NF-κB is the transcription factor for FGF-2 that causes endothelial mesenchymal transformation in cornea.
OBJECTIVE
To determine the role of nuclear factor-κB (NF-κB) during FGF-2-mediated endothelial mesenchymal transformation (EMT) in response to interleukin (IL)-1β stimulation in corneal endothelial cells (CECs).


METHODS
Expression and/or activation of IL-1 receptor-associated protein kinase (IRAK), TNF receptor-associated factor 6 (TRAF6), phosphatidylinositol 3-kinase (PI 3-kinase), IκB kinase (IKK), IκB, NF-κB, and FGF-2 were analyzed by immunoblot analysis. Cell proliferation was measured by 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. NF-κB activity was measured by NF-κB ELISA 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 upregulated expression of IRAK and TRAF6 and activated PI 3-kinase; expression of IRAK and TRAF6 reached maximum within 60 minutes, after which the expression disappeared, while PI 3-kinase activity was observed up to 4 hours after 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) or 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 combines the canonical pathway and the PI 3-kinase signaling to upregulate FGF-2 production through NF-κB, which plays a key role as a transcription factor of FGF-2 gene.
Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California, USA.
Jeong Goo
JG
Lee
EunDuck P
EP
Kay
D000818:Animals; D049109:Cell Proliferation / Q000187:drug effects; D002478:Cells, Cultured; D002843:Chromatin / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D006801:Humans; D051550:I-kappa B Kinase / Q000378:metabolism; D015151:Immunoblotting; D053592:Interleukin-1 Receptor-Associated Kinases / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology; D016328:NF-kappa B / Q000378:metabolism / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D011817:Rabbits; D048029:TNF Receptor-Associated Factor 6 / Q000378:metabolism
D002843:Chromatin; D053583:Interleukin-1beta; D016328:NF-kappa B; D048029:TNF Receptor-Associated Factor 6; D016222:Fibroblast Growth Factor 2; D019869:Phosphatidylinositol 3-Kinases; D053592:Interleukin-1 Receptor-Associated Kinases; D051550:I-kappa B Kinase