cAMP inhibits the proliferation of retinal pigmented epithelial cells through the inhibition of ERK1/2 in a PKA-independent manner.
Retinal pigmented epithelial (RPE) cell integrity is critical to the maintenance of retina functions and RPE cells do not proliferate in adults. The activation of RPE results in cell proliferation which may be associated with proliferative retinopathy and choroidal melanoma. Mitogen-activated protein kinase (MAPK) is believed to be a key participant in the response to mitogenic stimuli. We therefore investigated the involvement of the extracellular signal-regulated protein kinase (ERK) 1 and 2 during the induction of RPE cell proliferation. After foetal calf serum (FCS) stimulation activation of the Ras/Raf/ERK signalling pathway was detected by Western blotting and immunochemistry, with specific anti-phosphosignalling protein antibodies. Pharmacological and antisense (AS) oligonucleotide (ODN) strategies were used to analyse the signalling involved in FCS-induced RPE cell proliferation. Activation of the small G protein Ras and, to a lesser extent of Raf-1, the kinase directly downstream from Ras, was necessary to FCS-induced cell proliferation. MEK1/2 and ERK1/2 were activated during cell proliferation. Inhibition of MEK1/2 with UO 126 completely abolished ERK1/2 activation and reduced cell proliferation by 33-43%. ERK1/2 depletion by an AS ODN approach reduced cell proliferation by 27-33%, confirming the role of ERK1/2 in the FCS stimulation of RPE cells. We also investigated the role of PKA/cAMP, one of the major inhibitory pathways of ERK1/2. PKA blockade did not modify ERK1/2 activation or cell proliferation. In contrast, agents that increased cAMP concentration, abolished RPE proliferation, and MEK/ERK activation. Moreover, inhibition of the cAMP-activated small G protein Rap1, partially reversed the inhibitory effects of cAMP on cell proliferation and MEK/ERK activation. The requirement for Ras and ERK1/2, the lack of ERK1/2 regulation by PKA and the cAMP/Rap1 counter-regulatory pathway for ERK-mediated cell proliferation suggest complex regulation of signalling in RPE cells. These data may have important implications for the development of more selective models for retinal anti-proliferative therapies.
Centre Biomédical des Cordeliers, INSERM U. 450, affiliée CNRS, 15 rue de l'Ecole de Médecine 75006 Paris, France.
Christiane
C
Hecquet
Gaëlle
G
Lefevre
Monika
M
Valtink
Katrin
K
Engelmann
Frederic
F
Mascarelli
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002455:Cell Division / Q000187:drug effects; D000242:Cyclic AMP / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006801:Humans; D007124:Immunoenzyme Techniques; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; D019950:Mitogen-Activated Protein Kinase 1 / Q000037:antagonists & inhibitors*; D048052:Mitogen-Activated Protein Kinase 3; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors*; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000187:drug effects* / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D019908:Proto-Oncogene Proteins c-raf / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D020827:rap1 GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism; D019061:src-Family Kinases / Q000378:metabolism
D004791:Enzyme Inhibitors; D016376:Oligonucleotides, Antisense; D000242:Cyclic AMP; C482128:MAP2K2 protein, human; D011505:Protein-Tyrosine Kinases; D019061:src-Family Kinases; D017346:Protein Serine-Threonine Kinases; D019908:Proto-Oncogene Proteins c-raf; D017868:Cyclic AMP-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D048369:MAP Kinase Kinase 1; D048370:MAP Kinase Kinase 2; C482125:MAP2K1 protein, human; D020929:Mitogen-Activated Protein Kinase Kinases; D020827:rap1 GTP-Binding Proteins; D018631:ras Proteins