Heparin-binding EGF-like growth factor mediates the biological effects of P450 arachidonate epoxygenase metabolites in epithelial cells

Abstract

In addition to its important functions in detoxification of foreign chemicals and biosynthesis of steroid hormones, the cytochrome P450 enzyme system metabolizes arachidonate to 14,15-epoxyeicosatrienoic acid (14,15-EET). This study demonstrates that a P450 arachidonate epoxygenase metabolite can activate cleavage of heparin-binding epidermal growth factor-like growth factor (HB-EGF) and delineates an essential role for HB-EGF in the mitogenic effects of this lipid mediator. Blockade of HB-EGF processing or EGF receptor (EGFR) inhibited 14,15-EET-stimulated early mitogenic signals and DNA synthesis. 14,15-EET failed to induce mitogenesis in cell lines expressing minimal HB-EGF, whereas 14,15-EET induced soluble HB-EGF release into the conditioned media of cell lines that both express high levels of HB-EGF and display mitogenic response to this lipid mediator. Moreover, transfection of a bacterial 14,15-epoxygenase established intracellular endogenous 14,15-EET biosynthesis in cultured cell systems, which allowed direct confirmation of involvement of EGFR transactivation in the endogenous 14,15-EET-mediated mitogenic signaling pathway. This mechanism involves EET-dependent activation of metalloproteinases and release of the potent mitogenic EGFR ligand, HB-EGF.

Arachidonic acid is an important constituent of cellular membranes that is esterified to the sn-2 position of glycerophospholipids and is released from selected lipid stores after activation of specific phospholipases. Metabolism of released arachidonic acid by the cytochrome P450-dependent monooxygenase pathway produces biologically active compounds in two ways: epoxidation, producing 5,6-, 8,9-, 11,12-, 14,15-epoxyeicosatrienoic acids (EETs) and ω/ω-1 hydroxylation, resulting in the formation of 19- and 20-hydroxyeicosatetraenoic acids (1, 2). EETs play important roles in regulating vascular tone, mitogenesis, platelet aggregation, tissue and body homeostasis, Ca²⁺ signaling, and steroidogenesis (1–7). In addition, EETs have been suggested to be an endothelium-derived hyperpolarizing factor (8, 9) and to serve as intracellular second messengers in vasculature (10) and epithelia (11, 12). Recently, EETs have also been reported to play an anti-inflammatory role by inhibiting NF-κB-mediated vascular cell adhesion molecule 1 expression (13). EETs are produced predominantly by epoxygenases of the 2C gene subfamily of cytochrome P450s, which have been localized to the proximal tubule cells in the mammalian kidney. In this segment of the nephron, cytochrome P450 is the predominant arachidonic acid metabolic pathway, whereas cyclooxygenase and lipoxygenase are expressed at nearly undetectable levels (14, 15).

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the EGF family originally identified in conditioned medium (CM) of the human histiotic lymphoma cell line, U-937 (16), is expressed as a single transmembrane-spanning precursor protein composed of signal peptide, heparin-binding, EGF-like, juxtamembrane, transmembrane, and cytoplasmic domains, in a variety of tissues and cultured cells including vascular endothelial cells, smooth muscle cells (17), and renal epithelial cells (18, 19). Several intracellular signaling events including protein kinase C activation, calcium signaling, and protein tyrosine phosphorylation are able to induce proteolytic cleavage of pro-HB-EGF at the juxtamembrane site leading to the “ectodomain shedding” of a soluble EGF receptor (EGFR) ligand (sHB-EGF) with 76–86 aa that activates EGFR (HER1) as well as HER4 in an autocrine/paracrine manner (17, 20, 21). sHB-EGF is a potent mitogen for several cells including NIH 3T3 cells, smooth muscle cells, keratinocytes, and renal tubule cells (17–19). HB-EGF has been implicated in wound healing, blastocyst implantation, smooth muscle cells hyperplasia, atherosclerosis, and tumor growth (22–25). Recently, Prenzel et al. (26) reported that G protein-coupled receptor (GPCR) ligand-stimulated tyrosine phosphorylation of the EGFR involves metalloproteinase cleavage of pro-HB-EGF.

In the mammalian kidney, HB-EGF is predominantly expressed in the proximal tubules and the arterial smooth muscle cells (18, 27). Although the renal proximal tubule contains the highest concentration of EGFR in the tubules, EGF has not been found to be expressed in the normal proximal tubules (28). Therefore, HB-EGF might be the major ligand for EGFRs in the proximal tubules and might play an important role in regulation of the proximal tubule functions. In the present study, we demonstrated that an essential step in the mitogenic signaling mechanism of EETs involves transactivation of EGFR by induction of HB-EGF processing and release.

Materials and Methods

Reagents and Antibodies.

(±)-11,12-EET and (±)-14,15-EET were synthesized as described (4, 29). Arachidonic acid was obtained from Nu Chek Prep (Elysian, MN). EGF (receptor grade) was purchased from Collaborative Research. Tyrphostin AG1478 and phenanthroline were obtained from Calbiochem. Polyclonal and monoclonal antiphosphotyrosine antibodies were purchased from Zymed. Polyclonal antiextracellular signal-regulated kinase (ERK) antibodies, monoclonal antiphospho-ERK antibodies, anti-EGFR antibodies, and Protein A/G Agarose beads were from Santa Cruz Biotechnology. Polyclonal anti-HB-EGF antibody 2998 was a generous gift from Judith Abraham (Scios, Mountain View, CA). Heparin-Sepharose CL-6B column was from Amersham Pharmacia. Monoclonal neutralizing EGFR antibody clone 528 was a generous gift from Robert Coffey (Vanderbilt University). Batimastat (BB-94) was from British Biotechnology (Oxford, U.K.). CRM197, monoclonal anti-actin antibody, and all other chemicals were from Sigma.

Cell Culture.

LLCPKcl4, an established adherent proximal tubule-like epithelial cell line derived from pig kidney (30), was cultured in DMEM/F-12 mixture; LLC-PK1 cells were cultured with medium 199; human monocytic U937 cells were maintained in RPMI medium 1640; COS-7 cells were grown in DMEM at 37°C in a 5% CO₂-cell culture incubator. All media were supplemented with 100 units/ml penicillin, 100 μg/ml streptomycin, and 10% FBS (HyClone), and the media were changed every 2–3 days. Stable transfectants expressing the bacterial P450 epoxygenase F87V BM₃ (BM₃ cells) were maintained as described (12).

[³H]Thymidine Incorporation Assay, Immunoprecipitation, and Immunoblotting.

These assays were performed as previously described (4).

Cell Subcloning.

By using the cloning-ring technique, several single-cell-derived individual clones were isolated from the porcine renal epithelial cell line LLC-PK1 and screened by [³H]thymidine incorporation assay for their responsiveness to 14,15-EET. A range of individual clones with various sensitivity to 14,15-EET was obtained. Two clones mitogenically sensitive to 14,15-EET (with 5- to 7-fold stimulation), named EET⁺-1 and EET⁺-2, and another two clones that negatively responded to 14,15-EET, EET⁻-1 and EET⁻-2, were used in the present study.

Purification of Secreted HB-EGF.

Secreted soluble HB-EGF was purified by a modification of heparin affinity chromatography as described (19). Before exposure to vehicle or the indicated agents in serum-free media, confluent cells were rendered quiescent and washed twice with PBS. CM were centrifuged and filtered through a 0.45-mm filter. Each sample of CM was immediately applied to a heparin-Sepharose column that was preequilibrated with 10 mM Tris⋅HCl (pH 7.4) containing 0.2 M NaCl and 1 mM benzamidine. After extensive wash with equilibration buffer, bound proteins were eluted with 2.0 M NaCl in 10 mM Tris⋅HCl (pH 7.4) and dialyzed against 10 mM Tris⋅HCl (pH 7.4) at 4°C overnight. The dialyzed eluent was lyophilized and used for subsequent analysis.

Statistics.

Data are presented as means ± standard errors for at least three separate experiments (each in triplicate or duplicate). An unpaired Student's t test was used for statistical analysis and multiple group comparisons, ANOVA, and Bonferroni t tests were used. A P value of <0.05 compared with control was considered statistically significant.

Results

Our previous studies demonstrated that 14,15-EET is a potent mitogen for the renal proximal tubule cell line LLCPKcl4, which was cloned from the parental cell line LLC-PK1 and selected for its proximal tubule characteristics (30), and that the mitogenic effect of 14,15-EET is mediated by initiation of a tyrosine kinase phosphorylation cascade that activates the p44/p42 ERKs and phosphatidylinositol 3-kinase (4). EGFR tyrosine phosphorylation was noted to be increased within 5 min of 14,15-EET addition (4). As shown in Fig. 1A, 14,15-EET induced tyrosine phosphorylation of EGFR and ERKs in a concentration-dependent fashion, with a significant stimulation clearly detected at ≥100 nM. Further experiments were designed to determine the importance of EGFR activation in the mitogenic signaling of 14,15-EET in LLCPKcl4 cells by using the specific EGFR tyrosine kinase inhibitor tyrphostin AG1478 (32). First, we tested effects of different concentrations of AG1478 on EET-induced ERK tyrosine phosphorylation and found that AG1478 dose-dependently inhibited the effect of EET, with an IC₅₀ of ≈20 nM, and ≥200 nM AG1478 completely blocked 20 μM 14,15-EET-induced ERK tyrosine phosphorylation (data not shown). To prevent possible nonspecific inhibition with high concentrations, we chose to use 100 nM AG1478 to perform the subsequent studies. As shown in Fig. 1B, pretreatment of the cells with tyrphostin AG1478 not only blocked EGF-induced tyrosine phosphorylation of ERK1 and ERK2 but also that induced by 14,15-EET (20 μM). Tyrphostin AG1478 also blocked EGFR tyrosine phosphorylation stimulated by 14,15-EET (Fig. 1C) and abrogated 14,15-EET-stimulated [³H]thymidine incorporation (Fig. 1D). Preincubation of LLCPKcl4 cells with a monoclonal neutralizing EGFR antibody significantly inhibited the tyrosine phosphorylation of EGFR induced by 14,15-EET (Fig. 1E Top) and inhibited 14,15-EET-induced ERK tyrosine phosphorylation (Fig. 1E Middle); in contrast, an unrelated mAb against actin had no effect (data not shown), indicating that the neutralizing EGFR antibody is specifically blocking the EGFR. These data indicated that EGFR transactivation is an essential event in the mitogenic signaling pathway of 14,15-EET and that EGFR is upstream of ERK activation.

Figure 1.

(A) EGFR and ERK tyrosine phosphorylation in response to dosing of 14,15-EET. Quiescent LLCPKcl4 cells were treated with vehicle (Me₂SO) alone (lane 1), or different concentrations of 14,15-EET (lanes 2–10: 60, 40, 20, 10, 5, 1, 0.1, 0.01, and 0.001 μM, respectively). Cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies. (B–E) Effect of EGFR inhibition on 14,15-EET-stimulated mitogenic signaling (B, C, and E) and DNA synthesis (D) in LLCPKcl4 cells.

To identify the mechanism by which 14,15-EET activates EGFR, quiescent LLCPKcl4 cells were pretreated with two different metalloproteinase inhibitors, Phenanthroline or Batimastat, before exposure to 14,15-EET (20 μM). Immunoprecipitation and immunoblotting analysis revealed that both inhibitors blocked 14,15-EET-induced tyrosine phosphorylation of EGFR and ERK tyrosine phosphorylation (Fig. 2 A and B). In contrast, neither inhibitor had any effect on EGF-induced tyrosine phosphorylation of EGFR or ERKs (Fig. 2 A and B). These data suggested that metalloproteinase activation was involved in EGFR transactivation induced by 14,15-EET.

Figure 2.

Effect of metalloproteinase inhibition on 14,15-WWR-induced tyrosine phosphorylation of EGFR and ERKs. (B, a) Quantification of four separate experiments by densitometer, with representative blots (B, b and c) beneath this bar graph.

The transmembrane precursor of HB-EGF (pro-HB-EGF) also serves as the unique high-affinity receptor for diphtheria toxin (33). Binding of CRM197, the nontoxic and catalytically inactive [Glu-52] mutant of diphtheria toxin, to the extracellular HB-EGF domain potently and specifically inhibits the mitogenic activity of HB-EGF (34). Unlike in mice and rats, the amino acid residues critical for diphtheria toxin binding, Phe-115, Leu-127, and Glu-141 in the binding domain of the porcine and monkey proHB-EGF are identical with those of the human pro-HB-EGF, enabling CRM197 to bind and interact with the porcine and monkey pro-HB-EGF (35, 36). To determine whether HB-EGF shedding contributes to 14,15-EET-mediated activation of the EGFR-ERK signaling cascade, we assessed whether pretreatment with CRM197 could affect 14,15-EET-mediated tyrosine phosphorylation of EGFR and ERKs. As shown in Fig. 3A, preincubation with CRM197 inhibited 14,15-EET-stimulated tyrosine phosphorylation of both EGFR and ERKs. In contrast, EGF-induced EGFR tyrosine phosphorylation and ERK activation were not altered by CRM197. Furthermore, 14,15-EET also transactivated EGFR and induced tyrosine phosphorylation of ERK1 and ERK2 in the monkey kidney cell line COS-7, which were blocked by pretreatment with CRM197; phorbol 12-myristate 13-acetate (PMA) was included in these experiments to serve as a positive control, because PMA can induce cleavage of HB-EGF in COS-7 cells (Fig. 3B).

Figure 3.

Effects of the nontoxic mutant of diphtheria toxin CRM197 on 14,15-EET-induced tyrosine phosphorylation of EGFR and ERKs. (A) LLCPKcl4 cells. (B) COS-7 cells.

To examine whether 14,15-EET administration could initiate proteolytic processing of pro-HB-EGF, CM were collected from vehicle- or 14,15-EET-treated cells, and heparin-binding proteins were purified by heparin-Sepharose chromatography. PMA, a potent inducer of pro-HB-EGF processing, was added as a positive control. Immunoblotting with the anti-HB-EGF antibody 2998 revealed that 14,15-EET stimulated release of a 14-kDa and a 19-kDa soluble HB-EGF species into the CM of LLCPKcl4 cells (Fig. 4A). As shown in Fig. 4B, 14,15-EET also induced pro-HB-EGF processing in the human macrophage-like cell line U-937, which expresses high levels of pro-HB-EGF (16), indicating that the 14,15-EET-triggered shedding of pro-HB-EGF is not limited to epithelial cells. To examine activity of the EET-released HB-EGF, we treated quiescent A431 cells, which express high levels of EGFR, with or without the partially purified HB-EGF from CM. As demonstrated in Fig. 4C, compared with A431 cells treated with vehicle alone (lane 1), the partially purified HB-EGF from the CM of LLCPKcl4 cells treated with 14,15-EET significantly induced EGFR tyrosine phosphorylation in A431 cells (lane 3), whereas the HB-EGF purification product from the CM of LLCPKcl4 cells treated with Me₂SO alone had no effect (lane 2). These data indicate that 14,15-EET activates EGFR and its downstream signaling by induction of pro-HB-EGF shedding.

Figure 4.

(A and B) 14,15-EET-induced release of soluble HB-EGF. CM were collected after treatment of LLCPKcl4 cells (A) or the human macrophage-like cell line U-937 (B) with or without 14,15-EET and applied to a heparin-Sepharose column to purify heparin-binding proteins. PMA, a potent inducer of pro-HB-EGF processing, was added as a positive control. Bound proteins were eluted and analyzed by immunoblotting with the anti-HB-EGF antibody 2998. (C) EGFR activation in A431 cells by 14,15-EET-released HB-EGF partially purified from CM. Quiescent A431 cells were treated with vehicle alone (lane 1) or partially purified HB-EGF from CM of Me₂SO- or EET-treated LLCPKcl4 cells (lanes 2 and 3), EET⁺ cells (lanes 4 and 5), or EET⁻ cells (lane 6 and 7) (Me₂SO: lane 2, 4, and 6; EET: lane 3, 5, and 7). A431 cell lysates were subjected to immunoprecipitation and immunoblotting with the indicated antibodies.

Unlike in LLCPKcl4, 14,15-EET failed to stimulate [³H]thymidine incorporation in the parental LLC-PK1 cells (Fig. 5A). We examined HB-EGF expression levels in these two cell lines. HB-EGF is expressed as a membrane-anchored HB-EGF precursor (pro-HB-EGF) in the form of heterogeneous translation products with a relative molecular mass of 20–30 kDa, which are derived from multiple N-terminal truncations and heterogeneous glycosylation (20, 37). As indicated by the arrows in Fig. 5B, LLC-PK1 expressed significantly lower levels of pro-HB-EGF than LLCPKcl4. We therefore isolated individual clones from the parental LLC-PK1 cells that either would or would not respond to 14,15-EET, and screened these individual clones with [³H]thymidine incorporation assay. In response to 14,15-EET, positive clones (designated EET⁺-1 and EET⁺-2) increased [³H]thymidine incorporation 5- to 7-fold, whereas negative clones EET⁻-1 and EET⁻-2 had decreased [³H]thymidine incorporation (Fig. 5A). Immunoblotting revealed high expression levels of pro-HB-EGF in EET⁺-1 and EET⁺-2 (Fig. 5C); in contrast, minimal pro-HB-EGF could be detected in EET⁻-1 and EET⁻-2 cells (Fig. 5C). Consistent with the pro-HB-EGF expression profile of these subclones, the HB-EGF purification product from CM that was collected from EET-treated EET⁺-cells (Fig. 4C, lane 5) markedly activated EGFR in A431 cells; in contrast, the HB-EGF purification product from CM that was collected from EET-treated EET⁻-cells had no effect on EGFR tyrosine phosphorylation in A431 cells (Fig. 4C, lane 7). Furthermore, the purified HB-EGF from CM of EET-treated LLCPKcl4 and EET⁺-cells significantly stimulated [³H]thymidine incorporation in EET-cells (data not shown), indicating that the EET-cells retained their responsiveness to HB-EGF.

Figure 5.

(A) DNA synthesis in LLCPKcl4, parental LLC-PK1, and individual clones isolated from the parental LLC-PK1 in response to 14,15-EET. (B and C) Pro-HB-EGF protein expression in LLCPKcl4 and the parental LLC-PK1 (B), and in individual clones isolated from the parental LLC-PK1 (C).

In addition to the indicated results with 14,15-EET, we examined effects of 11,12-EET on tyrosine kinase phosphorylation and found dose-dependent tyrosine phosphorylation of both EGFR and ERKs, which were blocked by the specific HB-EGF-processing blocker, CRM197 (data not shown).

Synthetic eicosanoids have been widely used for the experimental analysis of their cellular and organ functions. However, in many cases, this approach does not address the enzymatic steps responsible for their biosynthesis from endogenous precursors, activation, and disposition. This approach is of special relevance regarding P450-derived eicosanoids, because in most cultured cells a rapid and progressive decrease occurs in the expression of the P450 isoforms found in vivo. Therefore, to determine whether endogenous 14,15-EET could also transactivate EGFR by activation of the HB-EGF-shedding process, we used an LLCPKcl4 cell line transfected with an engineered P450 epoxygenase of bacterial origin, F87V BM3. This enzyme converts arachidonic acid selectively (>98% of total products) to 14(S),15(R)-EET, the enantiomer that predominates in vivo in the kidney (12, 31). Our previous studies demonstrated that addition of exogenous arachidonic acid to F87V BM3-transfected cells (BM3 cells) increased endogenous 14,15-EET levels more than 100-fold, whereas no measurable increase occurred in vector-transfected cells (Vector cells) (12), and indicated that exogenously added arachidonic acid-induced tyrosine phosphorylation of EGFR and ERKs in the BM3 cells but not in the Vector cells (12). Accordingly, quiescent BM3 cells were pretreated with or without the metalloproteinase inhibitor Batimastat before exposure to arachidonic acid (30 μM), followed by immunoprecipitation and immunoblotting analysis. As shown in Fig. 6A, Batimastat (5 μM) almost completely blocked arachidonic acid-induced tyrosine phosphorylation of EGFR in the BM3 cells.

Figure 6.

(A) Effect of the metalloproteinase inhibitors, Batimastat, or the nontoxic mutant of diphtheria toxin CRM197 on arachidonic acid- or EGF-induced activation of EGFR and ERKs in the F87V BM-3-transfected cells. Empty vector-transfected cells (Vector) and the F87V BM-3-transfected cells (BM3) were treated with the indicated agents, then cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies.

14,15-EET is an intracellular second messenger in response to EGF in cells expressing epoxygenase activity (12), and in the BM3 cells, EGF increased [³H]thymidine incorporation to a significantly greater extent than in the Vector cells. In these studies, we found that administration of EGF to the BM3 cells increased tyrosine phosphorylation of EGFR (Fig. 6B) and ERKs (Fig. 6C) to a significantly greater degree than in the Vector cells. CRM197 reduced EGF-stimulated tyrosine phosphorylation of both EGFR and ERK in the BM3 cells to the levels observed in the Vector cells (Fig. 6 B and C). In contrast, CRM197 did not inhibit the EGF-stimulated EGFR and ERK tyrosine phosphorylation in the Vector cells, suggesting that this inhibitor did not nonspecifically inhibit EGFR or ERK activation (Fig. 6 B and C). Similarly, Batimastat partially blocked EGF-stimulated tyrosine phosphorylation of EGFR in the BM3 cells (Fig. 6A). These data indicated that the augmentation of EGFR and ERK activation was the result of HB-EGF processing induced by the increased 14,15-EET biosynthesis from endogenous arachidonic acid release secondary to EGF administration.

Discussion

Although studies have suggested that cytochrome P450 arachidonic acid metabolites are potent mitogens for both mesenchymal and epithelial cells (11, 38, 39), the mechanisms by which cytochrome P450 arachidonic acid metabolites induce mitogenesis have only recently been examined. Our recent studies have determined that a cytochrome P450 epoxygenase metabolite 14,15-EET induces DNA synthesis and cell proliferation by a signaling pathway involving activation of a tyrosine kinase cascade (4). In this study, we demonstrated that activation of HB-EGF cleavage and release is an essential step in the mitogenic signaling pathway of 14,15-EET. As depicted in Fig. 7, 14,15-EET induces EGFR activation and downstream signaling by induction of pro-HB-EGF processing through activation of a metalloproteinase that causes release of soluble HB-EGF. The released HB-EGF then binds to EGFR and activates its intrinsic receptor tyrosine kinase, leading to autophosphorylation of the EGFR. These data also provide evidence that insufficient expression of pro-HB-EGF, inhibiting release or activity of soluble HB-EGF ligand, or blocking the availability of functioning EGFR each is sufficient to inhibit 14,15-EET-stimulated intracellular signaling.

Figure 7.

Involvement of EGFR transactivation in 14,15-EET-mediated mitogenic signaling.

Endogenous ligands of the EGFR are expressed as single transmembrane precursors that undergo proteolytic cleavage by metalloproteinases to release soluble growth factors (40). Although earlier studies on the mechanisms of EGFR transactivation failed to elucidate an autocrine/paracrine mechanism (41, 42), and significant heterogeneity exists in the signaling mechanisms used by GPCR ligands to activate ERKs in different cell systems, GPCR agonists such as lysophosphatidic acid and endothelin have recently been demonstrated to stimulate ERKs through transactivation of the EGFR involving metalloproteinase-mediated release of soluble HB-EGF (26). In COS-7 cells, insulin-like growth factor 1 also induces EGFR transactivation by metalloproteinase-dependent release of HB-EGF (43). Our studies demonstrate EGFR transactivation through a lipid-derived second messenger. In addition, we demonstrated that augmentation of EGF-mediated signaling by production of endogenous EETs was mediated by HB-EGF release.

The identity of the metalloproteinase responsible for 14,15-EET-induced cleavage of the pro-HB-EGF will require further investigation. In other systems, the “a disintegrin and metalloproteinase” (ADAM) family of matrix metalloproteinases has been shown to mediate the proteolysis of the HB-EGF precursor (44). Among the ADAM family members, ADAM 9 has been reported to mediate protein kinase C-dependent HB-EGF cleavage in response to phorbol esters (45). However, GPCR-induced pro-HB-EGF processing is not sensitive to inhibitors of protein kinase C (26), suggesting that protein kinase C-dependent modulation of metalloproteinase activity may not be responsible for GPCR-induced EGFR transactivation.

Prostaglandins, leukotrienes, and other polar arachidonic acid metabolites exert various functions mainly through their specific GPCRs on the cell membrane; in contrast, the mechanisms mediating the biological effects of the less polar cP450 arachidonate metabolites such as EETs are incompletely understood. Specific binding sites for 14,15-EET in monocytes (46–48) and 12(R)-HETE in microvessel endothelial cells (49) have been reported. Recent studies have also indicated that EETs activate calcium-activated K⁺ channels in vascular smooth muscle cells but have no effect when added directly to the cytoplasmic surface of excised inside-out patches (50). More recently, it has been suggested that EET-mediated channel activation requires intermediate signaling steps involving G proteins (51). However, the detailed mechanism by which EETs initiate signaling events remains to be determined.

In summary, our studies reveal that induction of the EGFR transactivation is a crucial event in the mitogenic signaling transmission of the P450 arachidonate epoxygenase metabolites EETs, and that EGFR is upstream of both ERK1 and ERK2 in the EET-signaling pathway. We demonstrate that EETs induce transactivation of the EGFR by metalloproteinase activation that releases pro-HB-EGF from the cell membrane, and the released soluble HB-EGF subsequently activates the EGFR and downstream ERKs in the EET-stimulated mitogenic signaling pathway.

Abbreviations

EET

epoxyeicosatrienoic acid

EGF

epidermal growth factor

HB-EGF

heparin-binding EGF-like growth factor

EGFR

epidermal growth factor receptor

GPCR

G protein-coupled receptor

PMA

phorbol 12-myristate 13-acetate

CM

conditioned medium

ERK

extracellular signal-regulated kinase