Yamaguchi Tamio, Nagao Shizuko, Wallace Darren P, Belibi Franck A, Cowley Benjamin D, Pelling Jill C, Grantham Jared J
Kidney Institute, Kansas University Medical Center, Kansas City, Kansas 66160-7382, USA.
Kidney Int. 2003 Jun;63(6):1983-94. doi: 10.1046/j.1523-1755.2003.00023.x.
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.
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.
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.
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.
壁层上皮细胞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。3',5'-环磷酸腺苷（cAMP）通过激活蛋白激酶A（PKA）、丝裂原活化蛋白激酶激酶（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）细胞的增殖。在本研究中，我们检测了ADPKD和HKC细胞中PKA与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法确定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。
8-溴-cAMP增加了ADPKD肾细胞中磷酸化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水平未改变。H89抑制PKA显著降低了cAMP刺激的ERK和B-Raf磷酸化，而PD98059抑制MAPK则阻断了核苷酸激活ERK的作用。相比之下，在HKC细胞中，8-溴-cAMP未激活B-Raf和ERK。EGF刺激了ADPKD和HKC细胞中ERK和Raf-1的磷酸化，但对B-Raf无影响。在ADPKD细胞中，8-溴-cAMP和EGF联合作用使ERK激活水平高于单独使用任何一种激动剂时的水平，并且这种联合作用被PD98059消除，这表明ERK是由在MAPK处汇聚的EGF和cAMP反应级联激活的。
cAMP通过在一条与经典受体酪氨酸激酶级联不同但互补的途径中依次磷酸化PKA、B-Raf和MAPK来激活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。因此，cAMP和EGF具有极大的潜力加速肾囊肿的进行性增大。
