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.

Thymosin beta(4), a small ubiquitous protein containing 43 aa, has structure/function activity via its actin-binding domain and numerous biological affects on cells. Since it is the major actin-sequestering molecule in eukaryotic cells and is found essentially in all cells and body fluids, thymosin beta(4) has the potential for significant roles in tissue development, maintenance, repair, and pathology. Several active sites with unique functions have been identified, including the amino-terminal site containing 4 aa (Ac-SDKP) that generally blocks inflammation and reduces fibrosis. Another active site at the amino terminus contains 15 aa, including Ac-SDKP, and promotes cell survival and blocks apoptosis, while a short sequence containing LKKTETQ, the central actin-binding domain (aa 17-23) plus 1 additional amino acid (Q), promotes angiogenesis, wound healing, and cell migration. Several additional biological activities have been identified but not yet localized in the molecule, including its antimicrobial activity, the induction of various genes (including laminin-5, MMPs, TGF beta, zyxin, terminal deoxynucleotidyl transferase, and angiogenesis-related proteins), and the ability to activate ILK/PINCH/Akt, and other signaling molecules important in both apoptosis and inflammatory pathways. This review details these important physiologically and pathologically active sites and their potential therapeutic uses.

The mechanisms by which thymosin β 4 (Tβ(4)) regulates the inflammatory response to injury are poorly understood. Previously, we demonstrated that ectopic Tβ(4) treatment inhibits injury-induced proinflammatory cytokine and chemokine production. We have also shown that Tβ(4) suppresses TNF-α-mediated NF-κB activation. Herein, we present novel evidence that Tβ(4) directly targets the NF-κB RelA/p65 subunit. We find that enforced expression of Tβ(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 Tβ(4). Tβ(4) blocks RelA/p65 nuclear translocation and targeting to the cognate κB site in the proximal region of the IL-8 gene promoter. Tβ(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 Tβ(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 Tβ(4) modulates inflammation. In addition, the results of this study serve as a foundation for developing Tβ(4) as a new anti-inflammatory therapy.