April 2010
Volume 51, Issue 13
Free
ARVO Annual Meeting Abstract  |   April 2010
Hyperosmolarity Mediated Mitochondrial Dysfunction Requires Transglutaminase-2 in Human Corneal Epithelial Cells
Author Affiliations & Notes
  • E. Png
    Singapore Eye Research Institute, Singapore, Singapore
  • K. Samivelu
    Singapore Eye Research Institute, Singapore, Singapore
  • S. Yeo
    Singapore Eye Research Institute, Singapore, Singapore
  • J. Chew
    Singapore Eye Research Institute, Singapore, Singapore
  • S. S. Chaurasia
    Singapore Eye Research Institute, Singapore, Singapore
  • L. Tong
    Singapore Eye Research Institute, Singapore, Singapore
Investigative Ophthalmology & Visual Science April 2010, Vol.51, 5197. doi:
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      E. Png, K. Samivelu, S. Yeo, J. Chew, S. S. Chaurasia, L. Tong; Hyperosmolarity Mediated Mitochondrial Dysfunction Requires Transglutaminase-2 in Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):5197.

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

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Abstract

Purpose: : Hyperosmolar-induced ocular surface cell death is a key mitochondria-mediated event in inflammatory eye diseases such as dry eye. Transglutaminase-2 (TGM-2), a cross-linking enzyme, is purported to mediate cell death, but its link to mitochondria in hyperosmolar stress is unclear.We evaluated the role of TGM-2 and its involvement in hyperosmolarity-stimulated mitochondrial cell death in human corneal epithelial cells.

Methods: : Human corneal epithelial cell lines expressing either shRNA targeting TGM-2 or scrambled shRNA were constructed. Overexpression of TGM-2 was achieved by electoporating cells with cDNA plasmid coding for TGM-2. In medium supplemented with serum, sodium chloride was added to achieve hyperosmolarity (460 mOsm), and was confirmed by VAPOR Pressure Osmometer 5520 (Wescor, Utah, USA). Various techniques such as western blotting, mitochondrial membrane potential determination, real time cell proliferation monitoring, caspase activity determination and transglutaminase colorimetric microassay were employed.

Results: : Hyperosmolar conditions reduced viability and increased mitochondrial depolarization in scrambled shRNA transfected cells. However, hyperosmolarity failed to induce mitochondrial depolarization in TGM-2 silenced cells. TGM-2 silencing was also associated with increased proliferation. Transient overexpression of TGM-2 elevated transamidase activity and reduced viability. It also induced mitochondrial depolarization, increased caspase 3/7 and 9 activity, which was prevented by pan-caspase inhibitor Z-VAD-FMK.

Conclusions: : Hyperosmolar-stimulation in corneal epithelial apoptosis via caspase-dependent mitochondrial dysfunction is dependent on TGM-2. Protection against mitochondrial stress in the ocular surface targeting TGM-2 may have important implications in the treatment of inflammatory eye diseases.

Keywords: cornea: tears/tear film/dry eye • mitochondria • cell survival 
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