ACTIVATION OF NUCLEAR FACTOR KAPPA-B AND ONCOGENE EXPRESSION BY 12(R)-HYDROXYEICOSATRIENOIC ACID, AN ANGIOGENIC FACTOR IN MICROVESSEL ENDOTHELIAL-CELLS

12(R)-Hydroxy-5,8,14(Z,Z,Z)-eicosatrienoic acid (12(R)-HETrE) is an ar achidonic acid metabolite formed by the corneal epithelium of several species, porcine leukocytes, and human and rat epidermal cells. It is a potent, stereospecific proinflammatory and angiogenic factor and its synthesis is increased manyfold in inflamed tissues, e.g. cornea and skin. It is possible that the angiogenic activity of 12(R)-HETrE is du e to a direct mitogenic effect on microvessel endothelial cells via ye t to be elucidated cellular and molecular mechanisms. In the present s tudy, we demonstrated the ability of 12(R)-HETrE to stimulate the grow th of quiescent endothelial cells in a time- and concentration-depende nt manner with a maximal effect at 0.1 nM. This effect was highly ster eospecific since its enantiomer, 12(S)-HETrE, had no effect within the same concentration range. Northern blot analysis and transient transf ection experiments with chloramphenicol acetyltransferase constructs o f oncogene promoter regions demonstrated significant increases over co ntrol (0.5% fetal calf serum) in c-myc, c-jun, and c-fos mRNA levels a nd expression in cells treated with 0.1 nM 12(R)-HETrE. Electrophoreti c mobility shift assay of nuclear protein extracts from cells treated with 12(R)-HETrE with specific radiolabeled oligonucleotides correspon ding to known transcriptional binding sites, including AP-1, AP-2, SP1 , TRE, NF kappa B, TFIID, OKT1, CREB, CTF/NF1, and GRE demonstrated a markedly rapid and specific increase in the binding activity of NF kap pa B and to a lesser extent, AP-1. No significant increase was observe d in the binding of other transcription factors assayed as compared to control (untreated) cells. Since the protooncogenes (c-fos, c-jun, an d c-myc) are immediate early response genes that are implicated in the process of cell proliferation and differentiation, and activation of certain transcription factors, in particular NF kappa B, is associated with the immediate response of the cell to an injury, we propose that 12(R)HETrE's mitogenic and angiogenic activities are mediated, in par t, via the activation of NF kappa B and expression of these protooncog enes,