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J. Biol. Chem., Vol. 275, Issue 49, 38831-38841, December 8, 2000

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Eicosanoid Activation of Extracellular Signal-regulated Kinase1/2 in Human Epidermoid Carcinoma Cells^*

Charles K. Szekeres^§, Keqin Tang^§, Mohit Trikha^§, and Kenneth V. Honn^§¶

From the  Department of Radiation Oncology and the ^¶ Departments of Pathology and Chemistry, Wayne State University, Detroit and the ^§ Karmanos Cancer Institute, Detroit, Michigan 48202

Received for publication, March 29, 2000, and in revised form, August 17, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

12(S)-Hydroxyeicosatetraenoic acid (12(S)-HETE), a 12-lipoxygenase metabolite of arachidonic acid, has multiple effects on tumor and endothelial cells, including stimulation of invasion and angiogenesis. However, the signaling mechanisms controlling these physiological processes are poorly understood. In a human epidermoid carcinoma cell line (i.e. A431), 12(S)-HETE activates extracellular signal-regulated kinases 1/2 (ERK1/2), which is mediated by upstream kinases MEK and Raf. 12(S)-HETE stimulates phosphorylation of phospholipase C1 and activity of protein kinase C (PKC). In addition, independent of PKC 12(S)-HETE increases tyrosine phosphorylation of Shc, and Grb2, stimulates association between Shc and Src, and increases the activity of Ras, via Src family kinases. Furthermore, at low (10-100 nM) concentrations 12(S)-HETE counteracts epidermal growth factor-stimulated activation of ERK1/2 via stimulating protein tyrosine phosphatases. We also present evidence that 12(S)-HETE stimulates ERK1/2 via G proteins and that A431 cells have multiple binding sites for 12(S)-HETE. Finally, inhibition of 12-lipoxygenase induced apoptosis of A431 cells, which was reversed by addition of exogenous 12(S)-HETE. Collectively we demonstrate that the activation of ERK1/2 by 12(S)-HETE may be regulated by multiple receptors triggering PKC-dependent and PKC-independent pathways in A431 cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Tumor cell-host interactions are fundamentally influenced by bioactive lipids produced by the tumor cells themselves, as well as by the infiltrating leukocytes, monocytes, and by aggregation with platelets (1). Previous studies have demonstrated that one of the most important lipid metabolites to influence tumor progression is the lipoxygenase metabolite 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE).¹ This eicosanoid stimulates several steps of tumor invasion and motility (2-4), protects tumor cells from apoptosis (5), and promotes angiogenesis (6). Initial studies suggest that the pleiotropic effects of 12(S)-HETE on tumor cells are mediated by an eicosanoid receptor which activates protein kinase C (PKC) (7).

The extracellular signal-regulated kinases, ERK1 and -2, also known as p44 and p42 mitogen-activated protein kinases (p42/44 MAPK), respectively, are well characterized as convergence points of numerous signal transduction pathways. Through their diverse substrates, ERKs modulate nuclear, as well as cytoplasmic events in cells resulting in increased proliferation (8), differentiation (9), changes in cell morphology (9), and motility (10). It is well established that receptor-tyrosine kinases activate ERK via the consecutive stimulation of the guanine nucleotide exchange factor Sos, monomeric G protein Ras, and the Raf-MEK-MAPK cascade of protein kinases. In addition to growth factor receptors with tyrosine kinase activity, G protein-coupled receptors also are stimulators of ERK. G proteins can influence ERK activity via multiple mechanisms (11), which include activation of tyrosine kinases such as the EGF receptor (12) or Src (13), stimulating the early stages of the conventional cascade. Alternatively, G proteins may activate ERK by promoting the production of lipid second messengers via phospholipase C (PLC) or phosphatidylinositide 3-kinase resulting in the activation of PKC. Protein kinase C in turn can stimulate the ERK cascade through Raf and MEK.

An earlier study, reported that 12(S)-HETE increased the tyrosine phosphorylation of cellular proteins migrating in the 40-50-kDa range (14), which led us to hypothesize that ERK1/2 may be mediators of 12(S)-HETE-induced cellular responses.

In this study we identify ERK1/2 as signaling targets of exogenous 12(S)-HETE in A431 epidermoid carcinoma cells, and we describe the signaling mechanisms leading to this stimulation through the following: (a) the Src family-mediated activation of PLC1 and PKC, (b) Src family-mediated tyrosine phosphorylation of Shc and subsequent stimulation of the Raf/MEK/ERK cascade via Ras. Furthermore, we provide evidence for the involvement of G proteins in 12(S)-HETE signaling.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Antibodies and Reagents-- Anti-phospho-specific ERK, anti-phospho-specific MEK, anti-phospho-specific Elk, and phospho-specific epidermal growth factor (EGF) receptor antibodies were purchased from New England Biolabs (Beverly, MA). Anti-phospho-specific PKC was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-pan-ERK, Ras, MEK, PY20, Shc, Grb2, anti-PKC, and PKC sampler kit antibodies to screen PKC isotype expression were from Transduction Laboratories (Lexington, KY). Anti-actin was from ICN (Costa Mesa, CA), and horseradish peroxidase-conjugated secondary antibodies were purchased from Amersham Pharmacia Biotech. Anti-Raf-1, PLC2, PLC1, and Src (SRC 2, which recognizes Src, Yes, and Fyn) were from Santa Cruz Biotechnology (Santa Cruz, CA). 5-, 11-, 12-, and 15(S)-HETE were purchased from Cayman Chemicals (Ann Arbor, MI). Protein G-Sepharose 4B was from Zymed Laboratories Inc. (South San Francisco, CA). The inhibitors Go6976 (inhibitor of conventional PKC isozymes, IC₅₀ 2.3 nM (15), solvent: ethanol), PP2 (inhibitor of Src family kinases (16), solvent: ethanol), PD98059 (inhibitor of MEK, IC₅₀ 2 µM (17) solvent: ethanol), Tyrphostin51 (inhibitor of EGF receptor tyrosine kinase, IC₅₀ 800 nM (18), solvent: Me₂SO), FTase inhibitor II (inhibitor of farnesyltransferase, IC₅₀ 50 nM (19) solvent: H₂O), and suramin (inhibitor of G protein-receptor coupling (20) solvent: H₂O) were from Calbiochem. The specific peptide substrate for Src kinase assay, KVEKIGEGTYGVVYK, was purchased from Upstate Biotechnology, Inc. The 12-lipoxygenase-selective inhibitor N-benzyl-N-hydroxy-5-phenylpentanamide, BHPP (21), was a generous gift from Biomide Corp (Grosse Pointe Farms, MI). All other chemicals were obtained from Sigma.

Cell Culture-- The human epidermoid carcinoma cell line A431 (American Tissue Culture Collection, Manassas, VA) was cultured in Dulbecco's modified Eagle's media (DMEM) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 25 mg/liter gentamicin (Life Technologies, Inc.). Cells were passaged with 0.05% trypsin-EDTA.

Platelet type 12 lipoxygenase-transfected A431 cells were described earlier (22).

For drug treatments, A431 cells (1.5 × 10⁶) were plated in 6-well plates, cultured for 1 day in DMEM supplemented with 10% fetal bovine serum, and then serum-starved overnight (20 h). Fresh serum-free DMEM was added 1 h prior to treatments. Inhibitors (at the concentrations indicated in the figure legends) or vehicle (0.1%) treatment was 15 min before stimulation with 12(S)-HETE or other chemicals, with the exception of farnesyltransferase inhibitor (FTase inhibitor II, 1 h) and Src family kinase inhibitor (PP2, 30 min).

Immunoblotting-- Cells were rinsed twice with ice-cold phosphate-buffered saline (PBS) and lysed with 200 µl of boiling gel loading buffer (20% glycerol; 2% SDS; 2.5 × 10²mg/ml bromphenol blue; 125 mM Tris base, pH 6.8; 5% 2-mercaptoethanol). Cell lysates were sonicated briefly and boiled for 5 min. Aliquots (20 µl) were resolved on 10 or 4-20% SDS-polyacrylamide gels (the latter from Fisher) and electrotransferred onto nitrocellulose membrane (Bio-Rad). In gels used to detect the mobility shift of phosphorylated proteins, the acrylamide:bisacrylamide ratio was 118:1. Membranes were probed with the antibodies indicated in the text, and bands were visualized with the Supersignal system (Pierce). Blots were routinely stripped at 50 °C for 30 min in stripping buffer (0.7% 2-mercaptoethanol; 2% SDS; 62.5 mM Tris-HCl, pH 6.7) and reprobed with other antibodies. Densitometric analysis of the Western blots was performed with an LKB 2222-010 UltroScan XL laser densitometer (Bromma, Sweden).

Immunoprecipitation-- Cells were serum-starved overnight, pretreated with drugs, followed by washing with ice-cold PBS. For immunoprecipitation under denaturing conditions, cells were lysed (200 µl boiling 1% SDS; 10 mM Tris, pH 7.4), boiled for 5 min, and briefly sonicated. Cell lysates were centrifuged (13,000 × g, 5 min), and equal amounts of debris-free supernatant was mixed with 1 ml of IP buffer (1% Triton X-100; 150 mM NaCl; 10 mM Tris, pH 7.4; 1 mM EDTA; 1 mM EGTA; 0.2 mM Na₃VO₄; 0.2 mM PMSF; 0.5% Nonidet P-40) and anti-phosphotyrosine (3 µg) or other antibodies (1 µg). In the case of non-denaturing immunoprecipitation, cells were rinsed with ice-cold PBS and scraped off into 1 ml of ice-cold IP buffer, sonicated, incubated with agitation for 30 min at 4 °C, and centrifuged (13,000 × g, 10 min) to remove insoluble material. Equal amounts of protein were mixed with 2 µg of antibody for 3 h and then with 50 µl of protein G-Sepharose. After overnight incubation (4 °C), beads were washed twice with dilution buffer (10 mM Tris-HCl, pH 8.0; 140 mM NaCl; 0.1% Triton X-100; 0.1% BSA; 0.025% NaN₃) and once with TSA solution (10 mM Tris-HCl; 140 mM NaCl; 0.025% NaN₃). For PLC2 coprecipitation with G antibody anti-G (2 µg) was mixed with protein G-Sepharose (50 µl) for 3 h and then washed (2 times) with IP buffer before mixing with cell lysates. Immunocomplexes were denatured by boiling in gel loading buffer.

ERK Kinase Assay-- Kinase assays were performed using an MAPK assay kit from New England Biolabs according to the manufacturer's recommendations. Briefly, activated ERK was precipitated from cell lysates using anti-phospho-ERK antibody, and precipitates were incubated with a specific substrate, Elk-1, and ATP. The reaction was terminated by adding boiling gel loading buffer. ERK activity was detected by immunoblotting the products of the kinase reaction with anti-phospho-Elk antibody.

Raf Kinase Assay-- Raf kinase assay was performed using the c-Raf1 immunoprecipitation kinase cascade assay kit from Upstate Biotechnology, Inc., according to the protocol provided by the manufacturer. Briefly, Raf was precipitated and incubated with GST-MEK and GST-ERK1 in [³²P]ATP (PerkinElmer Life Sciences) containing buffer. ERK1 was recovered on phosphocellulose paper, and the incorporated radioactivity was measured with a 1900TR Liquid Scintillation Analyzer (Packard Instrument Co.).

Src Kinase Assay-- After various treatments, A431 cells were lysed, and Src was precipitated under non-denaturing conditions with anti-Src polyclonal antibody (SRC2; 3 µg/sample). Precipitates were washed (1 time) with lysis buffer and then (3 times) with 0.5 M LiCl, Tris-HCl, pH 7.5, and 1 time in 25 mM Tris-HCl, pH 7.5, then resuspended and incubated at room temperature for 30 min in 15 µl of kinase buffer (50 mM Tris-HCl, pH 7.5; 5 mM MgCl₂; 25 µM ATP; 1 µg/sample Src substrate; 5 µCi of [³²P]ATP). The reaction was terminated by adding 20 µl of 2× gel loading buffer and boiling for 5 min. Samples were run on 15% SDS-polyacrylamide, and incorporated radioactivity was quantitated with a Storm 940 PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Ras Assay-- Ras activity was assessed using Raf-1 RBD (Upstate Biotechnology, Inc.) according to the manufacturer's protocol. Briefly, beads conjugated to the Ras binding domain (RBD) of Raf were used to precipitate GTP-bound Ras. The amount of precipitated Ras was determined by Western blotting, with a pan antibody that recognizes all human isoforms of Ras.

PKC Translocation-- Translocation of PKC was determined as described previously (7, 23). Following 12(S)-HETE treatment, cells were rinsed (twice) with cold PBS, scraped off, and homogenized with 26-gauge needle in 2 ml of buffer A (25 mM Tris-HCl, pH 7.6; 1 mM EGTA; aprotinin (5 µg/ml); leupeptin (10 µg/ml) and 1 mM PMSF). Membrane and cytosolic fractions were separated by centrifugation (100,000 × g; 1 h, 4 °C). The membrane fraction was rinsed (twice) with buffer A and resuspended in buffer A containing 1% Nonidet P-40. Samples were applied to a 1-ml DEAE-Sepharose column (Sigma) equilibrated with buffer B (20 mM Tris-HCl, pH 7.5; 2 mM EDTA; 1 mM 2-mercaptoethanol; 0.2 mM PMSF; 0.15 mM pepstatin A). PKC was eluted with 2 ml of buffer B containing 120 mM NaCl. Samples were concentrated using a Centricon (Amicon, Beverly, MA), and the protein concentration was determined with the BCA protein assay kit (Pierce).

Inhibition of PKC Activity-- PKC activity was inhibited using two approaches. First, cells were treated with PKC inhibitor as described above. Second, PKC protein level in A431 cells was down-regulated with chronic PMA treatment (22 h, 100 nM) in serum-free media (24). Phorbol ester-containing media were removed 1 h prior to initiating treatment with various drugs, and cells were washed (twice) with serum-free DMEM.

Binding Assay-- 2 × 10⁵ cells were plated per well of 24-well plates (Corning Glass), cultured for 1 day under regular growth conditions, and then serum-starved overnight. Prior to performing the binding assay, cell count/well was determined, and media were changed to 12.5 mM HEPES, pH 7.4, containing 4 °C cold DMEM, and plates were placed at 4 °C for 30 min. Binding assay was initiated by adding [³H]12(S)-HETE with or without 1000× concentration of cold 12(S)-HETE or other eicosanoids in DMEM/HEPES at final volume of 400 µl. The plates were kept under constant and gentle swirling at 4 °C for the times indicated in the text. Following the incubation period 50% of the binding media was set aside and the rest decanted, and plates were washed 4 times with PBS, and cells were solubilized with 300 µl of 0.1 M NaOH for 10 min at room temperature. Cell lysates or 200 µl of the binding media were mixed with Ultima Gold scintillation liquid (Packard Instrument Co.), and radioactivity was measured with a Packard 1900TR scintillation counter. Each sample was counted for 20 min to accumulate at least 500 counts. Data points under 3 nM [³H]12(S)-HETE concentration represent the mean of triplicate determinations, and at higher concentrations duplicates were used.

Analysis of [³H]12(S)-HETE Incorporation into Membrane Fraction-- 3 × 10⁶ cells were incubated similar to binding assay with 3 nM [³H]12(S)-HETE in the presence or absence of 300 nM (100 times) cold 12(S)-HETE for 30 min and rinsed 4 times with PBS; cells were scraped off and homogenized with brief sonication in 2 ml of buffer A (25 mM Tris-HCl, pH 7.6; 1 mM EGTA; aprotinin (5 µg/ml); leupeptin (10 µg/ml) and 1 mM PMSF). Membrane and cytosolic fractions were separated by centrifugation (100,000 × g; 1 h, 4 °C). The membrane fraction was rinsed (4 times) with buffer A and solubilized with sonication in 1% SDS, 10 mM Tris, pH 7.4. Lipids were extracted with 1 ml of chloroform:methanol (1:1). Lipids dissolved in the chloroform layer were loaded on Whatman silica gel 60A thin layer chromatography plates and developed with ethyl acetate:methyl chloride:glacial acetic acid (5:5:1). Plates were dried, and position of lipids was determined following treatment with iodine vapor. Ten identical areas of each lane were scraped into scintillation vials, and Ultima Gold scintillation fluid added and radioactivity measured with a Packard 1900TR scintillation counter.

DNA Fragmentation Assay-- Cells (2.5 × 10⁶) were grown in 10-cm tissue culture dishes in DMEM until 90% confluent and serum-starved (18 h) prior to experimental use. Cells were washed with PBS (3 times) and then either treated with BHPP at different concentrations (25, 50, 100 µM) for 24 h or pretreated with 12(S)-HETE at 1 µM for 5 h. Equal amounts of ethanol were used as a vehicle control. Subsequently, cells were harvested with a rubber policeman, and fragmented DNA was extracted with 200 µl of the lysis buffer (50 nM Tris-HCl, pH 7.5, 20 mM EDTA, 1% Nonidet P-40) for 5 min. Samples were then centrifuged at 500 × g for 5 min. The resultant supernatants were transferred to a clean set of Eppendorf tubes, and pellets were dissolved in 200 µl of lysis buffer and extracted for 2 min. Samples were centrifuged again, and the resultant supernatants were combined with previous supernatants. Subsequently, SDS and DNase-free RNase (Ambion, Austin, TX) were added to the pooled supernatants to the final concentration of 0.1% and 5 mg/ml, respectively, and samples were incubated at 56 °C for 2 h. At the end of RNase treatment, proteinase K (2.5 ml/ml) was added, and samples were further incubated for 2 h at 37 °C. Samples were extracted once with alkaline phenol:chloroform:isoamyl alcohol (25:24:1) and DNA precipitated with 0.3 M NaAc, pH 5.2. DNA from equal number of cells or equal amounts of DNA (20 µg) were run on a 1.2% agarose gel, and the DNA ladder formation was visualized by ethidium bromide staining.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

12(S)-HETE Activates ERK1/2-- Following 12(S)-HETE treatment both ERK1 and -2 were transiently activated, as determined by Western blot analyses with an antibody to the activated forms of ERK1/2. A single peak of activation (10-fold) was observed 10 min after stimulation with 12(S)-HETE (300 nM) (Fig. 1A). The increase in ERK1/2 phosphorylation was detected as early as 2 min, returned to basal level 30 min after stimulation, and was concentration-dependent in the 0-500 nM 12(S)-HETE range (Fig. 1B). These results were confirmed with an in vitro kinase assay where an increase in phosphorylation of a MAPK-specific substrate, i.e. Elk, paralleled the increase in ERK1/2 activity (Fig. 1, A and B).

View larger version (28K): [in this window] [in a new window]   Fig. 1.   12(S)-HETE activates ERK1 and ERK2. A, 12(S)-HETE activates ERK1/2 in a time-dependent manner. ERKs were precipitated at the indicated time points from 12(S)-HETE (300 nM)-treated A431 cells. Elk was used as a specific substrate for in vitro kinase assay (inset, upper panel). ERK activity was monitored by probing blots for phosphorylated Elk. 12(S)-HETE also increased the phosphorylated (activated) state of ERK1/2 as revealed by Western blots of whole cell lysates with phospho-specific ERK1/2 antibody (inset, middle panel). The blot was reprobed with non-phospho-specific ERK antibody to demonstrate equal loading (inset, lower panel). Western blots of three independent experiments probed for phospho-ERK1/2 were analyzed by densitometric scanning, and the results are represented in the graph as a percent of maximal activation. B, 12(S)-HETE activates MAPK in a dose-dependent manner. A431 cells were treated for 10 min with increasing concentration of 12(S)-HETE, and ERK1/2 activity was determined either by its ability to phosphorylate Elk in an in vitro kinase assay (upper panel) or by probing Western blots of whole cell lysates for activated ERK1/2 (middle panel). The blot was stripped and reprobed with non-phospho-specific ERK antibody for loading control (lower panel). The bar graph represents densitometric analysis of three independent anti-phospho-ERK Western blots. Results are expressed in arbitrary units. C, HETEs affect on ERK1/2 activity. A431 cells were treated for 10 min with 300 nM vehicle, 5(S)-HETE, 11(S)-HETE, 12(S)-HETE, or 15(S)-HETE, and ERK1/2 phosphorylation was evaluated by Western blotting (upper panel). Blot was stripped and reprobed with non-phospho-specific ERK antibody for loading control (lower panel). The bar graph represents densitometric analysis of the anti-phospho-ERK Western blot.

To test whether 12(S)-HETE is unique or whether other eicosanoids also activate ERK1/2 in A431 cells in similar dose and time ranges, cells were treated with 5(S)-HETE, 11(S)-HETE, 12(S)-HETE, and 15(S)-HETE (300 nM, 10 min). Anti-phospho-ERK probed Western blots revealed (Fig. 1C) that under these conditions 12(S)-HETE is the most potent activator of ERK1/2, whereas 11(S)-HETE was 50% less potent. 5- and 15(S)-HETE stimulated ERK1/2 only to the extent of 5-15% of 12(S)-HETE, respectively. These results indicate that multiple HETEs may activate ERK1/2 in A431 cells, but 12(S)-HETE is the most potent, suggesting that this function is mediated by a specific response.

To investigate whether activation of ERK kinase by 12(S)-HETE was restricted to the A431 cell line, we tested human melanoma (WM983B and HT168), human prostate carcinoma (DU145), mouse melanoma (B16a), mouse lung carcinoma (3LL), endothelial (RVECT and HUVEC) as well as Chinese hamster ovary and African green monkey kidney (COS-1) cell lines. In these cell lines, with the exception of HT168, Chinese hamster ovary, and COS-1 cells, 12(S)-HETE also increased ERK1/2 activity within 10 min of treatment (data not shown). These findings indicate that 12(S)-HETE activation of ERK1/2 is not unique to A431 cells, but neither is it a ubiquitous phenomenon.

Other investigators (25) have reported that 12(S)-HETE increases the activity of another MAPK, i.e. c-Jun N-terminal kinase. In contrast, by using A431 cells, we found no evidence that either p38 or JNK mitogen-activated protein kinases were stimulated by this lipid mediator in a similar time and dose range as observed for ERK (data not shown).

The activity of ERK can be modulated at several levels in the evolutionarily conserved ERK cassette, i.e. either by enhancing GTP loading of Ras, by phosphorylation of Raf and MEK (26), or by binding non-enzymatic scaffold proteins (27). Some studies also suggest MEK-independent mechanisms for ERK activation, such as phosphatidylinositide 3-kinase-dependent or conventional PKC-dependent pathways (28). Therefore, we tested whether MEK, the immediate upstream dual specificity kinase to ERK, was involved in 12(S)-HETE-stimulated ERK1/2 activity.

12(S)-HETE Activates MEK-- 12(S)-HETE increased (8-fold) the activity of MEK in a dose-dependent manner in the 0-600 nM range (Fig. 2A). Furthermore, PD98059, a specific inhibitor of MEK1/2 dose dependently abolished MAPK activation by 12(S)-HETE (Fig. 2B), thus ruling out the possibility that 12(S)-HETE modulates ERK activity by a MEK-independent mechanism.

View larger version (40K): [in this window] [in a new window]   Fig. 2.   12(S)-HETE activates ERK1/2 through MEK. A, MEK is activated following 10 min of 12(S)-HETE treatment (0-600 nM), as determined by probing Western blots of whole cell lysates for activated MEK (upper panel). The blot was reprobed for actin as loading control (lower panel). The bar graph represents densitometric analysis of the Western blot. Results are expressed as arbitrary units. 12(S)-HETE dose-dependently increases phosphorylation of MEK (lower blot). Phosphorylated MEK runs slower than the non-phosphorylated form in low bisacrylamide SDS-polyacrylamide gel electrophoresis. In Western blots probed for MEK after 12(S)-HETE treatment, the upper band represents the phosphorylated form of MEK and the lower band represents the non-phosphorylated form of MEK. B, a MEK inhibitor blocks 12(S)-HETE stimulated activation of MAPK. MEK inhibitor PD98059 (15 min, at the indicated doses)-pretreated cells were exposed to 12(S)-HETE for 10 min. Whole cell lysates were probed for activated ERK1/2 on a Western blot (upper panel), stripped, and reprobed for ERK1/2 as a loading control (lower panel). The bar graph represents densitometric analysis of the Western blot with the results expressed as arbitrary units.

12(S)-HETE Activates Raf-- Since MEK is a convergence point for several signaling pathways, we next tested for the involvement of Raf1 in 12(S)-HETE signaling. By utilizing an in vitro kinase assay, we observed that Raf kinase activity was increased (3-fold) 5 min after cells were treated with 300 nM 12(S)-HETE (Fig. 3) and that this activation was decreased (75%) in cells pretreated with the PKC inhibitor, Go6976 (at 10× IC₅₀ dose (15)). These data suggest that PKC participates in 12(S)-HETE activation of Raf1.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Raf is stimulated by 12(S)-HETE in a conventional PKC-dependent manner. A431 cells were pretreated with the conventional PKC inhibitor Go6976 (at 1 and 10× IC50, 2.3 and 23 nM, respectively) for 15 min and then with 12(S)-HETE (300 nM, 5 min). Raf was immunoprecipitated and used for an in vitro kinase assay where it activated GST-MEK which in turn phosphorylated ([32P]ATP) GST-ERK2. Proteins were bound on phosphocellulose paper, and after extensive washing incorporated radioactivity was determined. Pretreatment of cells with a PKC inhibitor reduced activation of ERK2. Error bars represent S.D. of triplicate determinations, and the graph is a representative of three independent experiments.

12(S)-HETE Activates PKC-- The inhibition of 12(S)-HETE-stimulated Raf activity by Go6976 led us to examine the extent to which conventional PKCs were involved in the observed ERK1/2 stimulation. Treatment with 12(S)-HETE induced a dose-dependent translocation (5-fold at 600 nM) of PKC from the cytosol to the membrane fraction accompanied by increased phosphorylation (5-fold at 300 nM) (Fig. 4, A and B). Enzyme activity in the subcellular fractions was confirmed with a kinase assay using myelin basic protein as a substrate for immunoprecipitated PKC (data not shown). Phosphorylation of PKC reached a maximum at approximately 2.5 min (Fig. 4C). Therefore, the time course of PKC activation by 12(S)-HETE, in A431 human epidermoid carcinoma cells, is similar to that observed in B16a murine melanoma cells (7).

View larger version (28K): [in this window] [in a new window]   Fig. 4.   12(S)-HETE activates PKC in a time- and concentration-dependent manner. A, 12(S)-HETE dose dependently activates PKC. After 3 min 12(S)-HETE treatment, cytosolic and particulate fractions were isolated from A431 cells. Proteins from the particulate fractions were analyzed by Western blotting with an antibody directed to PKC. 12(S)-HETE dose dependently increased translocation of PKC to the particulate fraction. The bar graph represents densitometric analysis of 3 Western blots, with the results expressed as arbitrary units. B, 12(S)-HETE dose dependently induces phosphorylation of PKC. A431 cells were treated with the indicated doses of 12(S)-HETE for 3 min. Western blots of whole cell lysates were probed for the phosphorylated form of PKC (upper panel). As a loading control the blot was reprobed with a non-phospho-specific PKC antibody (lower panel). The bar graph represents densitometric analysis of the Western blot, with the results expressed as arbitrary units. C, PKC is transiently activated by 12(S)-HETE. Probing blots from 12(S)-HETE (300 nM)-stimulated whole cell lysates with anti-phospho-PKC antibody revealed that PKC is maximally activated at 2.5 min after stimulation (upper panel). As a loading control the blot was stripped and reprobed with a non-phospho-specific PKC antibody (lower panel). The graph represents densitometric analysis of the Western blot, with the results expressed as arbitrary units.

12(S)-HETE-stimulated ERK1/2 Activity Is Mediated in Part by PKC-- Western blotting of A431 cells revealed that they express seven isoforms of PKC, i.e. , , , , , µ, and  (Fig. 5A). Chronic exposure to phorbol esters leads to degradation of the activated PKC species. In A431 cells PKC , , and  but not PKC , µ, , and  were eliminated by chronic phorbol ester exposure. To determine whether 12(S)-HETE activation of ERK1/2 was mediated by PKC, we tested whether PKC depletion could block ERK1/2 activation. Depletion of PKC resulted in a partial (40%) inhibition of 12(S)-HETE activation of ERK1/2 (Fig. 5B). In contrast, this treatment completely abolished ERK1/2 activation by PMA, a direct and potent activator of PKC (Fig. 5B). Chronic exposure to another, non-related PKC activator, bryostatin, also resulted in a partial inhibition of 12(S)-HETE activation of ERK1/2 (data not shown). Chemical inhibition of conventional PKC isoforms by Go6976 partially (60%) reduced 12(S)-HETE activation of ERK in a dose-dependent manner (Fig. 5C). Even at 50 times the IC₅₀ concentration, Go6976 failed to completely block the 12(S)-HETE activation of ERK1/2. Together, these results suggest that the conventional PKC pathways are important but are not the exclusive mechanism for 12(S)-HETE activation of ERK.

View larger version (29K): [in this window] [in a new window]   Fig. 5.   ERK1/2 activation by 12(S)-HETE is dependent, in part, on conventional PKC activity. A, PKC , , , , , , and µ are expressed in A431 cells at a detectable level, as determined by Western blotting. Chronic PMA exposure depletes PKC , , and . c, positive control, mouse brain lysate (, , , , , , , ) or Jurkat cell lysate ( and µ); + and  represent PMA-depleted and non-depleted A431 cell lysates, respectively. B, conventional PKCs are partially responsible for 12(S)-HETE induced ERK1/2 activation. Chronic exposure to phorbol ester (100 nM PMA, 20 h) eliminated PKC from A431 cells (lower panel). In PKC-depleted cells, ERK1/2 activation was not induced by PMA treatment (10 min, 100 nM). 12(S)-HETE (10 min, 300 nM)-stimulated ERK1/2 activation is decreased when compared with that of cells in which PKC was not depleted; however, it was not eliminated completely (upper panel). Blot was stripped and reprobed with an antibody to ERK that recognizes these enzymes independent of their phosphorylation state (middle panel). The bar graph represents densitometric analysis of the Western blot, with the results expressed as arbitrary units. C, pretreatment of cells with a specific chemical inhibitor Go6976 (23 nM (+) and 115 nM (++)) for conventional PKCs dose dependently inhibited 12(S)-HETE-mediated activation of ERK1/2. Western blots of whole cell lysates were probed for activated ERK1/2 (upper panel), stripped, and reprobed with non-phospho-specific ERK antibody as a loading control (lower panel). The bar graph represents densitometric analysis of the Western blot, with the results expressed as arbitrary units.

12(S)-HETE Activates PLC1-- Activation of PKC by 12(S)-HETE in B16a murine melanoma cells can be blocked by either PLC inhibitors or by an inhibitor of G_i proteins, i.e. pertussis toxin (7). The PLC family of isoforms are activated by G proteins (29), and stimulation of PLC2 is sensitive to pertussis toxin treatment (30). Therefore, we tested for a role for this PLC isoform in 12(S)-HETE signaling. PLC2 co-immunoprecipitated with the G subunit after a stimulus with exogenous ATP, but not after 12(S)-HETE, or in non-stimulated cell lysates (Fig. 6B), suggesting that this PLC isoform is not involved in 12(S)-HETE signaling events.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Involvement of phospholipases in 12(S)-HETE signaling. A, PLC1 is rapidly phosphorylated on tyrosine residues after exposure to 12(S)-HETE (300 nM). Tyrosine-phosphorylated proteins were immunoprecipitated and probed for PLC1 by Western blot (upper panel). Maximal phosphorylation was observed between 0.5 and 2 min. Pretreatment with an inhibitor of Src family kinases (PP2, 5 µM, 30 min) abolished the 12(S)-HETE effect on tyrosine phosphorylation of PLC1 (lower panel). B, PLC2 is not involved in 12(S)-HETE signal transduction. PLC2 coprecipitated with G subunits in ATP (1 µM, 30 s) but not in 12(S)-HETE (300 nM, 30 s)-treated cells.

Members of the PLC family were thought to be activated by receptors with inherent tyrosine kinase activity (29). However, recent publications demonstrate that angiotensin II (31) and leukotriene B4 (32), both of which exert their effects through G protein-coupled receptors, stimulate tyrosine phosphorylation and activity of PLC1 as rapidly as 15 s after exposure. Therefore, we examined if PLC1 is involved in mediating the 12(S)-HETE effect (Fig. 6A). Tyrosine-phosphorylated proteins were immunoprecipitated, and Western blots were probed for PLC1. In 12(S)-HETE-challenged cells PLC1 is tyrosine-phosphorylated within 30 s and remains phosphorylated for 4 min. The time course for this phosphorylation precedes that of PKC, thereby supporting a role for PLC1 in the activation of PKC during 12(S)-HETE signaling.

Growth Factor Receptors Are Not Activated by 12(S)-HETE-- The finding that activation of PLC1 is an early event after 12(S)-HETE exposure suggests that the putative 12(S)-HETE receptor may be coupled to tyrosine kinases. A number of publications illustrate that G protein-coupled receptors can trans-activate receptor-tyrosine kinases, such as the EGF-, insulin-like growth factor-, and PDGF receptors (33-35). The EGF receptor is expressed at high level in A431 cells; therefore, we tested whether this receptor was responsible for the early tyrosine phosphorylation events induced by 12(S)-HETE. Tyrphostin 51, a specific inhibitor of the EGF receptor kinase, did not affect 12(S)-HETE signaling (Fig. 7A). In contrast, under similar experimental conditions it blocked the EGF-induced activation of ERK1/2 (Fig. 7A). In addition, Western blots using an anti-active EGFR antibody could not detect EGF receptor activation by 12(S)-HETE (Fig. 7B). No increase in tyrosine phosphorylation was apparent in precipitates of the PDGF receptor or the FGF receptor substrate FRS2 following 12(S)-HETE treatment (data not shown). We conclude that the trans-activation of EGF, PDGF, or FGF receptors does not appear to play a role in 12(S)-HETE signaling.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Interaction between EGF and 12(S)-HETE signaling. A, EGF receptor (EGFR) is not involved in 12(S)-HETE signaling in A431 cells. Tyrphostin 51, a specific inhibitor of EGF receptor kinase, inhibits ERK1/2 activation by EGF but does not effect 12(S)-HETE activation. Cells were treated with the inhibitor (at IC50, 800 nM, 20 min) and then with either EGF (1 ng/ml, 10 min) or 12(S)-HETE (300 nM, 10 min). Western blots of whole cell lysates were probed for active ERK1/2 (upper panel), stripped, and reprobed for ERK as a loading control (lower panel). B, EGF receptor is not activated by 12(S)-HETE. A431 cells were treated with the indicated concentrations of 12(S)-HETE or EGF (0.5 ng/ml for 1 min). Western blots were probed with a phospho-EGF receptor antibody to assess the receptor activation (upper panel) and with an actin antibody as a loading control. No activation of the EGF receptor is apparent after eicosanoid treatment. C, influence of 12(S)-HETE on EGF signaling. Cells pretreated with 12(S)-HETE (1 min, 0-600 nM) were challenged with EGF (50 ng/ml, 5 min). ERK1/2 activation in response to EGF was attenuated by 12(S)-HETE exposure in the 10-100 nM range and enhanced by 600 nM 12(S)-HETE (upper panel). Blots were stripped and reprobed with anti-ERK antibody to demonstrate even loading (lower panel). Results from the densitometric analysis of the middle panel is shown in the bar chart (arbitrary units). D, 12(S)-HETE affect on EGF signaling is mediated by protein tyrosine phosphatases. Cells were pretreated with protein tyrosine phosphatases inhibitor Na3VO4 (2 mM, 30 min), then exposed to 100 nM 12(S)-HETE or vehicle (1 min), and treated with EGF (50 ng/ml, 5 min). ERK1/2 activation identified with Western blotting (upper panel), stripping, and reprobing the membrane for ERK served as demonstration of even loading (lower panel). Results from the densitometric analysis are shown in the bar chart (arbitrary units).

12(S)-HETE Affects EGF Signaling-- Since G protein-coupled receptors may activate protein tyrosine phosphatases (36), next we tested whether 12(S)-HETE may have an opposing rather than synergistic effect on EGF signaling. Serum-starved cells were challenged with 0-600 nM 12(S)-HETE for 1 min and then with 50 ng/ml EGF for 5 min, and cell lysates were tested for ERK1/2 activity. We found a biphasic response. Lower concentrations of 12(S)-HETE (10-100 nM) reduced EGF-stimulated ERK1/2 phosphorylation, and higher (600 nM) concentrations of 12(S)-HETE further increased EGF-stimulated ERK1/2 activity (Fig. 7C).

One possibility is that at the 10-100 nM concentration 12(S)-HETE stimulates a phosphatase. To evaluate this, cells were pretreated with or without sodium orthovanadate (2 mM, 30 min); similar to the above described experiments, cells were treated with EGF in the presence or absence of 100 nM 12(S)-HETE. ERK1/2 activation was tested by Western blotting (Fig. 7D). In the absence of the protein tyrosine phosphatase inhibitor sodium orthovanadate, as earlier, 100 nM 12(S)-HETE interfered with EGF-stimulated ERK1/2 activation (35% reduction). In contrast, in the presence of the protein tyrosine phosphatase inhibitor, pretreatment with 100 nM 12(S)-HETE resulted in a slight (15%) increase in ERK1/2 phosphorylation. Taken together these results suggest that at lower doses 12(S)-HETE stimulates ERK1/2 activity, as well as stimulates a protein tyrosine phosphatase that antagonizes EGF signaling. However, at higher concentrations, 12(S)-HETE does not activate the protein tyrosine phosphatase, only ERK1/2, hence its effect is additive to that of EGF on ERK1/2.

Src Family Kinases Are Involved in Early 12(S)-HETE Signaling-- The Src family of non-receptor tyrosine kinases are implicated in signaling events downstream of G proteins (13). Pretreatment of A431 cells with PP2, a specific inhibitor of Src kinases, abolished tyrosine phosphorylation of PLC1 stimulated by 12(S)-HETE (Fig. 6A). Therefore, we questioned whether Src family kinases are activated by 12(S)-HETE. Western blot analysis of proteins precipitated from 12(S)-HETE treated A431 cells revealed a time-dependent increase in tyrosine phosphorylation of Src family kinases (Fig. 8A) as rapidly as 10 s following exposure to 12(S)-HETE. However, tyrosine phosphorylation of these enzymes can either stimulate or inhibit their activity, depending on the site of phosphorylation (37). Therefore, we assayed for Src kinase activity in SRC2 antibody-generated precipitates and found that 12(S)-HETE stimulated Src activity. Pretreatment with PP2 (5 µM) was sufficient to reduce kinase activity below basal levels (Fig. 8B). As revealed with immunoprecipitation and Western blotting, 12(S)-HETE increased association of Src with Shc (Src homology and collagen) adapter proteins, which was abolished by pretreating the cells with PP2 (Fig. 8C). Furthermore, PP2 (5 µM) significantly inhibited, but did not entirely abolish, ERK1/2 activation by 12(S)-HETE (Fig. 10B).

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Src family kinases are involved in 12(S)-HETE signaling. A, Src family kinases are phosphorylated in response to 12(S)-HETE. Tyrosine-phosphorylated proteins were precipitated 10 and 30 s or 1, 2, or 5 min after 12(S)-HETE treatment. Western blot (IB) was probed with an antibody that recognizes multiple members of Src family. B, Src family kinases are activated by 12(S)-HETE. A431 cells were pretreated with Src inhibitor PP2 (5 µM, 30 min) and with 12(S)-HETE (100 nM, 1 min). Immunoprecipitated (IP) Src family kinases were used in an in vitro kinase assay. Radioactivity incorporated into a specific substrate was determined by PhosphorImager analysis. C, Src family kinases associate with Shc adapter proteins in response of 12(S)-HETE treatment in an Src kinase activity dependent manner. A431 cells were pretreated with Src inhibitor PP2 (5 µM, 30 min) and then with 12(S)-HETE (100 nM, 1 min). Shc proteins were immunoprecipitated under non-denaturing conditions, and the associated proteins were analyzed by Western blotting.

12(S)-HETE Stimulates Phosphorylation of Shc-- The potential involvement of Src family kinases in 12(S)-HETE signaling suggested an alternative pathway to the previously established PLC-PKC route of ERK1/2 activation. Adapter proteins, such as Grb2 (growth factor receptor-bound protein 2; pp24) and Shc (pp66, pp52, and pp46), are tyrosine-phosphorylated by activated growth factor receptor and oncogene-tyrosine kinases and link these kinases to the ERK cascade. Therefore, we tested whether adapter proteins are phosphorylated in response to 12(S)-HETE treatment. Tyrosine phosphorylation of Shc adapter proteins was detectable as early as 15 s after 12(S)-HETE stimulation (Fig. 9A) and peaks at 30-60 s. The tyrosine phosphorylation of Shc is maximal in the 100-300 nM range of 12(S)-HETE (3.5-fold over basal level). Another adapter protein, Grb2 (growth factor receptor-bound), also was tyrosine-phosphorylated, and it associated with Shc in response to 12(S)-HETE treatment (Fig. 9B). Pretreatment with the Src family kinase inhibitor, PP2, completely abolished activation of these adapter proteins. Shc and Grb2 are well characterized as activators of Ras through the guanine nucleotide exchange factor, Sos. Pretreatment with a PKC inhibitor (Go6976) did not affect the increase in tyrosine phosphorylation of Shc (Fig. 9C). Therefore, Shc may represent an alternative pathway to PKC activation of ERK following 12(S)-HETE stimulation.

View larger version (21K): [in this window] [in a new window]   Fig. 9.   Adapter proteins are involved in 12(S)-HETE signaling. A, Shc is tyrosine-phosphorylated after 12(S)-HETE exposure in a time- (upper panel) and dose (lower panel)-dependent manner. Tyrosine-phosphorylated proteins were immunoprecipitated (IP) under denaturing conditions, and Western blots (IB) were analyzed with anti-Shc antibody. Maximal phosphorylation was observed at approximately 1 min (upper panel) and at 300 nM concentration (lower panel). B, Shc phosphorylation is dependent on Src family kinase activity but not on Gi proteins. A431 cells were pretreated with an inhibitor of Src family kinases (PP2, 5 µM, 30 min) and then with 12(S)-HETE (300 nM, 1 min) or EGF (0.5 ng/ml, 2 min). Shc was immunoprecipitated under non-denaturing conditions, and Western blots of the precipitated proteins were probed for phosphotyrosine (left, upper panel), stripped, and reprobed for Grb2 (left, middle panel), stripped again and probed for Shc as loading control (left, lower panel). Inhibition of Src family kinases completely blocked Shc tyrosine phosphorylation and its association with Grb2. Inhibition of Gi proteins with pertussis toxin pretreatment (PTX, 100 ng/ml, overnight) did not effect Shc phosphorylation (right panel) after 12(S)-HETE exposure (300 nm, 1 min) as determined by analyzing Western blots of tyrosine-phosphorylated proteins that were precipitated under denaturing conditions. C, 12(S)-HETE-stimulated phosphorylation of Shc is independent of conventional PKC activity. Pretreatment with PKC inhibitor Go6976 (115 nM, 15 min) did not influence the 12(S)-HETE (300 nM, 1 min)-induced phosphorylation of Shc. Treatment with EGF (0.5 ng/ml, 2 min) served as a positive control.

12(S)-HETE Activates Ras-- To test whether Ras was stimulated in our system, we took advantage of the fact that only GTP-Ras (the activated form) binds its effector, Raf. We found that 300 nM 12(S)-HETE stimulated Ras binding 3 min after 12(S)-HETE exposure (Fig. 10A), which was blocked by pretreatment with Src inhibitor, PP2 (5 µM, 30 min), but not by an inhibitor of conventional PKCs, Go6976 (115 nM, 30 min). Next we questioned the contribution of Ras to 12(S)-HETE-induced ERK activation. Inhibitors of farnesyltransferase (FTase inhibitor II, 5 µM, 1 h) interfere with Ras function. Farnesyltransferase inhibitor pretreatment partially blocked ERK activation by 12(S)-HETE (Fig. 10B).

View larger version (12K): [in this window] [in a new window]   Fig. 10.   Ras is involved in 12(S)-HETE signaling. A, Ras is activated by 12(S)-HETE in an Src-dependent but cPKC-independent manner. Cells were treated with PP2 (5 µM) or Go6976 (115 nM) for 30 min and then with 12(S)-HETE (300 nM) for 3 min. Cells were lysed, and activated Ras was precipitated (IP) with Raf-Ras binding domain (Raf-RBD)-conjugated beads. Western blots were probed for Ras. B, inhibitors of both Ras (FTase inhibitor I, 5 µM, 1 h) and Src family kinase (PP2, 5 µM, 30 min) reduced, but did not completely block, 12(S)-HETE (600 nM, 10 min)-stimulated ERK1/2 activation as revealed by probing Western blot (IB) for activated ERK1/2 (upper panel). Blot was stripped and reprobed for ERK as loading control (lower panel).

G Proteins Are Involved in 12(S)-HETE Signaling-- Since previous reports suggested involvement of G proteins in 12(S)-HETE signaling (7, 38), we tested whether they were involved in activation of ERK using two inhibitors of serpentine receptor/G protein signaling. Uncoupling seven transmembrane receptors from G proteins with suramin (5 min, 150 µM) (20) abolished the 12(S)-HETE effect on ERK phosphorylation (Fig. 11B). At higher concentrations suramin may block the interaction of growth factors with their receptor, such as EGF with EGFR. However, the suramin dose applied in our experiments did not affect the ERK response to EGF (Fig. 11B). Previous studies suggested the involvement of G_i in 12(S)-HETE signaling (7); therefore, we exposed A431 cells (18 h) to various doses of pertussis toxin (a specific inhibitor of G_i). The results demonstrated that 12(S)-HETE stimulates ERK1/2 in both a G_i-dependent and -independent manner (Fig. 11A). Pertussis toxin inhibition of ERK1/2 reached its maximum at 100 ng/ml and reduced 12(S)-HETE (300 nM) activation of ERK1/2 to 60% of the maximum at 10 min. Collectively, these results suggest that ERK1/2 activation by 12(S)-HETE is mediated by more than one heterotrimeric G protein.

View larger version (36K): [in this window] [in a new window]   Fig. 11.   The putative 12(S)-HETE receptor is G protein-coupled. A, Gi proteins are involved in 12(S)-HETE signaling. A431 cells were incubated with the indicated concentrations of pertussis toxin (PTX) overnight and then challenged with 300 nM 12(S)-HETE for 10 min. Western blotting with anti-active ERK1/2 reveals that ADP-ribosylation of Gi only partially block ERK1/2 activation (upper panel). Blot was stripped and reprobed for ERK as loading control (lower control). The bar graph represents densitometric analysis of the Western blot, with the results expressed as arbitrary units. B, suramin (5 min, 150 µM), which uncouples G proteins from receptors, reduced ERK1/2 activity to basal level after 12(S)-HETE stimulus (300 nM, 10 min), whereas it did not inhibit EGF (5 ng/ml) signaling (upper panel). Blot was stripped and reprobed for ERK as loading control (lower panel).

A431 Cells Have Multiple Binding Sites for 12(S)-HETE-- The inconsistency between the concentration of 12(S)-HETE necessary to saturate Shc phosphorylation (~100 nM, Fig. 9A) and ERK1/2 activation (>500 nm, Fig. 1) suggested that more than one receptor might be involved in mediating the 12(S)-HETE response in A431 cells. To substantiate this hypothesis A431 cell monolayers were incubated with [³H]12(S)-HETE in the presence or absence of competing 1000× non-labeled 12(S)-HETE. As shown in Fig. 12A, specific association between 12(S)-HETE and A431 cells was maximal at 100 min. Binding was further characterized by incubating cells with 0.4-10 nM [³H]12(S)-HETE with or without competing 1000× non-labeled 12(S)-HETE for 120 min. These studies reproducibly showed (Fig. 12B) a binding site that was saturated at approximately 0.8 nM concentration and another, which was not completely saturated by 10 nM 12(S)-HETE. Scatchard transformation of the binding data resulted in a curve plot rather than a linear plot (Fig. 12C) suggesting that 12(S)-HETE binds to A431 cells through multiple binding sites, with different binding affinities.

View larger version (22K): [in this window] [in a new window]   Fig. 12.   There are multiple 12(S)-HETE-binding sites on A431 cells. A, time-dependent binding. Cell monolayer was incubated at 4 °C with 2 nM 3[H]12(S)-HETE with (lower line) or without (upper line) competing 2 µM cold 12(S)-HETE for the indicated periods. Cell-bound radioactivity is expressed as disintegrations per min. Specific binding site is saturated at 100 min at 4 °C. The figure is representative of 3 independent experiments, and points are triplicate determinations. B, concentration-dependent binding. Cells were incubated for 120 min at 4 °C with 0.4-10 nM [3H]12(S)-HETE with or without competing 1000× cold 12(S)-HETE. The difference between specific and nonspecific binding was charted. Inset, plot of the specific binding at the high affinity binding region. The figure is representative of 4 independent experiments, and points are triplicate determinations. C, Scatchard plot representation of data generated from the concentration-dependent binding assays. Specific binding is plotted against specific binding/concentration of free 12(S)-HETE. The non-linear data suggests multiple binding sites. D, competition for the specific binding site with eicosanoids. Cells were incubated for 120 min at 4 °C with 2 nM 3[H]12(S)-HETE with or without competing 1000× cold eicosanoids. Results are expressed as percent of the radioactivity without competing eicosanoid (0). U46619, thromboxane A2-mimetic. E, radioactivity (3 nM [3H]12(S)-HETE) incorporated into plasma membrane with (right column) or without (left column) competing 100× cold 2(S)-HETE. F, thin layer chromatography analysis of membrane incorporated [3H]12(S)-HETE. Cells were incubated with 3 nM [3H]12(S)-HETE, and the membrane fraction was isolated. Lipids extracted from this fraction (lower line) or [3H]12(S)-HETE standard (upper line) were separated on a TLC plate, and radioactivity in even size rectangles from top (left side) to bottom (right size) of the plate was determined. Exposure to iodine vapor revealed that the majority of membrane lipids remained in the 2nd to 4th rectangles from the bottom, whereas the majority of the radioactivity corresponded to the position of free 3[H]12(S)-HETE.

Competition between Binding of 12(S)-HETE and Other Eicosanoids-- In order to evaluate the specificity of 12(S)-HETE binding, A431 cells were incubated with 3 nM [³H]12(S)-HETE in the presence of various eicosanoids (Fig. 12D). One thousand-fold non-labeled 12(S)-HETE was the best competitor, decreasing incorporated radioactivity by 70% and 11(S)-HETE by 55%. Of the HETEs tested 5(S)-HETE and 15(S)-HETE were the least potent, reducing binding by 30%. Other eicosanoids, prostaglandin E₂ and prostaglandin F₂, or thromboxane A₂ analog U46619 did not affect [³H]12(S)-HETE binding to A431 cells.

12(S)-HETE Binds to Cell Membrane-- Previous experiments suggested involvement of trimeric G proteins in 12(S)-HETE signaling. Since G protein-coupled receptors reside in the plasma membrane, we next questioned whether there is specific 12(S)-HETE binding to this cell compartment. Cells were exposed to 3 nM [³H]12(S)-HETE with or without competing 100-fold non-labeled 12(S)-HETE and then the membrane fraction was isolated. Radioactivity was recovered from the membrane fractions, which was displaced by excess non-labeled 12(S)-HETE (Fig. 12E). It is conceivable that the recovered 12(S)-HETE is in an esterified form. Therefore, following [³H]12(S)-HETE incubation, lipids were extracted from the membrane fraction and analyzed by thin layer chromatography. The majority of the radioactivity showed an identical migration pattern on TLC as authentic standard [³H]12(S)-HETE (Fig. 12F), suggesting that the 12(S)-HETE recovered from the membrane is non-esterified under these experimental conditions.

12(S)HETE Rescues A431 Cells from Apoptosis Induced by a 12-LOX Inhibitor-- Previous studies showed that 12-LOX functions as a survival factor in several tumor cell lines and that exogenous 12(S)-HETE blocks apoptosis induced by 12-LOX inhibitors (5). First, we showed by DNA laddering assay that the 12-LOX-specific inhibitor, BHPP, induced apoptosis in A431 cells in dose-dependent manner (Fig. 13A), similar to the effects found in an earlier study with W256 cells (5). When A431 cells, 12-LOX-transfected A431 cells, or vector control cells were treated (37 °C; 24 h) with BHPP at 25, 50, or 100 µM, the cells transfected with platelet-type 12-LOX were more resistant to apoptosis induced by BHPP than 3.1+ vector control cells as shown by the density of the DNA ladder (Fig. 13A). Prior to the treatment with BHPP, A431 cells were incubated with 12(S)-HETE for 2 h. As shown in Fig. 13B, 12(S)-HETE pretreatment completely prevented A431 cells from undergoing apoptosis triggered by BHPP at low dose and significantly reduced the response to high dose BHPP (Fig. 13B). The results suggest that the 12-LOX product, 12(S)-HETE, provides cells with resistance to apoptosis and also that 12(S)-HETE may play a role in an anti-apoptotic signaling pathway.

View larger version (51K): [in this window] [in a new window]   Fig. 13.   12(S)-HETE effect on BHPP-induced apoptosis in A431 cells by DNA laddering assay. A, comparison of A431 12-LOX transfectants with vector control 3.1+. Cells were treated with BHPP at the concentrations indicated for 24 h and were low molecular weight DNA-extracted, run on a 1.2% agarose gel, and visualized with ethidium bromide. B, A431 cells were pretreated with 12(S)-HETE (1 µM) before incubation in DMEM in the presence of BHPP, see details under "Experimental Procedures." Aliquots of DNA extracts were subjected to 1.2% agarose gel and visualized with ethidium bromide. M, DNA marker; C, ethanol as vehicle control; 3.1+, empty vector control; 12-LOX, A431 cells transfected with human full-length platelet-type 12-LOX; Control, without 12(S)-HETE treatment.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The platelet-type 12-lipoxygenase is ectopically expressed in a variety of human and rodent cancer cells, and 12-lipoxygenase expression has been correlated positively with metastatic potential in a rodent tumor model (39). More importantly, in a clinical study of 132 tumors from prostate cancer patients, the expression of 12-lipoxygenase message correlated with tumor stage, grade, and the presence of cancer cells in the surgical margins (40). The sole product of the metabolism of arachidonic acid by platelet type 12-lipoxygenase is 12(S)-HETE. This bioactive lipid is reported to induce a plethora of cellular responses when added exogenously to tumor cells or endothelial cells and to alter tumor growth when overexpressed endogenously. The latter effect may be due to the suppression of apoptosis and stimulation of angiogenesis (5, 6). For example, exogenously added 12(S)-HETE alters the metastatic phenotype by inducing alterations in the cancer cell cytoskeleton (41), thereby enhancing tumor cell motility (4), secretion of proteinases (42, 43), expression of integrins (2, 44), and increased invasion (46). In endothelial cells, 12(S)-HETE induces the non-destructive retraction of monolayers (47) and promotes tumor cell adhesion (46). The motility of isolated endothelial cells and tube formation is also enhanced by 12(S)-HETE (6). Given the numerous and varied cellular responses to 12(S)-HETE, we delineated in this study the signaling pathways utilized by this bioactive lipid in A431 human epidermoid carcinoma cells. Data presented in this study demonstrate that exogenous 12(S)-HETE induces a transient activation of ERK1/2. We report for the first time that 12(S)-HETE stimulates phosphorylation of PLC1, which in turn is responsible for activation of a conventional PKC isoform (i.e. PKC). We show that PKC plays a significant but not exclusive role in ERK1/2 activation. Furthermore, we demonstrate that the 12(S)-HETE-induced activation of Src family kinases and the subsequent phosphorylation of adapter proteins (i.e. Shc and Grb2) lead to activation of ERK1/2 via Ras. Furthermore, the data presented suggest that protein tyrosine phosphatases are involved in eicosanoid signaling and that multiple receptors might be involved in the A431 response to 12(S)-HETE. Finally, we show that inactivation of endogenous 12(S)-HETE production with 12-lipoxygenase inhibitors leads to apoptosis of A431 cells, which is counteracted by overexpression of 12-lipoxygenase or by exogenously added 12(S)-HETE.

Many of the above-mentioned signaling molecules are implicated in cellular functions known to be affected by 12(S)-HETE. For example, 12(S)-HETE-stimulated PKC activity may be responsible for altered cellular morphology, since 12(S)-HETE induces phosphorylation of cytoskeletal proteins including actin, vimentin, and myosin light chain (48) and stimulates cellular spreading (4). 12(S)-HETE-stimulated PKC may promote the cellular migratory phenotype (46). All of the aforementioned 12(S)-HETE stimulated responses can be inhibited or significantly reduced with select PKC inhibitors (42, 46).

The above results demonstrate that 12(S)-HETE also protects tumor cells from the induction of apoptosis, but the exact mechanism of this effect needs further investigation. However, one may speculate that since ERK can activate p90^rsk, which in turn can phosphorylate BAD and CREB (49), two proteins demonstrated to have an anti-apoptotic effect, this may be a plausible mechanism to explain the anti-apoptotic effect of 12(S)-HETE. Alternatively, PKC may prevent apoptosis independent of ERK. Ruvolo et al. (50) suggested that mitochondrial protein kinase C may inhibit apoptosis through phosphorylation of Bcl2.

The findings that 12(S)-HETE signaling can be inhibited by suramin or pertussis toxin suggests the existence of G protein-coupled receptor(s). However, no HETE receptors have yet been cloned. Receptors for arachidonate-lipoxygenase products that have been identified to date are the lipoxin A₄ (51) and leukotriene B₄ (52) and cysteinyl leukotriene receptors (53, 54). These receptors, similar to the receptors for prostaglandins, are members of the family of G protein-coupled seven transmembrane receptors. Several attempts to identify a 12(S)-HETE receptor have resulted only in the identification of a cytoplasmic binding complex, which is not well characterized (55, 56). Several lines of evidence suggests that 12(S)-HETE might have multiple receptors on A431 cells. 1) Binding studies revealed at least two binding sites on intact cells. 2) Shc tyrosine phosphorylation is maximal around 100 nM 12(S)-HETE concentration, but ERK activation is not saturated even at 500 nM. 3) 12(S)-HETE effect on EGF-stimulated activation of ERK is bi-phasic; at 100 nM it is negative, but at 600 nM, ERK phosphorylation is augmented. Considering these data we speculate that 12(S)-HETE may have a high and a low affinity receptor. The high affinity receptor stimulates a protein tyrosine phosphatase as well as a protein tyrosine kinase, i.e. Src. This is not necessarily conflicting, since Src family kinases are inhibited by phosphorylation of a C-terminal residue. The proposed phosphatase may remove phosphate from this residue, lifting the inhibition, which then leads to activation of Src. The observed increase in the overall tyrosine phosphorylation of Src following 12(S)-HETE exposure might reflect autophosphorylation of the activated Src on the two positive regulatory tyrosine residues. In this pathway Src is positioned upstream of Shc, Grb2, Ras, and ultimately of the ERK cascade. The low affinity receptor does not appear to stimulate either the protein tyrosine phosphatase, Src, or Shc. Possibly the low affinity receptor activates ERK1/2 via stimulation of PKC (hence increased translocation of PKC at 600 nM 12(S)-HETE concentration). However, that does not exclude the possibility that low dose 12(S)-HETE activates PKC via the high affinity receptor coupled to phospholipases.

The notion of multiple 12(S)-HETE receptors is supported by the fact that high affinity (K_d 1 nM (7)) as well as low affinity (K_d 658 nM (38)) binding sites were found on different cell lines, and by our observation that human prostate carcinoma cell lines express both high and low affinity binding sites simultaneously.² Furthermore, the Shc phosphorylation response curve is shaped like the curve of functional responses to 12(S)-HETE by melanoma cells that express the high affinity binding site (7).

It is not surprising that the utilization of labeled 12(S)-HETE for isolation of its receptor has been unsuccessful, considering that the serpentine receptors are sensitive to solubilization and may lose binding of their ligands once extracted from the plasma membrane or dissociated from G proteins (57). Therefore, functional cloning is a method that may lead to the isolation of the 12(S)-HETE receptor(s). The present report identifies downstream effector molecules of 12(S)-HETE signaling which can be monitored and hence provide useful tools for future cloning of the 12(S)-HETE receptor(s).

Following activation of the putative, G protein-coupled 12(S)-HETE receptor, we observed activation of Src kinases and a bifurcation of the signaling pathway. The concept of a G protein-coupled signaling system utilizing parallel pathways to activate ERK is not unique to 12(S)-HETE. For example, angiotensin II can stimulate ERK through activation of PKC or, alternatively, by trans-activating the EGF receptor (12).

Although this study describes two novel pathways for 12(S)-HETE-induced activation of ERK, it raises some interesting questions. The two pathways identified in the present study emanate from the activation of Src kinases. However, inactivation of Src family kinases with PP2 completely inhibited tyrosine phosphorylation of both PLC1 and Shc, following 12(S)-HETE treatment, yet this was insufficient to suppress completely ERK1/2 stimulation, suggesting the possibility of a Src-independent pathway in 12(S)-HETE signaling.

Both mitogenic and motogenic cellular responses are elicited by 12(S)-HETE (46). The different responses to the same signaling molecule may be due to the duration of the signal stimulated by 12(S)-HETE and whether or not 12(S)-HETE stimulation results from exogenously or endogenously generated eicosanoid. The duration of ERK activation has a profound effect on the cellular response. Depending on the cell type, the short time period activation of ERK might lead to differentiation, and long term activity of ERK may result in proliferation or vice versa (58). In this study, the addition of exogenous 12(S)-HETE to A431 cells stimulated ERK activity for approximately 30 min. However, phorbol ester or EGF can stimulate ERK activity for a period of hours. The short duration of the ERK response observed in the present study may result from esterification of exogenously added 12(S)-HETE into membrane lipids (59), limiting the availability of free eicosanoid, and hence, dampening the duration of the ERK response. It is possible that continuous, endogenous production of 12(S)-HETE could result in a constitutively elevated level of ERK activity. The addition of a single bolus of exogenous 12(S)-HETE to tumor cells, as utilized in this study, may mimic a situation that occurs during metastasis when tumor cells are exposed to exogenously produced 12-HETE during their aggregation with platelets, a rich source of 12(S)-HETE (1). However, the biological response may be different in the case of continuous, endogenous production of 12(S)-HETE, such as in epidermal growth factor (60) or autocrine motility factor (61)-stimulated cells.

Downstream signaling pathways activated by other lipoxygenase products are not well characterized. Nevertheless, there are similarities among the findings described in the literature and those in the present study. For example, neutrophil 5(S)-HETE, a positional isomer of 12(S)-HETE, also stimulates ERK via RAF and MEK (62). In eosinophils, leukotriene B₄ activates ERK1/2 but not JNK or p38 MAPK (63) and promotes hydrogen peroxide production in a PKC-dependent manner (64). Lipoxin A₄ increases intracellular calcium concentration in a manner inhibited by pertussis toxin (65). Leukotriene D₄ also increases calcium concentration in cells but by two pathways as follows: 1) by a pertussis toxin-sensitive pathway that results in the stimulation of extracellular calcium influx, and 2) by a pertussis toxin-insensitive pathway resulting in the mobilization of the intracellular calcium pool (45). Therefore, both leukotriene D₄ and 12(S)-HETE promote a simultaneous effect (i.e. an increase in intracellular calcium concentration or ERK activity, respectively), in a pertussis toxin-dependent and -independent manner. These examples suggest that lipoxygenase generated eicosanoids signal via pathways that are in common with other paracrine effectors.

Fig. 14 depicts a model of 12(S)-HETE-mediated signaling consistent with the data presented in this report. Stimulation of a putative high affinity G protein-coupled receptor leads to the activation of Src family kinases and a protein tyrosine phosphatase. The pathway bifurcates with tyrosine phosphorylation of two Src family substrates, Shc and PLC1. In one arm of the pathway Shc recruits Grb2 and activates Sos and Ras which in turn activates MEK and ERK. In the parallel pathway, PLC1 activates PKC, leading to activation of Raf, which stimulates the downstream kinases MEK and ERK. A low affinity receptor may also stimulate PKC and ultimately ERK1/2.

View larger version (68K): [in this window] [in a new window]   Fig. 14.   Schematic representation of the proposed 12(S)-HETE signal transduction pathways. 12(S)-HETE binds to a putative high affinity, trimeric G protein-coupled receptor that results in activation of a protein tyrosine phosphatase and Src family kinases. These kinases phosphorylate PLC1 and the adapter protein Shc. PLC activation results in stimulation of PKC, whereas Shc phosphorylation leads to recruitment of Grb2, Sos, and ultimately to increased GTP loading of Ras. Ultimately both of these pathways converge on Raf. A putative low affinity receptor also activates PKC via PLC. DAG, diacylglycerol; IP3, inositol 1,4,5-trisphosphate; PTP, protein tyrosine phosphatase.

The goal of this study was to identify signaling molecules that may be responsible for the pleiotropic effects of 12(S)-HETE on tumor cells. We identified PKC, Ras, and ERK1/2 as targets of 12(S)-HETE stimulation. These molecules are at the intersections of multiple signaling pathways, activation of which can lead to diverse changes in cellular behavior. Stimulation of these signaling molecules may help to explain the many and varied effects of 12(S)-HETE on tumor metastasis, i.e. adhesion, spreading, migration, and invasion (2-4), stimulation of angiogenesis (6), and protection against the induction of apoptosis (5). Since the presence of 12-lipoxygenase has been correlated with prostate cancer progression in a clinical setting (40), it is important to identify the signaling pathways utilized by its principal arachidonate metabolite (i.e. 12(S)-HETE). Future studies are directed toward the identification of the 12(S)-HETE receptor(s).

	ACKNOWLEDGEMENTS

We thank Yinlong Cai for excellent help with the apoptosis studies; Dr. Debra Skafar for most helpful suggestions on the binding studies; and Drs. Jozsef Timar, Daotai Nie, Clem Stanyon, and Avraham Raz for helpful discussion.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant CA 29997 (to K. V. 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.

To whom correspondence should be addressed: 431 Chemistry Bldg., Wayne State University, Detroit, MI 48202. Tel.: 313-577-1018; Fax: 313-577-0798; E-mail: k.v.honn@wayne.edu.

Published, JBC Papers in Press, August 21, 2000, DOI 10.1074/jbc.M002673200

² K. V. Honn, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: 12(S)-HETE, 12(S)-hydroxyeicosatetraenoic acid; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; PLC, phospholipase C; PKC, protein kinase C; Shc, Src homology collagen; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; Grb2, growth factor-bound protein 2; Me₂SO, dimethyl sulfoxide; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; RBD, Ras binding domain; PMA, phorbol myristate acetate; PDGF, platelet-derived growth factor; FGF, fibroblast growth factor; FTase, farnesyltransferase; MEK, MAPK/ERK kinase; 12-LOX, 12-lipoxygenase; BHPP, N-benzyl-N-hydroxy-5-phenylpentanamide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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