Endotherial mesenchymal transformation mediated by FGF–2 and interleukin–1ß in corneal endotherial cells

Abstract

Abstract: : Purpose: To investigate the molecular mechanism of endothelial mesenchymal transformation (EMT) in corneal endothelial cells (CECs). Methods: Proteomics were used to identify the protein factor released by inflammatory cells as interleukin–1ß (IL–1ß). FGF–2 mRNA levels were measured using real–time PCR, and FGF–2 at protein level was determined by immunoblotting analysis. PI 3–kinase activity was determined by measuring production of phosphatidylinositol–3–phosphate. Subcellular localization of FGF–2 and F–actin were determined by immunofluorescent staining. Results:The partially purified protein factor obtained from inflammatory cells was further identified using proteomics: protein fractions containing 17 kDa peptide were isoelectrofocused using pH gradient IPG strips (5.5 to 6.4); the 17 kDa peptide band was subjected to in–gel trypsin digestion and mass spectrometry using SELDI–TOF followed by sequencing and data base analysis. The sequence of 17 kDa peptide band was identified as IL–1ß and confirmed by immunoblotting. The role of IL–1ß was further determined during EMT. IL–1ß altered the cell shape of CECs from a polygonal to a fibroblastic morphology in a time– and dose–dependent manner, while neutralizing antibody to FGF–2 and LY294002 blocked the action of IL–1ß on cell shape changes. IL–1ß induces mRNA levels of FGF–2 in a dose– and time–dependent manner and the cytokine also induces the expression of nuclear and extracellular matrix (ECM) isoforms of FGF–2 at the protein level. IL–1ß initially induced nuclear accumulation of FGF–2 but further facilitated the subcellular localization of FGF–2 to membrane and ECM in addition to the nuclei. IL–1ß also activates PI 3–kinase activity. Conclusions: These observations suggest that IL–1ß induces de novo synthesis of FGF–2 at both transcriptional and translational levels and that IL–1ß is involved in EMT through its action on FGF–2, which is the direct mediator of EMT.

Keywords: cornea: endothelium • inflammation • growth factors/growth factor receptors