MAP kinase and beta-catenin signaling in HGF induced RPE migration.
OBJECTIVE
Hepatocyte growth factor (HGF) has been implicated in retinal pigment epithelial (RPE) cell proliferation and migration that occurs in proliferative retinal diseases such as proliferative vitreoretinopathy (PVR). The aim of this study is to investigate HGF induced signaling pathways that lead to RPE cell migration.


METHODS
Localization of beta-catenin was determined by immunofluorescence. HGF induced migration of ARPE-19 cells was studied using a quantitative migration assay after wounding in the presence of a DNA polymerase inhibitor, and in the presence or absence of a mitogen activated protein kinase (MAP kinase) kinase inhibitor. C-jun expression was determined by semi-quantitative RT-PCR and by Northern blot analysis. P42/p44 MAP kinase activity was determined by western blot and by an immunoprecipitation kinase assay. Tyrosine phosphorylation of the HGF receptor (HGFR or c-met) and beta-catenin was determined by immunoprecipitation and western blot analysis. Transactivation activity of beta-catenin was determined by luciferase reporter gene analysis.


RESULTS
Beta-catenin and E-cadherin were co-localized on the basal surface of the RPE in vivo. Diffusion of the cell surface-localized beta-catenin occurs in migratory cells in vitro in the presence of HGF. HGF induced a MAP kinase dependent ARPE-19 cell migration, which is accompanied with a transient increase of c-jun expression and concomitant increases of MAP kinase activity, tyrosine phosphorylation of HGFR and beta-catenin, increased cytosolic levels of beta-catenin, and transactivation activity of beta-catenin. Tyrosine phosphorylation of HGFR and beta-catenin occurs in the primary or passaged RPE cultures or proliferative ARPE-19 cells, but not freshly isolated RPE or differentiated ARPE-19 cells.


CONCLUSIONS
This study defines the signal transduction pathways activated by HGF in RPE cells, leading to an increase in the MAP kinase activity and free pool of beta-catenin, and changes in gene expression. These findings are consistent with the hypothesis that both beta-catenin and MAP kinases are components of the HGF induced RPE migration that occurs in proliferative retinal diseases.
Liou
Gregory I
GI
Department of Ophthalmology, Medical College of Georgia, Augusta, GA 30912, USA. giliou@mail.mcg.edu
Matragoon
Suraporn
S
Samuel
Sara
S
Behzadian
M Ali
MA
Tsai
Nai-Tse
NT
Gu
Xiaolin
X
Roon
Penny
P
Hunt
D Margaret
DM
Hunt
Richard C
RC
Caldwell
Ruth B
RB
Marcus
Dennis M
DM
D000368:Aged; D000369:Aged, 80 and over; D000818:Animals; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism; D002465:Cell Movement / Q000187:drug effects*; D002478:Cells, Cultured; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D006801:Humans; D008875:Middle Aged; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010766:Phosphorylation; D010857:Pigment Epithelium of Eye / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D011233:Precipitin Tests; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis / Q000235:genetics; D019859:Proto-Oncogene Proteins c-met / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology*; D015534:Trans-Activators / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism; D051176:beta Catenin
C495270:CTNNB1 protein, human; D015820:Cadherins; D003598:Cytoskeletal Proteins; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D015534:Trans-Activators; D051176:beta Catenin; D014443:Tyrosine; D017228:Hepatocyte Growth Factor; D019859:Proto-Oncogene Proteins c-met; D020928:Mitogen-Activated Protein Kinases