ECM-stimulated signaling and actin reorganization in embryonic corneal epithelia are Rho dependent.
OBJECTIVE
The goal of this study was to investigate the role of the small guanosine triphosphatase (GTPase), Rho, in the corneal epithelial response to extracellular matrix (ECM) molecules. The avian corneal epithelial model was used to establish that Rho is required for actin reorganization and tyrosine phosphorylation of integrin-mediated signal pathway proteins.


METHODS
Whole embryonic corneal epithelia were isolated without the basal lamina and either transfected with Rho-specific antisense oligonucleotides or treated with Clostridium botulinum C3 exoenzyme and then stimulated with fibronectin (FN) or collagen (COL). The epithelia were evaluated for actin reorganization and protein production including Rho protein levels and tyrosine phosphorylation with Western blot analysis.


RESULTS
After an overnight transient transfection with antisense oligonucleotides, Rho protein levels were decreased more than 80%, and tyrosine phosphorylation of all integrin-mediated signal transduction proteins was decreased compared with control epithelia. Intracellular Rho distribution did not change in the presence of antisense oligonucleotides; however, the amount of immunolabeled Rho decreased. Disrupting the signaling cascade with Rho antisense also blocked FN- and COL-stimulated actin cortical mat reformation. C. botulinum C3 exoenzyme, a pharmacologic agent that specifically causes adenosine diphosphate (ADP) ribosylation and inactivation of Rho, also blocked actin reorganization and tyrosine phosphorylation. In contrast, decreasing Raf protein levels did not change FN-mediated actin reorganization or tyrosine phosphorylation.


CONCLUSIONS
Decreasing Rho protein or blocking its function inhibited ECM-stimulated actin reorganization and signal transduction, as measured by tyrosine phosphorylation.
D000199:Actins / Q000502:physiology*; D000818:Animals; D001483:Base Sequence / Q000235:genetics; D002642:Chick Embryo / Q000502:physiology; D003599:Cytoskeleton / Q000502:physiology; D019573:Epithelium, Corneal / Q000196:embryology*; D005109:Extracellular Matrix / Q000502:physiology*; D008969:Molecular Sequence Data; D009924:Organ Culture Techniques; D010766:Phosphorylation; D015398:Signal Transduction / Q000502:physiology*; D014443:Tyrosine / Q000378:metabolism; D020741:rho GTP-Binding Proteins / Q000502:physiology*
D000199:Actins; D014443:Tyrosine; D020741:rho GTP-Binding Proteins