Abstract
Purpose: :
To investigate the relationship of Tβ4, actin/cytoskeleton dynamics, and membrane raft function in TNF-α signaling and inflammation.
Methods: :
Human corneal epithelial cells and fibroblasts were pre-treated or not with Tβ4 (1 µg/ml) for one hour prior to treatment with cyotchalasin D (cytoD, 5 µM) or jasplakinolide (JP, 1 µM)(actin/cytoskeleton disrupters). Additional experiments also included treatment with the membrane raft inhibitor MβCD. The cells were then stimulated with TNF-α (10 ng/ml). Cell culture supernatants were assayed for IL-8 using ELISA and the cells were assayed for the effects on actin stress fibers (phalloidin staining) and NFΚB nuclear translocation and binding activity.
Results: :
Disrupting the actin cytoskeleton with Tβ4, cytoD or with JP resulted in a significant inhibition of TNF-α-stimulated IL-8 secretion (P < 0.0001 for all comparisons). Disrupting membrane rafts before stimulating cells with TNF-α also significantly inhibited the secretion of IL-8 (** P < 0.0001). Treatment of the cells with MβCD caused a significant disruption of the actin cytoskeleton and decreased NFΚB nuclear translocation. In addition, Tβ4 treatment also disrupted TNF-α-stimulated actin stress fiber formation.
Conclusions: :
Taken together, these results provide support for our hypothesis that the actin cytoskeleton interacts with membrane rafts to play a central role in TNF-α-mediated signaling involving the activation and translocation of NFΚB and resulting in IL-8 production. Along with our other studies demonstrating that Tβ4 suppresses TNF-α-mediated IL-8 production and NFΚB activation, these findings show that Tβ4 treatment also disrupts TNF-α-induced actin cytoskeletal changes and provide a basis for our hypothesis that Tβ4’s inhibitory effects on TNF-α-mediated inflammation may be related to the actin cytoskeleton changes seen.
Keywords: cornea: epithelium • signal transduction • cytokines/chemokines