Roles of polycystin and calcium in B-Raf signaling

描述（由申请方提供）：在常染色体显性遗传性多囊肾病（ADPKD）中，肾小管上皮细胞的异常增殖是肾囊肿形成的主要因素。我们已经表明，激动剂，提高细胞内cAMP加速ADPKD细胞的增殖速度。相比之下，发现cAMP在正常人肾（NHK）细胞中是抗促有丝分裂的。ADPKD和NHK细胞之间cAMP促有丝分裂性表型差异的机制尚不清楚。最近，我们发现cAMP依赖性蛋白激酶A（PKA）激活的丝裂原活化蛋白（MAP）激酶途径涉及细胞外信号调节激酶（ERK）在ADPKD细胞，但不是NHK细胞。PKA刺激ERK的基础被认为依赖于B-Raf丰度。含有高水平B-Raf的细胞，如神经元细胞，被cAMP刺激，而含有很少B-Raf的细胞，如星形胶质细胞，被抑制。ADPKD细胞对cAMP的增殖反应是不寻常的，因为通常被cAMP抑制的肾细胞由于多囊蛋白的突变而被刺激增殖。最近的研究表明，多囊蛋白1（PC 1）和多囊蛋白2（PC 2）是调节细胞内Ca 2+内流的伴侣。我们认为，PC 1或PC 2的缺陷和多囊蛋白功能的丧失改变了细胞内Ca 2+，并继发性地导致对cAMP的增殖反应的表型转换。在初步的实验中，我们发现用Ca 2+通道阻断剂抑制Ca 2+内流，降低细胞外Ca 2+，或通过过表达PC 1的C-尾破坏PC 1/PC 2信号转导，可使M1细胞（小鼠皮质集合管细胞系）的表型从cAMP抑制型转变为cAMP刺激型，模拟NHK和ADPKD细胞之间的差异。M1细胞模型提供了一种研究特定细胞内cAMP依赖性增殖中表型转换的分子机制的方法。为了阐明这些机制，我们将研究以下几个方面：1）确定细胞内[Ca 2+]减少改变M1细胞cAMP依赖的B-Raf信号的分子机制。2)确定M1细胞中PC 1 C端片段的过表达（一种被认为破坏PC 1/PC 2功能的条件）改变cAMP依赖性B-Raf信号传导的机制。

DESCRIPTION (provided by applicant): In autosomal dominant polycystic kidney disease (ADPKD), aberrant proliferation of tubular epithelial cells is a major factor in renal cyst formation. We have shown that agonists that elevate intracellular cAMP accelerate the rate of proliferation of ADPKD cells. By contrast, cAMP was found to be anti-mitogenic in normal human kidney (NHK) cells. The mechanism for the phenotypic difference in cAMP mitogenicity between ADPKD and NHK cells is unknown. Recently, we found that cAMP-dependent protein kinase A (PKA) activated the mitogen-activated protein (MAP) kinase pathway involving extracellular signal-regulated kinase (ERK) in ADPKD cells, but not NHK cells. The basis for PKA stimulation of ERK is thought to be dependent on B-Raf abundance. Cells that contain high levels of B-Raf, such as neuronal cells, are stimulated by cAMP, while cells with little B-Raf, such as astrocytes, are inhibited. The proliferative response to cAMP by ADPKD cells is unusual in that renal cells, which are normally inhibited by cAMP, are stimulated to proliferate owing to mutations in polycystins. Recent studies indicate polycystin-1 (PC1) and polycystin-2 (PC2) are partners in the regulation of Ca 2+ entry into cells. We propose that defects in PC1 or PC2 and the loss of polycystin function alter intracellular Ca 2+ and secondarily cause a phenotypic switch in the proliferative response to cAMP. In preliminary experiments, we found that inhibition of Ca 2+ entry with Ca 2+ channel blockers, reducing extracellular Ca 2+, or disrupting PC1/PC2 signaling by overexpressing the C-tail of PC1 switched the phenotype of M1 cells (a mouse cortical collecting duct cell line) from cAMP-inhibitory to cAMP-stimulatory phenotype, mimicking the difference between NHK and ADPKD cells. The M1 cell model provides a method for investigating the molecular mechanisms responsible for the phenotypic switch in the cAMP-dependent proliferation within a particular cell. To elucidate these mechanisms, we will investigate the following aims: 1) Determine the molecular mechanism by which a reduction in the intracellular [Ca 2+] alters cAMP-dependent B-Raf signaling in M1 cells. 2) Determine the mechanism by which the overexpression of the C-terminal fragment of PC1 in M1 cells, a condition thought to disrupt PC1/PC2 function, alters cAMP-dependent B-Raf signaling.