FGF-2-Induced Wound Healing in Corneal Endothelial Cells Requires Cdc42 Activation and Rho Inactivation through the Phosphatidylinositol 3-Kinase Pathway
Abstract
PURPOSE: Acquisition of elongated cells with pseudopodia is observed when corneal endothelial cells (CECs) are simultaneously treated with basic fibroblast growth factor (FGF)-2 and RhoA inhibitors. This study was designed to determine whether these phenotypes are migratory and whether Cdc42 activation and RhoA inactivation are involved in cell migration. METHODS: A scratch-induced directional migration assay was used to measure migratory rates. Activation of Cdc42 was determined by GTP pull-down assay. Transfection was performed using constitutively active (ca) or dominant negative (dn) Rho guanosine triphosphatase (GTPase) vectors. RESULTS: Stimulation with basic FGF-2 alone resulted in a 43% recovery of the wound area, whereas CECs treated with FGF-2 and Y27632 (inhibitor of Rho-associated kinase) achieved an 84% recovery of the wound area with a fast migratory speed (0.72 microm/min). The synergistic effects of FGF-2 and Y27632 were completely blocked by LY294002 (PI 3-kinase inhibitor). Under these conditions, activation of PI 3-kinase and Cdc42 were observed in the migratory cells. The involvement of activated Cdc42 and inactivated Rho in endothelial migration was determined by transfecting CECs with ca- or dnRho GTPase vectors. A high migratory rate (0.52 microm/min) was seen in CECs expressing caCdc42, whereas endothelial migration was completely inhibited in CECs expressing caRho. When cells expressing caCdc42 were treated with FGF-2, migration reached the maximum rate (0.69 microm/min), similar to that observed in cells treated with FGF-2 and Y27632. CONCLUSIONS: These findings suggest that endothelial migration is induced by activated Cdc42 and inactivated Rho via PI 3-kinase after FGF-2 stimulation and that Cdc42 activation is crucial for CECs to acquire the characteristic migratory phenotypes.
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All scratch assays were conducted in the presence of 5 μg/mL mitomycin-C to suppress cellular proliferation.
“All experiments were conducted in the presence of 5 μg/mL of mitomycin-C (Sigma) to suppress cellular proliferation133.”
From Neuropeptides Substance P and Calcitonin Gene Related Peptide Accelerate the Development and Fibrogenesis of Endometriosis (2019) · cites this paper as [133]
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- Authors
- Lee, J. G., & Kay, E. P.
- Affiliations
- From the Doheny Eye Institute and the
- University of Southern California (US)
- Venue
- Investigative Opthalmology & Visual Science
- Published
- 2006-04-01
- Volume
- 47
- Issue
- 4
- Pages
- 1376
- ISSN
- 0146-0404 (ISSN-L)
- 1552-5783
- OpenAlex
- W2001414744
- Cited by
- 79
- References
- 37
- Citations by year
- 2026: 1 · 2025: 1 · 2024: 2 · 2023: 4 · 2022: 5 · 2021: 3 · 2020: 5 · 2019: 3 · 2018: 3 · 2017: 1 · 2016: 2 · 2015: 7 · 2014: 5 · 2013: 6 · 2012: 8
- FWCI
- 2.69
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Investigative Ophthalmology & Visual Science (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Eye Institute (P30EY003040, #EY03040, EY06431, R01EY006431)
- Topics
- Health Sciences › Medicine › Radiology, Nuclear Medicine and Imaging › Corneal Surgery and Treatments
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Kinase Regulation and GTPase Signaling
- Health Sciences › Medicine › Rehabilitation › Wound Healing and Treatments
- Keywords
- corneal endothelial cells, wound healing, basic fibroblast growth factor, Cdc42, RhoA, Rho kinase, phosphatidylinositol 3-kinase, cell migration, endothelial cell migration, Y-27632, LY294002, pseudopodia
- MeSH terms
- Animals, Cell Movement, Electrophoresis, Polyacrylamide Gel, Endothelium, Corneal, Enzyme Inhibitors, Genetic Vectors, Rabbits, Transfection, Wound Healing, Fibroblast Growth Factor 2, Microscopy, Confocal, Cell Culture Techniques, Fluorescent Antibody Technique, Indirect, Phosphatidylinositol 3-Kinases, rhoA GTP-Binding Protein, cdc42 GTP-Binding Protein, Cell Proliferation
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