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G. Sosne, P. Qiu, M. Kurpakus-Wheater; Lipid Rafts and Actin Are Important for Thymosin Beta 4- Mediated Anti-Inflammatory Effects in Cornea. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3397.
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To investigate the relationship of Tβ4, actin/cytoskeleton dynamics, and membrane raft function in TNF-α signaling and inflammation.
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.
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.
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.
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