Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys

Tamio Yamaguchi, Shizuko Nagao, Darren P. Wallace, Franck A. Belibi, Benjamin D. Cowley, Jill C. Pelling, Jared J. Grantham

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Abstract

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.

Original languageEnglish
Pages (from-to)1983-1994
Number of pages12
JournalKidney International
Volume63
Issue number6
DOIs
Publication statusPublished - 01-06-2003

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Autosomal Dominant Polycystic Kidney
Kidney Cortex
Cyclic AMP
Cysts
Epithelial Cells
Epidermal Growth Factor
Cyclic AMP-Dependent Protein Kinases
Mitogen-Activated Protein Kinase Kinases
Phosphorylation
Proto-Oncogene Proteins B-raf
MAP Kinase Kinase Kinases
Kidney
Proto-Oncogene Proteins c-akt
Extracellular Signal-Regulated MAP Kinases
Receptor Protein-Tyrosine Kinases
Nephrectomy
Adenosine
Phosphotransferases
Nucleotides
Western Blotting

All Science Journal Classification (ASJC) codes

  • Nephrology

Cite this

Yamaguchi, T., Nagao, S., Wallace, D. P., Belibi, F. A., Cowley, B. D., Pelling, J. C., & Grantham, J. J. (2003). Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys. Kidney International, 63(6), 1983-1994. https://doi.org/10.1046/j.1523-1755.2003.00023.x
Yamaguchi, Tamio ; Nagao, Shizuko ; Wallace, Darren P. ; Belibi, Franck A. ; Cowley, Benjamin D. ; Pelling, Jill C. ; Grantham, Jared J. / Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys. In: Kidney International. 2003 ; Vol. 63, No. 6. pp. 1983-1994.
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abstract = "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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Yamaguchi, T, Nagao, S, Wallace, DP, Belibi, FA, Cowley, BD, Pelling, JC & Grantham, JJ 2003, 'Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys', Kidney International, vol. 63, no. 6, pp. 1983-1994. https://doi.org/10.1046/j.1523-1755.2003.00023.x

Cyclic AMP activates B-Raf and ERK in cyst epithelial cells from autosomal-dominant polycystic kidneys. / Yamaguchi, Tamio; Nagao, Shizuko; Wallace, Darren P.; Belibi, Franck A.; Cowley, Benjamin D.; Pelling, Jill C.; Grantham, Jared J.

In: Kidney International, Vol. 63, No. 6, 01.06.2003, p. 1983-1994.

Research output: Contribution to journalArticle

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.

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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