受体拮抗剂，其可能降低肾cAMP。然而，虽然这些实验支持cAMP是重要的观点，但尚不清楚多囊蛋白的缺陷如何导致与细胞增殖增加和囊肿扩大相关的cAMP介导机制的失调。多囊蛋白被认为调节细胞内Ca 2+水平，以响应配体介导的或机械感觉刺激。最近我们 显示单独的多囊蛋白-1能够通过异源三聚体G蛋白偶联机制升高细胞内Ca 2+。我们还表明，使用三种细胞培养模型系统，PKD样细胞增殖依赖于cAMP介导的Ras/MAPK通路的激活和Ca 2+依赖性基因表达的变化。所有三个系统都显示出从cAMP抑制到cAMP刺激的细胞增殖的相同表型转换。然而，尽管有这些观察结果，仍然不清楚多囊蛋白是否以及如何参与。因此，我们计划确定PKD样cAMP刺激的表型是否是一种 Pkd 1缺陷细胞的特征。我们将使用Pkd 1（m1 Bei）小鼠，它具有单个氨基酸错义突变和完全成熟的PKD表型，我们计划产生两种新的小鼠模型，Pkd 1（DeltaL）和Pkd 1（Gpro），它们将分别携带多囊蛋白-1的异源三聚体G蛋白结合区中的单个氨基酸（aa）缺失突变，模拟已知的人类突变，或多囊蛋白-1的异源三聚体G蛋白结合区中的52个氨基酸移码突变。我们还计划利用这些小鼠的后肾培养物来确定在Pkd 1缺陷小鼠模型和人类ADPKD中囊肿开始生长时胚胎肾细胞中是否发生PKD样表型转换。最后，我们计划在液流介导的纤毛弯曲条件下测试多囊蛋白-1介导的信号传导活性，以确定多囊蛋白-1是否可以产生足以改变Ca 2 +-的Ca 2+信号。 依赖基因表达。

Several observations have suggested that cAMP is central to the pathogenesis of PKD, by activating the Ras/MAPK pathway resulting in the cell proliferation required for cyst formation and cyst growth, and by activating CFTR to stimulate fluid secretion to fill cysts. The importance of cAMP was recently further confirmed by the successful treatment of PKD in four animal models, using a vasopressin V2 receptor antagonist which presumably lowered renal cAMP. However, while these experiments support the view that cAMP is important, it is not known how a defect in the polycystins causes misregulation of cAMP-mediated mechanisms associated with increased cell proliferation and cyst enlargement. The polycystins are thought to regulate intracellular Ca2+ levels in response to ligand-mediated or mechanosensory stimuli. Recently, we showed that polycystin-1 alone is capable of elevating intracellular Ca2+ through a heterotrimeric G protein-coupled mechanism. We have also shown, using three cell culture model systems, that PKD-like cell proliferation is dependent on cAMP-mediated activation of the Ras/MAPK pathway and to changes in Ca2+-dependent gene expression. All three systems displayed the same phenotypic switch from cAMP-inhibited to cAMP-stimulated cell proliferation. Yet, despite these observations, it is still not clear whether and how the polycystins may be involved. As such, we plan to determine if the PKD-like cAMP-stimulated phenotype is a characteristic of Pkd1 deficient cells. We will use the Pkd1(m1Bei) mouse, which has a single amino acid missense mutation and a full-blown PKD phenotype, and we plan to generate two new mouse models, Pkd1(DeltaL) and Pkd1(Gpro), which will carry, respectively, a single amino acid (aa) deletion mutation in the heterotrimeric G protein binding region of polycystin-1 mimicking a known human mutation, or a 52 aa frameshift mutation in the heterotrimeric G protein binding region of polycystin-1. We also plan to utilize metanephric kidney cultures from these mice to determine whether the PKD-like phenotypic switch occurs in embryonic kidney cells at a time when cysts begin to grow in Pkd1-deficient mouse models and in human ADPKD. Finally, we plan to test polycystin-1 mediated signaling activity under conditions of fluid flow mediated ciliary bending to determine if polycystin-1 can generate a Ca2+ signal sufficient to alter Ca2+- dependent gene expression.