May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Distinct Sub-cellular Localization of 15-lipoxygenase-1 and -2 in Human Corneal Epithelial Cells
Author Affiliations & Notes
  • M.S. Chang
    Department of Ophthalmology, Vanderbilt University, Nashville, TN, United States
  • C. Schneider
    Department of Pharmacology, Vanderbilt University, Nashville, TN, United States
  • R.L. Roberts
    Department of Pathology, Vanderbilt University, Nashville, TN, United States
  • F.R. Haselton
    Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, United States
  • A.R. Brash
    Department of Biomedical Engineering, Vanderbilt University, Nashville, TN, United States
  • Footnotes
    Commercial Relationships  M.S. Chang, None; C. Schneider, None; R.L. Roberts, None; F.R. Haselton, None; A.R. Brash, None.
  • Footnotes
    Support  5K08EY013592-02
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 1336. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      M.S. Chang, C. Schneider, R.L. Roberts, F.R. Haselton, A.R. Brash; Distinct Sub-cellular Localization of 15-lipoxygenase-1 and -2 in Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):1336.

      Download citation file:


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

      ×
  • Supplements
Abstract

Abstract: : Purpose: 15-Lipoxygenase (15-LOX) has been implicated in cellular proliferation and differentiation. There are two 15-LOX enzymes in humans, 15-LOX-1 and 15-LOX-2, and both are expressed in corneal epithelial cells. The two lipoxygenases share only 35-40% amino acid identity, yet both produce 15(S)-hydroxy-eicosatetraenoic acid (15(S)-HETE) from arachidonic acid. In this study we investigated how the expression of these two enzymes is coordinated within the cell to mediate their physiologic actions. Methods: Full-length cDNA for human 15-LOX-1 and -2 was subcloned into the pEGFP vector (Clontech) to express GFP-15-LOX-1 and -2 fusion proteins. The plasmids were transfected separately into SV40- immortalized human corneal epithelial cells using Lipofectamine 2000 (Invitrogen). After 18 to 24 hours, the transfected cells were viewed by confocal microscopy. Time-lapsed phase contrast microscopy was also performed over an 8 to 12 hour period. Results: The subcellular expression of GFP-15-LOX-1 is diffuse and occurs only in the cytoplasm, while GFP-15-LOX-2 is expressed both in the cytoplasm and in the nucleus. By fluorescence recovery after photo-bleaching (FRAP), the GFP-15-LOX-2 appears to be actively accumulated in the nucleus. By time-lapsed phase contrast microscopy, the cells expressing the GFP-LOX proteins appear to undergo blebbing followed by cell death. The specificity of this response to overexpression of lipoxygenase is unclear. Conclusions: These results demonstrate that 15-LOX-1 and 15-LOX-2 have different subcellular localization indicating compartmentalization of 15S-HETE produced by the two 15-lipoxygenases. This suggests distinct physiologic roles for the two enzymes in human corneal epithelium despite their similar enzymatic activity.

Keywords: cornea: epithelium • eicosanoids • cornea: basic science 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×