Epoxyeicosatrienoic Acids and Their Sulfonimide Derivatives Stimulate Tyrosine Phosphorylation and Induce Mitogenesis in Renal Epithelial Cells*

In our present studies utilizing a well characterized proximal tubule cell line, LLCPKcl4, we determined that all four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET, and 14,15-EET) stimulated [3H]thymidine incorporation, with 14,15-EET being the most potent. In contrast, no mitogenic effects were seen with arachidonic acid, other cP450 arachidonate metabolites (12R-hydroxyeicosatetraenoic acid (12R-HETE), 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), or 20-HETE), or lipoxygenase metabolites (5S-HETE, leukotriene B4, or lipoxin A4). We found that their metabolically more stable sulfonimide (SI) analogs (11,12-EET-SI and 14,15-EET-SI) were also potent mitogens. In addition 14,15-EET-SI also increased cell proliferation as well as expression of both c-fos and egr-1 mRNA. The protein kinase C and A inhibitors, H-7 and H-8, or the cyclooxygenase inhibitor, indomethacin, had no effect upon 14,15-EET-induced [3H]thymidine incorporation, but the selective tyrosine kinase inhibitor, genistein, significantly inhibited it. Immunoprecipitation and immunoblotting demonstrated increased tyrosine phosphorylation of PI3-kinase and epidermal growth factor receptor (EGFR) within 1 min of EET administration. EETs also stimulated association of PI3-kinase with EGFR. PI3-kinase inhibitors, wortmannin and LY 294002, markedly inhibited 14,15-EET-SI-stimulated [3H]thymidine incorporation. In addition, 14,15-EET-SI administration stimulated tyrosine phosphorylation of src homologous and collagen-like protein (SHC) and association of SHC with both growth factor receptor-binding protein (GRB2) and EGFR. Mitogen-activated protein kinase was also activated within 5 min. Pretreatment of the cells with the mitogen-activated protein kinase kinase inhibitor, PD98059, inhibited the 14,15-EET-SI-stimulated [3H]thymidine incorporation. Moreover, immunoblotting indicated that 14,15-EET stimulated tyrosine phosphorylation of the specific pp60c- src substrate p120 and c-Src association with EGFR. 14,15-EET increased src kinase activity within 1 min. Our data indicate that EETs are potent mitogens for renal epithelial cells, and the mitogenic effects of the EETs are mediated, at least in part, by the activation of Src kinase and initiation of a tyrosine kinase phosphorylation cascade.

Cytochrome P450 epoxygenase catalyzes the NADPHdependent epoxidation of arachidonic acid to 5, 6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids (EETs) 1 in a regio-and stereo-selective manner. The EETs are produced predominantly by epoxygenases of the 2C family of cP450s, which have been localized in the kidney to the mammalian proximal tubule. The proximal tubule contains the highest concentration of cP450 within the mammalian kidney (1) and expresses minimal cyclooxygenase or lipoxygenase activity (2). The EETs or their hydration products have been implicated in modulation of vascular tone and renal glomerular hemodynamics (3), renal proximal tubule function, and regulation of mitogenesis (4,5). Recently, EETs have been suggested to be an endothelialderived hyperpolarizing factor (6), although there is controversy about this issue (7,8). Direct administration of EETs inhibits amiloride-sensitive sodium transport (9) and 86 Rb uptake in LLC-PK1, a nontransformed, immortalized cell line from pig kidney with certain proximal tubule characteristics (10).
EETs have also been proposed as second messengers for hormones and growth factors in the proximal tubule. We have shown that epidermal growth factor (EGF) stimulates EET production in rat proximal tubule suspensions and primary cultured rabbit proximal tubule cells, and EETs may mediate both EGF-stimulated calcium influx and mitogenesis in proximal tubules (11). Omata et al. also demonstrated EET production upon stimulation with angiotensin II (12). EETs may mediate the effect of high (Ͼ10 Ϫ7 M) angiotensin II to increase cytosolic calcium ([Ca 2ϩ ] i ) and decrease Na ϩ /H ϩ exchange activity in cultured rabbit proximal tubule cells and isolated rat proximal tubules (13)(14)(15). In the present studies, we examined mitogenic signaling mechanisms of EETs in renal epithelial cells. We determined the mitogenic effects of all four EET regioisomers and utilized metabolically more stable sulfonimide analogs to determine potential intracellular signaling mechanisms mediating mitogenic effects. We demonstrate here that EETs activate pp60 c-src and initiate a tyrosine kinase cascade that mediates their mitogenic effects. This is the first evidence that cP450-mediated arachidonic acid metabolites can signal through such pathways.
Cell Culture-LLCPKcl4, an established proximal tubule epithelial cell line derived from pig kidney (17), was routinely cultured in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 100 units/ml penicillin, 100 g/ml streptomycin, and 10% fetal calf serum (Hyclone Laboratories, Logan, UT) at 37°C in a 5% CO 2 cell culture incubator. The medium was changed every 2-3 days. For studies of [ 3 H]thymidine incorporation, cells were seeded into 24-well plates. For cellular RNA isolation and immunoprecipitation, cells were grown in 100-and 60-mm plates, respectively.
[ 3 H]Thymidine Incorporation Assay and Cell Growth Curves-Confluent cells in 100-mm dishes were detached by trypsinization, resuspended in growth medium, seeded at a density of 3 ϫ 10 4 /well in 24-well plates, and incubated for 2-3 days until they were 80% confluent. The medium was then changed to serum-free medium and incubated for an additional 3 days. EETs and/or the indicated drugs in the corresponding data were routinely added to the quiescent cells in triplicate and incubated for 19 h, followed by the addition of 2 Ci/ml [ 3 H]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, lysed with 0.2 N NaOH, 0.5% SDS and incubated at 37°C for at least 30 min, and radioactivity was determined by liquid scintillation spectrometry (Beckman Instruments). 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 the effect of the 14,15-EET-SI on cell number, 1 ϫ 10 4 /well LLCPKcl4 cells were seeded into 24-well plates.
After 48 h of growth, the medium was changed to serum-free medium and incubated for an additional 24 h. Cells were then exposed to 20 M 14,15-EET-SI or vehicle for the indicated times.
RNA Isolation and Northern Blot Hybridization-Confluent LLCP-Kcl4 cells grown in 100-mm dishes were made quiescent for 3 days, exposed to vehicle (Me 2 SO), 14,15-EET-SI, phorbol 12-myristate 13acetate, or EGF for the indicated times and concentrations, then total cellular RNA was isolated by the single-step acid guanidinium thiocyanate-phenol-chloroform method (18). 15 g of total RNA was sizefractionated by formaldehyde-agarose (1%) gel electrophoresis and transferred onto Nytran nylon membranes (Schleicher & Schuell). [␣-32 P]cDNA probes were labeled to a specific activity of Ͼ2 ϫ 10 8 dpm/g by random priming (Megaprime DNA Labeling System; Amersham Pharmacia Biotech). After overnight hybridization, membranes were washed twice with 2ϫ SSC (0.3 M NaCl and 0.03 M sodium citrate), 0.1% SDS at room temperature for 15 min and 0.2ϫ SSC, 0.1% SDS at 65°C for 30 min. Autoradiography was performed at Ϫ70°C by exposing the washed membranes to Hyperfilm (Amersham Pharmacia Biotech) with intensifying screens. To assess the uniformity of RNA loading, blots were stripped and reprobed with a cDNA probe for glyceraldehyde-3-phosphate dehydrogenase labeled with [␣-32 P]dCTP.
Immunoprecipitation and Western Blotting-Quiescent cultures of LLCPKcl4 cells were treated with agonists for the indicated times, washed twice with ice-cold Ca 2ϩ /Mg 2ϩ -free phosphate-buffered saline, and lysed on ice for 30 min in lysis buffer (0.5% Nonidet P-40, 50 mM NaCl, 10 mM Tris-HCl, pH 7.4, 2 mM EDTA, 2 mM EGTA, 0.5% sodium deoxycholate, 0.1% SDS, 100 M Na 3 VO 4 , 100 mM NaF, 30 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin). Cell lysates were clarified at 10,000 ϫ g for 15 min at 4°C, and protein concentrations were determined by the bicinchoninic acid assay (Pierce). Target proteins were immunoprecipitated at 4°C for 2 h with appropriate antibodies. In some experiments, immobilized antibodies were used to minimize recovery of immunoglobulin heavy chains. Immune complexes were then captured with 50 l of protein A-agarose beads and washed four times with wash buffer (20 mM HEPES, pH 7.2, 100 mM NaCl, 0.1% Triton X-100, 10% glycerol, and 100 M Na 3 VO 4 ).
Immunoprecipitation samples were resuspended and boiled in sample buffer before separation on 5-15% SDS-polyacrylamide gels and immunoblotted onto polyvinylidene difluoride membranes. After blocking with 3% bovine serum albumin in 150 mM NaCl, 50 mM Tris-HCl, pH 7.4 (TBS) for 1 h at room temperature, blots were probed with the indicated primary antibody overnight at 4°C. The blots were washed 3 times at room temperature with 0.05% Tween 20 in TBS, incubated with the appropriate secondary antibody conjugated with biotin, washed with TBS-Tween, incubated with preformed avidin-biotinhorseradish peroxidase complex using a commercially available kit (ABC kit; Pierce), and detected by a peroxidase-catalyzed enhanced chemiluminescence detection system (ECL; Amersham Pharmacia Biotech).
Src Kinase Activity Assay-Src immunoprecipitates were prepared as described above and washed twice with kinase buffer (20 mM Tris-HCl, pH 7.5, 1 mM EDTA, 10 mM MgCl 2 , 1 mM dithiothreitol, 250 M Na 3 VO 4 ). Phosphorylation reactions were initiated by the addition of 10 Ci of [␥-32 P]ATP and 1 mg/ml enolase as a substrate and allowed to proceed for 15 min at room temperature. The reactions were stopped by the addition of 10 l of acetic acid. The amount of enolase phosphorylation was then determined by spotting an aliquot (25 l) of the reaction mix onto P81 cation exchange paper (Whatman), washing three times in 0.5% phosphoric acid, and measuring radioactivity in a scintillation counter.
Statistics-Data are presented as means ϮS.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 Ͻ 0.05 compared with control was considered statistically significant.

EET Induced Mitogenesis in LLCPKcl4
Cells-We have previously shown that EGF increased EET production in rat or rabbit renal proximal tubule cells (1,11). In the present studies we utilized a nontransformed cell line, LLCPKcl4, cloned from the parental LLC-PK1 cell line and selected for its expression of high levels of proximal tubule markers (17). These cells increased [ 3 H]thymidine incorporation in response to EGF, with EC max ϳ 256 nM and an EC 50 of ϳ8 nM ( Fig. 2A). Arachidonic acid in concentrations from 1 to 40 M failed to stimulate [ 3 H]thymidine incorporation in LLCPKcl4 (n ϭ 14). Administration of the product of P450 arachidonic acid -hydroxylation, 20-HETE, did not stimulate [ 3 H]thymidine incorporation in LLCPKcl4 (n ϭ 7). Similarly, no mitogenic effects were seen with the cP450 arachidonate metabolite, 12R-HETE (n ϭ 6). The lipoxygenase metabolites, 5S-HETE, LTB4, and lipoxin A4, also failed to stimulate [ 3 H]thymidine incorporation in LLCPKcl4 (n ϭ 4 -6). However, as indicated in EETs are susceptible to oxidative degradation; to block acetyl CoA-dependent activation and lysophospholipid acylation, the EET carboxylate was blocked by condensation with methanesulfonimide to generate the corresponding sulfonimide derivatives (11,12-EET-SI and 14,15-EET-SI). Shown in Fig. 1 are structures of 14,15-EET and its sulfonimide derivative, 14,15-EET-SI. These sulfonimide analogs were also found to be potent mitogens for LLCPKcl4 cells, as determined by increased [ 3 H]thymidine incorporation (Fig. 2C). As with the parent compound, 14,15-EET-SI was found to be relatively more potent than 11,12-EET-SI. Therefore, 14,15-EET-SI was utilized for further investigation of the mitogenic signaling pathways. In addition to [ 3 H]thymidine incorporation, 14,15-EET-SI increased cell proliferation in the absence of fetal calf serum (Fig. 2D).
14,15-EET-SI Activated Immediate Early Gene Transcription-Quiescent LLCPKcl4 cells were exposed to 20 M 14,15-EET-SI for the indicated times, the cells were lysed, total cellular RNA was isolated, and levels of mRNA expression were determined by Northern blot hybridization. 14,15-EET-SI increased steady state levels of both c-fos and egr-1 mRNA within  (Fig. 3). In contrast, 14,15-EET-SI did not induce any significant increases in COX-2 mRNA levels, nor did it induce expression of acyl CoA oxidase mRNA, a marker of peroxisomal proliferator activator receptor activation (not shown).
14,15-EET-SI Induced Activation of Tyrosine Phosphorylation Cascade-To investigate signaling mechanisms of the mitogenic effects of 14,15-EET, we tested a range of inhibitors. The isoquinoline sulfonimides, H-7 and H-8, are potent inhibitors of serine/threonine kinases and of cyclic nucleotide-dependent protein kinases. Although neither is specific, H-7 has relative specificity for PKC, whereas H-8 is most effective for protein kinase A and cGMP-dependent protein kinase (19,20). No inhibition of 14,15-EET-SI-induced [ 3 H]thymidine incorporation into DNA of LLCPKcl4 cells was seen with either H-7 or H-8. In addition, the cyclooxygenase inhibitor, indomethacin, did not alter 14,15-EET-SI-induced [ 3 H]thymidine incorporation. However, the tyrosine kinase inhibitor, genistein, blocked [ 3 H]thymidine incorporation in a concentration-dependent manner, with an IC 50 of ϳ10 M (Fig. 4A). Similar inhibitory effects were seen for 11,12-EET-SI-stimulated [ 3 H]thymidine incorporation (not shown). After treatment with 20 M 14,15-EET-SI, the cells were lysed and immunoprecipitated with polyclonal anti-phosphotyrosine (PY), then immunoprobed with monoclonal anti-phosphotyrosine (PY-20). We found that administration of 14,15-EET-SI led to increases in tyrosine phosphorylation of several LLCPKcl4 proteins, including bands of of molecular mass ϳ175, ϳ120, ϳ85, and ϳ60 kDa (Fig. 4B).
To characterize the 85-kDa tyrosine-phosphorylated protein, cell lysates were subjected to either immunoprecipitation with anti-phosphotyrosine and immunoblotting with an anti-PI3kinase antibody or immunoprecipitation with anti-PI3-kinase and immunoblotting with anti-phosphotyrosine. The results indicated in Fig. 5A confirmed that 14,15-EET-SI administra-tion induced tyrosine phosphorylation of the 85-kDa regulatory subunit of PI3-kinase. Increased tyrosine phosphorylation of this protein was detectable within 1 min of 14,15-EET-SI addition and remained elevated for up to 30 min (the longest time period tested). The PI3-kinase inhibitor, wortmannin, was found to inhibit 14,15-EET-SI-stimulated DNA synthesis in a concentration-dependent manner. Pretreatment of the cells with 1 nM wortmannin for 30 min inhibited 21% of the stimulation, and 10 nM wortmannin almost totally (95%) abolished the stimulatory effect of 20 M 14,15-EET-SI, with IC 50 ϳ 5 nM (Fig. 5B). At the concentrations used in the present studies, wortmannin has been shown to inhibit PI3-kinase specifically and to have no inhibitory effect upon phospholipase C, phospholipase D, phospholipase A 2 , or adenylate cyclase (21). Furthermore, similar effects were shown by another structurally and mechanistically distinct, highly specific PI3-kinase inhibitor, LY294002 (22), with an IC 50 value of ϳ5 M (Fig. 5C). Wortmannin and LY294002 administration resulted in similar inhibition of 11,12-EET-SI-stimulated [ 3 H]thymidine incorporation (not shown).
Immunoblotting with an anti-EGF receptor antibody identified the ϳ175-kDa protein that was tyrosine-phosphorylated in response to 14,15-EET-SI (Fig. 6A). Increases in EGF receptor tyrosine phosphorylation were noted within the first 5 min of 14,15-EET-SI addition. Moreover, immunoprecipitation with a PI3-kinase antibody and immunoblotting with an anti-EGFR antibody indicated that 14,15-EET-SI induced the association of PI3-kinase with EGF receptors (Fig. 6B).
Tyrosine phosphorylation of the adaptor protein SHC has been reported to occur in response to activation of both tyrosine kinase receptors and nontyrosine kinase receptors. To determine if 14,15-EET-SI stimulates SHC tyrosine phosphorylation, we probed anti-PY immunoprecipitates with SHC antibody and determined that 14,15-EET-SI stimulated tyrosine phosphorylation of the three SHC isoforms: 66 kDa, 52 kDa, and 46 kDa. Initial increases in tyrosine phosphorylation were detected in 52-kDa and 66-kDa SHC, with subsequent tyrosine phosphorylation of 46-kDa SHC (Fig. 7A). Phosphorylated SHC is known to bind to SH2 domains of tyrosine kinase receptors and to recruit the adaptor protein, GRB2, to the plasma membrane. By immunoprecipitating with anti-SHC and then immunoblotting with antibodies against GRB2 or EGF receptors, we further corroborated that 14,15-EET-SI-activated SHC simultaneously co-immunoprecipitated both GRB2 and EGF receptors (Fig. 7, B and C), i.e. 14,15-EET-SI induced association of SHC not only with GRB2 but also with EGF receptors.
GRB2 has been shown to bind to the guanine nucleotide exchanging factor, SOS, leading to Ras activation and Ras-dependent activation of the mitogen-activated protein kinase cascade. In this regard, we determined that 14,15-EET-SI induced tyrosine phosphorylation of both ERK1 and ERK2. As shown in Fig. 8A, both ERK1 and ERK2 were phosphorylated on tyrosine residues after treatment with 20 M 14,15-EET-SI. To determine whether there was a role for the mitogen-activated protein kinase signaling pathway in EET-stimulated [ 3 H]thymidine incorporation, we utilized the mitogen-activated protein kinase kinase inhibitor, PD98059 (23,24). Pretreatment of the cells with PD98059 dose-dependently inhibited the 14,15-EET-SI-stimulated [ 3 H]thymidine incorporation, with IC 50 ϳ5 M (Fig. 8B). In other experiments, PD98059 also inhibited 11,12-EET-SI-stimulated [ 3 H]thymidine incorporation (not shown).
14,15-EET-SI Activated c-Src Kinase-Further studies were undertaken to determine the mechanism by which 14,15-EET-SI increased tyrosine phosphorylation in LLCPKcl4 cells. Immunoblotting with antibodies against the specific pp60 c-src substrate, p120, indicated increased tyrosine phosphorylation of  6), then the cells were lysed, total cellular RNA was isolated, and mRNA expression of c-Fos or Egr-1 was determined by Northern blot analysis as described under "Experimental Procedures." To assess the uniformity of RNA loaded into each gel well, the blots were stripped and reprobed with a cDNA probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). this protein within 1 min after 14,15-EET-SI addition (Fig. 9A).
In additional experiments, cell lysates were immunoprecipitated with anti-EGFR antibody and immunoblotted with a polyclonal antibody that recognizes the kinase catalytic region highly conserved in c-Src, c-Fyn, and c-Yes members of the Src kinase family. Immunoreactive c-Src, or Src-like kinases, were found to be associated with the EGF receptor within 1 min of 14,15-EET-SI administration to LLCPKcl4 cells (Fig. 9B). In contrast, there was minimal, if any, association of Src-like kinases with the EGF receptor in the first 5 min after EGF addition (Fig. 9B).
To demonstrate 14,15-EET-SI stimulation of Src-like kinase activity and to further define the identity of the kinase involved, we utilized a specific monoclonal antibody to immunoprecipitate pp60 c-src for an in vitro Src kinase activity assay. Within 1 min, 14,15-EET-SI increased Src kinase activity (Fig.  9C). The stimulated enzymatic activity peaked at 45 min and was elevated for up to 1.5 h, confirming that pp60 c-src kinase was activated in LLCPKcl4 cells by 14,15-EET-SI. DISCUSSION Previous studies suggested that cP450 arachidonic acid metabolites were mitogens for both mesenchymal and epithelial cells (4,5,11,25,26). The mechanisms by which cP450 AA metabolites induced mitogenesis were not explored in these earlier studies. In the present studies, we determined that all four regioisomers of EETs induced [ 3 H]thymidine incorpora-tion in LLCPKcl4, a nontransformed proximal tubule-like cell line, and that the signaling pathway involved activation of a tyrosine kinase cascade. These mitogenic effects appeared to be relatively specific for EETs in these cells, since no increases in [ 3 H]thymidine incorporation were seen with the parent compound, arachidonic acid, with the hydration product of 14,15-EET, 14,15-DHET, with nonepoxygenase cP450 arachidonate metabolites (12R-HETE, 20-HETE), or with lipoxygenase metabolites (5S-HETE, LTB4, lipoxin A4).
It has been established that prostaglandins and leukotrienes mediate their biologic actions at least in part through specific cell surface G-protein-coupled receptors. On the other hand, little is known about the signaling mechanisms responsible for most of the biological activities, including mitogenic effects, attributed to EETs. There have been reports of a specific binding site for 14,15-EET in monocytes (27,28) and for 12(R)-HETE in microvessel endothelial cells (29). Studies have also indicated that EETs activate calcium-activated K ϩ channels in vascular smooth muscle cells but have no effect when added directly to the cytoplasmic surface of excised inside-out patches (30). More recent studies have suggested that channel activation requires intermediate signaling steps involving G-proteins (31). The addition of GTP to excised patches restored 11,12-EET activation, which was blocked by GDP␤S. These studies implicated G s␣ in this response (30). Whether EETs interact through a G-protein-coupled receptor or directly activate het-  3 and 7), or 30 min (lanes 4 and 8), and tyrosine-phosphorylated proteins were immunoprecipitated with polyclonal anti-phosphotyrosine antibodies (anti-PY) and immunoblotted with monoclonal anti-phosphotyrosine antibodies (PY-20). I.P., immunoprecipitation; I.B., immunoblotting. erotrimeric G-proteins remains to be determined. There is evidence that unsaturated fatty acids, including arachidonic acid, inhibit G z activation by binding directly to G za ; similar effects have been reported for arachidonic acid on the small Gproteins, Ras and Rac (32)(33)(34). 14,15-EET has also been reported to stimulate ADP-ribosylation of a yet-to-be characterized 52-kDa protein in rat liver cytosol (35), but to date, no studies have directly investigated the effects of EETs on G-protein activation.
In LLCPKcl4 cells, the EET-stimulated tyrosine kinase-signaling cascade activated mitogen-activated protein kinase. Our data suggest that c-Src (pp60 c-src ) is the cytosolic tyrosine kinase that initiated the tyrosine phosphorylation cascade. pp60 c-src and the structurally related members of the Src family (Fyn, Yes, Fgr, Lyn, Hck, Lck, Blk, Yrk) are nonreceptor tyrosine kinases that are normally associated with cell membranes. An increasing number of intracellular signaling pathways and cellular responses have been shown to be dependent upon Src activation, including morphological changes and cell proliferation (36). Recent studies have found that seven-transmembrane receptors, which signal through G-protein activation, mediate Ras-dependent activation of mitogen-activated protein kinases by activation of c-Src or Src-like kinases, phosphorylation of the SHC adapter protein, SHC⅐GRB2 complex formation, and recruitment of Ras guanine nucleotide exchange factor activity (SOS) (37)(38)(39), which then leads to activation of mitogen-activated protein kinase signaling pathways. Agonists known to utilize this pathway include, among others, angiotensin II, catecholamines, thrombin, and endothelin (40 -43). The mechanism of c-Src activation has still not been clarified; for G i -activating receptors, it appears that G ␤␥ subunits are involved (38), whereas for other receptors, G ␣q /G ␣11 may be involved (44). These G-protein receptor-mediated tyrosine-phosphorylated proteins utilize growth factor receptors such as EGFRs and platelet-derived growth factor receptors as "scaffolds" (38,45); unlike the sequence of events occurring when EGFRs and platelet-derived growth factor receptors bind their ligands, these receptors 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 the present studies indicate that 14,15-EET-SI also activated Src kinase and initiated a tyrosine phosphorylation cascade that utilized the EGF receptor as a scaffold and resulted in mitogen-activated protein kinase activation.
Our data also demonstrated a role for PI3-kinase in the mitogenic pathway of EETs. Src kinase can activate PI3-kinase by tyrosine phosphorylation of the 85-kDa subunit (46 -48), and PI3-kinase associates with tyrosine kinase receptors (49). PI3-kinase is an important mediator of mitogenic signals for a variety of growth factors (50). There are also data suggesting roles for PI3-kinase in the regulation of transport functions. The PI3-kinase inhibitor, wortmannin, inhibits both bulk endocytosis (51,52) and specific receptor-mediated endocytosis (53). In colonic cells, the inhibitory effect of EGF on calciumdependent chloride secretion may be mediated by the lipid products generated by PI3-kinase (54). Furthermore, in intestinal epithelial cells, PI3-kinase is involved in EGF-mediated stimulation of Na ϩ /H ϩ exchange activity and NaCl absorption, and PI3-kinase translocates specifically to the brush border after EGF treatment (55). Given that previous studies have indicated that EETs modulate transport functions in a variety of epithelial cells (56), it will be of interest to determine whether these effects are mediated by activation of a similar tyrosine kinase cascade.  9. A, 14,15-EET induced tyrosine phosphorylation of a specific pp60 c-src substrate (p120) in LLCPKcl4 cells. Lanes and timing are as in Fig. 5A. Cell lysates were immunoprecipitated with an anti-PY antibody and immunoblotted with a specific monoclonal anti-pp60 -src substrate p120 antibody. B, 14,15-EET-SI induced association of Src with EGF receptor. Lanes and timing are as in A, except that lane 9 is a positive control (A431 lysate). C, 14,15-EET induced Src kinase activity. Quiescent LLCPKcl4 cells were treated with vehicle (Me 2 SO) alone, 20 M 14,15-EET-SI, or 100 nM EGF for the indicated times and then solubilized in lysis buffer. The pp60 c-src kinase was immunoprecipitated with a specific monoclonal antibody to pp60 c-src (clone 327). Src immune complex was then washed twice with kinase buffer. As described under "Experimental Procedures," Src kinase activity was determined by measuring incorporation of [␥-32 P] into enolase. I.P., immunoprecipitation; I.B., immunoblotting.
Although the role of this tyrosine kinase cascade in stimulation of mitogenesis is based in large part on the use of inhibitors, at the concentrations we employed genistein is a potent and highly tyrosine-specific protein kinase inhibitor, with minimal inhibition of serine/threonine-specific protein kinases (57). Similarly, at the concentrations utilized in the present studies, wortmannin and LY294002 have been shown to be highly specific for PI3-kinase (21,22), and PD98059 is highly specific for mitogen-activated protein kinase kinase (23,24).
In summary, these results demonstrate that epoxyeicosatrienoic acids are potent mitogens for renal epithelial cells and indicate that the mitogenic effects are mediated by activation of Src kinase and initiation of a tyrosine kinase phosphorylation cascade. This is the first evidence that these lipid mediators can signal through tyrosine kinase activation; further studies will be required to elucidate the role of the EETs in agonistmediated activation of tyrosine phosphorylation cascades.