NF-κB Is the Transcription Factor for FGF-2 That Causes Endothelial Mesenchymal Transformation in Cornea

The retrocorneal fibrous membrane (RCFM), first described by Fuchs in 1901,1 has been observed in various clinical conditions associated with disease and damage of the corneal endothelium.2–4 The presence of RCFM (or posterior collagenous layer) posterior to the Descemet's membrane is thought to represent an end-stage disease process of the corneal endothelium, which results in functional alteration of the corneal endothelium and leads to corneal opacity and blindness. An in vitro model to elucidate the molecular mechanism of RCFM formation led us to the finding that fibroblast growth factor 2 (FGF-2) is the direct mediator of endothelial mesenchymal transformation (EMT) observed in RCFM: first, FGF-2 signaling directly regulates cell cycle progression by degrading p27Kip1 (p27), leading to a marked stimulation of cell proliferation5–7; second, FGF-2 signaling upregulates the steady state levels of α1(I) collagen RNA by stabilizing the message and subsequently facilitates synthesis and secretion of type I collagen into the extracellular space8; and third, FGF-2 signaling induces a change in cell shape from a polygonal to a fibroblastic morphology through regulation of the Rho family of small GTPases and subsequent reorganization of actin.9,10 We also reported that interleukin (IL)-1β exerts a critical role as a switch of the FGF-2–mediated EMT; IL-1β signaling greatly upregulated FGF-2 production through phosphatidylinositol 3-kinase (PI 3-kinase)/p38 pathways in corneal endothelial cells (CECs).11,12 We further confirmed such is the case in vivo, in which polymorphonuclear leukocytes (PMNs) infiltrating the anterior chamber are a major source of IL-1β.13 IL-1β, a potent proinflammatory cytokine, plays an important role in acute and chronic inflammatory diseases14–16 and a crucial role in the regulation of inflammation and wound healing on the ocular surface.17–19 Numerous studies have reported that IL-1α and IL-1β both orchestrate the inflammatory process by inducing the production and release of secondary cytokines; IL-1β stimulates the expression of a variety of genes necessary for the wound repair processes.20–22 Both IL-1α and IL-1β markedly stimulate synthesis and release of FGF-2 in a variety of cell types.23–25 Likewise, CECs both in vivo and in vitro produce all isoforms of FGF-2 in response to IL-1β stimulation through PI 3-kinase/p38 signaling.11–13 A recent study demonstrated that PI 3-kinase/AKT-dependent pathway upstream to the transcription factor nuclear factor (NF)-κB is partly involved in IL-6 gene transcription in response to IL-1.26 It is likely that the induction of FGF-2 by IL-1β is mediated by NF-κB in CECs. Activation of NF-κB by IL-1β requires the type I IL-1 receptor (IL-1R1), which recruits specific cytoplasmic proteins to transmit its signals, such as IL-1 receptor–associated protein kinase (IRAK) and TNF receptor–associated factor 6 (TRAF6).27,28 There is evidence that activation of PI 3-kinase in response to IL-1β depends on the presence of IRAK-1.28 A recent study showed that IL-1β–induced IL-6 production is mediated by both PI 3-kinase and IRAK-4.29 Thus, PI 3-kinase is involved in IL-1β–induced NF-κB activation signaling. NF-κB signaling pathway functions in essentially all mammalian cell types and regulates genes involved in the inflammatory and immune responses.30–32 NF-κB is kept inactive in the cytoplasm through association with an inhibitory protein of the IκB family. In response to multiple stimuli, IκB becomes phosphorylated and polyubiquitinated, subsequently leading to degradation by ubiquitin-proteasome complex. As a consequence, the released free NF-κB is further activated and enters the nucleus to activate transcription of its target genes. We, therefore, hypothesized that one of the target genes of NF-κB is FGF-2, in response to IL-1β stimulation. In the present study, we showed that the IL-1β/IL-1R signaling for FGF-2 production employs PI 3-kinase signaling as part of the canonical NF-κB signaling: IRAK and TRAF6 activate PI 3-kinase, which is subsequently involved downstream of the canonical NF-κB signaling to activate IκB kinase (IKK) and NF-κB. We further demonstrated that NF-κB directly binds to the putative κB binding site of the promoter region of human FGF-2 gene. It is the first demonstration that NF-κB is indeed the transcription factor of FGF-2, the direct mediator of endothelial to mesenchymal transformation observed in RCFM.