Modulation of the Rho mediated signaling pathways in embryonic corneal fibroblasts
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Purpose. The Ras protein family is irvolved in a variety of events including cell signaling, cell shape, motility and control of actin cytosketeton. The Rho protein may be responsible for regulating a signal transduction pathway linking extracellular matrix (ECM) to the assembly of focal adhesions and actin stress fibers The specificproteins and series of phosphorylaiion steps involved in the signaling sequence is currently unknown. We compared corneal embryonic avian fibroblasts (CEAF) to newborn dermal fibroblasts (DF) to determine the sequence of signaling events in response to extracellular matrix (ECM) We addressed the following questions. 1) Is the Rho signaling pathway involved ii actin reorganization? 2) Can the signaling pathway be modulated by blocking Rho? 3) Is there a difference in responsiveness to ECM or bombesin between DF and CEAF? friethods, Serum starved DF and CEAF were stimulated at specific times with soluble fibronectin (50 (J.g/ml), collagen (100 (ig/ml) or bombesin (10(0.g/ml). Total proteins were analyzed by western analysis to determine the sequence of jihosphorylation events from 2 min to 2 hrs. Confocal microscopy visually confirmed the signaling sequence. Antisense transfection with a 20mer oligonucleotii.e directed to the 5' region of the Rho mRNA was used to block Rho expression for 24-48 hrs before ECM or bombesin stimulation for 2hrs. Results. The data suggests that ECM stimulates FAK, GRb2/SOS, Rho, PLCy, MARK with possible crosstalk to other signal events. Antisense transfection directed against i'.ho mRNA decreased Rho protein levels and disrupted this signaling sequence. The signaling sequence and response to antisense Rho appeared to be similar in both DF ,ind CEAF cells. In addition, the transfected cells became round and decreased adhesiveness in response to ECM or bombesin. Conclusion. We demonstrated that ,iho is necessary for the ECM stimulated signaling cascade, cell shape and attachment.
