Abstract
Abstract: :
Purpose: To investigate whether FGF–2 plays a role in the morphogenetic pathway by reorganizing actin cytoskeleton through the actions of PI 3–kinase and the Rho family of small GTPases. Methods:Organization of actin cytoskeleton and focal adhesions were analyzed by immunofluorescent staining. Expression of proteins was determined by immunoblot analysis. Pharmacologic inhibitors or neutralizing antibody to FGF–2 were respectively used to block individual pathways. Results: CECs treated with FGF–2 lost the characteristic polygonal cell morphology; such cell shape change was completely blocked by treatment with LY294002. CECs in culture have abundant stress fibers. However, FGF–2 caused a loss of these stress fibers and focal adhesions; the modulated cells contain 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. Expression of vinclulin was not altered 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. The contact–inhibited ex vivo endothelial monolayer demonstrated a circumferential actin ring, and no stress fibers were observed. When CE was treated with FGF–2, actin cortex was greatly disrupted in the modulated cells. Both neutralizing antibody to FGF–2 and LY294002 completely impeded the modulating activity of FGF–2. When CE was simultaneously treated with FGF–2 and Y27632, the circumferential actin cortex was greatly disrupted; 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 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.
Keywords: cornea: endothelium • cytoskeleton • signal transduction