J Biol Chem. 1999 Feb 19;274(8):4764-9.  

Transfection of an active cytochrome P450 arachidonic acid epoxygenase indicates
that 14,15-epoxyeicosatrienoic acid functions as an intracellular second
messenger in response to epidermal growth factor.

Chen JK, Wang DW, Falck JR, Capdevila J, Harris RC.

Department of Medicine, Vanderbilt University, Nashville, Tennessee 37232, USA.

A common feature of most isolated cell systems is low or undetectable levels of
bioactive cytochrome P450. We therefore developed stable transfectants of the
renal epithelial cell line, LLCPKcl4, that expressed an active regio- and
enantioselective arachidonic acid (AA) epoxygenase. Site-specific mutagenesis
was used to convert bacterial P450 BM-3 into an active regio- and
stereoselective 14S,15R-epoxygenase (F87V BM-3). In clones expressing F87V BM-3
(F87V BM-3 cells), exogenous AA induced significant 14S,15R-epoxyeicosatrienoic
acid (EET) production (241. 82 ng/10(8) cells, >97% of total EETs), whereas no
detectable EETs were seen in cells transfected with vector alone. In F87V BM-3
cells, AA stimulated [3H]thymidine incorporation and increased cell
proliferation, which was blocked by the tyrosine kinase inhibitor, genistein, by
the phosphatidylinositol 3 (PI-3) kinase inhibitors, wortmannin and LY294002,
and by the mitogen-activated protein kinase kinase inhibitor, PD98059. AA also
induced tyrosine phosphorylation of extracellular signal-regulated kinase (ERK)
and PI-3 kinase that was inhibited by the cytochrome P450 BM-3 inhibitor,
17-ODYA. Epidermal growth factor (EGF) increased EET production in F87V BM-3
cells, which was completely abolished by pretreatment with either 17-ODYA or the
phospholipase A2 (PLA2) inhibitor, quinacrine. Compared with vector-transfected
cells, F87 BM-3 transfected cells demonstrated marked increases in both the
extent and sensitivity of DNA synthesis in response to EGF. These changes
occurred in the absence of significant differences in EGF receptor expression.
As seen with exogenous AA, EGF increased ERK tyrosine phosphorylation to a
significantly greater extent in F87V BM-3 cells than in vector-transfected
cells. Furthermore, in these control cells, neither 17-ODYA nor quinacrine
inhibited EGF-induced ERK tyrosine phosphorylation. On the other hand, in F87V
BM-3 cells, both inhibitors reduced ERK tyrosine phosphorylation to levels
indistinguishable from that seen in cells transfected with vector alone. These
studies provide the first unequivocal evidence for a role for the AA epoxygenase
pathway and endogenous EET synthesis in EGF-mediated signaling and mitogenesis
and provide compelling evidence for the PLA2-AA-EET pathway as an important
intracellular-signaling pathway in cells expressing high levels of cytochrome
P450 epoxygenase.

PMID: 9988714 [PubMed - indexed for MEDLINE]