May 2007
Volume 48, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2007
UVB-Stimulated Apoptosis in Human Corneal Epithelial Cells Is Promoted by the Multi-Functional BH3-Only Protein Transglutaminase-2
Author Affiliations & Notes
  • L. M. Tong
    Singapore National Eye Center, Singapore, Singapore
  • L. Zhou
    Singapore Eye Research Institute, Singapore, Singapore
  • Y.-W. Wong
    Singapore Eye Research Institute, Singapore, Singapore
  • Z. Chen
    Ocular Surface Center, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas
  • C. De Paiva
    Ocular Surface Center, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas
  • D.-Q. Li
    Ocular Surface Center, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas
  • S. C. Pflugfelder
    Ocular Surface Center, Cullen Eye Institute, Baylor College of Medicine, Houston, Texas
  • R. W. Beuerman
    Singapore Eye Research Institute, Singapore, Singapore
  • Footnotes
    Commercial Relationships L.M. Tong, None; L. Zhou, None; Y. Wong, None; Z. Chen, None; C. De Paiva, None; D. Li, None; S.C. Pflugfelder, None; R.W. Beuerman, None.
  • Footnotes
    Support NIH Grants, EY11915 (SCP) and EY014553 (DQL), a fellowship from Agency for Science Technology and Research, Singapore, an unrestricted Grant from Research to Prevent Blindness, the Oshman Foundation
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 546. doi:
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      L. M. Tong, L. Zhou, Y.-W. Wong, Z. Chen, C. De Paiva, D.-Q. Li, S. C. Pflugfelder, R. W. Beuerman; UVB-Stimulated Apoptosis in Human Corneal Epithelial Cells Is Promoted by the Multi-Functional BH3-Only Protein Transglutaminase-2. Invest. Ophthalmol. Vis. Sci. 2007;48(13):546.

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

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Abstract

Purpose:: Apoptosis in the ocular surface plays an important role in diseases such as dry eye. Transglutaminase (TGM)-2 belongs to the class of BH3-only pro-apoptotic proteins that senses external stress and feeds into the mitochondria apoptotic pathway. This study investigates the characteristics of human corneal epithelial cell death induced by ultraviolet radiation (UVB) and the possible roles of TGM-2.

Methods:: Human corneal epithelial cells cultured from limbal explants were exposed to a single dose of UVB radiation (20 mJ/cm2). Electron microscopy, MTT and TUNEL assays were performed. Fluorescent-cadaverine uptake was used to evaluate transamidation activity. Propidium iodide stained nuclear morphology was evaluated by digital imaging. Short interfering (si) RNA against TGM-2 was used to reduce expression of TGM-2. Changes in TNFR-I distribution and caspase-3 activation were evaluated at various times after UVB. Caspase-3 activation was detected by an antibody that detected active caspase. Intracellular delivery of exogenous TGM-2 was performed using a peptide carrier system (Chariot). TGM-2 was immunoprecipitated, and elutes analysed by nano-LC MS/MS.

Results:: Corneal epithelial cell viability was reduced after UVB, and nuclear chromatin condensation was detected on microscopy. UVB up-regulated TGM-2 transcript and protein levels, as well as transamidase activity. Monodansyl-cadaverine (MDC), a cell permeable competitive inhibitior of TGM, was able to reduce the UVB-induced cell death. Transfection with siRNA significantly reduced basal and UVB-stimulated TGM transcript levels. Such transfection or pre-incubation with MDC reduced caspase activation after UVB. Cell surface clustering of TNF Receptor-I was evident after UVB, and silencing of TGM-2 or addition of MDC inhibited this effect. Delivery of exogenous TGM-2 to cells increased caspase activation. Nuclear shapes were more irregular and ovoid after UVB compared to control, and incubation with MDC reduced the UVB-induced changes in nuclear morphology. Mitochondrial ATP synthase and ADP/ATP translocases were found to bind TGM-2.

Conclusions:: UVB-stimulated corneal epithelial cell death shows features of apoptosis, and UVB-stimulated TGM-2 up-regulation is pro-apoptotic. TGM-2 regulates key stages of apoptosis. Modulation of TGM-2 pathways to target apoptosis may be beneficial in dry eye.

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