Interleukin‐1β enhances cell migration through AP‐1 and NF‐κB pathway‐dependent FGF2 expression in human corneal endothelial cells

Abstract

Background Information Interleukin 1β is a major pro-inflammatory cytokine that plays a crucial role in the regulation of inflammation and wound healing in the cornea. Elucidation of Interleukin1β signaling may help identify therapeutic targets for corneal wound healing; however, mechanisms such as cell migration, a component of Interleukin 1β induced wound healing response in human corneal endothelial cells, have not been well characterized. Results Stimulation of human corneal endothelial cells with Interleukin 1β activated expression of fibroblast growth factor 2 and resulted in enhanced cell migration. This, in turn, was abolished by treatment with either Interleukin-1 receptor antagonist or SU-5402, a pan fibroblast growth factor signaling inhibitor. Phosphatidyl inositol 3-kinase or interleukin receptor-associated kinase 1/4 antagonists demonstrated that interleukin receptor-associated kinase 1/4 activates phosphatidyl inositol 3-kinase, which in turn phosphorylates p38 and inhibitor κB kinase α/β, leading to fibroblast growth factor 2 expression through activation of activator protein 1 and nuclear factor kappa-light-chain-enhancer of activated B cells in human corneal endothelial cells. Treatment of interleukin 1β stimulated human corneal endothelial cells with either activator protein 1 or nuclear factor kappa-light-chain-enhancer of activated B cells antagonists decreased fibroblast growth factor 2 expression and resulted in reduced interleukin 1β enhanced cell migration. Co-treatment of interleukin 1β stimulated human corneal endothelial cells with both inhibitors completely blocked fibroblast growth factor 2 expression and interleukin 1β enhanced cell migration. Chromatin immunoprecipitation assays demonstrated that activator protein 1 and nuclear factor kappa-light-chain-enhancer of activated B cells directly bind to the fibroblast growth factor 2 promoter following interleukin 1β stimulation. Conclusion The results show that binding of interleukin 1β to its receptor in human corneal endothelial cells leads to parallel activation of activator protein 1 and nuclear factor kappa-light-chain-enhancer of activated B cells pathways, leading, in turn, to fibroblast growth factor 2 expression and enhanced cell migration.

Interleukin-1β-Induced Wnt5a Enhances Human Corneal Endothelial Cell Migration through Regulation of Cdc42 and RhoA

“…Immortalized human CEC line hCEC-B4G12 (DSMZ, Braunschweig, Germany) was cultured as previously described (7). Briefly, hCEC-B4G12 cells were cultured in human endothelial serumfree medium supplemented with 10 ng/ml human recombinant FGF2 without antibiotics.…”

“…Cell migration assay was performed using our previously published protocols with some modifications (7,46). Briefly, human CECs were plated in 24-well tissue culture dishes at a concentration of 4 ϫ 10 4 cells and maintained until the cells reached greater than 95% confluence.…”

“…All assays were performed following previously reported protocols (7,8,48,49). The following gel concentrations were used to separate proteins: 15% polyacrylamide gel for Cdc42, Rac1, RhoA, cofilin, and p-cofilin; 10% polyacrylamide gel for Wnt5a, lamin B, ␣-tubulin, and ␤-actin; 8% polyacrylamide gel for Fzd 5, ␤-catenin, LIMK2, p-LIMK2; 6% polyacrylamide gel for Ror2, DAAM1, SSH1, and p-SSH1.…”

“…A variety of soluble factors have previously been shown to enhance cell migration, including interleukin 1␤ (IL-1␤), a major mediator of inflammation and wound healing in the cornea (4)(5)(6). We previously reported that IL-1␤ stimulation induces fibroblast growth factor 2 (FGF2) expression through NF-B and AP-1 pathways, leading to enhanced migration of human and rabbit CECs (7)(8)(9). However, IL-1␤ and FGF2 have also been shown to be important mediators of the endothelial-mesenchymal transition (EMT) that leads to retrocorneal fibrous membrane (RCFM) formation (4,8,10), which represents end-stage pathology for the eye, where restoration of vision is no longer possible.…”

Asbestos-Induced Mesothelial to Fibroblastic Transition Is Modulated by the Inflammasome

“…In fact, IL-1b induces FGF2 secretion in HMCs and corneal endothelial cells. 34,35 The dose of IL-Ra used in these experiments can be said to be the limiting factor because the use of an approximately sevenfold higher concentration (1 mg/mL) led to a complete blockage of MFT with a slight reduction in FGF2 secretion (greatest reduction was observed in the lower-molecularweight isoforms). Because cells were only exposed to IL-1Ra at this dose for 72 hours with and without asbestos exposure, further studies are needed to ascertain the exact mechanism by which IL-1b initiates or induces MFT in mesothelial cells.…”