Comparison and modulation of the Rho mediated signaling pathways in dermal and embryonic corneal fibroblasts.
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abstract
The Ras protein family is involved in a variety of events including cell signaling, cell shape, motility and control of actin cytoskeleton. 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 specific proteins and series of phosphorylation steps involved in the signaling sequence is currently unknown. We compared newborn dermal fibroblasts (DF) and corneal embryonic avian fibroblasts (CEAF) to determine the sequence of signaling events. We addressed the following questions. 1) Is the Rho signaling pathway involved in 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? Serum starved DF and CEAF were stimulated at specific times with soluble fibronectin, collagen or bombesin. Total proteins were analyzed by western analysis to determine the sequence of phosphorylation events from 2 min to 2 hrs. Confocal microscopy visually confirmed the signaling sequence. The data suggests that ECM stimulates FAK, GRb2/SOS, Rho, PLC, MAPK with possible crosstalk to other signal events. Antisense transfection with a 20mer oligonucleotide directed to the 5 region of the Rho 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 and CEAF cells. In addition, transfected cells became round and decreased adhesiveness in response to ECM or bombesin. In conclusion we demonstrated Rho is necessary for the signaling cascade, cell shape and attachment.
