Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys
Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys
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DOI：
10.1046/j.1523-1755.2003.00023.x
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发表时间：
2003-06-01
影响因子：
19.6
通讯作者：
Grantham, JJ
中科院分区：
文献类型：
--
作者：
Yamaguchi, T;Nagao, S;Grantham, JJ
背景。壁上皮细胞的增殖是常染色体显性多囊肾病（ADPKD）中囊肿进行性增大的主要原因。腺苷 3', 5' 环单磷酸 (cAMP) 通过激活蛋白激酶 A (PKA)、丝裂原激活蛋白激酶激酶 (MEK) 和细胞外信号调节激酶 (ERK/MAPK) 刺激 ADPKD 囊肿细胞的增殖，但不刺激正常人肾皮质 (HKC) 的细胞增殖。在本研究中，我们检测了ADPKD和HKC细胞中PKA和MEK之间的信号通路。从肾切除标本中制备人 ADPKD 和 HKC 细胞的原代培养物。我们通过免疫激酶测定和蛋白质印迹测定了cAMP和表皮生长因子（EGF）对ADPKD和HKC细胞中ERK、B-Raf和Raf-1激活的影响。结果。 8-Br-cAMP 增加 ADPKD 肾脏细胞中磷酸化 ERK（2.7-+/-0.6 倍，N=7）和 B-Raf 激酶活性（3.6-+/-1.1 倍，N=5）；磷酸化 Raf-1 的水平没有改变。 H89 抑制 PKA 显着降低 cAMP 刺激的 ERK 和 B-Raf 磷酸化，PD98059 抑制 MAPK 阻断核苷酸激活 ERK 的作用。相比之下，在 HKC 细胞中，8-Br-cAMP 不会激活 B-Raf 和 ERK。 EGF刺激ADPKD和HKC细胞中ERK和Raf-1的磷酸化，但对B-Raf没有影响。在 ADPKD 细胞中，8-Br-cAMP 和 EGF 联合增加 ERK 激活，高于单独任一激动剂的激活，并且这种联合效应被 PD98059 消除，表明 ERK 被汇聚于 MAPK 的 EGF 和 cAMP 响应级联激活。结论。 cAMP 通过 PKA、B-Raf 和 MAPK 的顺序磷酸化，激活 ERK 并增加 ADPKD 上皮细胞的增殖，但不增加正常人肾皮质细胞的增殖，该途径与经典受体酪氨酸激酶级联独立但互补。因此，cAMP 和 EGF 具有加速肾囊肿进行性增大的巨大潜力。
Background. The proliferation of mural epithelial cells is a major cause of progressive cyst enlargement in autosomal-dominant polycystic kidney disease (ADPKD). Adenosine 3', 5' cyclic monophosphate (cAMP) stimulates the proliferation of cells from ADPKD cysts, but not cells from normal human kidney cortex (HKC), through the activation of protein kinase A (PKA), mitogen-activated protein kinase kinase (MEK), and extracellular signal-regulated kinase (ERK/MAPK). In the current study, we examined the signaling pathway between PKA and MEK in ADPKD and HKC cells.Methods. Primary cultures of human ADPKD and HKC cells were prepared from nephrectomy specimens. We determined the effects of cAMP and epidermal growth factor (EGF) on the activation of ERK, B-Raf and Raf-1 in ADPKD and HKC cells by immune kinase assay and Western blot.Results. 8-Br-cAMP increased phosphorylated ERK (2.7- +/-0.6-fold, N=7), and B-Raf kinase activity (3.6-+/-1.1-fold, N=5) in cells from ADPKD kidneys; levels of phosphorylated Raf-1 were not changed. Inhibition of PKA by H89 strikingly decreased cAMP-stimulated phosphorylation of ERK and B-Raf, and MAPK inhibition by PD98059 blocked the effect of the nucleotide to activate ERK. By contrast, in HKC cells 8-Br-cAMP did not activate B-Raf and ERK. EGF stimulated the phosphorylation of ERK and Raf-1 in both ADPKD and HKC cells, but had no effect on B-Raf. 8-Br-cAMP and EGF conjointly increased ERK activation above that of either agonist alone in ADPKD cells, and this combined effect was abolished by PD98059, indicating that ERK was activated by EGF- and cAMP-responsive cascades that converge at MAPK.Conclusion. cAMP activates ERK and increases proliferation of ADPKD epithelial cells, but not cells from normal human kidney cortex, through the sequential phosphorylation of PKA, B-Raf and MAPK in a pathway separate from, but complementary to, the classical receptor tyrosine kinase cascade. Consequently, cAMP and EGF have great potential to accelerate the progressive enlargement of renal cysts.