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*

Jian-Kang ChenDagger , Dao-Wen WangDagger , John R. Falck§, Jorge CapdevilaDagger , and and Raymond C. HarrisDagger parallel

From the Departments of Dagger  Medicine and  Biochemistry, Vanderbilt University, Nashville, Tennessee 37232 and the § Department of Biochemistry, University of Texas Southwestern, Dallas, Texas 75235

    ABSTRACT
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Abstract
Introduction
References

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/108 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.

    INTRODUCTION
Top
Abstract
Introduction
References

Release of arachidonic acid secondary to phospholipase A2 activation is a well recognized cellular response to a variety of growth factors, hormones, and cytokines (1, 2). Metabolites of arachidonic acid play important roles as intracellular second messengers, with well documented autocrine effects of cyclooxygenase and lipoxygenase metabolites (1, 3-5). There is now abundant suggestive evidence that arachidonate metabolites generated by cytochrome P450, the third pathway of arachidonic acid metabolism, may also serve as second messengers (6). These compounds have been implicated in the regulation of peptide secretion, distal nephron Na+ fluxes, cell Ca2+ influx, and activation of Ca2+-dependent K+ channels (6). However, previous studies have relied upon the use of inhibitors of limited selectivity for cP450 or upon the exogenous administration of relatively high concentrations of cP450 arachidonate metabolites; in addition, a significant limitation to studies of EET1 mechanisms and sites of actions is the well documented loss and redistribution of cP450 isoforms that occur soon after the initiation of cell culture. To overcome these limitations, we have used stable transfection of a mammalian proximal tubule-like cell line, LLCPKcl4, with F87V BM-3, an active and regio- and stereoselective 14S,15R arachidonic acid expoxygenase of bacterial origin containing a cP450 domain fused to a cP450 reductase domain (7). We report studies of the mechanism of EET-dependent EGF-mediated mitogenesis and provide unequivocal evidence that in these cells, cP450 arachidonic acid metabolite production increases in response to agonist activation, and cP450 arachidonic acid metabolites can serve as intracellular second messengers to mediate signal transduction and functional responses.

    EXPERIMENTAL PROCEDURES

Antibodies and Chemicals-- Polyclonal rabbit anti-BM-3 antibodies were purified using protein G chromatography (7). Polyclonal and monoclonal anti-phosphotyrosine antibodies were purchased from (San Francisco, CA). Monoclonal anti-PI-3 kinase antibody and anti-EGF receptor antibody were from Transduction Laboratories (Lexington, KY). Polyclonal anti-ERK antibodies and protein A-agarose beads were from Santa Cruz Biotechnology (Santa Cruz, CA). Arachidonic acid was obtained from NuCheck-Prep, Inc. (Elysian, MN). The sulfonimide analog of 14,15-EET was synthesized as described previously (8). EGF (receptor grade) was purchased from Collaborative Research (Bedford, MA). Genistein, wortmannin, PD98059, and quinacrine were from Calbiochem. 17-ODYA was from Biomol (Plymouth Meeting, PA). All other chemicals were from Sigma.

Cell Culture-- LLCPKcl4, an established proximal tubule epithelial cell line derived from pig kidney (9), was routinely cultured in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal calf serum (Hyclone Laboratories, Logan, UT) at 37 °C in a 5% CO2 incubator. The medium was changed every 2-3 days.

Stable Transfection of Mutant BM-3-- The entire coding region of mutant BM-3 (F87V BM-3) cDNA was cloned into the HindIII and XbaI sites in the mammalian expression vector pCB6+. 1 µg/ml F87V BM-3 cDNA in pCB6+ or empty pCB6+ vector alone was used for stable transfection. Cultured LLCPKcl4 cells were grown to 80% confluence and were transfected using LipofectAMINE (Life Technologies, Inc.). After 6 h, the DNA-LipofectAMINE mixture was removed and the cells incubated in medium containing 10% fetal calf serum for 24 h, then cultured for 7 passages in medium containing 600 µg/ml of G418. G418-resistant clones were then isolated and screened by immunoblotting with the affinity purified polyclonal antibody against BM-3. Two of the clones that expressed detectable levels of immunoreactive F87V BM-3, C9, and C23 (Fig. 1A), along with LLCPKcl4 cells transfected with vector alone, were used for subsequent functional studies.

Immunoprecipitation and Western Blot Analysis-- Quiescent LLCPKcl4 cells were treated with arachidonic acid and/or other drugs as indicated, then washed twice with ice-cold Ca2+/Mg2+-free phosphate-buffered saline and lysed on ice for 30 min with radioimmune precipitation buffer (8). Cell lysates were clarified at 10,000 × g for 15 min at 4 °C, and protein concentrations were determined by the bicinchoninic acid assay (Pierce). Tyrosine-phosphorylated proteins were immunoprecipitated with anti-phosphotyrosine antibodies, immune complexes captured with protein A-agarose beads, washed four times with wash buffer (20 mM HEPES, pH 7.2, 100 mM NaCl, 0.1% Triton X-100, 10% glycerol, and 100 µM Na3VO4), and eluted by boiling in sample buffer.

Total cell lysates or anti-phosphotyrosine immunoprecipitates, as indicated in the corresponding data, were subjected to 7.5-15% SDS-polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride membranes, probed with the indicated primary antibody and the appropriate secondary antibody conjugated with biotin, incubated with preformed avidin-biotin-horseradish peroxidase complex using a commercially available kit (ABC kit; Pierce), and the immune complexes detected by a peroxidase-catalyzed enhanced chemiluminescence detection system (ECL; Amersham, United Kingdom).

Endogenous EET Production Measurement and Stereochemical Analysis-- Quiescent C9, C23, and empty vector-transfected cells were treated with or without 30 µM arachidonic acid, 10 nM EGF, and/or indicated inhibitors for the indicated times, scraped with a 1:1 mixture of media and CH3OH, and then mixed with two volumes of CHCl3 containing 1 mM triphenylphosphine, and an equimolar mixture of synthetic 14C-labeled 8,9-, 11,12-, and 14,15-EET (55-56 mCi/mmol, 30 ng each). After acidification, the samples were extracted twice and the organic phases evaporated under argon. To the resulting residue, 0.5 ml of 0.4 N KOH in 80% CH3OH was added and the mixture incubated at 50 °C for 60 min. Acidification was followed by extraction into ethyl ether and chromatography in SiO2 as described (10). The EETs were resolved into 14,15-EET, and a mixture of 8,9- and 11,12-EET by reversed-phase high-pressure liquid chromatography, and then derivatized to the corresponding pentafluorobenzyl (PFB) esters by reaction with pentafluorobenzyl bromine. Aliquots of the purified EET-PFB regioisomers were individually dissolved in dodecane and analyzed and quantified by NICI/gas chromatography/mass spectrometry, utilizing CH4 as reagent gas, as described (10).

For stereochemical analysis, samples of enzymatically derived [14C]14,15-epoxyeicosatrienoic acid (25 µg, 1 µCi/µmol) and of synthetic 14R,15S-EET and 14S,15R-EET were catalytically hydrogenated over PtO2 and esterified using excess pentafluorobenzyl bromine, as described (10). The resulting PFB esters were purified by reversed-phase high-pressure liquid chromatography and, after solvent evaporation, the optical antipodes of the purified PFB-14,15-epoxyeicosatrienoic acid were resolved by high-pressure liquid chromatography on a Chiralcel OD column (4.6 × 250 mm) (J.T. Baker Inc.) with an isocratic mixture of 0.11% isopropanol, 99.89% n-hexane at 1 ml/min with UV monitoring at 210 nm. The retention times for the PFB esters of synthetic 14R,15S- and 14S,15R-EET were 70.6 and 78.9 min, respectively (7).

[3H]Thymidine Incorporation Assay-- Subconfluent cells in 24-well plates were made quiescent with serum-free medium. Agonists and antagonists were routinely added to the quiescent cells in triplicate and incubated for 19 h, followed by addition of 2 µCi/ml of [3H]thymidine to pulse the cells for an additional 2 h. Cells were then washed four times with ice-cold phosphate-buffered saline, precipitated twice (30 min each time on ice) with ice-cold 10% trichloroacetic acid, briefly washed once with ice-cold ethanol, then lysed with 0.2 N NaOH, 0.5% SDS lysis buffer, incubated at 37 °C for at least 30 min, and radioactivity of incorporated [3H]thymidine was determined by liquid scintillation spectrometry (Beckman). Results were plotted as the number of counts/min/well. Each experimental data point represents triplicate or duplicate wells from at least four different experiments. For determination of cell proliferation, cells were plated in replicate dishes at a density of 105 cells in 60-mm dishes in Dulbecco's modified Eagle's/F12 + 10% fetal calf serum. 24 h later, the medium was changed to Dulbecco's modified Eagle's/F12 + 0.2% fetal calf serum and 5 µM arachidonic acid.

Statistics-- Data are presented as means ± S.E. for at least four separate experiments (each in triplicate or duplicate). Unpaired Student's t test was used for statistical analysis and for multiple group comparisons, analysis of variance and Bonferroni t tests were used. A value of p < 0.05 compared with control was considered statistically significant.

    RESULTS

Previous studies indicated that synthetic 14,15-EET was a potent mitogen for renal epithelial cells and that the mitogenic effects of this EET were mediated, at least in part, by Src kinase and the initiation of a tyrosine kinase phosphorylation cascade (8). To reproduce more physiologic conditions in which the putative lipid mediator is synthesized endogenously from endogenous AA pools, we have used a novel strategy to investigate the role of the 14,15-AA epoxygenase in endogenous EET production and cell signaling. LLCPKcl4 cells, a well characterized proximal tubule cell line with undetectable endogenous cP450 expression levels, were stably transfected with a mutant form of bacterial cP450 BM-3. cP450 BM-3, isolated from Bacillus megaterium, is a single polypeptide containing fused cP450 and cP450 reductase domains that catalyzes NADPH-dependent AA oxidation as a self-contained catalytic unit (7). Replacement of phenylalanine 87 for valine converts this protein into a regio- and stereoselective 14S,15R-epoxygenase (14S,15R-EET, 99% of total products, 98% optical purity) (7). We identified two clones, C9 and C23, that expressed immunoreactive F87V BM-3 proteins (Fig. 1A).


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Fig. 1.   A, expression of F87V BM-3 protein in the transfected LLCPKcl4 cells. LLCPKcl4 cells were transfected with the entire coding region of F87V BM-3 cDNA using LipofectAMINE (Life Technologies, Inc.). Sixty-three G418-resistant clones were isolated and screened by Western immunoblotting with the affinity purified polyclonal antibody against BM-3. C9, C21, C22, C23, C24, and C25 were representative G418-resistant clones of these isolated 63 clones. M was the pooled G418-resistant multiple clone cells used for selecting single-cell clones. B, EET production following administration of exogenous arachidonic acid. C9, C23, and empty vector-transfected cells (empty vector) were rendered quiescent with serum-free medium for 24 h and treated with or without 30 µM arachidonic acid for 30 min, then scraped into the culture medium, and EET production was measured as described under "Experimental Procedures." C, DNA synthesis. Arachidonic acid induced [3H]thymidine incorporation in the F87V BM-3-transfected clones, C9 and C23, but not in cells transfected with empty vector alone (n = 3-7). D, cell counts. In cells grown in the absence of other exogenous growth factors, the addition of arachidonic acid (5 µM) increased cell proliferation in C23 but not in cells transfected with the empty vector.

As shown in Fig. 1B, in the absence of exogenous arachidonic acid, EET levels were low or undetectable in quiescent C9 or C23 or in control vector-transfected cells. Following incubation with exogenous arachidonic acid (30 µM), EET production increased significantly in both C9 (67.28 ng/108 cells) and C23 (241.82 ng/108 cells), whereas there was no increase in the cells transfected with empty vector alone. The greater increase in EET production in C23 was consistent with the higher levels of immunoreactive protein expression in this clone (Fig. 1A). The predominant EET present in arachidonic acid-treated F87V BM-3-transfected cells was 14S,15R-EET (>97% optical purity), indicating enzymatic production by the expressed F87V BM-3 (Table I).

                              
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Table I
Stereochemical analysis of the EET produced in arachidonic acid-treated cells
C23 and empty vector-transfected cells (empty vector) were made quiescent with serum-free medium for 24 h and treated with 30 µM arachidonic acid for 30 min, then scraped into the culture medium and subjected to stereochemical analysis (values in ng/108 cells).

Together with increased EET production, the AA addition stimulated [3H]thymidine incorporation in a concentration-dependent manner in the F87V BM-3-transfected cells but not in cells transfected with the empty vector (Fig. 1C). In the absence of added growth factors, 5 µM arachidonic acid also stimulated cell proliferation in F87V BM-3-transfected cells but not in vector-transfected cells (Fig. 1D). There were no differences in growth rate in the absence of exogenous arachidonic acid administration (not shown). Similar to what was previously observed in LLCPKcl4 cells in response to exogenous administration of 14,15-EET (8), arachidonic acid stimulation of [3H]thymidine incorporation in F87V BM-3-transfected cells was blocked by the tyrosine kinase inhibitor, genistein (11) (Fig. 2A), by the PI-3 kinase inhibitors wortmannin (12) and LY294002 (13) (not shown), and by the MEK inhibitor, PD 98059 (14) (Fig. 2B), suggesting an important role for PI-3 kinase and mitogen-activated protein kinases (ERKs) in the EET-mediated mitogenic response.


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Fig. 2.   A, effect of tyrosine kinase inhibition on DNA synthesis. Arachidonic acid (30 µM), genistein (30 µM) (n = 6). B, effect of MEK inhibition on DNA synthesis. Arachidonic acid (30 µM), MEK inhibitor, PD98059 (10 µM) (n = 8). C, arachidonic acid induced ERK activation in the F87V BM-3-transfected cells. Quiescent wild type (nontransfected) cells, empty vector-transfected cells, and the F87V BM-3-transfected clones, C9 and C23, were treated with or without 14,15-EET (20 µM) for 15 min or arachidonic acid (AA, 30 µM) for 30 min, then tyrosine-phosphorylated proteins in the cell lysates were immunoprecipitated with anti-phosphotyrosine antibodies (anti-PY) and immunoblots probed with an anti-ERK antibody, which recognizes p44 ERK1 and p42 ERK2 proteins. D, effect of the cP450 inhibitor, 17-ODYA, on ERK activation in empty vector-transfected cells and the F87V BM-3-transfected clone, C23. Quiescent cells were pretreated with or without 17-ODYA (20 µM), then treated with the appropriate vehicle (Me2SO for 14, 15-EET and Tris for arachidonic acid) or 14,15-EET (20 µM) or arachidonic acid (AA, 30 µM), and then immunoprecipitated and immunoprobed as described in C.

Arachidonic acid produced significant increases in tyrosine phosphorylation of ERK in F87V BM-3 cells (Fig. 2C) and also increased tyrosine phosphorylation of the p85 subunit of PI-3 kinase (not shown). On the other hand, no such effects were observed in vector-transfected cells. Pretreatment of the cells with 17-ODYA (15), which is a potent inhibitor of BM-3 P450 enzyme activity2 did not alter the tyrosine phosphorylation of ERKs by exogenously administered 14,15-EET in either empty vector-transfected cells or F87V BM-3-transfected LLCPKcl4 cells. However, 17-ODYA inhibited completely ERK tyrosine phosphorylation induced by arachidonic acid in F87V BM-3 cells (Fig. 2D).

To study hormonal regulation of EET production in these transfected cells and to investigate the role of this pathway in EGF signaling, LLCPKcl4 cells were exposed to EGF, a documented mitogen for these cells (8). EGF/EGF receptor interactions markedly increased EET production to 387.59 ng/108 cells in C23, whereas minimal increases were seen in empty vector-transfected LLCPKcl4 cells (Fig. 3A). The EGF-induced EET production in C23 was completely inhibited by 17-ODYA, and the PLA2 inhibitor, quinacrine, completely abolished the EGF-stimulated EET production in these cells (Fig. 3A).


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Fig. 3.   A, effect of the cP450 inhibitor, 17-ODYA, or the PLA2 inhibitor, quinacrine, on EET production in response to EGF application. Quiescent cells were exposed to EGF (10 nM) with or without 17-ODYA (20 µM) or quinacrine (250 µM) pretreatment, then scraped into the culture medium and EET production was measured as described under "Experimental Procedures." B, increase in [3H]thymidine incorporation in the F87V BM-3-transfected clones, C9 and C23, in response to increasing concentrations of EGF (n = 4-10). C, EGF receptor expression in wild type and transfected LLCPKcl4 cells. Cells were made quiescent with serum-free medium, lysed, and protein concentrations determined with the BCA assay (Pierce). An equal amount of total cellular proteins (50 µg/lane) was resolved on 5% SDS-polyacrylamide gel electrophoresis, transferred, and immunoprobed with an anti-EGF receptor antibody. The 170-kDa EGF receptor is indicated. D, effect of 17-ODYA or quinacrine on EGF-induced ERK tyrosine phosphorylation in empty vector-transfected cells and the F87V BM-3-transfected clone, C23. Quiescent cells were exposed to EGF (10 nM) with or without 17-ODYA or quinacrine pretreatment and then immunoprecipitated and immunoprobed as described in Fig. 2D.

EGF-stimulated [3H]thymidine incorporation was significantly greater in the F87V BM-3-transfected LLCPKcl4 cells than in cells transfected with empty vector alone (Fig. 3B). F87V BM-3 transfection resulted in (a) increased sensitivity to EGF, i.e. stimulation of [3H]thymidine incorporation could be observed at lower concentrations of EGF in the transfected cells; and (b) augmentation of the mitogenic potency, i.e. maximal stimulation seen with 1 µM EGF was greater in the F87V BM-3-transfected LLCPKcl4 cells. These effects appeared to be associated with the changes in EGF-signaling efficiency, because no changes in the levels of immunoreactive EGF receptor were observed after cell transfection (Fig. 3C), whereas administration of EGF to F87V BM-3 cells increased ERK tyrosine phosphorylation to a significantly greater extent than in cells transfected with the vector alone (Fig. 3D). In cells transfected with the vector alone, as well as in wild type LLCPKcl4 cells (not shown), 17-ODYA had no effect on EGF-induced ERK tyrosine phosphorylation. Similarly, quinacrine did not affect EGF-induced ERK tyrosine phosphorylation in the vector-transfected cells (Fig. 3D). In contrast, either 17-ODYA or quinacrine reduced EGF-stimulated ERK tyrosine phosphorylation in the F87V BM-3-transfected LLCPKcl4 cells to levels not different from that observed in the empty vector-transfected cells (Fig. 3D).

A further indication of intracellular responses induced by endogenously produced EETs in the F87V BM-3-transfected cells was the finding that administration of arachidonic acid to these cells induced tyrosine phosphorylation of the EGF receptor (Fig. 4). These results are consistent with our previous findings of increased EGF receptor tyrosine phosphorylation following administration of exogenous 14,15-EET to wild type LLCPKcl4 cells (8).


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Fig. 4.   Arachidonic acid induced tyrosine phosphorylation of EGF receptors in the F87V BM-3-transfected cells. Quiescent cells, nontransfected empty vector-transfected cells, and the F87V BM-3-transfected clones, C9 and C23, were treated with or without 14,15-EET (20 µM) or arachidonic acid (30 µM), then cell lysates were immunoprecipitated with a polyclonal anti-phosphotyrosine antibody and immunoblotted with a monoclonal anti-EGFR antibody.


    DISCUSSION

Cytochrome P450 epoxygenase catalyzes the NADPH-dependent epoxidation of arachidonic acid to 5,6-, 8,9-, 11,12-, and 14,15-EET, in a regio- and stereoselective manner (16). The early demonstration of endogenous EET biosynthesis in several rat and human organs (6, 16) and of the presence of EETs in human plasma and urine (17, 18) established this reaction as a formal metabolic pathway and provided further credence to its postulated functional roles. Previous studies have indicated that EETs, or their dihydroxy derivatives, have potent biological activities, including modulation of vascular tone (19), glomerular hemodynamics (20, 21), and regulation of mitogenesis (22). EETs have been suggested to be an endothelial-derived hyperpolarizing factor (23). In addition, recent studies have also suggested that EETs may serve as intracellular second messengers in vasculature (24) and in epithelia (25).

Most studies implicating a role for EETs as intracellular second messengers have been based on the effects of cP450 inhibitors and/or the use of synthetic EETs. The cP450 inhibitors currently available do not selectively inhibit epoxygenase activity, so these studies cannot definitively rule out the possibility that other cP450 arachidonate metabolites, other cellular activities of cP450, or even non-cP450-derived arachidonate metabolites, mediate the observed effects. Although reproduction of the effect with the addition of exogenous EETs is suggestive evidence, the high concentrations of exogenously administered EETs required to elicit biological responses has also raised the possibility of nonspecific effects. In addition, it is well documented that most lipid mediators are only one component of what are usually complex signaling processes that involve, in addition to the temporally and spatially controlled biosynthesis of the putative lipid mediator, the coordinated participation of additional signaling pathways to elicit the indicated cellular response. Therefore, addition of synthetic EETs or other lipid moieties bypasses the integrated cellular response seen in response to EGF or similar agonists. To overcome these limitations of previous studies, the present studies used stable transfection of a cP450 that has been genetically engineered to be a regio- and stereoselective epoxygenase to investigate the potential role of EETs as intracellular second messengers.

The findings in the present study demonstrate that 14,15-EET can serve as an intracellular second messenger for EGF in cells expressing epoxygenase activity and that these signaling mechanisms involve the activation of kinase-associated mitogenic pathways. Endogenous production of EETs significantly augmented the mitogenic effects of EGF, indicated both by augmented stimulation of early signaling responses such as mitogen-activated protein kinase and PI-3 kinase and by increases in DNA synthesis. In F87V BM-3-transfected cells, administration of exogenous arachidonic acid activated a tyrosine kinase cascade and increased [3H]thymidine incorporation. This activation required conversion of the arachidonic acid to 14S,15R-EET, because addition of 17-ODYA blocked 14,15-EET production and subsequent cellular responses. The lack of significant EET formation in the absence of either exogenous arachidonic acid or agonist stimulation, and the inhibition of EGF-mediated EET production and functional activity by PLA2 inhibition, indicate that, as with most enzymes of the AA cascade, the rate-limiting step for the cP450 pathway is substrate availability.

In addition to autophosphorylation following ligand binding, there has been increasing evidence for tyrosine phosphorylation of intrinsic tyrosine kinase receptors by activation of seven-transmembrane receptors (26-28). These G-protein receptor-mediated tyrosine-phosphorylated proteins use growth factor receptors such as EGF receptor and platelet-derived growth factor receptor as "scaffolds" (29, 30); unlike the sequence of events occurring when these receptors bind their ligands, they are not activated by autophosphorylation in this process but are tyrosine phosphorylated by Src-like kinases. Src, GRB2, and SOS may be associated via interactions with Shc, which interacts with SH2 domains of the receptor. It is therefore of interest that, together with our previous findings (8), our results indicate that the endogenous AA epoxygenase metabolite, 14,15-EET, also appears to activate Src kinase and initiate a tyrosine phosphorylation cascade that uses the EGF receptor as a scaffold and results in mitogen-activated protein kinase activation. cP450 arachidonic acid metabolites have been implicated as second messengers for a number of agonists that activate seven-transmembrane receptors, such as angiotensin II, bradykinin, and parathyroid hormone (31-33); whether EETs are also involved in activation of tyrosine kinase-mediated pathways by these agonists has not yet been determined.

The biological effects of prostaglandins, leukotrienes, and other polar arachidonic acid metabolites are largely mediated through specific cell surface G-protein-coupled receptors; in contrast, the mechanisms of activation of the less polar cP450 arachidonate metabolites is unclear. There have been reports of a specific binding site for 14,15-EET in monocytes (34, 35). In addition, recent studies have indicated that EETs activated calcium-activated K+ channels in vascular smooth muscle cells. EETs did not activate channels when added directly to cytoplasmic surface of excised inside-out patches but required intermediate signaling steps involving G-proteins (36, 37). It is also possible that these compounds may directly activate or modulate enzymatic activity or could become incorporated into phospholipid and thereby modulate function. The availability of cultured cells with robust functional epoxygenase activity should provide a valuable tool for elucidation of the mechanisms of action of EETs.

In summary, the lack of significant functional cP450 activity in cultured cell systems has contributed to the failure heretofore to appreciate fully the signaling capabilities of these compounds. The present studies provide direct and definitive evidence that cP450 arachidonate metabolites can serve as intracellular second messengers in cells expressing functional cP450 activity and should stimulate further research to explore the range of functions and molecular mechanisms of cP450 arachidonate metabolites.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK38226 (to R. C. H., J. C., and J. R. F.) and funds from the Department of Veterans Affairs (to R. C. H.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

parallel To whom correspondence should be addressed: Div. of Nephrology, S 3322, MCN, Vanderbilt University School of Medicine, Nashville, TN 37232. Tel.: 615-343-0030; Fax: 615-343-7156; E-mail: Ray.Harris{at}mcmail.vanderbilt.edu.

    ABBREVIATIONS

The abbreviations used are: EET, epoxyeicosatrienoic acid; EGF, epidermal growth factor; ERK, extracellular signal-regulated kinase; PFB, pentafluorobenzyl; PLA2, phospholipase A2.

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Abstract
Introduction
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