Thymosin beta 4 suppression of corneal NFκB: A potential anti-inflammatory pathway

Abstract

The purpose of this study was to determine the effect of thymosin beta 4 (Tβ 4) on NFκB protein levels, activation, phosphorylation, and nuclear translocation in a model of tumor necrosis factor (TNF)-α-mediated corneal inflammation. Transformed and primary (HCET and HCEC) human corneal epithelial cells were stimulated with the pro-inflammatory cytokine TNF-α and treated or not with Tβ 4. Nuclear NFκB p65 subunit protein levels were assayed using ELISA, and activity was measured by determining NFκB binding to consensus oligonucleotides. NFκB p65 protein phosphorylation was also measured by ELISA. Nuclear translocation of NFκB p65 subunit was assayed by immunofluorescence microscopy. Compared to non-treated controls, Tβ 4 treatment significantly decreased nuclear NFκB protein levels, NFκB activity and p65 subunit phosphorylation in corneal epithelial cells after TNF-α stimulation. In TNF-α-stimulated corneal epithelial cells, NFκB p65 subunit translocation to the nucleus was observed using immunofluorescence microscopy. In contrast, Tβ 4 blocked nuclear translocation of the NFκB p65 subunit in TNF-α-stimulated corneal epithelial cells. TNF-α initiates cell signaling pathways that converge on the activation of NFκB, thus both are known mediators of the inflammatory process. Tβ 4, a protein with diverse cellular functions including wound healing and suppression of inflammation, inhibits the activation of NFκB in TNF-α-stimulated cells. These results have important clinical implications for the potential role of Tβ 4 as a corneal anti-inflammatory agent.