Roles of polycystin and calcium in B-Raf signaling

描述（由申请人提供）：在常染色体显性多囊肾病（ADPKD）中，小管上皮细胞的异常增殖是肾囊肿形成的主要因素。我们已经证明，提高细胞内cAMP的激动剂可以加速ADPKD细胞的增殖速度。相反，在正常人肾（NHK）细胞中发现cAMP具有抗有丝分裂作用。ADPKD和NHK细胞之间cAMP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）激活了ADPKD细胞中涉及细胞外信号调节激酶（ERK）的丝裂原活化蛋白（MAP）激酶途径，而NHK细胞则没有。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，受到cAMP的刺激，而含有少量B-Raf的细胞，如星形胶质细胞，则受到抑制。ADPKD细胞对cAMP的增殖反应是不寻常的，因为肾细胞通常受到cAMP的抑制，但由于多囊素的突变而被刺激增殖。最近的研究表明，多囊蛋白-1 （PC1）和多囊蛋白-2 （PC2）是调控ca2 +进入细胞的伙伴。我们认为，PC1或PC2的缺陷以及多囊蛋白功能的缺失改变了细胞内ca2 +，并继发导致cAMP增殖反应的表型转换。在初步实验中，我们发现用ca2 +通道阻滞剂抑制ca2 +进入，减少细胞外ca2 +，或通过过度表达PC1的c尾破坏PC1/PC2信号传导，可以将M1细胞（小鼠皮质收集管细胞系）的表型从camp抑制型转变为camp刺激型，模拟了NHK和ADPKD细胞之间的差异。M1细胞模型为研究特定细胞内camp依赖性增殖中表型转换的分子机制提供了一种方法。为了阐明这些机制，我们将研究以下目标：1)确定细胞内[ca2 +]减少改变M1细胞中camp依赖性B-Raf信号的分子机制。2)确定M1细胞中PC1 c端片段的过表达（一种被认为会破坏PC1/PC2功能的情况）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.