Cross-talk among Rho GTPases acting downstream of PI 3-kinase induces mesenchymal transformation of corneal endothelial cells mediated by FGF-2.
OBJECTIVE
Endothelial-mesenchymal transformation (EMT), in which the contact-inhibited corneal endothelial cells (CECs) become multilayers of spindle-shaped cells containing protrusive processes, is mediated by fibroblast growth factor (FGF)-2. The involvement in EMT of cross-talk among Rho GTPases mediated by FGF-2 was also investigated.


METHODS
GTP pull-down assays were performed to identify the activated Rho GTPases. Transfection of CECs with either constitutively active (ca) or dominant negative (dn) Rho GTPase vectors was performed. Protein-protein interaction was investigated by coimmunoprecipitation and a yeast two-hybrid assay.


RESULTS
The alteration of morphology and actin cytoskeleton caused by FGF-2 was mediated by active Rac and inactive Rho. Prolonged treatment of CECs with FGF-2 induced formation of protrusive processes through activated Cdc42. All FGF-2 actions were blocked by the phosphatidylinositol (PI) 3-kinase inhibitor LY294002. Cells transfected with caRacG12V acquired elongated morphology; the actin cytoskeleton was reorganized to the cortex. Formation of protrusive processes was observed in the elongated cells expressing caCdc42G12V or dominant negative (dn)RhoT19N, whereas polygonal cells expressing dnRacT17N, caRhoG14V, or dnCdc42T17N had stress fibers. Further analysis demonstrated that Rac was associated with Cdc42 or Rho through a 32- or 30-kDa Dbl homology/pleckstrin homology-containing protein.


CONCLUSIONS
These findings suggest that alteration of cell shape and actin cytoskeleton are closely linked to the sequential activation of Rho GTPases through PI 3-kinase in response to FGF-2 stimulation. Cortical actin is formed via active Rac and inactive Rho followed by formation of protrusive processes mediated by active Cdc42 and inactive Rho.
Lee
Jeong Goo
JG
Doheny Eye Institute, Department of Ophthalmology, Keck School of Medicine, University of Southern California, Los Angeles 90033, USA.
Kay
EunDuck P
EP
D000199:Actins / Q000378:metabolism; D000818:Animals; D048430:Cell Shape / Q000187:drug effects; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D005822:Genetic Vectors; D007124:Immunoenzyme Techniques; D008648:Mesoderm / Q000166:cytology* / Q000201:enzymology; D018613:Microscopy, Confocal; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011817:Rabbits; D014162:Transfection; D020798:Two-Hybrid System Techniques; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism; D020744:rac GTP-Binding Proteins / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D002867:Chromones; D004791:Enzyme Inhibitors; D016222:Fibroblast Growth Factor 2; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D020764:cdc42 GTP-Binding Protein; D020744:rac GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one