Caspase-9 activation in hypoxic human corneal epithelial cells.
The purpose of this study was to determine the effect of hypoxia on caspase-8 and -9 gene and protein expression and activity in corneal epithelium. Non-transformed human corneal epithelial cells (HCEC) were cultured in 2% oxygen. A cDNA expression array coupled with densitometric analysis was used to compare relative mRNA expression levels of 96 apoptosis-related genes in hypoxic and normoxic HCEC. Caspase-8, caspase-9, FLIP, Fas, FasL, and TNFalpha protein expression was assessed further using Western blot analysis and ELISA. Caspase-8 and -9 activities were measured using a fluorometric activity assay. Hypoxia did not affect caspase-8 or -9 gene or protein expression in HCEC, however caspase-9 activity was significantly increased. Hypoxia significantly suppressed the activity of caspase-8. FLIP and Fas gene and protein expression were not significantly altered in hypoxic cells compared to normoxic controls. mRNA and protein levels of TNFalpha and TNFR-1 were significantly decreased, while FasL mRNA and proteins levels were significantly increased in hypoxic HCEC. In corneal epithelium stressed by hypoxia caspase-9 activity is upregulated, suggesting that apoptosis proceeds via the mitochondrial pathway. Caspase-8 activity may be suppressed because the loss of TNFalpha and TNFR-1 gene and protein expression inhibits the initial formation of a death signaling complex.
Kurpakus-Wheater
M
M
Department of Anatomy and Cell Biology, Kresge Eye Institute, Wayne State University School of Medicine, Detroit, Michigan, USA. mkurpaku@med.wayne.edu
Sexton
R
R
McDermott
M L
ML
Mrock
L K
LK
Sosne
G
G
D017209:Apoptosis / Q000502:physiology; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000235:genetics / Q000378:metabolism*; D003315:Cornea / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D000860:Hypoxia / Q000378:metabolism*
C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases