Differential regulation of mesangial cell mitogenesis by cAMP phosphodiesterase isozymes 3 and 4.
Mesangial cell (MC) mitogenesis is regulated through "negative cross talk" between cAMP-PKA and ERK signaling. Although it is widely accepted that cAMP inhibits mitogenesis through PKA-mediated phosphorylation of Raf-1, recent studies have indicated that cAMP-mediated inhibition of mitogenesis may occur independently of Raf-1 phosphorylation or without inhibiting ERK activity. We previously showed that MCs possess functionally compartmentalized intracellular pools of cAMP that are differentially regulated by cAMP phosphodiesterases (PDE); an intracellular pool directed by PDE3 but not by PDE4 suppresses mitogenesis. We therefore sought to determine whether there was a differential effect of PDE3 vs. PDE4 inhibitors on the Ras-Raf-MEK-ERK pathway in cultured MC. Although PDE3 and PDE4 inhibitors activated PKA and modestly elevated cAMP levels to a similar extent, only PDE3 inhibitors suppressed MC mitogenesis (-57%) and suppressed Raf-1 kinase and ERK activity (-33 and -68%, respectively). Both PDE3 and PDE4 inhibitors suppressed B-Raf kinase activity. PDE3 inhibitors increased phosphorylation of Raf-1 on serine 43 and serine 259 and decreased phosphorylation on serine 338; PDE4 inhibitors were without effect. Overexpression of a constitutively active MEK-1 construct reversed the antiproliferative effect of PDE3 inhibitors. PDE3 inhibitors also reduced cyclin A levels (-27%), cyclin D and cyclin E kinase activity (-30 and -50%, respectively), and induced expression of the cell cycle inhibitor p21 (+90%). We conclude that the antiproliferative effects of PDE3 inhibitors are mechanistically related to inhibition of the Ras-Raf-MEK-ERK pathway. Additional cell cycle targets of PDE3 inhibitors include cyclin A, cyclin D, cyclin E, and p21.
Mayo Clinic and Foundation, 200 First Street SW, Rochester, MN 55905, USA.
Jingfei
J
Cheng
Michael A
MA
Thompson
Henry J
HJ
Walker
Catherine E
CE
Gray
Montserrat M
MM
Diaz Encarnacion
Gina M
GM
Warner
Joseph P
JP
Grande
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000037:antagonists & inhibitors / Q000502:physiology*; D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D015153:Blotting, Western; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism; D019927:Cyclin E / Q000378:metabolism; D005920:Glomerular Mesangium / Q000166:cytology* / Q000502:physiology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000502:physiology; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008938:Mitosis / Q000502:physiology*; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D019927:Cyclin E; D007527:Isoenzymes; D010726:Phosphodiesterase Inhibitors; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases