Decreased plasminogen activator inhibitor-1 secretion in hypoxic corneal epithelial cells is associated with increased urokinase plasminogen activator activity.
The aim of this study was to determine the effects of hypoxia on mRNA levels, cell-associated and -secreted protein concentration, activity, and protein complex formation of urokinase-type plasminogen activator, its receptor, and plasminogen activator inhibitor type-1 in corneal epithelium. Non-transformed human corneal epithelial cells were cultured in 20% oxygen (normoxic conditions) or 2% oxygen (hypoxic conditions) for 1, 3, 5, or 7 days. Relative changes in mRNA levels of plasminogen activator, receptor, and plasminogen activator inhibitor-1 were determined using a cDNA expression array, chemiluminescence, and densitometry. Protein concentrations were determined using enzyme linked immunosorbent assays. Activity assays were also used. Protein complex formation was assayed using cell surface biotinylation, immunoprecipitation, and Western blot analysis. Hypoxic corneal epithelial cells demonstrated no significant differences in plasminogen activator or receptor mRNA. Cell-associated plasminogen activator and membrane-associated receptor protein levels were unchanged. In contrast decreases in mRNA and secreted plasminogen activator inhibitor-1 protein were observed in hypoxic cells. Concurrently, increased cell-associated plasminogen activator activity was observed in hypoxic cells. The formation of plasminogen activator/receptor/plasminogen activator inhibitor-1 complex at the cell surface was not inhibited by hypoxia. However, in hypoxic cells less plasminogen activator inhibitor-1 was associated with receptor. It is concluded that in corneal epithelium cultured in 2% oxygen plasminogen activator inhibitor-1 may be an important regulatory factor of the plasminogen activator system resulting in increased urokinase plasminogen activator activity.
Wang
Zhiyu
Z
Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 8263 Scott Hall, 540 E. Canfield, Detroit, MI 48201, USA.
Kurpakus-Wheater
Michelle
M
D019921:Biotinylation; D015153:Blotting, Western; D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000378:metabolism*; D018076:DNA, Complementary / Q000378:metabolism; D003720:Densitometry; D015536:Down-Regulation; D004797:Enzyme-Linked Immunosorbent Assay; D004847:Epithelial Cells / Q000378:metabolism*; D005780:Gelatin / Q000494:pharmacology; D006801:Humans; D000860:Hypoxia; D020411:Oligonucleotide Array Sequence Analysis; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism*
D018076:DNA, Complementary; D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D005780:Gelatin; D014568:Urokinase-Type Plasminogen Activator