©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation of Mitogen-activated Protein Kinase by Arachidonic Acid in Rat Liver Epithelial WB Cells by a Protein Kinase C-dependent Mechanism (*)

(Received for publication, November 14, 1994; and in revised form, December 27, 1994)

Charles S. T. Hii (1)(§) Antonio Ferrante (1) Yasmin S. Edwards (3) Zhi H. Huang (1) Perry J. Hartfield (3) Deborah A. Rathjen (1) Alf Poulos (2) Andrew W. Murray (3)

From the  (1)Departments of Immunology and (2)Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia 5006 and the (3)School of Biological Sciences, Flinders University of South Australia, Bedford Park, South Australia 5042, Australia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Arachidonic acid (20:4(n-6)), which is released by cells responding to a wide range of stimuli, may play an important role in intracellular signaling. We now report that incubation of WB cells with 20:4(n-6) resulted in the appearance of several tyrosine-phosphorylated cytosolic proteins. Two of the phosphotyrosine-containing proteins, migrating in SDS-polyacrylamide gels of approximately 43 and 45 kDa, corresponded in mobility to phosphorylated species of the 42- and 44-kDa mitogen-activated protein kinase (MAPK) isoforms. Immunoblots of soluble fractions from unstimulated WB cells with anti-MAPK antibodies revealed the presence of the 42- and 44-kDa isoforms of MAPK. Upon incubation with 20:4(n-6), the mobility of both isoforms was retarded, consistent with their activation by phosphorylation. Chromatography of soluble fractions from these cells on Mono Q columns revealed early and late eluting peaks of myelin basic protein kinase activity, which contained the 42- and 44-kDa MAPK isoforms, respectively. Activation of MAPK was transient, peaking at 5 min, and was detectable at 5 µM 20:4(n-6). Further studies into the mechanisms by which MAPK was activated by 20:4(n-6) strongly suggested the involvement of protein kinase C (PKC). Not only did incubation of WB cells with 20:4(n-6) result in the translocation of PKC alpha, , and to a particulate fraction, it was found that the fatty acid failed to activate MAPK in cells pretreated for 26 h with phorbol 12-myristate 13-acetate, which depleted WB cells of PKC alpha, , and . In addition, fatty acids of the n-3 series were effective activators of MAPK. The present study, to our knowledge, is the first to report that polyunsaturated fatty acids can cause the activation of MAPK.


INTRODUCTION

Exogenous polyunsaturated fatty acids (PUFA) (^1)such as arachidonic acid (20:4(n-6)) exert a wide range of effects on cells of diverse origin. These include the regulation of gap junctional permeability between adherent cells(1) , neutrophil secretory and NADPH oxidase activities(2, 3) , neutrophil migration(4) , insulin secretion (5) , expression of cell-surface receptors and adhesion molecules(3) , gene transcription(6) , and cytotoxic T cell function(7) . Although a number of studies have reported that exogenous 20:4(n-6) can modulate the activities of components of intracellular signaling (8, 9, 10, 11, 12, 13, 14) , the mechanisms by which the fatty acid acts still need further clarification. In addition, 20:4(n-6) is liberated in response to a diverse range of stimuli through the activation of phospholipase A(2), and 20:4(n-6) thus released and its eicosanoid metabolites have been proposed to participate in cell signaling(14, 15) .

The mitogen-activated protein kinase (MAPK) is a family of kinases which has been reported to play an important role in intracellular signaling(16) . Members of this family of kinases, originally found to be activated by mitogens, have now been found to be activated by a wide variety of mitogenic and non-mitogenic agents via a cascade of kinases/effector molecules which, in mammalian cells, include protein kinase C (PKC) and/or p21, Raf-1, and MEK (MAPK/extracellular signal-regulated protein kinase (ERK) kinase)(16) . In order to determine whether some of the actions of 20:4(n-6) could be mediated by the MAPK cascade, we investigated whether 20:4(n-6) and PUFA of the n-3 series could alter the activity of MAPK. We report here the novel finding that the n-3 and n-6 series of PUFA stimulated the activity of MAPK in WB cells.


EXPERIMENTAL PROCEDURES

Materials

Fatty acids, phorbol 12-myristate 13-acetate (PMA), lysophosphatidic acid (LPA), epidermal growth factor (EGF), myelin basic protein (MBP), kinase A peptide inhibitor, and general reagents for kinase assays were from Sigma. [-P] ATP (specific activity 4000 Ci/mmol) was obtained from Bresatec, Adelaide. Antibodies against MAPK, R1 and R2, were kind gifts from Dr. S. Pelech, and their specificities have been described(17) . Monoclonal anti-phosphotyrosine antibody 4G10 was obtained from Upstate Biotechnology Inc., and isozyme-specific antibodies to PKC were from Boehringer Mannheim. Enhanced chemiluminescence (ECL) solutions and reinforced nitrocellulose were from DuPont NEN and Schleicher & Schuell, respectively.

Cell Culture and Preparation of Extracts

The source and maintenance of WB cells were as described previously(18) . Prior to use, cells were incubated for up to 2 h in serum-free medium. PUFA, PMA, and LPA were dissolved in ethanol, dimethyl sulfoxide (Me(2)SO), and 1% fat-free bovine serum albumin, respectively. The final concentrations of the vehicles were: ethanol, 0.01% (v/v); Me(2)SO, 0.01% (v/v); and fat-free serum albumin, 0.001% (w/v). Control cells received vehicle(s) that did not affect kinase activity. To pretreat with phorbol ester, WB cells were incubated for 26 h in the presence of 300 nM PMA, the last 2 h in the absence of serum. Cells were incubated in the presence of 20:4(n-6) for the times indicated. Incubations were terminated by removing the incubation medium and washing the cells once with Hank's balanced salt solution (4 °C). The cells were sonicated (3 times 10 s, output of 2 units, Soniprobe) in buffer A(18) , centrifuged (100,000 times g times 20 min), and the supernatants (termed cytosolic fractions) were collected. These fractions were either adsorbed onto phenyl-Sepharose CL4B to partially purify MAPK (18) or applied onto Mono Q columns (Pharmacia Biotech Inc.), which were developed with a linear salt gradient(18) . To probe for immunoreactive MAPK, partially purified enzyme or enzyme in whole cell homogenates was mixed with Laemmli buffer, separated on polyacrylamide gels, and immunoblotted as described below. To characterize the presence of PKC isozymes, WB cells were lysed in buffer B (25 mM Tris/HCl (pH 7.5), containing 1 mM dithiothreitol, 5 mM EGTA, 2 mM EDTA, 10 mM benzamidine, 10 mM phenylmethylsulfonyl fluoride, 0.01% leupeptin, and 2% Triton X-100) and incubated on ice for 30 min. Samples were centrifuged (100,000 times g times 30 min), and the identity of PKC isozymes in the supernatants was determined using isozyme-specific anti-PKC antibodies. To study PKC translocation, WB cells were sonicated in buffer B without Triton X-100 and centrifuged. The pellets were resuspended in Buffer B, incubated for 30 min on ice, and centrifuged, and samples were immunoblotted as described below.

MAPK Assay

Kinase activity was assayed as described previously (18) by monitoring the incorporation of P(i) into myelin basic protein (MBP) in the presence of EGTA and protein kinase A peptide inhibitor. Assays were terminated by spotting aliquots of the reaction mixture onto P-81 filter paper followed by extensive washing with 75 mM orthophosphoric acid. Radioactivity was determined by liquid scintillation spectrometry.

Western Blotting

Denatured proteins (1-2 µg for MAPK blots, 20 µg for PKC blots) were separated on either 10 or 12% polyacrylamide gels, transferred to nitrocellulose (100 V, 1.2 h), and immunoreaction and detection were carried out as described earlier (18) . Affinity-purified polyclonal anti MAPK antibodies R1 and R2, monoclonal anti-phosphotyrosine antibody (4G10), and PKC isozyme-specific antibodies were used to detect MAPK isoforms, phosphotyrosine-containing proteins, and PKC isozymes, respectively. Immunocomplexes were detected by ECL(18) .

Data Analysis

PKC immunoblots from 20:4(n-6)-treated samples were analyzed by laser densitometric scanning (LKB 2202 Ultroscan). Differences in density were analyzed by Student's paired t test, and data are expressed as -fold increase over respective paired control.


RESULTS AND DISCUSSION

Incubation of WB cells with 20:4(n-6) (20 µM) for 5 min resulted in tyrosine phosphorylation of a number of cytosolic proteins, including those of 43 and 45 kDa (Fig. 1). Several proteins, notably those of approximately 76, 108, and 138 kDa, reacted strongly with the anti-phosphotyrosine antibody, while others, including those of 43 and 45 kDa, reacted less strongly. The pattern of tyrosine-phosphorylated proteins detected in cells incubated with LPA, EGF, or PMA (Fig. 1) showed no major observable quantitative differences between the four compounds. We have previously reported the presence and activation of the 42- and 44-kDa isoforms of MAPK by LPA, EGF, and PMA in WB cells (18) , raising the possibility that the 43- and 45-kDa proteins shown in Fig. 1could be tyrosine-phosphorylated MAPK isoforms. Members of the MAPK family are phosphorylated on tyrosine and serine/threonine by MEK when activated(16) . To determine whether incubation of WB cells with 20:4(n-6) led to the phosphorylation of the 42- and 44-kDa MAPK isoforms cytosolic fractions obtained from cells incubated with 20:4(n-6) (20 µM) for 5 min were immunoblotted with anti-MAPK antibody R1, which we had used previously to detect phosphorylated MAPK isoforms in WB cells(18) . Incubation of WB cells in the presence of 20:4(n-6) resulted in the appearance of immunoreactive material, which migrated between the 42- and 44-kDa bands, and an additional band of approximately 45 kDa (Fig. 2, lanes 2 and 4). When the same sample was probed with anti-MAPK antibody R2, which detected the 44-kDa MAPK more strongly than the 42-kDa MAPK, the 45-kDa material was revealed more clearly (Fig. 2, lane4). The data thus suggest that the 43- and 45-kDa tyrosine-phosphorylated proteins are likely to be phosphorylated forms of MAPK. To determine whether these fractions contained activated MAPK samples were chromatographed on Mono Q columns. Fig. 3(panelA) shows that cytosolic fractions from WB cells incubated for 5 min with 20:4(n-6) could be resolved into two peaks of MBP kinase activity which corresponded to the peaks induced when cells were incubated with PMA. Immunoblots of the peak fractions revealed that the early and late eluting peaks contained predominately the presence of the 42- and 44-kDa MAPK isoforms (Fig. 3, panelB).


Figure 1: Induction of tyrosine phosphorylation of cytosolic proteins by 20:4(n-6). Cells were incubated with vehicles (lane2), LPA (20 µM, lane3), EGF (10 ng/ml, lane4), PMA (100 nM, lane5), or 20:4(n-6) (20 µM, lane6) for 5 min. Cytosolic fractions were prepared and immunoblotted with 4G10 as described under ``Experimental Procedures.'' Lane1, molecular mass markers. Similar data were obtained in a repeat experiment.




Figure 2: Detection of MAPK isoforms. Cytosolic fractions were prepared as described in legends to Fig. 1, and samples were immunoblotted with anti-MAPK antibodies R1 (lanes1 and 2) or R2 (lanes3 and 4). Lanes1 and 3, vehicle; lanes2 and 4, 20:4(n-6). The 42-kDa MAPK isoform was also detectable in lanes3 and 4 upon longer exposure (not shown). Results are representative of three experiments.




Figure 3: Chromatography of MAPK isoforms on Mono Q columns. A, WB cells were incubated with vehicle (circle), 20:4(n-6) (20 µM) () or PMA (100 nM) (box) for 5 min, and cytosolic fractions were prepared and chromatographed on Mono Q columns as described under ``Experimental Procedures.'' Fractions were assayed for MBP kinase activity. B, peak fractions from 20:4(n-6)-stimulated samples were immunoblotted with anti-MAPK antibody R1. Lane1, early eluting peak; lane2, late eluting peak.



To characterize further the activation of MAPK by 20:4(n-6) cytosolic fractions from fatty acid-treated cells were partially purified by adsorption onto phenyl-Sepharose CL4B. These studies showed that the enhancement of kinase activity was transient, peaking at 5 min following the addition of 20:4(n-6) (Fig. 4, panelA). Activation of MAPK was also dependent on the concentration of 20:4(n-6) with a detectable increase in kinase activity observed at a fatty acid concentration of 5 µM (Fig. 4, panelB).


Figure 4: Activation of MAPK by 20:4(n-6). Cells were incubated for 0-60 min (A) with 20:4(n-6) (20 µM) or for 5 min (B) with 0-20 µM 20:4(n-6) and kinase activity in partially purified samples was assayed as described under ``Experimental Procedures.'' (circle), control; (), 20:4(n-6). Similar data were obtained in two other repeat experiments.



Since PUFA have been shown to activate PKC (12, 19) and that PKC is one of the upstream regulators of the MAPK cascade(16) , we investigated whether PKC was involved in the activation of MAPK by 20:4(n-6) in WB cells. These cells contain PKC alpha, , , and (Fig. 5, panelsA-C) but not beta or (data not shown). Visualization of the isozymes was prevented by preincubation of the antibodies with isozyme-specific peptide antigens (data not shown). Both 20:4(n-6) and PMA stimulated the translocation of PKC alpha, , and to a particulate fraction (Fig. 5, panels B and C). Densitometric scanning revealed that 20:4(n-6) increased membrane-associated PKC alpha, , and by (mean ± S.E., n = 3) 2.9 ± 0.7 (p < 0.05), 3.1 ± 1.0 (p = 0.054), and 1.8 ± 0.3 (p < 0.05) fold, respectively, over control without affecting PKC (1.14 ± 0.1) (Fig. 5, panelC). Pretreatment of WB cells with PMA (300 nM) for 26 h led to a loss of the majority of alpha, , and immunoreactivity but not (Fig. 5, panelA), consistent with PKC being a PMA-unresponsive isozyme(12) . This treatment was associated with a loss of activation of MAPK by both 20:4(n-6) and PMA (Table 1). Recent studies have implicated PKC alpha and as upstream regulators of the MAPK cascade (20) . (^2)Activation of MAPK by EGF, acting via p21(21) , was only slightly reduced in these PKC-depleted cells (Table 1). These data suggest that activation of MAPK by 20:4(n-6) involved the activation of PMA-responsive PKC isozymes, and that 20:4(n-6) and EGF acted through different pathways.


Figure 5: Depletion and translocation of PKC isozymes. WB cells were preincubated with PMA (300 nM) or Me(2)SO (0.01%, v/v) for 26 h to deplete PKC (A), incubated with PMA (100 nM) for 5 min (B) or with 20:4(n-6) (20 µM) for 2 min (C). Total PKC (A) or PKC in particulate fractions (B and C) were immunoblotted with isozyme-specific antibodies to PKC as described under ``Experimental Procedures.'' Results are representative of three experiments for panelsB and C.





Activation of MAPK by PUFA was not restricted to the n-6 series of PUFA. Both 20:5(n-3) and 22:6(n-3), found in abundance in fish oils and in the brain and retina(22) , were also capable of enhancing the activity of MAPK as determined by the ability of phenyl-Sepharose CL4B-purified kinase to phosphorylate MBP. Kinase activities (fmol of P(i)/µg of protein/20 min) after a 5-min incubation with the PUFA (20 µM) were: ethanol, 478; 20:4(n-6), 1134; 20:5(n-3), 1010; 22:6(n-3), 1010. Certain effects of 20:4(n-6) have been proposed to be dependent on the oxidation of 20:4(n-6) to eicosanoid products by lipoxygenase(15) . Although both the n-3 PUFA and 20:4(n-6) were capable of enhancing the activity of MAPK, these PUFA give rise to different eicosanoid products with different biological actions (see (23) , and references therein). This, therefore, makes it unlikely that the activation of MAPK by PUFA was dependent on the formation of eicosanoids.

Both PKC and p21 have been demonstrated to regulate the MAPK cascade in mammalian cells(16) . While p21 has recently been reported to activate the MAPK kinase kinase activity of Raf-1 through the mediation of 14-3-3(24, 25) , it is not clear whether PKC activates the MAPK cascade via Raf-1, MEK kinase, the mammalian homologue of yeast STE11(26) , or both. A recent study has demonstrated phosphorylation of Raf-1 by PKC alpha on Ser-499 and possibly Ser-259, resulting in the activation of Raf-1 autokinase and kinase activity toward a peptide substrate(20) . However, the MAPK kinase kinase activity of Raf-1 was not determined. Another study failed to detect activation of Raf-1 kinase activity despite phosphorylation of the kinase by PKC(27) . A Raf-1-independent pathway has also been identified in a BALB/c-derived cell line stimulated by phorbol esters (28) . PKC may also activate Raf-1 in a p21-dependent manner in certain cell types(29) . Despite these uncertainties regarding the site of PKC action, the observations that intact PKC was required for the activation of MAPK by 20:4(n-6), and that the fatty acid stimulated the translocation of PKC alpha, , and to the particulate fraction, are consistent with the suggestion that activation of MAPK by 20:4(n-6) proceeded via PKC. However, we stress that not all cellular responses to 20:4(n-6) are blocked by PMA pretreatment since PUFA-induced inhibition of gap junctional permeability in WB cells was unaffected by PMA pretreatment, (^3)suggesting involvement of either PKC or PKC-independent mechanisms.

Activation of MAPK by PUFA appears to be a general response in cells exposed to the fatty acids, since we have also observed activation of MAPK by 20:4(n-6) in other cells including human neutrophils, human macrophages, human mesangial cells, human umbilical vein endothelial cells, Monomac 6 cells, and Jurkat cells (data not shown). This raises the possibility that some of the actions of PUFA could be mediated via the MAPK cascade.

Many studies have reported that stimulation with a diverse range of agonists activates phospholipase A(2), resulting in release of 20:4(n-6)(14, 15, 32) . In stimulated pancreatic B cells, the concentration of unesterified 20:4(n-6) has been reported to be in excess of 50 µM(30) . Furthermore, unesterified fatty acids are known to be released in pathological conditions such as cardiac ischemia(31) . This study therefore raises the possibility that liberated PUFA may be capable of stimulating the activity of MAPK. Since many of the agonists that activate the MAPK cascade also activate phospholipase A(2)(32) , possibly by direct phosphorylation of phospholipase A(2) by MAPK(33) , we speculate that unesterified fatty acids released during agonist stimulation have the potential to modify or prolong the activation of MAPK in response to primary stimulants.

This study, to our knowledge, is the first to report the finding that 20:4(n-6) and PUFA of the n-3 series were capable of activating MAPK. We also demonstrated that 20:4(n-6) induced the appearance of several tyrosine-phosphorylated cytosolic proteins. The mechanisms by which 20:4(n-6) induced the tyrosine phosphorylation of these proteins are unknown. Activation of MAPK by 20:4(n-6) was dependent on PKC.


FOOTNOTES

*
This work was supported by funds from the National Health and Medical Research Council, Anti-Cancer Foundation of South Australia, and Peptide Technology Limited. Portions of this work were presented at the 9th Molecular Cellular Developmental Biology/Iowa State University Symposium, Ames, IA in September, 1994. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 61-8-204-6293; Fax: 61-8-204-6046.

(^1)
The abbreviations used are: PUFA, polyunsaturated fatty acid(s); MAPK, mitogen-activated protein kinase; MEK, MAPK/extracellular signal-regulated protein kinase (ERK) kinase); PKC, protein kinase C; 20:4(n-6), arachidonic acid; 20:5(n-3), eicosapentaenoic acid; 22:6(n-3), docosahexaenoic acid; PMA, phorbol 12-myristate 13-acetate; MBP, myelin basic protein; LPA, lysophosphatidic acid; EGF, epidermal growth factor.

(^2)
Clark, K. J., and Murray, A. W.(1995) J. Biol. Chem.270, in press.

(^3)
C. S. T. Hii, A. Ferrante, Y. Schmidt, D. A. Rathjen, B. S. Robinson, A. Poulos, and A. W. Murray, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. S. L. Pelech for the anti-MAPK antibodies R1 and R2 and A. J. Bilney for maintaining the WB cells.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.