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Arachidonate 12-lipoxygenase in the platelet was the first mammalian lipoxygen
ase discovered. It catalyzes the transformation of arachidonic acid into 12(S
)-hydroperoxyeicosatetraenoic acid, which is subsequently converted to 12-(S)-
hydroxyeicosatetraenoic acid (12(S)-HETE) by a glutathione-dependent peroxide.
Epidermal growth factor (EGF) increases 12-lipoxygenase mRNA level by about
2-fold with a lag period of 4 to 8 h, which precedes the increase in 12-lipoxy
genase activity by 2 to 4 h in human epidermoid carcinoma A431 cells, and EGF-
induced 12-lipoxygenase expression is in part mediated by the protein kinase C
(PKC) signal transduction pathway. The present report in this paper describ
es that in addition to the involvement of PKC pathway, the possible role of Ra
s signal pathway in the EGF-induced expression of 12-lipoxygenase in A431 cell
s.The EGF-induced expression of 12-lipoxygenase mRNA was inhibited by transcri
ption inhibitors: actinomycin D and 5, 6-dichlorobenzimidazole riboside, and
the degradation rate of 12-lipoxygenase mRNA was no difference between EGF-tre
ated and control cells. GF109203X and calphostin C, which are two PKC inhibit
ors, inhibited PMA-induced enzyme activities of 12-lipoxygenase and mitogen-ac
tivated protein (MAP) kinase. Calphostin C inhibited EGF-induced 12-lipoxygen
ase activity and slightly inhibited EGF-induced MAP kinase activity. GF109203
X did not affect EGF-induced 12-lipoxygenase and MAP kinase activities. T
hese results indicate that other signal transduction pathways may be involved
in EGF-induced expression of 12-lipoxygenase in addition to the PKC pathway.Th
e possible involvement of Ras signalling in EGF-induced expression of 12-lipo
xygenase was studied. A431 cells were transiently cotransfected with lucifera
se vectors bearing different lengths of promoter of human 12-lipoxygenase and
pSV2ras with reduced GTPase activity. Analysis of a series of deletion constr
ucts with 5'-flanking region before -175 bp in the upstream from translation s
tarting site showed a 40-fold increase in luciferase activity stimulated by pS
V2ras. It was remarkably reduced to half (20-fold) when the sequence was dele
ted to -145 bp, and in SPM4, SPM5, and SPM45 in which Sp1 sites were point-mut
ated between -169 and -150 bp. No luciferase activity induced by pSV2ras was
observed in SPM6, SPM9, SPM8 and SPM7 in which the Sp1 site between -145 and -
100 bp was directly mutated. Taken together, the present results indicate tha
t Ras signal pathway activated the transcription of 12-lipoxygenase gene and t
he Sp1 played a pivotal role in the up-regulation of 12-lipoxygenase gene in H
a-ras-transfected A431 cells.
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