May 2004
Volume 45, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2004
Hyperosmolarity induces apoptosis through a cytochrome c–mediated death pathway and MAPK activation in human corneal epithelial cells
Author Affiliations & Notes
  • L. Luo
    Ophthalmology, Baylor College of Medicine, Houston, TX
  • D.–Q. Li
    Ophthalmology, Baylor College of Medicine, Houston, TX
  • S.C. Pflugfelder
    Ophthalmology, Baylor College of Medicine, Houston, TX
  • Footnotes
    Commercial Relationships  L. Luo, None; D. Li, None; S.C. Pflugfelder, None.
  • Footnotes
    Support  NIH Grant EY11915 (SCP), an unrestricted Grant from Research to Prevent Blindness
Investigative Ophthalmology & Visual Science May 2004, Vol.45, 3758. doi:
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      L. Luo, D.–Q. Li, S.C. Pflugfelder; Hyperosmolarity induces apoptosis through a cytochrome c–mediated death pathway and MAPK activation in human corneal epithelial cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3758.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: To investigate whether hyperosmolarity induces apoptosis through a cytochrome c–mediated death pathway and activation of mitogen–activated protein kinase (MAPK) signaling pathways in human corneal epithelial cells. Methods: Primary human corneal epithelial cells cultured in normal osmolar media (312 mOsM) were switched to a high salt media (150–200 mOsM higher osmolarity), with or without SB202190, an inhibitor of c–jun N–terminal kinase (JNK), PD98059, an inhibitor of extracellular–regulated kinase (ERK), or doxycycline for 15–60 min and 24 hours. Cell apoptosis was assessed by nuclear morphology with Hoechst 33342 staining and ApopTag® In Situ Oligo Ligation (ISOL) assay. Immunofluorescent staining and confocal microscopy with mitotracker probes were performed to detect caspase 3 and cytochrome c. Western blots were performed on cell extracts lysed in RIPA buffer for phospho–MAPK or in cytosolic fractions for the apoptogenic molecules cytochrome c and Smac/DIABLO. Total RNA was extracted and subjected to semiquantitative RT–PCR and gene array for apoptosis–related genes. Results: Compared with control, hyperosmolarity significantly induced cell apoptosis evidenced by ISOL(18.9 ± 4.8 v.s 3.3 ± 1.6%, P<0.001) and condensed nuclear detected by Hoechst staining with increased release of cytochrome c and Smac/DIABLO from mitochondria, activated caspase 3, suppressed bcl2/bax ratio, stimulated expression of interleukin–1ß converting enzyme (ICE) and other apoptosis related genes including caspase 2, 8, p53, bak and fas. Hyperosmolarity strongly activated JNK and ERK, while SB202190 and PD98059 suppressed JNK and ERK activation, respectively, and also inhibited hyperosmolarity–induced apoptosis, caspase 3 production, and expression of ICE, caspase 2, 8, p53,and bak, fas mRNA. PD98059 inhibited the release of cytochrome c and Smac/DIABLO. Interestingly, doxycycline markedly inhibited hyperosmolarity–induced apoptosis by inhibiting cytochrome c and Smac/DIABLO release, caspase 3 production, and expression of ICE, caspase 2, 8, p53, bax, bak and fas mRNA. Conclusions: These findings demonstrate for the first time that hyperosmolarity induces apoptosis through a cytochrome c–mediated death pathway in human corneal epithelial cells which may be involved in the pathogenesis of dry eye. This apoptosis may be mediated by JNK and ERK MAPK signaling pathways and could be inhibited by MAPK inhibitors and doxycycline.

Keywords: cornea: epithelium • apoptosis/cell death • signal transduction: pharmacology/physiology 
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