科研之友
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摘要
PURPOSE. To determine the role of nuclear factor-kappa B (NF-kappa B) during FGF-2-mediated endothelial mesenchymal transformation (EMT) in response to interleukin (IL)-1 beta 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), I kappa B kinase (IKK), I kappa B, NF-kappa B, 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-kappa B activity was measured by NF-kappa B ELISA kit, while binding of NF-kappa B to the promoter region of FGF-2 gene was determined by chromatin immunoprecipitation. RESULTS. Brief stimulation of CECs with IL-1 beta 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-1 beta stimulation. Use of specific inhibitor to PI 3- kinase or IRAK demonstrated that IRAK activates PI 3- kinase, the signaling of which phosphorylates IKK alpha/beta and degrades I kappa B, subsequently leading to activation of NF-kappa B. The induction of FGF-2 by IL-1 beta was completely blocked by inhibitors to NF-kappa B activation (sulfasalazine) or PI 3- kinase (LY294002), and both inhibitors greatly blocked cell proliferation of CECs. Chromatin immunoprecipitation further demonstrated that NF-kappa B is the transcription factor of FGF-2 as NF-kappa B binds the putative NF-kappa B binding site of the FGF-2 promoter. CONCLUSIONS. These data suggest that IL-1 beta signaling combines the canonical pathway and the PI 3- kinase signaling to upregulate FGF-2 production through NF-kappa B, which plays a key role as a transcription factor of FGF-2 gene. (Invest Ophthalmol Vis Sci. 2012;53:1530-1538) DOI:10.1167/iovs.11-9102
- 出版日期2012-3
