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JeongGoo Lee, EunDuck P. Kay; IL-1β Induces FGF-2 Production via PI 3-kinase/p38/NF-B Pathways in Rabbit Corneal Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):6438.
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In response to IL-1β stimulation, corneal endothelial cells (CECs) produce all isoforms of FGF-2, the direct mediator of endothelial mesenchymal transformation of CECs. We investigated the signaling pathways for FGF-2 production by IL-1β and further studied the regulatory role of NF-ΚB in this inductive pathway.
Cell proliferation was measured by MTT assay. Expression of FGF-2, p65 and p-p65 of NF-ΚB was analyzed by immunoblotting. NF-ΚB activity was measured using transcription assay kit. Pharmacological inhibitors were used to block PI 3-kinase, p38, ERK1/2, or NF-ΚB. To identify NF-ΚB binding site of FGF-2 promoter, chromatin immunoprecipitation (ChIP) assay was used.
Brief stimulation of cells with IL-1β activated PI 3-kinase and p38; FGF-2 production mediated by IL-1β was completely blocked by treatment of specific inhibitors for PI 3-kinase and p38. Inhibitor for NF-ΚB activation also blocked FGF-2 production in response to IL-1β stimulation. Inhibitors for PI 3-kinase and p38 blocked phosphorylation of the p65 subunit of NF-ΚB. Using an NF-ΚB transcription factor assay, we further confirmed that activation of NF-ΚB by IL-1β is regulated by PI 3-kinase and p38 pathways. These results show that NF-ΚB is the downstream effecter of the PI 3-kinase/p38 pathways for the inductive activity of IL-1β on FGF-2. We also defined that NF-ΚB is a transcription factor for induction of FGF-2 using ChIP assay in human retinal pigment epithelial cells, in which IL-1β upregulated FGF-2 production similar to human and rabbit CECs. The -685 region of the FGF-2 promoter contains a functional NF-ΚB binding site composed of the decamer sequence 5'-GGGATTTACA-3'.
These findings suggest that CECs utilize NF-ΚB as a major transcription factor to produce FGF-2 in response to IL-1β stimulation through PI 3-kinase and p38 and that ChIP assay further shows that NF-ΚB directly binds to the FGF-2 promoter.
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