Detection and subcellular localization of two 15S-lipoxygenases in human cornea

Invest Ophthalmol Vis Sci. 2005 Mar;46(3):849-56. doi: 10.1167/iovs.04-1166.


Purpose: There are two human 15-lipoxygenases (LOX), 15-LOX-1 and -2, which convert arachidonic acid to 15S-hydroxyeicosatetraenoic acid (15S-HETE). The presence of both 15-LOXs in the human cornea prompted this study to delineate their roles in the human corneal epithelium.

Methods: Human corneal epithelia from donor corneas and a human corneal epithelial (HCE) cell line were used in [1-(14)C]arachidonic acid incubations, Western blot analysis, and quantitative real-time RT-PCR. Cell cultures of HCE were treated with 15S-HETE to measure its effect on cell growth. HCE cells were transfected with plasmids to express green fluorescent (GFP) fusion proteins of 15-LOX-1 and -2, and in vivo laser confocal microscopy was performed to determine the subcellular localization of the 15-LOX fusion proteins.

Results: [1-(14)C]Arachidonic acid incubations yielded 15S-HETE as the only LOX product. Treatment with 15S-HETE (5-10 microM) reduced growth rate and induced apoptosis in cultured HCE cells in a dose-dependent manner. 15-LOX-2 but not 15-LOX-1 was detected by Western blot analysis, although we were able to detect similar levels of both 15-LOX mRNAs by real-time quantitative RT-PCR. 15-LOX-1 and -2 proteins showed different subcellular expression patterns. 15-LOX-2 GFP was expressed in the cytoplasm and nucleus (actively taken up into the nucleus). 15-LOX-1 GFP fusion protein expression was restricted to the cytoplasm.

Conclusions: These findings indicate that 15-LOX-2 is the predominant 15-LOX protein in human cornea, and its product, 15S-HETE, plays a role in cellular proliferation. Because the two 15-LOXs have different subcellular compartmentalization, the authors hypothesize that their products are also compartmentalized and therefore exert different molecular effects in the human corneal epithelium.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Arachidonate 15-Lipoxygenase / analysis*
  • Arachidonate 15-Lipoxygenase / genetics
  • Arachidonic Acids / pharmacology
  • Blotting, Western
  • Cell Line
  • Cell Proliferation / drug effects
  • Cornea / enzymology*
  • Dose-Response Relationship, Drug
  • Epithelium, Corneal / enzymology
  • Humans
  • Hydroxyeicosatetraenoic Acids / metabolism
  • Hydroxyeicosatetraenoic Acids / pharmacology
  • Microscopy, Confocal
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Subcellular Fractions


  • Arachidonic Acids
  • Hydroxyeicosatetraenoic Acids
  • RNA, Messenger
  • 15-hydroxy-5,8,11,13-eicosatetraenoic acid
  • ALOX15B protein, human
  • Arachidonate 15-Lipoxygenase