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 (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.

The β‐thymosins: Intracellular and extracellular activities of a versatile actin binding protein family

“…However, the analysis of the recent observation that extracellular Tb4 induces cardiomyocyte migration and promotes their survival indicated that it is involved in the formation of an active complex between ILK (integrin linked kinase) and PINCH, a LIM domain protein which interacts with a number of receptor tyrosine kinases. In cardiomyocytes the PINCH-ILK complex activates the kinase Akt, which supports cell survival and possibly protects by this mechanism cardiac cells against an ischemic insult [Bock-Marquette et al, 2004;Sosne et al, 2007].…”

“…Additionally to intact Tb4, the N-terminal tetrapeptide (AcSDKP) as well as larger fragments of the peptide have been detected in human blood serum [Junot et al, 1999;Plavina et al, 2008]. Extracellular Tb4 has a number of other important physiological properties [Goldstein et al, 2005], such as the ability to down-regulate a number of key inflammatory chemokines and cytokines like TNF-a, whose proinflammatory effect on corneal epithelial cells is in vitro inhibited by extracellularly applied Tb4 [Sosne et al, 2005[Sosne et al, , 2006[Sosne et al, , 2007.…”

Thymosin Beta‐4 Is Elevated in Women With Heart Failure With Preserved Ejection Fraction

“…TB4 has been shown to inhibit the NF‐κB pathway in a mouse model of myocardial infarction53 and in tumor necrosis factor α–stimulated corneal epithelial cells 54. We also note that inhibition of NF‐κB resulted in suppression of TB4 expression in a prior study of melanoma cells 55…”

Thymosin β as an Actin-binding Protein with a Variety of Functions

“…It turned out that a reduction occurred of nuclear NFKB amount, its activity and the degree of phosphorylation of its key subunit, p65. It is suggested that a block takes place in these cells of translocation of the p65 subunit to the nucleus [33]. There are also reports that Tβ4 sulfoxide, which is formed by monocytes in the presence of glucocorticoids, also inhibits inflammatory responses [20].…”

“…There are reports that it has an impact on the multi-stage activation of NFKB, the transcription factor which activates many pro-inflammatory genes [33]. Experiments were performed in which the inflammatory process was induced by the cytokine TNF-α and then the cells were treated with Tβ4.…”