POLYCYSTIN-1 MEDIATED CALCIUM AND cAMP SIGNALING

一些观察表明，cAMP在PKD的发病机制中处于核心地位，它通过激活RAS/MAPK通路导致囊腔形成和生长所需的细胞增殖，并通过激活CFTR刺激液体分泌来填充囊腔。最近在四种动物模型中成功治疗PKD进一步证实了cAMP的重要性，使用了一种可能降低肾脏cAMP的加压素V2受体拮抗剂。然而，尽管这些实验支持cAMP是重要的这一观点，但目前尚不清楚多囊蛋白的缺陷如何导致cAMP介导的机制的错误调节，这些机制与细胞增殖和囊泡增大有关。多囊藻毒素被认为可以调节细胞内钙离子水平，以响应配体介导的刺激或机械感觉刺激。最近，我们 结果表明，多囊蛋白-1单独能够通过异源三聚体G蛋白偶联机制升高细胞内钙离子。我们还利用三种细胞培养模型系统表明，PKD样细胞的增殖依赖于cAMP介导的RAS/MAPK通路的激活和钙依赖基因表达的变化。这三个系统都显示了从cAMP抑制的细胞增殖到cAMP刺激的细胞增殖的相同的表型转换。然而，尽管有这些观察，仍不清楚多囊藻毒素是否以及如何参与其中。因此，我们计划确定PKD样cAMP刺激的表型是否是一种 PKD1缺陷细胞的特征。我们将使用PKD1(M1Bei)小鼠，它具有单一氨基酸错义突变和完整的PKD表型，我们计划建立两个新的小鼠模型，PKD1(DeltaL)和PKD1(GPRO)，它们将分别在多囊蛋白-1的异源三聚体G蛋白结合区携带一个氨基酸(AA)缺失突变，模仿已知的人类突变，或在多囊蛋白-1的异源三聚体G蛋白结合区具有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来确定当PKD1缺陷的小鼠模型和人类ADPKD模型中的囊开始生长时，胚胎肾脏细胞中是否发生了类似PKD的表型转换。最后，我们计划在流体介导的纤毛弯曲条件下测试多囊蛋白-1介导的信号活性，以确定多囊蛋白-1是否能产生足以改变钙离子的钙信号。 依赖的基因表达。

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.