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Mitogen-activated protein kinase activation mediates PDGF-directed migration of RPE cells
D R Hinton1, S He, K Graf
1Department of Pathology, University of Southern California School of Medicine, Los Angeles 90033, USA.
Abstract:
Growth factor-directed migration is a critical component of the wound healing response although little is known about the signaling pathways involved. We examined the effect of inhibiting the mitogen-activated protein kinase (MAPK) pathway on platelet-derived growth factor (PDGF) and fibronectin-induced cell migration of human retinal pigment epithelial (RPE) cells. Using transwell cell-culture chambers, the effect of PDGF-BB (10-50 ng/ml) and fibronectin on components of migration was measured with or without the MAPK pathway inhibitor PD98059 (10-30 microM) MAPK activation of serum-starved cells by PDGF-BB was demonstrated by an immunoprecipitation/kinase assay and by immunohistochemistry using antibody specific for phosphorylated MAPK. PDGF-BB (10 ng/ml) stimulated MAPK activity in RPE (10 min) and its nuclear localization (1 h). PD98059 inhibited the activation of MAPK by PDGF-BB or serum. PDGF-BB stimulated RPE chemokinesis, chemotaxis, and haptotaxis; chemokinesis was additively increased and chemotaxis synergistically increased by the presence of a fibronectin substratum. PD98059 potently inhibited fibronectin-induced haptotaxis and PDGF-BB-induced chemotaxis but inhibited chemokinesis only at higher PDGF-BB (50 ng/ml) concentrations in the presence of fibronectin substratum. These results demonstrate that MAPK is critically involved in multiple components of RPE migration in vitro and suggest the potential of targeting MAPK to inhibit RPE migration in vivo.
Insights
The mitogen-activated protein kinase (MAPK) pathway is crucial for platelet-derived growth factor (PDGF) and fibronectin-induced migration of retinal pigment epithelial (RPE) cells. Inhibiting MAPK significantly impacts cell movement, suggesting therapeutic potential for wound healing.
Area of Science:
- Cell Biology
- Molecular Biology
- Wound Healing Research
Background:
- Cell migration is vital for wound healing.
- Signaling pathways regulating growth factor-induced cell migration are not fully understood.
- Retinal pigment epithelial (RPE) cells play a key role in ocular wound healing.
Purpose of the Study:
- To investigate the role of the mitogen-activated protein kinase (MAPK) pathway in platelet-derived growth factor (PDGF) and fibronectin-mediated migration of human RPE cells.
- To determine the effect of inhibiting the MAPK pathway on different components of RPE cell migration.
Main Methods:
- Utilized transwell cell-culture chambers to assess RPE cell migration.
- Stimulated RPE cells with PDGF-BB and fibronectin.
- Measured migration with and without the MAPK pathway inhibitor PD98059.
- Assessed MAPK activation using immunoprecipitation/kinase assays and immunohistochemistry for phosphorylated MAPK.
Main Results:
- PDGF-BB stimulated MAPK activity and nuclear localization in RPE cells.
- PD98059 effectively inhibited PDGF-BB-induced MAPK activation.
- PDGF-BB enhanced RPE chemokinesis, chemotaxis, and haptotaxis, with fibronectin further augmenting these processes.
- PD98059 potently inhibited fibronectin-induced haptotaxis and PDGF-BB-induced chemotaxis.
- MAPK inhibition only partially affected chemokinesis at higher PDGF-BB concentrations with fibronectin.
Conclusions:
- The MAPK pathway is critically involved in multiple aspects of RPE cell migration in vitro.
- Targeting the MAPK pathway may offer a therapeutic strategy to modulate RPE cell migration in vivo, potentially aiding wound healing processes.