Detection and subcellular localization of two 15S-lipoxygenases in human cornea.
OBJECTIVE
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.
Chang
Min S
MS
Department of Ophthalmology and Visual Sciences, Vanderbilt Eye Institute, 8000 MCE, Vanderbilt University Medical Center, Nashville, TN 37232, USA. min.chang@vanderbilt.edu
Schneider
Claus
C
Roberts
Richard L
RL
Shappell
Scott B
SB
Haselton
Fredrick R
FR
Boeglin
William E
WE
Brash
Alan R
AR
D001093:Arachidonate 15-Lipoxygenase / Q000032:analysis* / Q000235:genetics; D001095:Arachidonic Acids / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D003315:Cornea / Q000201:enzymology*; D004305:Dose-Response Relationship, Drug; D019573:Epithelium, Corneal / Q000201:enzymology; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism / Q000494:pharmacology; D018613:Microscopy, Confocal; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013347:Subcellular Fractions
D001095:Arachidonic Acids; D006893:Hydroxyeicosatetraenoic Acids; D012333:RNA, Messenger; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; C458528:ALOX15B protein, human; D001093:Arachidonate 15-Lipoxygenase