Transglutaminase–2 Mediates p65 Translocation From Cytoplasm to Nucleus in the UVB–Activated NF–B Pathway

Abstract

Purpose: : Ultraviolet radiation (UVB) is a known stimulus for the activation of nuclear factor (NF–ΚB) in human corneal epithelial cells. The triggering mechanism for the NF–ΚB pathway is not known. We aimed to investigate whether transglutaminase (TGM)–2 mediates p65 cytoplasmic–nuclear translocation, a key step in NF–ΚB activation.

Methods: : A human corneal epithelial cell line (T–HCEC) was irradiated by a uniform single dose (20 mJ/cm²) of UVB with or without SN–50 or mono–dansyl cadaverine (MDC) pretreatment. Immunofluorescent staining (IF) and Western blot were performed using antibodies against p65 and TGM–2. IF with laser scanning confocal microscopy was used to assess p65 intra–cellular localization. A peptide–based non–covalent carrier system (Chariot^TM) was used to deliver guinea pig liver TGM–2 protein into T–HCEC without morphological evidence of toxicity. Short interfering RNA (SiRNA–TGM–2) was used to silence TGM–2 gene expression. Biological TGM activity was assessed by a fluorescein–cadaverine uptake assay.

Results: : UVB induced an increase in TGM–2 protein level and trans–amidation activity and also induced the translocation of p65 protein from cytoplasm to nucleus (IF) after 4 hours in T–HCEC. This translocation was verified by Western blots showing that the p65 immuno–reactivity increased in the nuclear fraction of the cell lysates of UVB–exposed cells relative to control. This UVB–induced p65 translocation was inhibited by SN–50 peptide, a cell–permeable chemical that inhibits nuclear translocation of active NF–ΚB, and by MDC, a competitive TGM inhibitor. We observed in non–irradiated cells that recombinant human TNF–α induced the p65 nuclear translocation, which was also inhibited by MDC or SiRNA–TGM–2. Furthermore, the exogenous TGM–2 protein introduced into non–irradiated T–HCEC by the Chariot system triggered p65 nuclear translocation, whereas cells incubated with either TGM–2 or Chariot reagent alone were not demonstrated to have p65 nuclear translocation.

Conclusions: : Our findings demonstrate that increase in TGM–2 protein mediates p65 nuclear translocation in the UVB–activated NF–ΚB pathway in human corneal epithelial cells. TGM–2 could be a potential therapeutic target in ocular surface inflammatory diseases where NF–ΚB activation is a common mechanism.

Keywords: cornea: epithelium • signal transduction • stress response