FGF-2 induced by interleukin-1 beta through the action of phosphatidylinositol 3-kinase mediates endothelial mesenchymal transformation in corneal endothelial cells.
Our previous work demonstrated that both polymorphonuclear leukocytes (PMNs) and protein fractions released from PMNs induced de novo synthesis of fibroblast growth factor 2 (FGF-2), which in turn becomes the direct mediator of endothelial mesenchymal transformation observed in corneal endothelial cells (CECs). To identify the protein factor, we used ProteinChip Array technology. Protein fractions obtained from the conditioned medium released by PMNs were resolved by two-dimensional electrophoresis with immobilized pH gradient strips. Most of the protein spots, with molecular masses of 17 kDa, were sequentially subjected to in-gel trypsin digestion and mass spectrometry. The 17-kDa peptide band was identified as interleukin-1 beta (IL-1 beta). Biological activities of IL-1 beta were further determined; IL-1 beta altered the shape of CECs from polygonal to fibroblastic morphologies in a time- and dose-dependent manner, whereas neutralizing IL-1 beta antibody, neutralizing antibody to FGF-2, and LY294002 blocked the action of IL-1 beta. IL-1 beta greatly increased the levels of FGF-2 mRNA in a time- and dose-dependent manner; IL-1 beta stimulated expression of all isoforms of FGF-2. IL-1 beta initially induced nuclear accumulation of FGF-2 and facilitated translocation of FGF-2 to plasma membrane and extracellular matrix. IL-1 beta activated phosphatidylinositol (PI) 3-kinase, the enzyme activity of which was greatly stimulated after a 5-min exposure to IL-1 beta. This early and rapid activation of PI 3-kinase greatly enhanced FGF-2 production in CECs; pretreatment with LY294002 hampered the induction activity of IL-1 beta. These observations suggest that IL-1 beta takes part in endothelial to mesenchymal transformation of CECs through its inductive potential on FGF-2 via the action of PI 3-kinase.
Lee
Hyung Taek
HT
Doheny Eye Institute, Keck School of Medicine of the University of Southern California, Los Angeles, California 90033, USA.
Lee
Jeong Goo
JG
Na
Moonseok
M
Kay
EunDuck P
EP
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002867:Chromones / Q000494:pharmacology; D003315:Cornea / Q000378:metabolism*; D017077:Culture Media, Conditioned / Q000494:pharmacology; D003599:Cytoskeleton / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D004727:Endothelium / Q000378:metabolism*; D005109:Extracellular Matrix / Q000378:metabolism; D016222:Fibroblast Growth Factor 2 / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007249:Inflammation; D007375:Interleukin-1 / Q000378:metabolism*; D013058:Mass Spectrometry; D008648:Mesoderm / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism; D010641:Phenotype; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D040081:Protein Array Analysis; D020033:Protein Isoforms; D012313:RNA / Q000378:metabolism; D011817:Rabbits; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors; D014158:Transcription, Genetic; D014357:Trypsin / Q000494:pharmacology; D015854:Up-Regulation; D014945:Wound Healing
D000199:Actins; D002867:Chromones; D017077:Culture Media, Conditioned; D007375:Interleukin-1; D009025:Morpholines; D020033:Protein Isoforms; D016222:Fibroblast Growth Factor 2; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D012313:RNA; D019869:Phosphatidylinositol 3-Kinases; D014357:Trypsin