TY - JOUR
T1 - Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys
AU - Yamaguchi, Tamio
AU - Nagao, Shizuko
AU - Wallace, Darren P.
AU - Belibi, Franck A.
AU - Cowley, Benjamin D.
AU - Pelling, Jill C.
AU - Grantham, Jared J.
N1 - Funding Information:
This paper was supported by grants from the HHS to J. Grantham (P01-DK53763, P50-DK57301) and J. Pelling (CA81518, CA72987), and the Polycystic Kidney Foundation (J. Grantham). We thank Gail Reif, Marcy Christensen, and Lorraine Rome for valuable assistance in the laboratory, and James Calvet, Robin Maser, and P.J.S. Stork for helpful discussions. George Pierce, Mark Aeder, Charles Shield, Brantley Thrasher, Chris Bryan, the Midwest Transplant Network, and the Polycystic Kidney Foundation assisted in the procurement of discarded kidneys.
PY - 2003/6/1
Y1 - 2003/6/1
N2 - 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.
AB - 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.
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U2 - 10.1046/j.1523-1755.2003.00023.x
DO - 10.1046/j.1523-1755.2003.00023.x
M3 - Article
C2 - 12753285
AN - SCOPUS:0037513435
SN - 0085-2538
VL - 63
SP - 1983
EP - 1994
JO - Kidney International
JF - Kidney International
IS - 6
ER -
