FGF-2 induced reorganization and disruption of actin cytoskeleton through PI 3-kinase, Rho, and Cdc42 in corneal endothelial cells.
OBJECTIVE
Corneal endothelial cells (CECs) undergo endothelial to mesenchymal transformation (EMT) in response to FGF-2 stimulation. One phenotypic change that occurs during EMT is a change in cell shape from polygonal to elongated fibroblast-like cells. We investigated whether FGF-2 plays a role in this morphogenetic pathway by reorganizing actin cytoskeleton through the actions of phosphatidylinositol (PI) 3-kinase and the Ras related Rho family of small guanosine triphosphatases (GTPases).


METHODS
Cell morphology was analyzed using phase contrast microscopy, and the organization of actin cytoskeleton and focal adhesions were analyzed by immunofluorescent staining. Expression of vinculin and beta-actin was determined by immunoblot analysis. Pharmacologic inhibitors (LY294002, C3 exoenzyme, Y27632, or PD98059) or neutralizing antibody to FGF-2, respectively, were used to block PI 3-kinase, Rho, Rho associated kinase, extracellular signal regulated kinase, or FGF-2 pathways.


RESULTS
CECs treated with FGF-2 became smaller and lost their characteristic polygonal cell morphology. Such cell shape change was completely blocked by treatment with LY294002. CECs in culture have abundant stress fibers that are oriented radially across the cell. However, FGF-2 caused a loss of these stress fibers and focal adhesions. The modulated cells contained a cortical actin ring while LY294002 completely abolished this action of FGF-2 on actin cytoskeleton. Treatment of cells with C3 exoenzyme or Y27632 in the presence of FGF-2 induced spindle shaped cells with prominent pseudopodia which were rapidly formed upon exposure to the inhibitor. The expression level of vinculin was found to be similar in all experimental conditions but vinculin was mostly translocated to the cytoplasm in response to FGF-2 stimulation. CECs plated on Matrigel matrix demonstrated findings similar to those from cells plated on the conventional culture dishes, except that Matrigel facilitated the formation of pseudopodia. We further investigated in vivo actin organization using organ cultures of corneal endothelium (CE) on Descemet's membrane. The contact inhibited endothelial monolayer demonstrated a circumferential actin ring, and no stress fibers were observed. When CE was treated with FGF-2, a half population of CE lost its characteristic contact inhibited cobblestone morphology. Actin cortex was greatly disrupted in these modulated cells. Both neutralizing antibody to FGF-2 and LY294002 completely impeded the modulating activity of FGF-2 on the endothelial monolayer. When CE was simultaneously treated with FGF-2 and Y27632, the circumferential actin cortex was greatly disrupted and the endothelial monolayer was transformed into multi-layers of fibroblastic cells containing pseudopodia. Both LY294002 and neutralizing antibody to FGF-2 antagonized the actions of FGF-2 and Y27632.


CONCLUSIONS
These data indicate that CECs in culture have constitutively active Rho activity as evidenced by stress fiber formation and that PI 3-kinase negatively regulates the formation of stress fibers and focal adhesions, perhaps antagonizing the Rho pathways. Formation of pseudopodia in response to FGF-2 and Y27632 may suggest that the Rho/ROCK pathway negatively regulates Cdc42.
Lee
Hyung Taek
HT
Doheny Eye Institute, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.
Kay
EunDuck P
EP
D000199:Actins / Q000378:metabolism*; D000818:Animals; D048429:Cell Size / Q000187:drug effects; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology*; D019084:Fluorescent Antibody Technique, Indirect; D015151:Immunoblotting; D047908:Intracellular Signaling Peptides and Proteins; D018613:Microscopy, Confocal; D008858:Microscopy, Phase-Contrast; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011817:Rabbits; D016596:Vinculin / Q000378:metabolism; D020764:cdc42 GTP-Binding Protein / Q000378:metabolism*; D020741:rho GTP-Binding Proteins / Q000378:metabolism*; D054460:rho-Associated Kinases
D000199:Actins; D004791:Enzyme Inhibitors; D016222:Fibroblast Growth Factor 2; D047908:Intracellular Signaling Peptides and Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D016596:Vinculin; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins; D054460:rho-Associated Kinases