ABSTRACT
The mechanisms by which thymosin ß 4 (Tß(4)) regulates the inflammatory response to injury are poorly understood. Previously, we demonstrated that ectopic Tß(4) treatment inhibits injury-induced proinflammatory cytokine and chemokine production. We have also shown that Tß(4) suppresses TNF-α-mediated NF-κB activation. Herein, we present novel evidence that Tß(4) directly targets the NF-κB RelA/p65 subunit. We find that enforced expression of Tß(4) interferes with TNF-α-mediated NF-κB activation, as well as downstream IL-8 gene transcription. These activities are independent of the G-actin-binding properties of Tß(4). Tß(4) blocks RelA/p65 nuclear translocation and targeting to the cognate κB site in the proximal region of the IL-8 gene promoter. Tß(4) also inhibits the sensitizing effects of its intracellular binding partners, PINCH-1 and ILK, on NF-κB activity after TNF-α stimulation. The identification of a functional regulatory role by Tß(4) and the focal adhesion proteins PINCH-1 and ILK on NF-κB activity in this study opens a new window for scientific exploration of how Tß(4) modulates inflammation. In addition, the results of this study serve as a foundation for developing Tß(4) as a new anti-inflammatory therapy.
Subject(s)
DNA-Binding Proteins/metabolism , Interleukin-8/metabolism , NF-kappa B/metabolism , Protein Serine-Threonine Kinases/metabolism , Thymosin/pharmacology , Tumor Necrosis Factor-alpha/metabolism , Actins/metabolism , Adaptor Proteins, Signal Transducing , Animals , Anti-Inflammatory Agents/pharmacology , COS Cells , Cells, Cultured , Chlorocebus aethiops , DNA-Binding Proteins/antagonists & inhibitors , DNA-Binding Proteins/genetics , Gene Expression Regulation/drug effects , Gene Expression Regulation/physiology , Humans , Interleukin-8/antagonists & inhibitors , Interleukin-8/genetics , LIM Domain Proteins , Membrane Proteins , Mutation , NF-kappa B/antagonists & inhibitors , NF-kappa B/genetics , Protein Binding , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Rats , Thymosin/genetics , Thymosin/physiology , Transcription Factor RelA/antagonists & inhibitors , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/geneticsABSTRACT
The purpose of this study was to determine the effect of thymosin beta 4 (Tbeta4) on NFkappaB protein levels, activation, phosphorylation, and nuclear translocation in a model of tumor necrosis factor (TNF)-alpha-mediated corneal inflammation. Transformed and primary (HCET and HCEC) human corneal epithelial cells were stimulated with the pro-inflammatory cytokine TNF-alpha and treated or not with Tbeta4. Nuclear NFkappaB p65 subunit protein levels were assayed using ELISA, and activity was measured by determining NFkappaB binding to consensus oligonucleotides. NFkappaB p65 protein phosphorylation was also measured by ELISA. Nuclear translocation of NFkappaB p65 subunit was assayed by immunofluorescence microscopy. Compared to non-treated controls, Tbeta4 treatment significantly decreased nuclear NFkappaB protein levels, NFkappaB activity and p65 subunit phosphorylation in corneal epithelial cells after TNF-alpha stimulation. In TNF-alpha-stimulated corneal epithelial cells, NFkappaB p65 subunit translocation to the nucleus was observed using immunofluorescence microscopy. In contrast, Tbeta4 blocked nuclear translocation of the NFkappaB p65 subunit in TNF-alpha-stimulated corneal epithelial cells. TNF-alpha initiates cell signaling pathways that converge on the activation of NFkappaB, thus both are known mediators of the inflammatory process. Tbeta4, a protein with diverse cellular functions including wound healing and suppression of inflammation, inhibits the activation of NFkappaB in TNF-alpha-stimulated cells. These results have important clinical implications for the potential role of Tbeta4 as a corneal anti-inflammatory agent.
