A role for 12(S)-HETE in the response of human lens epithelial cells to epidermal growth factor and insulin.
OBJECTIVE
To determine whether the 12-lipoxygenase pathway of arachidonic acid metabolism is present in the human lens and whether 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) plays a role in regulating proto-oncogene expression and DNA synthesis in human lens epithelial cells (HLECs).


METHODS
Second- and third-passage primary cultures of HLECs were used for analysis. Human cataract epithelia were obtained from surgery. 12-lipoxygenase mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR), and the PCR product was sequenced. The 12-lipoxygenase protein was detected by immunoblotting. 12(S)-HETE was detected in HLEC-conditioned medium by radioimmunoassay. For studies of growth factor-induced mitogenesis, HLECs were serum starved, then stimulated with 15 ng/ml epidermal growth factor (EGF) and 1 microgram/ml insulin or with 0.3 microM 12(S)-HETE. The 12-lipoxygenase inhibitor, cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate (CDC, 10 microM) was used to block endogenous 12-lipoxygenase activity. Expression of c-fos mRNA was determined by RT-PCR, and DNA synthesis was measured by 3H-thymidine incorporation.


RESULTS
12-lipoxygenase mRNA and protein were detected in HLECs and in human cataract tissues. 12(S)-HETE was released into the medium by HLECs in the presence of EGF-insulin. Stimulation of c-fos mRNA expression and DNA synthesis by EGF-insulin was inhibited when the 12-lipoxygenase pathway was blocked by CDC. This inhibition was reversed completely by exogenously added 12(S)-HETE. However, exogenous 12(S)-HETE was unable to stimulate HLEC DNA synthesis in the absence of growth factors.


CONCLUSIONS
The 12-lipoxygenase pathway of arachidonic acid metabolism is present in human lens epithelial cells. 12(S)-HETE does not stimulate HLEC DNA synthesis in the absence of growth factors but enables the cellular response to EGF and insulin.
Arora
J K
JK
National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2730, USA.
Lysz
T W
TW
Zelenka
P S
PS
D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D000328:Adult; D001092:Arachidonate 12-Lipoxygenase / Q000378:metabolism; D001483:Base Sequence; D002109:Caffeic Acids / Q000494:pharmacology; D002386:Cataract / Q000188:drug therapy / Q000378:metabolism; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D017931:DNA Primers / Q000737:chemistry; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004848:Epithelium / Q000187:drug effects / Q000502:physiology; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000494:pharmacology / Q000502:physiology*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007223:Infant; D007328:Insulin / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D016859:Lipoxygenase Inhibitors / Q000494:pharmacology; D008875:Middle Aged; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D000090063:Proto-Oncogene Mas; D016760:Proto-Oncogene Proteins c-fos / Q000096:biosynthesis; D012313:RNA / Q000302:isolation & purification; D012333:RNA, Messenger / Q000378:metabolism; D011863:Radioimmunoassay; D014158:Transcription, Genetic
D002109:Caffeic Acids; D017931:DNA Primers; D006893:Hydroxyeicosatetraenoic Acids; D007004:Hypoglycemic Agents; D007328:Insulin; D016859:Lipoxygenase Inhibitors; C000715470:MAS1 protein, human; D000090063:Proto-Oncogene Mas; D016760:Proto-Oncogene Proteins c-fos; D012333:RNA, Messenger; C099255:cinnamyl-3,4-dihydroxycyanocinnamate; D019377:12-Hydroxy-5,8,10,14-eicosatetraenoic Acid; D004815:Epidermal Growth Factor; D012313:RNA; D004247:DNA; D001092:Arachidonate 12-Lipoxygenase