Activation and role of MAP kinase-dependent pathways in retinal pigment epithelial cells: ERK and RPE cell proliferation.
OBJECTIVE
Retinal pigment epithelial (RPE) cell proliferation plays a key role in the pathogenesis of ocular diseases involving the posterior segment. Mitogen-activated protein kinases (MAPKs) are involved in the control of cell proliferation. This study was conducted to investigate the involvement of the extracellular signal-regulated kinase (ERK) pathway, the major MAPK pathway implicated in cell growth during the induction of RPE cell proliferation.


METHODS
RPE cell proliferation was stimulated with 10% fetal calf serum (FCS). Activation of the Ras/Raf/MAP kinase-ERK kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling pathway was detected by Western blot analysis and immunochemistry with specific anti-phosphosignaling protein antibodies. Pharmacologic and antisense (AS) oligonucleotide (ODN) strategies were used to analyze the ERK signaling involved in serum-induced cell proliferation.


RESULTS
FCS (10%) induced more vigorous RPE cell proliferation than did FGF2, VEGF, platelet-derived growth factor (PDGF), or epidermal growth factor (EGF), alone or in combination. Pharmacologic inhibition of Ras and Raf-1 reduced cell proliferation by 67% to 100% and by 62% to 79%, respectively, demonstrating that activation of the Ras/Raf-1 pathway was essential for FCS-induced RPE cell proliferation. MEK1/2, ERK2, and P90 ribosomal S6 kinase (P90(RSK)), the kinases downstream from ERK2, were strongly activated during cell proliferation. Pharmacologic inhibition of MEK1/2 abolished activation of ERK2, but reduced cell proliferation by only 32%, showing that MEK/ERK participates in the signaling involved in RPE cell growth. Both inhibition of ERK2 activation, which reduced cyclin D1 production, and inhibition of cyclin D1 by AS ODN decreased cell proliferation, suggesting that RPE cell proliferation is mediated by cyclin D1 through ERK2.


CONCLUSIONS
The requirement for Ras and the regulatory role of ERK2 in cyclin D1 production and in cell proliferation suggest that the Ras/Raf/MEK/ERK pathway plays a key role in the control of RPE cell proliferation. These data may have important implications for the development of more selective methods for the inhibition of retinal proliferation.
Cordeliers Biomedical Institut, National Institute of Health and Medical Research (INSERM) Unit 450, Center for Scientific Research, Paris, France.
Christiane
C
Hecquet
Gaëlle
G
Lefevre
Monika
M
Valtink
Katrin
K
Engelmann
Frederic
F
Mascarelli
D015153:Blotting, Western; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D019938:Cyclin D1 / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006133:Growth Substances / Q000494:pharmacology; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020929:Mitogen-Activated Protein Kinase Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000201:enzymology; D019908:Proto-Oncogene Proteins c-raf / Q000037:antagonists & inhibitors / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000037:antagonists & inhibitors / Q000378:metabolism
D006133:Growth Substances; D020319:Oligodeoxyribonucleotides, Antisense; D019938:Cyclin D1; D019908:Proto-Oncogene Proteins c-raf; D020928:Mitogen-Activated Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)