Overexpression of C-terminal Src kinase blocks 14, 15-epoxyeicosatrienoic acid-induced tyrosine phosphorylation and mitogenesis.

We have previously reported that 14,15-epoxyeicosatrienoic acid (14, 15-EET) is a potent mitogen for the renal epithelial cell line, LLCPKcl4. This mitogenic effect is dependent upon activation of a protein-tyrosine kinase cascade that results in activation of mitogen-activated protein kinase and phosphatidylinositol 3-kinase. Because of suggestive evidence that 14,15-EET also activated Src in these cells, we stably transfected LLCPKcl4 with an expression construct of the C-terminal Src kinase (CSK), which inhibits Src family kinase activity. In vitro Src kinase activity assays confirmed that in empty vector-transfected cells (Vector cells), 14, 15-EET increased Src kinase activity, while in clones overexpressing CSK mRNA and immunoreactive protein (CSK cells), 14,15-EET-induced activation of Src was almost completely blocked (94% inhibition). Of interest, epidermal growth factor (EGF) and fetal bovine serum (FBS) also increased Src activity in Vector cells, but not in CSK cells, further confirming the ability of CSK overexpression to prevent Src activation. CSK cells failed to increase [(3)H]thymidine incorporation in response to exogenous 14,15-EET. In contrast, both EGF and FBS significantly increased [(3)H]thymidine incorporation in CSK cells. Immunoprecipitation with anti-phosphotyrosine antibodies and immunoblotting with an antibody against extracellular signal-regulated kinase (ERK) indicated that in CSK cells, 14,15-EET failed to activate ERK1 and ERK2; however, EGF- and FBS-induced activation of ERKs was not different from that seen in Vector cells. In Vector cells, the 14,15-EET-stimulated tyrosine phosphorylation of ERKs was blocked by pretreatment with 1 microm PP2, a selective inhibitor of Src kinases. The present study demonstrates that 14, 15-EET exerts its mitogenic effects predominantly through a Src kinase-mediated pathway, which is the most upstream signaling step determined to date in the 14,15-EET-activated tyrosine kinase cascade in renal epithelial cells.

In addition to cyclooxygenase and lipoxygenase pathways, the cytochrome P450-dependent monooxygenase pathway also catalyzes the in vivo metabolism of arachidonic acid to biologically active compounds. This pathway metabolizes arachidonic acid in two ways: epoxidation, producing 5, 6-, 8,9-, 11,12-, and 14,15-epoxyeicosatrienoic acids and /-1 hydroxylation, re-sulting in the formation of 19-and 20-hydroxyeicosatetraenoic acids (1,2). Epoxyeicosatrienoic acids (EETs) have been demonstrated to play important roles in regulating vascular tone, mitogenesis, platelet aggregation, tissue and body homeostasis, and Ca 2ϩ signaling (1)(2)(3)(4)(5)(6)(7). The EETs are produced predominantly by epoxygenases of the 2C family of cP450s, which have been localized to the mammalian proximal tubule cells of the kidney. In this segment of the nephron, cyclooxygenase and lipoxygenase are expressed at nearly undetectable levels (8,9). pp60 c-src is the prototype of a family of nine cytoplasmic protein-tyrosine kinases that is activated by a number of receptor protein-tyrosine kinases, such as epidermal growth factor receptor and platelet derived-growth factor receptor (10,11), and a variety of extracellular stimuli-induced cellular responses, including DNA synthesis, mitosis, proliferation, hypertrophy, differentiation, adhesion and cytokine production (10,(12)(13)(14)(15)(16)(17). Another cytoplasmic protein-tyrosine kinase, the C-terminal Src kinase (CSK), is a mediator responsible for negative regulation of the Src family kinase activity (18,19). CSK phosphorylates Tyr-527 in the C-terminal tail of c-Src and thus creates a binding site for the Src homology 2 domain, locking the molecule in an inactive state. Dephosphorylation of Src Tyr-527, followed by autophosphorylation on Tyr-416 by the c-Src kinase activity, increases Src kinase activity up to 10 -20-fold (20 -24).
Our previous studies demonstrated that 14,15-EET is a potent mitogen for the renal epithelial cell line, LLCPKcl4, and that these effects are mediated by a tyrosine kinase phosphorylation cascade that activates the mitogen-activated protein kinases, p44/p42 extracellular signal-regulated kinases (ERKs), and phosphatidylinositol 3-kinase (4). We also found evidence for possible Src kinase involvement in the mitogenic signaling pathways of 14,15-EET (4). In certain systems, Src family members are mediators of cell division at multiple points in the cell cycle (25)(26)(27). The demonstration of an important role Src in cell division and the illustration of a negative regulatory role of the C-terminal Src kinase (CSK) on c-Src (23,25,28) stimulated us to investigate the potential role of Src kinases in the mitogenic signaling pathways of 14,15-EET.
Cell Culture-LLCPKcl4, an established renal proximal tubule epithelial cell line derived from pig kidney (29), was routinely cultured as described previously (4,30). cDNA Manipulation and Stable Transfection-In order to stably transfect CSK cDNA into LLCPKcl4 cells, the cDNAs containing the entire coding region of CSK were cut out from pcDNA-I vector (Invitrogen, San Diego, CA) by EcoRI digestion and then ligated into the EcoRI site of the neomycin resistant gene-containing vector pIRES1neo (CLONTECH Laboratories Inc., Palo Alto, CA). The sense orientation to the human cytomegalovirus (CMV) promoter was identified by restriction enzyme digestion analysis and further confirmed by sequencing analysis. One g/ml CSK cDNA in pIRES1neo or empty pIRES1neo vector alone was used for stable transfection into LLCPKcl4 cells using LipofectAMINE (Life Technologies, Inc.) as described previously (30). After 7 passages in medium containing 600 g/ml G418, G418-resistant clones were then isolated and screened by slot blot hybridization. Nine clones that expressed the highest levels of CSK mRNA were used to select further the highest levels of the immunoreactive CSK by immunoblotting with polyclonal antibodies against CSK. Two of the clones that expressed the highest levels of immunoreactive CSK, clones 13 and 26, along with LLCPKcl4 cells transfected with vector alone, were used for subsequent signaling studies.
Stable transfection and selection of stable transfectants of mutant bacterial cytochrome P450 BM3 were conducted as described (30).
RNA Isolation and Slot Blot Hybridization-Nontransfected, pIRES1neo vector-transfected cells and CSK-transfected individual clones were grown in 6-mm dishes and made quiescent for 3 days, and then total cellular RNA was isolated as described previously (4). 10 g of total RNA, as determined by absorbance at 260 nm, was slot blotted onto Nytran nylon membranes (Schleicher & Schuell) and immobilized with a UV cross-linker. [␣-32 P]cDNA probe was 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.
Immunoprecipitation and Immunoblotting-pIRES1neo vectortransfected and CSK-transfected cells were made quiescent and treated with indicated agents, washed twice with ice-cold Ca 2ϩ /Mg 2ϩ -free phosphate-buffered saline, and lysed on ice for 30 min in RIPA buffer (4). 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. Immune complexes were 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 ). Immunoprecipitates were resuspended and boiled in sample buffer before separation on a 7.5% or 12% SDS-PAGE, and transferred onto polyvinylidene difluoride membranes and immunodetected with the indicated primary antibodies and the appropriate secondary antibodies as described previously (4).
[ 3 H]Thymidine Incorporation Assay-Subconfluent cells in 24-well plates were made quiescent with serum-free medium. Agents were routinely added to the quiescent cells in triplicate or duplicate and incubated for 19 h, followed by 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, then lysed with 0.2 N NaOH, 0.5% SDS lysis buffer and incubated at 37°C for at least 30 min, and radioactivity of incorporated [ 3 H]thymidine was determined by liquid scintillation spectrometry (Beckman, Palo Alto, CA). Results were plotted as the number of counts per minute per well. Each experimental data point represents triplicate or duplicate wells from at least four different experiments.
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 acid-denatured 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 P-81 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 Ϯ S.E. for at least four separate experiments (each in triplicate or duplicate). Unpaired Stu- The pp60 c-src kinase was immunoprecipitated with a monoclonal antibody specifically against 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. dent'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.

RESULTS AND DISCUSSION
We stably transfected CSK cDNA, cloned in the mammalian expression vector pIRES1neo, into the LLCPKcl4 cell line, which was cloned from the parent LLC-PK1 cell line and selected for its expression of high levels of proximal tubule characteristics (31). G418-resistant clones were then isolated and screened by for mRNA expression levels (data not shown), and selected for immunoreactive CSK expression with polyclonal antibodies against CSK (Fig. 1). Two of the clones that overexpressed immunoreactive CSK, clones 13 and 26, along with LLCPKcl4 cells transfected with empty vector alone, were used for subsequent signaling studies.
Cells transfected with empty vector alone and CSK-overexpressing cells were rendered quiescent and exposed to the indicated agents. Cell lysates were prepared and subjected to immunoprecipitation with a monoclonal antibody specifically against pp60 c-src , and the immunoprecipitates were used for in vitro Src kinase activity assays. As shown in Fig. 2, 14,15-EET increased Src kinase activity 3.1-fold in empty vector-transfected cells, whereas in the CSK-overexpressing cells, 14,15-EET-induced activation of Src kinase activity was almost completely blocked (94% inhibition). Of interest, EGF and FBS also significantly increased Src kinase activity in empty vectortransfected cells, but not in CSK-overexpressing cells (Fig. 2). This inhibition of EGF-, FBS-and 14,15-EET-induced activation of Src kinase confirmed the ability of CSK overexpression to prevent Src activation.
14,15-EET has previously been demonstrated to be a potent mitogen in the nontransfected LLCPKcl4 cells, and Src has been suggested to involved in the signal transduction (4 We have reported that 14,15-EET activates both extracellular signal-regulated kinases (ERKs) and phosphatidylinositol 3-kinase in this cell line, and both mitogen-activated protein kinase pathway and phosphatidylinositol 3-kinase pathway are required for EET-induced mitogenesis (4). In order to ex- amine whether CSK overexpression affected the early signals activated by 14,15-EET, cell lysates were prepared and immunoprecipitated with anti-phosphotyrosine antibodies and immunoblotted with anti-ERK antibody (Fig. 4A). In CSK-overexpressing cells, 14,15-EET failed to activate either 44-kDa ERK1 or 42-kDa ERK2, whereas EGF-and FBS-induced activation of both ERK1 and ERK2 were not different from that observed in the cells transfected with empty vector alone. Similar results were obtained when cell lysates were probed with antibodies specific for phosphorylated ERKs (Fig. 4B).
In empty vector-transfected cells, pretreatment of the cells for 30 min with PP2, a selective inhibitor of kinase activity of pp60 c-src and other Src family kinases, at a concentration (1 M) that did not affect basal ERK expression, markedly inhibited 14,15-EET-increased tyrosine phosphorylation of both ERK1 and ERK2 (Fig. 5).
Synthetic eicosanoids have been widely utilized for the experimental analysis of their cellular and organ functions. However, in many cases, this approach provides only limited information with regard to their mechanisms of action and the enzymatic steps responsible for their biosynthesis from endogenous precursors, activation, and disposition. This is of special relevance regarding P450-derived eicosanoids, since in most cultured cells, there is a rapid and progressive decrease in the expression of the P450 isoforms found in vivo. LLCPKcl4 cells have no detectable endogenous P450 expression (30). In order to study further the involvement of Src kinase in endogenous EET signaling, we utilized stable LLCPKcl4 transfectants expressing a regio-and stereoselective 14S,15R-epoxygenase (14S,15R-EET, 99% of total products, 98% optical purity), the enantiomer that predominates in vivo in the kidney (30 -32). This protein is the bacterial cP450 BM3, in which phenylalanine 87 was replaced for valine (F87V BM3) (31) and catalyzes NADPH-dependent arachidonic acid oxidation as a self-contained catalytic unit (31). In the F87V BM3-transfected cells, EGF increased [ 3 H]thymidine incorporation to a significantly greater extent than in cells transfected with the vector alone, and 14,15-EET has been documented to function as an intracellular second messenger in response to EGF (30). F87V BM3 transfectants metabolize arachidonic acid to 14,15-EET after EGF administration (30). As shown in Fig. 6, in vector-transfected cells, exogenous arachidonic acid was unable to induce ERK tyrosine phosphorylation and the specific inhibitor of pp60 c-src kinase, PP2, did not affect EGF-induced tyrosine phosphorylation of ERKs. In contrast, in the F87V BM3-transfected cells, arachidonic acid addition dramatically stimulated ERK tyrosine phosphorylation, which was almost completely blocked by PP2 pretreatment (Fig. 6). Administration of EGF to F87V BM 3 -transfected cells increased ERK tyrosine phosphorylation to a significantly greater extent than in cells transfected with the empty vector alone. The Src kinase inhibitor, PP2, reduced EGF-stimulated ERK tyrosine phosphorylation in the F87V BM 3 -transfected LLCPKcl4 cells to the levels observed in the empty vector-transfected cells (Fig. 6). However, PP2 did not inhibit the EGF-stimulated ERK tyrosine phosphorylation in the vector-transfected cells, further indicating that this inhibitor did not nonspecifically inhibit ERK activation. These results indicate that Src kinase is activated by the EGF-induced increases in 14,15-EET biosynthesis from endogenous arachidonic acid pools.
In summary, the present studies indicate that in renal epithelial cells, the epoxygenase metabolites of arachidonic acid, epoxyeicosatrienoic acids, exert their mitogenic effects predominantly through a pathway mediated by pp60 c-src and/or other Src family kinases and that this Src kinase activity is the most upstream signaling protein determined to date in the 14,15-EET-activated tyrosine kinase cascade. EETs have also been shown to activate tyrosine kinase cascades and induce mitogenesis in smooth muscle, mesangial, and endothelial cells (33)(34)(35), but whether these effects are also mediated through Src kinase dependent pathways will require further study.