Hyperosmolarity-induced apoptosis in human corneal epithelial cells is mediated by cytochrome c and MAPK pathways.
OBJECTIVE
To study whether hyperosmolarity induces apoptosis in human corneal epithelial cells through cytochrome c-mediated death pathways and by activation of mitogen-activated protein kinases (MAPKs).


METHODS
Primary human corneal epithelial cells cultured in normal osmolar media (312 mOsM) were switched to hyperosmolar media (450, 500, and 550 mOsM) by adding 70, 90, and 120 mM NaCl, respectively, with or without the c-jun N-terminal kinase (JNK) inhibitor SB202190 or the extracellular-regulated kinase (ERK) inhibitor PD98059. Apoptosis was assessed by the ApopTag In Situ Oligo Ligation (ISOL) assay. Confocal microscopy was used to detect cytochrome c and active caspase-3. Total RNA was extracted and subjected to reverse transcriptase-polymerase chain reaction for apoptosis-associated genes. Western blots were performed on cell extracts for the apoptogenic molecules cytochrome c and Smac/DIABLO, and phospho-JNK and ERK.


RESULTS
ISOL-positive apoptotic cells significantly increased from 3.3 +/- 1.6% in control medium to 11.4 +/- 5.8%, 18.9 +/- 4.8%, and 43.9 +/- 8.8% in 70, 90, and 120 mM NaCl added media, respectively. The 90 mM NaCl high saline medium notably increased release of cytochrome c and Smac/DIABLO from mitochondria; activated caspase-3, JNK and ERK; stimulated mRNA expression of interleukin-1-converting enzyme and Bax; and reduced Bcl2 expression. SB202190 and PD98059 significantly suppressed hyperosmolarity-induced JNK/ERK activation and ISOL-positive cells. In addition, PD98059 inhibited the release of cytochrome c and Smac/DIABLO from mitochondria.


CONCLUSIONS
These findings show that hyperosmolarity induces apoptosis of human corneal epithelial cells through a cytochrome c-mediated death pathway, which may be mediated by JNK and ERK MAPK signaling pathways.
Luo
Lihui
L
Ocular Surface Center, Cullen Eye Institute, Department of Ophthalmology, Baylor College of Medicine, Houston, TX 77030, USA.
Li
De-Quan
DQ
Pflugfelder
Stephen C
SC
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020170:Caspase 1 / Q000235:genetics; D053148:Caspase 3 / Q000378:metabolism; D018929:Cell Culture Techniques; D045304:Cytochromes c / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019573:Epithelium, Corneal / Q000201:enzymology / Q000473:pathology*; D005419:Flavonoids / Q000494:pharmacology; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D007223:Infant; D020935:MAP Kinase Signaling System; D018613:Microscopy, Confocal; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009994:Osmolar Concentration; D011725:Pyridines / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012462:Saline Solution, Hypertonic / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein / Q000235:genetics
C494826:BAX protein, human; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D011725:Pyridines; D012333:RNA, Messenger; D012462:Saline Solution, Hypertonic; D051028:bcl-2-Associated X Protein; D045304:Cytochromes c; D020928:Mitogen-Activated Protein Kinases; D053148:Caspase 3; D020170:Caspase 1; C090942:4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)imidazole; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one