Purpose: 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.