June 1996
Volume 37, Issue 7
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Articles  |   June 1996
A role for 12(S)-HETE in the response of human lens epithelial cells to epidermal growth factor and insulin.
Author Affiliations
  • J K Arora
    National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2730, USA.
  • T W Lysz
    National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2730, USA.
  • P S Zelenka
    National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892-2730, USA.
Investigative Ophthalmology & Visual Science June 1996, Vol.37, 1411-1418. doi:
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    • Get Citation

      J K Arora, T W Lysz, P S Zelenka; A role for 12(S)-HETE in the response of human lens epithelial cells to epidermal growth factor and insulin.. Invest. Ophthalmol. Vis. Sci. 1996;37(7):1411-1418.

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

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Abstract

PURPOSE: 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.

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