Abstract
Purpose: :
To determine the role of NF-ΚB during FGF-2-mediated endothelial mesenchymal transformation (EMT) in response to IL-1β stimulation in corneal endothelial cells (CECs).
Methods: :
Expression and/or activation of IRAK, TRAF6, PI 3-kinase, IKK, IΚB, NF-ΚB, and FGF-2 were analyzed by immunoblotting. Cell proliferation was measured by MTT assay. NF-ΚB activity was measured by NF-ΚB ELISA assay kit, while binding of NF-ΚB to the promoter region of FGF-2 gene was determined by chromatin immunoprecipitation.
Results: :
Brief stimulation of CECs with IL-1β briefly upregulated expression of IRAK and TRAF6 and activated PI 3-kinase: expression of IRAK and TRAF6 reached maximum within 60 min, after which the expression disappeared, while PI 3-kinase activity was observed up to 4 hours following IL-1β stimulation. Use of specific inhibitor to PI 3-kinase or IRAK demonstrated that IRAK activates PI 3-kinase, the signaling of which phosphorylates IKKα/β and degrades IΚB, subsequently leading to activation of NF-ΚB. The induction of FGF-2 by IL-1β was completely blocked by inhibitors to NF-ΚB activation (sulfasalazine) and PI 3-kinase (LY294002), and both inhibitors greatly blocked cell proliferation of CECs. Chromatin immunoprecipitation further demonstrated that NF-ΚB is the transcription factor of FGF-2 as NF-ΚB binds the putative NF-ΚB binding site of the FGF-2 promoter.
Conclusions: :
These data suggest that IL-1β signaling employs both the canonical pathway and the PI 3-kinase pathway to upregulate FGF-2 production through NF-ΚB, which plays a key role as a transcription factor of FGF-2 gene. These findings link the inflammatory response to the FGF-2-mediated EMT.
Keywords: cornea: endothelium • signal transduction • wound healing