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Arachidonic Acid Mediates Interleukin-1 and Tumor Necrosis Factor-cuInduced Activation of the c-jun Amino-Terminal Kinases in Stromal Cells
By Maria Teresa Rizzo and Carmelo Carlo-Stella
We have previously shown that arachidonic acid mediates
interleukin-1 (IL-1) and tumor necrosis factor-a (TNF-a)-induced transcription of c-jun. The signaling pathway of arachidonic acid-induced c-jun transcription was independent
of protein kinase C activation and involved a tyrosine kinasedependent process. The present study was undertaken t o
further elucidate the signal transduction pathway of arachidonate-induced c-jun transcription. We used a glutathioneS-transferase-c-jun fusion protein containing the aminoterminal domain of c-jun (residues 5 t o 89) t o explore the
hypothesis that arachidonic acid stimulates c-jun amino-terminal kinase (JNK) activity in the murine stromal cell line
+/+.1 LDA 11. Extracts from arachidonic acid-treated cells
catalyzed phosphorylation of the c-jun fusion protein, indicating stimulation of JNK activity. Similar results were obtained when cells were challenged with IL-1 and TNF-a. The
effect of arachidonic acid was specific, because extracts from
stimulated cells failed t o phosphorylate a mutated fusion
protein in which serine 63 and 73 of e-jun were each substituted with leucine. Arachidonic acid induced JNK activation
in a time- and dose-dependent manner that was not mimicked by saturated fatty acids such as palmitic acid or other
unsaturated fatty acids from the n-3, n-6, or n-9 series. Furthermore, other lipids, such as diacylglycerol, phosphatidic
acid, and C,-ceramide, failed t o induce a significant increase
in JNK activity. Treatment of stromal cells with propyl gallate, a dual inhibitor of lipoxygenase and cyclooxygenase
enzymes, did not affect the ability of arachidonic acid t o
induce JNK activation. Moreover, ETVA (5,8,11,1Ceicosatetraynoic acid), a nonmetabolizable arachidonate analogue,
also induced JNK activation. These results are consistent
with the hypothesis that the signal transduction pathway by
which arachidonate stimulates c-jun transcription involves
activation of the JNK cascade. Furthermore, arachidonic acid
itself and not its cyclooxygenase or lipoxygenase metabolites is involved in stimulating JNK activity. Thus, arachidonic acid may act as a second messenger in mediating the
effects of IL-I and TNF-a in the activation of c-jun.
0 1996 b y The American Society of Hematology.
A
acid may act as a second messenger promoting the transfer
of signals from the cell surface to the nucleus by a pathway
involving a protein tyrosine kinase cascade." Downstream
intermediates in this activation cascade have not, as yet, been
identified.
A novel group of protein kinases, closely related to MAP
kinases, has been recently identified.I4 These protein kinases
known as stress-activated protein kinases (SAPKs) or c-jun
amino-terminal kinases (JNKs) bind to the amino-terminal
domain of c-jun and phosphorylate it specifically on Ser 63
and Ser 73, thereby increasing c-jun transcriptional activity
and, thus, its ability to induce transcription of AP- 1 -containing genes, including c-jun itself. 15-2n The JNK protein
kinases are proline-directed kinases that are activated by
phosphorylation at conserved tyrosine and threonine residues
by dual specificity protein kinases that have been recently
Upon phosphorylation, JNK protein kinases
translocate to the nucleus. where they participate in the activation of transcription factors such as c-jun and ATF~,lh.2U.25.26
Induction of JNK activity is detected upon cell
stimulation with a variety of agonists, including IL-1 and
T N F - ~16.27-29
,
Some of the steps of the signaling pathway
that lead to activation of JNK by IL-1 and TNF-a have been
recently e l u ~ i d a t e d . ~ ' -However,
~ ~ . ~ " the intracellular mediators involved in stimulation of JNK activity by IL-l and
TNF-(Uare not well defined. Based on these considerations,
we undertook this study to further investigate the signal
transduction pathway by which arachidonic acid mediates
IL-I and TNF-a-induced c-jun transcription. We have explored the hypothesis that arachidonic acid, released upon
stimulation of stromal cells with IL-1 and TNF-a, triggers
a signal transduction pathway that involves activation of
JNK leading to c-jun phosphorylation and thus to c-jun transcription.
RACHIDONIC ACID plays a pivotal role in a wide
array of cellular responses.',2 Although metabolites of
the lipid exert important effects by influencing many cellular
functions, a direct role for arachidonic acid has been implicated in the induction of certain cellular responses.'.'' Studies by us and other investigators have shown that arachidonate may be an important mediator of growth factor-induced
gene
Thus, we showed that interleukin-1 (IL-1)
and tumor necrosis factor-a (TNF-a)-induced granulocytemacrophage colony-stimulating factor (GM-CSF) gene expression involves activation of phospholipase A2 and was
mediated by arachidonic acid-induced activation of the transcription factor, c-jun.' Arachidonic acid enhanced c-jun
transcription by a signaling pathway that did not require
activation of protein kinase C (PKC), but instead resulted
from a cellular process that was blunted by the phosphotyrosine kinase inhibitor genistein and potentiated by the tyrosine
phosphatase inhibitor vanadate. l o Therefore, arachidonic
From the Bone Marrow Transplantation Laboratory, Methodist
Cancer Center, Methodist Hospital, Indianapolis, IN; and the Division of Hematology and Bone Marrow Transplantation, Pclrma Universify, P a m a , Italy.
Submitted March 25, 1996; accepted July 18, 1996.
Supported in part by funds from Methodist Cancer Center, by the
Showalter Research Foundation to M.T.R., and by a grant from
Consiglio Nazionale delle Ricerche (Progetto Finalizzato A. C.R.0.)
f0
c.c.-s.
Address reprint requests to Maria Teresa Riuo, MD, Bone Marrow Transplantation Laboratory, MPC 141 7, Methodist Research
Institute, Methodist Hospital, I701 N Senate Bhd, Indianapolis
46202, IN.
The publication costs of this article were defrayed in part by page
charge paymenr. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8810-0032$3.00/0
3192
MATERIALS AND METHODS
Materials. Human recombinant IL- lp and murine recombinant
TNF-a were purchased from Genzyme (Cambrige, MA). McCoy's
Blood, Vol 88, No 10 (November 15). 1996: pp 3792-3800
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3793
ACTIVATION OF JNK BY ARACHIDONIC ACID
5A modified medium and HL-I medium were purchased from
GIBCO BRL (Grand Island, NY) and from Ventrex Laboratories,
Inc (Portland, ME), respectively. Fetal bovine serum was obtained
from Hyclone (Logan, UT). [y-”P] adenosine triphosphate (ATP)
was purchased from Du Pont New England Nuclear (Boston, MA).
Hyperfilm was purchased from Amersham Life Science (Arlington
Heights, IL). Arachidonic acid, palmitic acid, oleic acid, linoleic
acid, linolenic acid, eicosatetraynoic acid (ETYA), propyl gallate,
indomethacin, nordihydroguaiaretic acid (NDGA), melittin, quinacrine, genistein, glutathione, glutathione agarose, and the lactate dehydrogenase (LDH) kit were obtained from Sigma Chemical Co (St
Louis, MO). Phosphatidic acid and diacylglycerol were from Avanti
Polar Lipids (Alabaster, AL). Ceramide was purchased from Biomol
Research Laboratories (Plymouth Meeting, PA). Anti-JNK1 antibodies were purchased from Santa CNZ Biotechnology, Inc (Santa Cruz,
CA). Protein G plus/protein A agarose was obtained from Oncogene
Science (Uniondale, NY).The glutathione-S-transferase (GST)-cjun fusion proteins (5-89) and (A 63/73) were a generous gift of Dr
A. Kraft (University of Alabama, Birmingham, AL).
Cell culture. The characteristics of the stromal cell line +/+.1
LDA 11 have been previously de~cribed.~’
Cells were grown in
McCoy’s 5A modified medium supplemented with 10% fetal bovine
serum and containing 50 U/mL penicillin and 50 pg/mL streptomycin. Cells were maintained in humidified 5% CO, at 37°C.
Solid-phase kinase assay. We used a GST-C-jun fusion protein
coupled to glutathione agarose beads to assay binding and substrate
phosphorylation by jun kinase in cell extracts. Serum-deprived stromal cells were stimulated under serum-free conditions with the indicated stimuli and for the indicated length of time. Arachidonic acid
was dissolved in 100% ethanol before use and was presented to cells
so that the final concentration of ethanol was less than 0.1 %. Control
cells received the same amount of ethanol as did treated cells. After
stimulation, cells were washed with cold phosphate-buffered saline
(PBS) containing 5 mmoVL NaF and 1 mmoVL Na3V04.Cells were
lysed in cold lysis buffer (20 mmoVL HEPES, pH 7.6, 300 mmoV
L NaCI, 1.5 mmoVL MgCl,, 1 mmoVL EDTA, 0.5 mmoVL dithiothreitol (DTT), 1 mmoVL Na3V04,0.1% sodium deoxycholate, 1%
Triton X-100 with 1 mmoVL phenylmethylsulfonylfluoride,10 pg/
mL aprotinine, 10 pg/mL leupeptin, and 10 pg/mL antipain) and
clarified by centrifugation at 10,000g for 10 minutes. C-jun kinase
activity was determined as described by Hibi et al,27using recombinant GST-c-jun fusion protein purified by glutathione affinity chrom a t o g r a p h ~Briefly,
.~~
cell lysates (200 pg) were mixed with 5 pg
of immobilized GST-c-jun (5-89) or, as a negative control, GST
protein alone and gently shaken for 3 hours at 4°C. Beads were then
collected by centrifugation and washed three times in washing buffer
containing 20 “OIL HEPES (pH 7.6), 50 mmom NaCI, 2.5 mmoV
L MgCl,, 0.1 mmoVL EDTA, and 0.05% Triton X-100. The activity
of JNK bound to GST-c-jun was determined by resuspending beads
in 20 pL of kinase assay buffer (20 mmom HEPES, pH 7.6, 20
mmoVL MgCl,, 20 mmol/L P-glycerophosphate, 5 mmoVL NaF,
0.1 mmoVL Na3V04, and 2 mmoVL DTT), and kinase reactions
were started by the addition of 50 pmoVL ATP and 10 pCi of [ y ”P-ATP]. Reactions were performed at 30°C and terminated after
20 minutes by the addition of 2X sodium dodecyl sulfate (SDS)
sample buffer. Samples were denaturated at 100°C by boiling for 5
minutes and eluted proteins were resolved in 12% SDS-polyacrylamide gel, followed by autoradiography. Phosphorylated GST-cjun bands were excised from the gel and quantitated by liquid scintillation counting.
Immune complex kinase assay. For JNK immune complex kinase assays, 500 pg of whole cell extracts from unstimulated and
stimulated cells was precleared by incubation with 1 pg/mL rabbit
antimouse IgG for 1 hour at 4°C and then for an additional 30
minutes with 10 pL of protein G plus/protein A. After preclearing
cell lysates, supematants were incubated overnight at 4°C with 1 pg
of a polyclonal anti-JNK1 antibody. A nonimmune rabbit IgG was
used as the negative control. Immune complexes were recovered by
the addition of 20 pL protein G pludprotein A agarose. The protein
complexes were extensively washed with washing buffer followed
by a washing with kinase assay buffer and resuspended in kinase
assay buffer containing 1 pg of GST-c-jun (5-89), GST-c-jun (A
63/73), or GST protein alone as substrates in the presence of 50
pmoVL ATP and 10 pCi [Y-~~PIATP.
The reaction mixture was
incubated for 20 minutes at 30”C, terminated by the addition of 2X
SDS sample buffer, and boiled for 5 minutes. Phosphorylated proteins were resolved by 12% SDS-polyacrylamide gel and subsequently detected by autoradiography. 32P-Piincorporation into GSTc-jun was quantitated as described above.
In-gel kinase assay. Assays of proteins renaturated in polyacrylamide gels were performed as described by Kameshita and Fuji~ a w a . ’Briefly,
~
lysates from control and stimulated cells were separated on a 10% SDS-polyacrylamide gel that was polymerized in
the absence or in the presence of GST-c-jun (40 pg/mL) added as
a substrate directly to the gel before polymerization. After electrophoresis, the gel was washed several times before denaturation in 6
m o m guanidine-HCI. The gel was then renaturated with five
changes of a buffer containing 50 mmoVL Tris-HCI (pH 8.0), 5
mmoVL 2P-mercaptoethano1, and 0.04% Tween 40 at 4°C for 16
hours. After renaturation, the gel was incubated for 1 hour at 25°C
in kinase assay buffer containing 50 p m o m ATP and 20 pCi/mL
of [y-”P]ATP. After incubation, the gel was washed with 5% trichloroacetic acid containing 1% sodium pyrophosphate. The gel was
dried and exposed to x-ray film at -80°C.
Protein determination. Protein concentrations of cell lysates
were determined by the method of Bradford using bovine serum
albumin as a standard.”
Cyroroxicity assay. LDH activity was determined according to
the manufacturer’s instructions from supematants after treatment of
cells with arachidonic acid (10 to 100 pmoVL) for 30 minutes.
RESULTS
Effect of arachidonic acid on JNK protein kinases. Our
recent studies suggested that a signal transduction pathway
involving a tyrosine kinase cascade mediated arachidonic
acid-induced c-jun transcription.” To further elucidate the
components of the signaling route to c-jun transcription by
arachidonic acid, we performed experiments to explore the
potential involvement of the JNWSAPK cascade. JNK activity was measured in cell lysates by a solid-phase in vitro
kinase assay using as a substrate the GST-c-jun fusion protein containing the amino-terminal domain of c-jun from
amino acid 5 to 89 immobilized on glutathione-agarose
beads.35Stromal cells were serum-deprived and subsequently
stimulated with arachidonic acid or IL-1 plus TNF-a. Cell
extracts were prepared and assayed for their ability to induce
phosphorylation of the GST-c-jun fusion protein. As shown
in Fig lA, JNK activity present in unstimulated cells substantially increased upon exposure of cells to 25 pmol/L arachidonic acid for 30 minutes. Exposure of stromal cells to IL1 (500 U/mL) plus TNF-a (500 U/mL) for 15 minutes also
potentiated JNK activity. Quantitation of radioactivity within
excised gel slices showed a 3.5- and 4.3-fold increase in cjun phosphorylation in cells treated with arachidonic acid or
with E-1 plus TNF-a, respectively. Similar results were
obtained when IL-1 and TNF-a were used individually (data
not shown). No phosphorylation was detected when the ki-
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RlZZO AND CARLO-STELLA
3794
A
om-cjun
b
C
oslr-cjun w
Fig 1. (A) Activation of JNK by arachidonic acid or IL-1 and TNF-a. Lysates from control cells or from cells stimulated with arachidonic acid
(25 pmollLl for 30 minutes or IL-1 (500 UlmL) plus TNF-a (500 UlmL) for 15 minutes were incubated with 5 p g of immobilized GST-c-jun or
GST protein alone as a negative control. The beads were washed and incubated in kinase assay buffer containing 50 pmollL ATP and 10 pCi
[y-BZPIATP
for 20 minutes at 30°C. Reactions were terminated by the addition of 2 x SDS sample buffer. Phosphorylated proteins were resolved
by 1296 SDS-polyacrylamide gel electrophoresis, dried, and analyzed by autoradiography. (B) Dose-response of JNK activation by arachidonic
acid. Cells were stimulated with different concentrations of arachidonic acid for 30 minutes and cell lysates were prepared. JNK activity was
measured by the solid-phase kinase assay, as described in the Materials and Methods. (C) Time course of JNK activation by arachidonic acid.
Cells were treated with arachidonic acid (25 pmollL) for varying times. JNK activity was assayed by the solid-phase kinase assay, as described
above. Results are from a representative experiment performed twice with similar results.
nase reaction was performed using agarose beads coated
with GST protein only, indicating that phosphorylation was
substrate (c-jun) dependent (Fig 1 A).
Arachidonic acid induced activation of JNK in a doseand time-dependent manner (Fig 1B and C). A 2.5-fold increase of GST-c-jun phosphorylation was observed with a
concentration of I O pmol/L arachidonic acid as compared
with vehicle-treated cell (Fig IB). A 3.2- and 4.5-fold increase of JNK activity was detected at 25 pmol/L and 50
pmoI/L arachidonic acid, respectively (Fig IB). Further increases in the concentration of arachidonic acid did not further potentiate JNK activity (data not shown). The effect of
arachidonic acid did not appear to be a result of generalized
cytotoxicity, because LDH was not detectable in supematants from cells stimulated with different concentrations of
arachidonic acid ( I O to 100 pmol/L) for 30 minutes (data
not shown). Phosphorylation of GST-c-jun was observed
within 15 minutes of stimulation with arachidonic acid (Fig
1C). Arachidonic acid-induced phosphorylation of GST-cjun increased over 30 minutes and started to decline thereafter at 60 minutes (Fig IC).
The JNK group of protein kinases include the 46-kD JNKl
and the 55-kD JNK2 isoforms, both of which are activated
by IL-I and TNF-a in different cell sy~tems.’~.’’~’‘
To characterize the JNK activity further, we performed in-gel kinase
assays. These experiments showed that stimulation of stromal cells with 25 pmol/L arachidonic acid for 30 minutes
or with IL-I (500 U/mL) plus TNF-a (500 U/mL) for 15
minutes induced activation of both the 46-kD and 55-kD
isoforms of JNKs. although the 46-kD form of JNKl was
the major c-jun kinase activated in response to arachidonic
acid or IL-I plus TNF-a (Fig 2). No activation was detected
when proteins were separated in gel polymerized in the absence of GST-c-jun (data not shown). The ability of arachidonic acid to induce JNK activation was further examined
by immune complex kinase assays. JNK activity was immunoprecipitated from lysates of arachidonic acid-treated cells
using a polyclonal antibody that recognizes the 46-kD JNKl
isoform. As shown in Fig 3, anti-JNKI immunoprecipitates
of arachidonic acid-stimulated cells induced phosphorylation
of GST-c-jun (5-89). A similar effect was observed when
immunoprecipitates from lysates of IL- 1 plus TNF-atreated cells were used (Fig 3). On the contrary, no phosphorylation was detected when immunoprecipitates prepared
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ACTIVATION OF JNK BY ARACHIDONIC ACID
Fig 2. Preferential activation of JNKl by arachidonic acid or IL-1
and TNF-a. Lysates from cells treated either with vehicle (Co) or with
25 pmol/L arachidonic acid (AA) for 30 minutes or with 11-1 (500 U /
mL) plus TNF-a (500 U/mL) for 15 minutes were subjected to 10%
SDS-polyacrylamide gel electrophoresis. GST-c-jun (40 pg/mL) was
added to the gel before polymerizationas a substrate. Protein renaturation and kinase assays were performed as described in the Materials and Methods. Shown by the arrows are both isoforms of the JNK
protein kinases. This experiment was repeated twice with similar
results.
with a nonreactive antibody were used. Phosphoamino acid
analysis confirmed that GST-c-jun (5-89) was phosphorylated on serine by JNKl immunoprecipitated from arachidonate-treated cells (data not shown). Furthermore, when immune complex kinase assays were performed using a GSTc-jun fusion protein in which Ser 63 and Ser 73 were each
replaced with leucine (GST-c-jun A63/73), a marked decrease of phosphorylation was observed (Fig 3).
Spec$cio of arachidonate-induced JNK activation. To
determine whether the effect of arachidonic acid on JNK
activation was specific, we tested the ability of several saturated and unsaturated long-chain fatty acids to activate JNK
(Fig 4A). Stimulation of cells with 25 pmol/L oleic acid (n9) for 30 minutes caused a mild increase of JNK activity as
compared with that induced by arachidonic acid. Similarly,
25 pmol/L linoleic acid (n-6) and linolenic acid (n-3) induced a modest activation of JNK (Fig 4A). Palmitic acid
(25 pmoVL) had a marginal effect on JNK activation as
compared with the effect of arachidonic acid (Fig 4A).
We next undertook experiments to determine whether
other lipid second messengers were able to stimulate JNK
activation (Fig 4B and C). Stimulation of stromal cells with
50 ymol/L phosphatidic acid for 30 minutes failed to induce
JNK activation (Fig 4B). Diacylglycerol, a known activator
of PKC, also failed to induce JNK activation under the conditions used (Fig 4B). Because ceramide has been proposed
to mediate the effects of TNF-a in many cellular functions,
including activation of JNK,36.37we undertook experiments
to determine its effect on induction of JNK activity in stromal
cells. As shown in Fig 4C, stimulation with 5 pmol/L Czceramide for 30 minutes induced a weak increase (0.8-fold)
of JNK activity as compared with that induced by stimulation
with 25 pmol/L arachidonate (3.0-fold) for the same length
of time. An equimolar concentration (25 pmol/L) of cera-
3795
mide also failed to induce an increase of JNK activity as
compared with that induced by arachidonic acid (data not
shown). Thus, in the stromal cell line used, arachidonic acid
appears to be the preferential candidate in mediating activation of JNK by IL-I and TNF-a.
Role of arachidonic acid metabolites on JNK activation.
Arachidonic acid is rapidly metabolized through the cyclooxygenase and the lipoxygenase pathways leading to formation
of several biologically active metabolites, including prostaglandins and leukotrienes.’.’ To determine whether JNK activation is a direct effect of arachidonic acid or one of its
metabolites, we assessed the effects of arachidonic acid metabolism inhibitors on JNK activation. As shown in Fig 5A,
pretreatment of cells with 100 pmolk propyl gallate, an
antioxidant that inhibits both lipoxygenase and cyclooxygenase enzymes,” before stimulation did not effect the ability
of arachidonic acid to induce JNK activation. The inhibitor
had no detectable effect, by itself, on activation of JNK (Fig
5A). Indomethacin (IO pmol/L), a cyclooxygenase inhibitor,
and nordihydroguaiaretic acid or NDGA (1 0 pmoVL), a lipoxygenase inhibitor, similarly failed to block JNK stimulation by arachidonic acid (data not shown).
We next examined the effect of ETYA on arachidonateinduced JNK activation. ETYA is a structural analogue of
arachidonic acid in which four alkyne bonds replace the four
alkene bonds present in arachidonic acid.99ETYA competitively inhibits the uptake of arachidonic acid into intracellular membranes and inhibits both cyclooxygenase and lipoxygenase enzymes by acting as a false s u b ~ t r a t e Exposure
.~~
of cells to arachidonic acid or ETYA alone for 30 minutes
GST-cjun(5-89) C
I
..--
GST-cjnn(A63D3) c
Fig 3. Specificity of GST-c-jun phosphorylation by arachidonic
acid or IL-1 and TNF-a. Lysates from control and arachidonic acidtreated (25 pmol/L; 30 minutes) or IL-1 (500 UlmL; 15 minutes) plus
TNF-a (500 UlmL; 15 minutes) -treated cells were subjected to immunoprecipitationwith an antiJNKl antibody or anti-lgG antibody.
The kinase activity of the immune complexes was determined by
phosphorylationof GST-c-jun (5-89) or the mutated GST-c-jun (A
63/73) in the presence of [y3‘P1ATP. Proteins were analyzed by 12%
SDS-gelelectrophoresis and autoradiography. Resultsare from a representative experiment.
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R l U O AND CARLO-STELLA
3796
A
500
B
-
400
h
S
E
V
0
S
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Y
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C
-a'I
E
I-
ul
u
100
om-cjpn
F
Co
m
o
o
*
AA
OA
LA
LnA
PA
Fig 4. (A) Effect of fatty acids on JNK activation. Cells were stimulated for 30 minutes with 25 pmol/L each of the following fatty acids:
arachidonic acid (AA), oleic acid (OA), linoleic acid (LA), linolenic acid (LnA), and palmitic acid (PA). Cell lysates were prepared and used for
the solid-phase kinase assay as described. Data represent the mean k SEM of three independent experiments, expressed as an increase with
respect t o unstimulated cells (Co). An autoradiograph corresponding t o the labeled substrate from a representative determination is shown.
(6) Effect of lipids on JNK activation. Stromal cells were stimulated with 25 pmol/L arachidonic acid (AA), 50 pmollL phosphatidic acid (PA),
or 50 pmol/L diacylglycerol (DAG) for 30 minutes. Cell lysates were assayed using the solid-phase kinase assay with GST-c-jun as a substrate,
as described under the Materials and Methods. Results are from a single experiment that was repeated twice. (C) Effect of ceramide on JNK
activation. Cells were stimulated with C,-ceramide (5 pmol/L; 30 minutes) or arachidonic acid (25 pmollL; 30 minutes), and lysates were
prepared and assayed in the solid-phase kinase assay as described above.
induced a 3.7- and 2.9-fold increase of JNK activity, respectively, as determined by scintillation counting of the phosphorylated GST-c-jun proteins excised from the gel (Fig
SB). When cells were stimulated with arachidonic acid in
the presence of ETYA, a 6.0-fold increase of GST-c-jun
phosphorylation was detected (Fig SB). Finally, the effects
of propyl gallate and ETYA were examined on IL-I plus
TNF-a-induced activation of JNK. As shown in Fig 5C,
pretreatment with propyl gallate did not inhibit JNK activity
induced by IL-l plus TNF-a. Similar results were obtained
when cells were stimulated with IL-l plus TNF-a in the
presence of ETYA (Fig SC).
Role of arachidonic acid on IL-l and TNF-a-induced
JNK activation. We have previously shown that c-jun gene
expression induced by IL-l and TNF-a was decreased by
treatment of cells with the PLA2 inhibitor, quinacrine.' Moreover, c-jun gene expression induced by IL- l plus TNF-a or
by arachidonic acid was sensitive to inhibition by the tyrosine kinase inhibitor, genistein."' To further determine the
potential role of arachidonic acid as an intracellular mediator
of IL-l plus TNF-a-induced JNK activation, we examined
the effect of quinacrine on cytokine-induced JNK activity.
Cells were stimulated with IL-I (500 U h L ) plus TNF-a
(500 U h L ) for 15 minutes in the presence of different concentrations of quinacrine. As shown in Fig 6, treatment of
cells with 1 pmol/L and 5 pmol/L quinacrine caused a 39%
and 54% decrease on IL-I plus TNF-a-induced JNK activity, respectively, consistent with the hypothesis that arachidonic acid mediates IL-1 plus TNF-a stimulation of JNK
activity. We next examined the effect of genistein on JNK
activation induced by exposure of cells to either IL-I plus
TNF-a or arachidonic acid. Stromal cells were preincubated
for 2 hours with 10 pmol/L genistein after stimulation with
IL-I (500 U/mL) plus TNF-a (500 U h L ) for 15 minutes or
with 25 pmol/L arachidonic acid for 30 minutes. As shown in
Fig 7, treatment with genistein resulted in a 46% and 64%
inhibition of JNK activity induced by IL-I plus TNF-a or
by arachidonic acid, respectively.
DISCUSSION
In recent years, a role for arachidonic acid in the regulation
of gene expression has been proposed."-'3 Our previous studies have identified arachidonic acid as a potential intracellular mediator of the effect of IL-I and TNF-a on c-jun gene
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ACTIVATION OF JNK BY ARACHIDONIC ACID
3797
expres~ion.~~'"
Stimulation of the murine stromal cell line +/
+.1 LDA 11 with arachidonic acid or with IL-I and TNFLY led to enhanced c-jun transcription by a mechanism independent of PKC activation." In addition, increased tyrosine
kinase activity was detected upon stimulation with arachidonic acid and treatment of cells with tyrosine kinase inhibitors markedly decreased arachidonic acid-induced c-jun transcription." These observations led us to postulate that a
tyrosine kinase-dependent process was involved in the signaling route of c-jun transcription by arachidonic acid.
In the present study, we have further clarified a portion
of the signal transduction pathway that leads to c-jun transcription when cells are exposed to arachidonic acid. Spe-
700
I
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I+T
Q=*c
A
co
U
eL
Fig 6.
+
mL) in the absence or in the presence of 1 pmolIL and 5 pmolIL
quinacrine. Lysates were prepared and analyzed for JNK activity as
described. Data represent the mean ? SEM of an experiment performed in triplicate. Autoradiography corresponding t o the labeled
substrate from a representative determination is shown.
GST-cjunb
cifically, we have presented evidence that regulation of cjun transcription by arachidonic acid involves activation of
the JNK signaling pathway. Consistent with this hypothesis.
extracts from cells stimulated with arachidonic acid induced
phosphorylation of a c-jun fusion protein containing Ser 63
and Ser 73, indicating the presence of activated JNK proteins. Similar effects were observed with extracts from cells
treated with IL-l and TNF-a. The results are consistent with
the hypothesis that these growth factors exert their effects
by inducing the release of arachidonate, which mediates activation of intracellular c-jun kinases.
We used in-gel kinase assays as well as kinase assays of
immunoprecipitates to identify the isoform of JNK activated
in response to arachidonic acid. In-gel kinase assays have
been previously shown to be able to detect both the JNKl
and JNK2 isoforms of JNK protein kinases." The major
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GSTcjunb
C
st;
LL
z
c
+
LL
Z
+
c
8 A
A
r
I
GST-cjun b
Effect of quinacrine on IL-1 plus TNF-a-induced JNK activ-
ity. Cells were stimulated with IL-1 (500 UImL) plus TNF-a (500 U I
+
r
I
Fig 5. Effect of arachidonic acid metabolism on JNK activation. (A)
Cells were pretreated with 100 pmolIL propyl gallate for 10 minutes
before stimulation with vehicle (PG) or with 25 pmolIL arachidonic
acid for 30 minutes (AA
PG). Control cells ICo) were exposed t o
vehicle alone. Lysates were prepared and incubated with immobilized GST-e-jun. Reactions were performed as described in the Materials and Methods. Phosphorylated proteins were separated by SDSgel electrophoresis and analyzed by autoradiography. IB) Cells were
stimulated with 25 pmolIL arachidonic acid (AA) or 50 pmolIL ETYA
alone or in combination with arachidonate IAA + ETYAI. Phosphorylation of GST-c-jun. was analyzed as described above. Bands corresponding t o phosphorylated c-jun were excised from gels and
counted by scintillation counting. (CI Cells were stimulated either
with vehicle (Co) or with IL-1 (500 UImL) plus TNF-a (500 UImLI in
the absence or in the presence of propyl gallate (100 pmol/L) or ETYA
150 pmolIL). Lysates were prepared and analyzed by the solid-phase
kinase assay using the immobilized GST-c-jun as described. An autoradiogram from a representative experiment is shown.
+
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R I U O AND CARLO-STELLA
3798
800
I
700
L
C
0
0
.-6m
-
600 ;
500
L
6
400
c
n
x
300
p.
.C
3
200
m
OST-cjuu
co
I+T
I+T+Q
0
AA
AA+O
Fig 7. Effact of genistein on 11-1 plus TNF-a- and arachidonic acidinduced JNK activity. Cells were preincubated for 2 hours with 10
pmol/L genistein (GI in serum-free conditions. Cells were either left
unstimulated or stimulated with 11-1 (500 UlmLI plus TNF-a (500 U/
mLJ for 15 minutes or with arachidonic acid (25 pmol/L; 30 minutest.
Lysates were made and assayed in the solid-phase kinase assay as
described. Shown is a representative experiment performed in triplicate (mean f SEMI. Autoradiography of phosphorylatedGST-c-jun
is from a representative determination.
JNK protein kinase activated in response to arachidonic acid
or IL-I and TNF-a appeared to be the 46-kD JNKl isoform.
Additional evidence that JNKl activated in response to arachidonic acid was involved in phosphorylating Ser 63 and
Ser 73 of the amino-terminal domain of c-jun was provided
by the fact that anti-JNKI immune complexes from arachidonate-treated cell were able to phosphorylate the GST-cjun fusion protein. Conversely, when the mutated GST-cjun fusion protein was used as a substrate, a marked decrease
in phosphorylation was detected. Taken together, these results suggest that JNKl or an immunologically related kinase
is induced after stimulation of stromal cells with arachidonic
acid. Thus, activation of the JNK proteins by arachidonic
acid may mediate IL-l and TNF-a-induced phosphorylation
of c-jun on Ser 63 and Ser 73.
The effect of arachidonic acid on JNK activation was
specific. Whereas other fatty acids induced JNK activation,
their effect on JNK activity was much smaller that the effect
observed after stimulation with arachidonic acid. Moreover,
several other lipid second messengers, including phosphatidic acid, diacylglycerol, and ceramide, failed to stimulate
JNK activity in these cells. Several reports have implied
ceramide as a mediator of TNF-a signalling in other systems?’ A recent study by Verheij et al” showed that, in the
human monocytic leukemia cell line, U937, and in bovine
aortic endothelial cells, ceramide induced apoptosis by activating the SAPWJNK cascade. Other lipids, including arachidonic acid, which failed to induce apoptosis in these cells,
also failed to stimulate the SAPWJNK cascade. The explanation for these divergent results is not presently clear. Signal-
ling mediators for IL-1 and TNF-a may differ in different
types of cells. Thus, in human umbilical vein endothelial
cells, TNF-a-induced activation of the JNK cascade was
not mediated by ceramide?’ Although other factors may be
involved, it seems clear in our system that arachidonic acid
is a relevant messenger mediating the IL- 1 and TNF-a effect
on JNK activation. Arachidonic acid rather than ceramide
appears to mediate other responses as well. Thus, Jaattella
et aI4’ found that, in a TNF-sensitive subclone of MCF7
breast carcinoma cells, TNF-a-induced apoptosis was mediated by PLAz activation, but not by ceramide. More recently,
a role for arachidonic acid metabolism in mediating
apoptosis it has been postulated?’.44 However, whether this
effect is mediated by activation of the JNK cascade or by
other effectors it is not yet known. Further investigations are
warranted to better define the role of ceramide and arachidonic acid in mediating the effects of IL-I and TNF-a on
cell function.
Inhibition of arachidonic acid metabolism by propyl gallate did not affect the ability of arachidonate to induce JNK
activation. Furthermore, the nonmetabolizable arachidonate
analogue, ETYA, stimulated JNK activity at levels similar
to that stimulated by arachidonic acid. We also observed
that stimulation of stromal cells with arachidonic acid in the
presence of ETYA enhanced the ability of arachidonic acid
to induce JNK activation. This effect may be ascribed to
increased intracellular availability of arachidonic acid due
to inhibition of its metabolism. In addition, we have observed
that ETYA stimulates c-jun gene expression in the stromal
cell line used (unpublished observation). Taken together,
these results suggest that metabolites of the cyclooxygenase
or lipoxygenase pathway do not mediate the effect of arachidonic acid on JNK activation. Although the involvement of
an alternative metabolic pathway cannot be excluded, the
results presented herein suggest that arachidonate itself is
involved in regulation of JNK activity.
In the stromal cell line used, JNK activation induced by IL
1 and TNF-a parallels that induced by arachidonic acid in many
ways. Neither response is affected by pretreatment of cells with
propyl gallate or ETYA. Moreover, genistein similarly affected
IL-f plus TNF-a-induced and arachidonate-induced JNK activation. Furthermore, quinacrine partially inhibited JNK activation by IL-1 plus TNF-a, suggesting that the effect of these
cytokines on JNK activity is mediated, in part, by the release of
arachidonic acid upon PLA2activation. Together with our previous observations?’’ these results are consistent with the hypothesis that arachidonic acid mimics the effects of IL-1 and TNF-a
on c-jun activation. Consistent with this possibility, stimulation
of stromal cells with IL-I and TNF-cy induced activation of
PLA2,the inhibition of which blunted growth factor-induced, but
not arachidonate-induced c-jun gene expression? These findings
strengthen our contention that arachidonic acid acts as a second
messenger of L-1and TNF-cy signaling network. The precise
mechanism by which arachidonic acid induces JNK activation
remains to be identified. Arachidonic acid may exert its effect
on JNK through other, as yet unidentified, kinases. Experiments
by others have shown that induction of JNK kinase (JNKK)
precedes JNK activation in growth factor-stimulated cells?’
Moreover, MEK kinase 1 (MEKK) is able to induce SAPKs
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ACTNATION OF JNK BY ARACHIDONIC ACID
activation by phosphorylating SAPK activator, SEKl?l Whether
these kinases are involved in activation of JNK by arachidonic
acid remains to be determined.
ACKNOWLEDGMENT
We are grateful to Dr A. Kraft for the generous gift of the GSTc-jun fusion proteins. We thank Dr B. Morimoto for helpful discussions and comments, Dr L. Williamson for her advice on the solidkinase assay, and Prof V. Rizzoli for his encouragement and support.
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1996 88: 3792-3800
Arachidonic acid mediates interleukin-1 and tumor necrosis
factor-alpha- induced activation of the c-jun amino-terminal kinases in
stromal cells
MT Rizzo and C Carlo-Stella
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