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J. Biol. Chem., Vol. 277, Issue 22, 19882-19888, May 31, 2002
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From the INSERM, Unité 388, Institut L. Bugnard, CHU
Rangueil, 31403 Toulouse Cedex 4, France
Received for publication, October 22, 2001, and in revised form, February 20, 2002
We have investigated the mechanisms whereby
The Like for other ARs (9), it is probable that the mechanisms
whereby Recent experiments carried out on rat proximal tubule cells in primary
culture and on LLC-PK1 cells transfected with the rat nonglycosylated
Drugs and Reagents--
UK14304, dexmedetomidine, and RX821002
were respectively donated by Pfizer (Sandwich, UK), Orion Pharma
(Turku, Finland), and Reckitt and Colman Laboratories
(Kingston-upon-Hull, UK). [3H]RX821002 (59 Ci/mmol),
[3H]AA (202 Ci/mmol), nitrocellulose membranes, and the
ECL Western blotting system were from Amersham Biosciences
(Courtaboeuf, France). Arachidonic acid, quinacrine, methyl arachidonyl
fluorophosphonate, [Glu52]diphtheria toxin (CRM 197), and
U0126 were obtained from Calbiochem. Indomethacin, ketoconazole,
nordihydroguaiaretic acid (NDGA), phorbol 12-myristate 13-acetate
(PMA), tyrphostin AG1478, EGF, staurosporine, 1,10-phenanthroline, and
all other chemicals were from Sigma. Fetal calf serum was
purchased from Invitrogen. Anti-ERK1 and anti-ERK2 polyclonal Ab
were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), and
anti-active MAPK was from Promega (Madison, WI). Anti-Shc polyclonal Ab
and fluorescein-conjugated goat anti-rabbit IgG were respectively
purchased from Upstate Biotechnology, Inc. (Lake Placid, NY) and Nordic
Immunological Laboratories (Tilburg, The Netherlands).
Culture of LLC-PK1- Detection of ERK1/2 and Shc--
Three days postseeding, cells
were placed for 24 h in culture medium free of serum. They were
then exposed to the compound to be tested, rapidly rinsed with ice-cold
phosphate-buffered saline, and harvested in 1 ml of radioimmune
precipitation buffer (10 mM Tris-HCl, pH 7.4, 1%
Triton-X100, 1% sodium deoxycholate, 0.1% SDS, 150 mM
NaCl, 2 mM sodium orthovanadate, 1 mM
phenylmethylsulfonyl fluoride, and 0.5 mM aprotinin).
Soluble proteins were extracted by centrifugation (15,000 × g, 15 min at 4 °C), separated by SDS-PAGE, and blotted
onto a nitrocellulose membrane. Phosphorylated forms of MAPKs were
revealed by chemiluminescence using anti-active MAPK Ab. Shc
phosphorylation was determined after immunoprecipitation. Briefly, 500 µl of cell lysate were incubated overnight at 4 °C with 5 µg of
rabbit polyclonal Shc-Ab and 50 µl of protein A-agarose beads. Immune
complexes were extensively washed with ice-cold radioimmune
precipitation buffer, dried, and denatured in Laemmli buffer. Samples
were subjected to SDS-PAGE, transferred onto a nitrocellulose membrane,
and probed with horseradish peroxidase-conjugated anti-phosphotyrosine
Ab. In all experiments, the membranes were stripped of Ig and reprobed
using either a mixture of anti-ERK1 Ab and anti-ERK2 Ab or anti-Shc Ab.
Films were analyzed by densitometry, and the extent of phosphorylation
was normalized to protein loading.
Measurement of AA Release--
Cells rendered quiescent by a
24-h period of serum deprivation were labeled for 10 h with 1 µCi/ml [3H]AA. They were carefully washed in
Dulbecco's modified Eagle's medium containing 10 mM Hepes
and 0.2% fatty acid-free bovine serum albumin and then exposed to the
drug to be tested. Aliquots of the incubation medium were collected
every 10 min over a period of 30 min and centrifuged (20,000 × g, 10 min, 4 °C), and the radioactivity was measured in
the supernatant.
Immunofluorescence Microscopy--
Cells plated on glass
coverslips were grown, rendered quiescent as indicated above, and
exposed or not to the compound to be tested. They were fixed in 4%
paraformaldehyde (15 min) and treated with 50 mM
NH4Cl in phosphate-buffered saline (10 min). The cells were
permeabilized first in phosphate-buffered saline buffer containing
0.05% saponin and 0.2% bovine serum albumin (15 min) and then in
methanol (10 min at Statistical Analysis--
Results are expressed as mean ± S.E. for the number of experiments indicated (n). The data
were analyzed using Student's t test, and a p
value <0.05 was considered statistically significant.
AA Release Is Involved in
In primary culture of rabbit proximal tubule cells, activation of MAPK
by angiotensin II receptor is the consequence of AA release (16). In a
first step to evaluate the putative role of AA as an intermediary
between activated MAPK Phosphorylation Depends on Generation of AA
Derivatives--
In rabbit proximal tubule, the effect of AA on MAPK
depends on the generation of an epoxy metabolite (16). The production of AA derivatives results, in most mammalian cells, from the activity of three distinct enzymatic systems, namely the lipoxygenase, cyclooxygenase, and cytochrome P450-dependent epoxygenase.
To evaluate the respective contribution of these pathways, MAPK
activation was examined in LLC-PK1-
Although metabolic products are responsible for many of the indirect
effects of AA, some are the direct consequence of PKC activation. To
determine whether PKC participated in MAPK activation by AA,
experiments were carried out in the presence of staurosporine (Fig.
5). Treatment of the cells with 200 nM staurosporine totally abolished the phosphorylation of
MAPK induced by PMA, proving that the different isoforms of PKC were
truly inhibited. By contrast, it did not prevent AA-induced MAPK
phosphorylation. Thus, as previously found for
MAPK Phosphorylation Requires Metalloproteinase Activity and EGF-R
Transactivation--
Previous studies carried out on rabbit proximal
tubule have shown that cell treatment with AA resulted in a significant
increase of EGF-R phosphorylation and its subsequent association with
Shc (19). In our model, the role of EGF-R activation was first
investigated using the specific inhibitor of EGF-R tyrosine kinase
activity, tyrphostin AG1478. As shown in Fig.
6A, the preincubation of
LLC-PK1- The phosphorylation of MAPKs (ERK1/2) by The mechanisms whereby G-protein-coupled receptors activate the MAPK
cascade are highly dependent upon the receptor considered and the cell
type it is expressed in. Although previous studies have shown that the
action of the An other major effort of the present work was to define the
pathway leading from stimulated In conclusion, our results provide evidence for a pathway by which
We thank Dr. C. Flordellis for
valuable discussion and F. Quinchon for excellent technical assistance.
*
This work was supported by the BIOMED 2 Program PL963373
(European Commission, Brussels, Belgium) and by a grant from the Fondation pour la Recherche Médicale (Paris, France).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.
Published, JBC Papers in Press, March 12, 2002, DOI 10.1074/jbc.M110142200
2B-Adrenergic Receptor Activates MAPK via a
Pathway Involving Arachidonic Acid Metabolism, Matrix
Metalloproteinases, and Epidermal Growth Factor Receptor
Transactivation*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2B-adrenergic receptor (2B-AR)
promotes MAPK activation in a clone of the renal tubular cell line,
LLC-PK1, transfected with the rat nonglycosylated 2-AR gene. Treatment of LLC-PK1-2B
with UK14304 or dexmedetomidine caused arachidonic acid (AA) release
and ERK2 phosphorylation. AA release was abolished by prior treatment
of the cells with pertussis toxin, quinacrine, or methyl arachidonyl
fluorophosphonate but not by the addition of the MEK inhibitor U0126.
The effects of 2-agonists on MAPK phosphorylation were
mimicked by cell exposure to exogenous AA. On the other hand,
quinacrine abolished the effects of UK14304, but not of AA, suggesting
that AA released through PLA2 is responsible for MAPK activation by
2B-AR. The effects of 2-agonists or AA
were PKC-independent and were attenuated by indomethacin and
nordihydroguaiaretic acid. Treatment with batimastat, CRM 197, or
tyrphostin AG1478 suppressed MAPK phosphorylation promoted by
2-agonist or AA. Furthermore, conditioned culture medium
from UK14304-treated LLC-PK1-2B induced MAPK
phosphorylation in wild-type LLC-PK1. Based on these data, we propose a
model whereby activation of MAPK by 2B-AR is mediated
through stimulation of PLA2, AA release, generation of AA derivatives,
activation of matrix metalloproteinases, release of heparin-binding
EGF-like growth factor, transactivation of epidermal growth factor
receptor, and recruitment of Shc. Whether this pathway is particular to 2B-AR and LLC-PK1 or whether it can be extended to other
cell types and/or other G-protein-coupled receptors remains to be established.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-adrenergic receptors (2-ARs)
are members of the G-protein-coupled receptor superfamily that mediate
physiological responses to the endogenous catecholamines, such as
reduction of blood pressure, sedation, platelet aggregation, and
inhibition of renin release or insulin secretion. Three subtypes of
2-ARs (namely 2A, 2B, and
2C) have been identified (1). Although recent studies,
conducted on mice with genetic alterations of 2-AR
expression, have clarified the respective roles of 2A- and 2B-ARs in the mediation of the cardiovascular and
sedative effects of 2-agonists, the precise functions of
each subtype are far from being definitively elucidated (2). Until
recently, the effects of 2-ARs were generally considered
as exclusively due to the modulation of effectors such as adenylyl
cyclase or phospholipase C
. There is now accumulating evidence that,
in addition to these pathways, 2-ARs are also involved
in the regulation of cell growth via stimulation of mitogen-activated
protein kinases (MAPKs). The phosphorylation of MAPKs has been observed
in transfected cells (3, 4) as well as in various types of cells
spontaneously expressing 2-ARs (5, 6). The three
receptor subtypes promoted phosphorylation of ERK1 and ERK2 in Chinese
hamster ovary cells (3). According to results obtained in HEK 293 and
COS cells (7, 8), this effect is independent of receptor
internalization via clathrin-coated pits.
2-ARs promote MAPK activation are highly
dependent upon the subtype considered and the particular cell type it
is expressed in. So far, the precise pathways of the mitogenic signal transmission were exclusively examined for 2A-AR. In HEK
293 cells (10), activation of ERK1/2 by 2A-AR is
primarily triggered through release of 
subunits from pertussis
toxin-sensitive G proteins, stimulation of phospholipase C,
phosphoinositide hydrolysis, increase of intracellular
Ca2+, and successive activation of Pyk2 and Src. Activation
of Src causes the formation of Shc-Grb2-Sos complex, which leads to ERK phosphorylation via the Ras/Raf/MEK cascade. In COS cells (11), 2A-AR-induced phosphorylation of ERK2 proceeds via two
distinct pathways, which are dependent ("transactivation pathway")
or not ("direct pathway") on the tyrosine kinase activity of the
EGF receptor (EGF-R). The early steps of both pathways involve the release of subunits from Gi proteins and the
activation of Src by an unknown process that is independent of inositol
1,4,5-trisphosphate production (12). Then, the phosphorylation of MAPKs
occurs either directly through recruitment of the MEK cascade via
phosphorylation of the adapter protein Shc or indirectly through
activation of unidentified matrix metalloproteinases, release of
heparin-binding EGF-like growth factor (HB-EGF), and subsequent
transactivation of EGF-R.
2-AR (RNG) gene (LLC-PK1-2B) have shown
that 2B-ARs promote MAPK activation and arachidonic acid
(AA) release (13). The sequential relationship between PLA2 and MAPK
activation was not investigated. As demonstrated in eosinophils during
the process of adhesion to fibronectin (14), PLA2 activation may result
from its phosphorylation by MAPKs. Conversely, as shown in rabbit renal
epithelial cells for angiotensin II receptor, the activation of MAPK
could be the consequence of AA release (15). Based on the use of
different inhibitors, the present work demonstrates that activation of
MAPK by 2B-AR is, in LLC-PK1-2B, primarily mediated
by a pathway involving stimulation of PLA2, generation of AA
derivatives, activation of matrix metalloproteinases, release of
HB-EGF, and transactivation of EGF-R.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2B Cells--
The clone of the
renal tubular cell line, LLC-PK1, permanently expressing the rat
2B-AR was obtained by transfection with a pcDNA3
vector containing the coding region of the RNG gene. LLC-PK1-2B cells were routinely grown in Dulbecco's
modified Eagle's medium containing 25 mM glucose, 100 µg/ml streptomycin, 100 IU/ml penicillin, and 5% fetal calf serum.
Binding experiments with [3H]RX821002 showed that the
level of receptor expression was 730 ± 51 fmol/mg of protein.
20 °C). All subsequent steps were carried out
in permeabilization buffer and were separated by several washes. The
cells were incubated with ERK2 polyclonal Ab (1:40) and then with
fluorescein-conjugated goat anti-rabbit IgG (1:400). The coverslips
were finally washed in phosphate-buffered saline, mounted in
fluorescent mounting medium (Dako Corp., Carpinteria, CA), and examined
under epifluorescence illumination. Digital images were captured using
the software CoolSNAP (Roper Scientific GmbH, Munich, Germany) and
processed with Adobe Photoshop 4 (Adobe Systems Inc., San Jose, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2B-AR-induced MAPK
Phosphorylation--
A previous study from our group has shown that
exposure of proximal tubule cells to 2-agonists resulted
in activation of MAPK and in an increase of AA release (13). As
depicted in Fig. 1A, the
treatment of LLC-PK1-2B with 1 µM UK14304
induced a time-dependent increase of the tyrosine
phosphorylation of p42 MAPK. The effect is maximal between 10 and 20 min and persists for at least 40 min. In addition, cell exposure to
UK14304 caused an acceleration of AA release, which was abolished by 20 µM quinacrine or 50 µM methyl arachidonyl
fluorophosphonate (not shown). Effects of the 2-agonist
on MAPK phosphorylation and AA release were abolished by pretreatment
of the cells with pertussis toxin (Fig. 1, B and C). On the other hand, the addition of the MEK inhibitor,
U0126, blunted phosphorylation of MAPK but did not affect the
augmentation of AA release induced by UK14304. Similar results were
obtained using dexmedetomidine, suggesting that stimulation of PLA2
activity by 2-agonists is not the consequence of MAPK
activation.
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Fig. 1.
Effects of pertussis toxin and U0126 on MAPK
phosphorylation and AA release induced by
2-agonists. A, kinetics
of MAPK activation. Serum-deprived LLC-PK1-2B cells were
treated for the indicated times with 1 µM UK14304 or for
5 min with 2 µM PMA (PMA). Phosphorylated and
total MAPKs were respectively revealed using anti-active MAPK Ab
(upper panel) and a mixture of anti-ERK1 Ab and
anti-ERK2 Ab (lower panel). B, effects
of pertussis toxin pretreatment and U0126 on MAPK phosphorylation.
Serum-deprived LLC-PK1-2B were exposed or not for 10 min
to 1 µM UK14304 (UK) or 1 µM
dexmedetomidine (dxm). The effects of the
2-agonists were assayed in cells pretreated for 20 h with 200 ng/ml pertussis toxin (PTX) or incubated in the
presence of 35 µM U0126. Phosphorylated and total MAPKs
were respectively revealed using anti-active MAPK Ab (upper
panel) and a mixture of anti-ERK1 Ab and anti-ERK2 Ab
(lower panel). C, effects of pertussis
toxin pretreatment and U0126 on AA release. Serum-deprived
LLC-PK1-2B were loaded with [3H]AA (1 µCi/ml) and extensively washed in Hepes-buffered Dulbecco's modified
Eagle's medium containing 0.2% fatty acid-free bovine serum albumin.
AA release was measured over a period of 30 min. The effects of 1 µM UK14304 (UK) or 1 µM
dexmedetomidine (dxm) were assayed in cells pretreated for
20 h with 200 ng/ml pertussis toxin (PTX) or incubated
in the presence of 35 µM U0126. Results are means ± S.E. from three experiments and are expressed as percentage relative to
control.
2B-AR and MAPK,
LLC-PK1-2B cells were treated with AA. As shown in Fig.
2A, AA induced a dose-dependent increase in MAPK phosphorylation. The effect
of AA was detectable at 200 nM and reached a maximum at 20 µM. Such concentrations are in the physiological range,
since the level of free AA was estimated to be 5 µM in
the rat kidney (17). Exposure to AA also resulted in the redistribution
of ERK2 from the cytoplasm to the nucleus (Fig. 2B), showing
that phosphorylation of MAPK could be correlated with the translocation
of ERK2. However, in contrast to that for UK14304, the effect of AA was
not affected by pretreatment of the cell with pertussis toxin (Fig.
2C). In a second step, the role of endogenous AA release in
the activation of MAPK by 2-agonist was evaluated using
PLA2 inhibitors. Of the compounds tested, quinacrine was the only one
with no side effect; all others, including methyl arachidonyl
fluorophosphonate and AACOCF3, caused by themselves a significant
increase in ERK2 phosphorylation. Such an undesirable effect was
previously reported for methyl arachidonyl fluorophosphonate in
macrophage (18). Preincubation of LLC-PK1-2B for 5 min
with 20 µM quinacrine totally abolished MAPK
phosphorylation induced by UK14304 but not by AA (Fig.
3A). Again, results from
Western blotting were confirmed by examination of the subcellular
distribution of ERK2. Indeed, translocation to the nucleus following
exposure to UK14304 is abrogated by quinacrine pretreatment (Fig.
3B). All together, these results are therefore consistent
with the implication of PLA2 and AA generation in MAPK activation
induced by 2B-AR.
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Fig. 2.
AA induces phosphorylation of MAPKs and
nuclear translocation of ERK2. A,
dose-dependent effect of AA on MAPK phosphorylation.
Serum-deprived LLC-PK1- 2B cells were treated for 10 min
with various concentrations of AA ranging from 20 nM to 200 µM. Phosphorylated and total MAPKs were
respectively revealed using anti-active MAPK Ab (upper
panel) and a mixture of anti-ERK1 Ab and anti-ERK2 Ab
(lower panel). B, effect of AA on the
subcellular distribution of ERK2. LLC-PK-2B cells were
grown on glass coverslips, rendered quiescent, and exposed for 15 min
to 20 µM AA (AA) or not (Control).
Cells were fixed and permeabilized, and the localization of ERK2 was
assessed by immunofluorescence using anti-ERK2 polyclonal Ab and
fluorescein-conjugated goat anti-rabbit IgG. Images shown
are representative of three independent experiments (scale
bar = 10 µM). C, effect of
pertussis toxin on MAPK phosphorylation induced by UK14304 or AA.
Serum-deprived LLC-PK1-2B cells were pretreated for
20 h with 200 ng/ml pertussis toxin (+) or not (). They were
then incubated or not for 10 min with either 1 µM UK14304
(UK) or 20 µM AA (AA).
Phosphorylated and total MAPKs were respectively revealed using
anti-active MAPK Ab (upper panel) and a mixture
of anti-ERK1 Ab and anti-ERK2 Ab (lower
panel).
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Fig. 3.
Quinacrine abolishes MAPK activation by
UK14304. A, effect of quinacrine on MAPK
phosphorylation induced by UK14304 or AA. Serum-deprived
LLC-PK1- 2B cells were incubated for 5 min in the
presence of vehicle or 20 µM quinacrine and then exposed
or not for 10 min to 1 µM UK14304 or 20 µM
AA. Phosphorylated and total MAPKs were respectively revealed using
anti-active MAPK Ab (upper panels) and a mixture
of anti-ERK1 Ab and anti-ERK2 Ab (lower panels).
B, effect of quinacrine on UK14304-induced translocation of
ERK2. LLC-PK-2B cells grown on glass
coverslips were rendered quiescent by serum deprivation and treated
(Qui) or not (Control) for 5 min with quinacrine
prior to 15-min exposure to 1 µM UK14304 (UK).
Cells were fixed and permeabilized, and the localization of ERK2 was
assessed by immunofluorescence using anti-ERK2 polyclonal Ab and
fluorescein-conjugated goat anti-rabbit IgG. Images shown
are representative of three independent experiments (scale
bar = 10 µM).
2B treated with
inhibitors prior to stimulation with UK14304. As shown in Fig.
4, the lipoxygenase inhibitor NDGA (10 µM) significantly diminished the MAPK phosphorylation
induced by UK14304. A decrease was also observed with the
cyclooxygenase inhibitor indomethacin (50 µM) but not
with the epoxygenase inhibitor ketoconazole (30 µM). Of
note, the combined pretreatment with NDGA and indomethacin completely
abolished the phosphorylation of ERK induced by UK14304 or exogenous
AA. These findings strongly suggest that cyclooxygenase and/or
lipoxygenase activities are essential in the mediation of the effects
of 2B-AR.
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Fig. 4.
Effects of inhibitors of AA metabolism on
MAPK phosphorylation. Serum-deprived LLC-PK1- 2B
cells were incubated for 5 min in the presence of 10 µM
NDGA (NDGA), 50 µM indomethacin
(INDO), 30 µM ketoconazole (KETO),
or 10 µM NDGA plus 50 µM indomethacin
(NDGA/INDO) and then exposed for 10 min to 1 µM UK14304 (UK) or 20 µM AA
(AA). MAPK phosphorylation was revealed using anti-active
MAPK Ab (upper panel), and protein loading was
assessed by reprobing the blot with a mixture of anti-ERK1 Ab and
anti-ERK2 Ab (middle panel). Band intensity was
semiquantified by densitometric scanning of the film (lower
panel). Reported results are mean ± S.E.
(n = 3). *, p < 0.05; **,
p < 0.01; ***, p < 0.001 versus respective control values.
2-agonists (13), the effect of AA is independent of
PKC.
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Fig. 5.
Staurosporine impairs MAPK phosphorylation by
PMA but not AA. Serum-deprived LLC-PK1- 2B cells
were incubated for 5 min in the presence of 200 nM
staurosporine (+) or not (). They were then exposed for 10 min to 20 µM AA (AA) or 2 µM PMA
(PMA). Phosphorylated and total MAPKs were respectively
revealed using anti-active MAPK Ab (upper panel)
and a mixture of anti-ERK1 Ab and anti-ERK2 Ab (lower
panel).
2B cells in culture medium containing 100 nM tyrphostin AG1478 prevented the phosphorylation of MAPK
caused by UK14304, dexmedetomidine, or AA. Thus, the transactivation of
EGF-R plays a critical role in the mediation of the effect of
2B-AR on MAPK. According to recent evidence, EGF-R
transactivation by G-protein-coupled receptor requires the cleavage of
pro-HB-EGF by matrix metalloproteinases. Therefore, we next
investigated whether MAPK phosphorylation was sensitive to the
inhibitor of the matrix metalloproteinases, batimastat (Fig.
6B). Pretreatment of the cells with 5 µM
batimastat neither affected the basal level of MAPK phosphorylation nor
inhibited the response to EGF but resulted in a blockade of the effect
of UK14304 or AA. Similar results were obtained with
1,10-phenanthroline (not shown). The implication of HB-EGF release was
examined using the diphtheria toxin mutant, CRM 197. Pretreatment of
LLC-PK1-2B cells with CRM 197 (200 ng/ml) had no effect
on the ability of exogenous EGF to activate MAPK (not shown) but
strongly inhibited the effect UK14304 or AA (Fig. 6C). The
release of a factor, with EGF activity and acting in an
autocrine/paracrine mode, was confirmed by experiments in which the
effect of conditioned medium from LLC-PK1-2B was assayed
on wild-type LLC-PK1 (Fig. 7). Incubation of wild-type cells in medium collected from nonstimulated
LLC-PK1-2B or their direct treatment with UK14304 did
not cause any change in the extent of MAPK phosphorylation (not shown).
In contrast, a clear increase was observed when medium came from
LLC-PK1-2B treated with UK14304. As expected, this
response was blocked by tyrphostin AG1478 but was unaffected by the
addition of quinacrine or batimastat or by the prior treatment of
wild-type LLC-PK1 with pertussis toxin. It is well established that
activation of MAPK by EGF-R occurs via the tyrosine phosphorylation of
adapter proteins such as Shc and the recruitment of Grb2-Sos complexes.
Previous experiments on rat proximal tubule cells in primary culture
have shown that 2B-AR stimulation resulted in tyrosine
phosphorylation of the p46 and p52 isoforms of Shc (13). Therefore, we
examined whether AA induces phosphorylation of Shc in
LLC-PK1-2B. As depicted in Fig.
8, treatment of the cells with AA caused
a time-dependent increase in the phosphorylation of the p52
isoform of Shc.
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Fig. 6.
Activation of MAPK depends on matrix
metalloproteinases and EGF-R tyrosine kinase activity.
A, effect of tyrphostin AG1478 on MAPK phosphorylation
induced by 2-agonists or AA. Serum-deprived
LLC-PK1-2B cells were incubated or not for 20 min in the
presence of 100 nM tyrphostin AG1478. They were then
exposed for 10 min to either 1 µM UK14304
(UK), 20 µM AA (AA), or 5 ng/ml EGF
(EGF). Phosphorylated and total MAPKs were respectively
revealed using anti-active MAPK Ab (upper panel)
and a mixture of anti-ERK1 Ab and anti-ERK2 Ab (lower
panel). B, effect of batimastat on MAPK
phosphorylation induced by UK14304 or AA. Serum-deprived
LLC-PK1-2B cells were incubated or not for 30 min in the
presence of 5 µM batimastat (BAT). They were
then exposed for 10 min to either 1 µM UK14304
(UK) or 1 µM dexmedetomidine (dxm)
or 20 µM AA (AA). Phosphorylated and total
MAPKs were respectively revealed using anti-active MAPK Ab
(upper panel) and a mixture of anti-ERK1 Ab and
anti-ERK2 Ab (lower panel). C, effect
of CRM 197 on MAPK phosphorylation induced by UK14304 or AA.
Serum-deprived LLC-PK1-2B cells were incubated or not
for 1 h in the presence of 200 ng/ml CRM 197. They were then
exposed for 10 min to either 1 µM UK14304 (UK)
or 20 µM AA (AA). Phosphorylated and total
MAPKs were respectively revealed using anti-active MAPK Ab
(upper panel) and a mixture of anti-ERK1 Ab and
anti-ERK2 Ab (lower panel).
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Fig. 7.
Conditioned medium from UK14304-stimulated
LLC-PK1- 2B causes phosphorylation of MAPKs in
wild-type LLC-PK1. Wild-type cells, rendered quiescent by serum
deprivation, were exposed for 10 min to either 5 ng/ml EGF or
conditioned medium from LLC-PK1-2B cells that were
previously treated for 10 min with 1 µM UK14304. Effects
of the conditioned medium were examined in the presence of 20 µM quinacrine (Qui), 5 µM
batimastat (BAT), or 100 nM tyrphostin AG1478
(AG1478) and after a 20-h pretreatment with 200 ng/ml
pertussis toxin (PTX). Phosphorylated and total MAPKs were
respectively revealed using anti-active MAPK Ab (upper
panel) and a mixture of anti-ERK1 Ab and anti-ERK2 Ab
(lower panel).
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Fig. 8.
AA promotes Shc phosphorylation.
Serum-deprived LLC-PK1- 2B cells were treated for the
indicated times with 20 µM AA. Shc proteins were
immunoprecipitated, and phosphorylation extent was estimated using
anti-phosphotyrosine Ab (upper panel). The
membrane was stripped of Ig and reprobed using anti-Shc Ab to assess
protein loading (lower panel).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2-agonists
has been reported in a variety of cells, including Chinese hamster
ovary cells transfected with the RNG gene (3) and COS or HEK 293 cells
transfected with the 2C2 gene (7, 8), which encode the rat and human
2B-AR subtypes, respectively. More recently, this effect
has also been observed in rat proximal tubule cells in primary culture
as well as in LLC-PK1-2B (13). In these two models,
receptor stimulation resulted in an acceleration of cell proliferation,
suggesting that the action of catecholamines on 2B-AR
may play, in rat, a role in the adaptive response to acute renal tissue
injury. Additionally, 2B-AR is known to enhance Na+ reabsorption as a consequence of increased activity of
the Na+/H+ exchanger, NHE3 (20). Since NHE3 was
recently found to be controlled by MAPK in mouse proximal tubule cells
(21), it is possible that MAPK activation is also involved in the
regulation of NHE3 by 2B-AR.
2B-AR is independent of receptor internalization (7, 8), the signaling pathway(s) accounting for the
phosphorylation of MAPK by this receptor subtype remains poorly
defined. The results obtained in this study provide substantial evidence that, in LLC-PK1-2B, the activation of ERK by
2-agonists is triggered via a mechanism comprising the
activation of matrix metalloproteinases, the release of HB-EGF, and the
subsequent activation of the EGF-R. This cascade was demonstrated by
the following observations. First, UK14304-induced phosphorylation of
MAPK is totally abrogated in the presence of the matrix
metalloproteinase inhibitors (batimastat or 1,10-phenanthroline) and by
prior treatment of the cells with CRM 197. Second, conditioned medium
from LLC-PK1-2B cells treated with UK14304 causes
activation of MAPK in wild-type LLC-PK1, even in the presence of
batimastat. Third, the consequences of LLC-PK1-2B exposure to
2-agonists or of wild-type LLC-PK1 exposure to
conditioned medium are abolished by prior treatment of the cells with
the inhibitor of EGF-R tyrosine kinase activity, tyrphostin AG1478.
Previous studies of lysophosphatidic acid receptor or
2A-AR have demonstrated that the contribution of the
EGF-R transactivation pathway is largely dependent on the cell type (10, 22). In HEK 293, the major pathway of MAPK activation by
2A-AR is via the activation of Pyk2, a
calcium-dependent tyrosine kinase of the focal adhesion
kinase family (10). On the other hand, the effects of
2A-AR in COS cells are mediated by both EGF-R
transactivation and direct recruitment of the MEK cascade by Src (9,
23). According to our results, the transactivation pathway is
predominant in LLC-PK1-2B; whether it is exclusive awaits definitive demonstration.
2B-AR to transactivation
of EGF-R. Because 2-agonists activate AA release in
LLC-PK1-2B and because AA is responsible for MAPK
phosphorylation following angiotensin II treatment in rabbit proximal
tubule cells (16), we sought to evaluate its implication. Consistent
with a crucial role of the lipid second messenger, exposure of
LLC-PK1-2B to exogenous AA resulted in tyrosine
phosphorylation of p52 Shc and in activation of MAPK with time courses
that show a striking parallel to those observed with
2-agonists. Like for 2-agonists, the
action of AA was prevented by batimastat, 1,10-phenanthroline, CRM 197, or tyrphostin AG1478. However, a major divergence was that, unlike the
effects of 2-agonists, those of AA are resistant to
pretreatment with pertussis toxin or to the addition of quinacrine.
Additional support for implication of AA in the mediation of
2B-AR signal came from the study of the effects of
inhibitors of AA metabolism. According to these experiments,
phosphorylation of MAPK by UK14304 was strongly inhibited by NDGA and
indomethacin. Ketoconazole was by contrast ineffective, indicating that
AA products generated by lipoxygenase and/or cyclooxygenase, but not
epoxygenase, are involved in 2-agonist effect. The
observation that AA causes MAPK phosphorylation in our model is in
opposition with results obtained on LLC-PK1/C14 (24). In this clone,
ERK phosphorylation was observed in response to epoxyeicosatrienoic
acids but not to AA. Moreover, AA became efficient after cell
transfection with an active form of cytochrome P450 epoxygenase of
bacterial origin, indicating eicosanoid-dependent
activation of MAPK. The reason for these discrepancies is enigmatic.
However, activation of MAPK by AA was repeatedly reported in rabbit
proximal tubules as well as in various cell types, including vascular
smooth muscle cells and neutrophils. In vascular smooth muscle cells,
efficacy of AA was dependent on its conversion into
15-hydroxyeicosatetraenoic acid and on PKC activation (25). Dependence
on lipoxygenase and PKC activity was also found in human neutrophils
(26). In this cell type, the effects of AA engaged a membrane receptor linked to Gi/o proteins (27). This is not the case in
LLC-PK1-2B, since neither staurosporine nor pertussis
toxin treatment abolished ERK phosphorylation caused by AA. Regarding
these points, LLC-PK1-2B resembles rabbit renal
epithelial cells. However, it is epoxy derivatives that mediate the
effects of AA on MAPK phosphorylation in these cells (16). Whereas
involvement of the cytochrome P450 pathway can be excluded in our
model, the respective contribution of COX and LOX is still unclear,
since it is difficult to reconcile why products from either pathway
could function similarly. The implication of COX activity is beyond
doubt, because the effects of 2-agonists and AA were
also blocked by aspirin (not shown). By contrast, that of LOX is more
questionable, since NDGA can also interfere with COX activity and act
as an antioxidant. Alternatively, the possibility that prostaglandins
and leukotrienes act in concert cannot be definitively ruled out.
Indeed, the combined action of COX and LOX was already demonstrated to
be necessary for some of the effects of angiotensin II in rat kidney
and bovine bronchi (28, 29). It is therefore clear that the
identification of the AA metabolites responsible for MAPK activation in
LLC-PK1-2B will require future study. In addition, the mechanism
whereby these products may affect matrix metalloproteinase activity has yet to be defined. In line with the existence of a relationship between
the two phenomena, constitutive expression of cyclooxygenase-2 in
human colon cancer cells results in increased activation of MMP-2 (30),
whereas inhibitors of PLA2 and cyclooxygenase-2 reduce the release
of matrix metalloproteinases in prostate tumor cells (31).
2B-AR activates MAPK through stimulation of PLA2,
generation of AA metabolites by cyclooxygenase and/or lipoxygenase,
stimulation of matrix metalloproteinases, release of HB-EGF, and
transactivation of the EGF-R (Fig. 9).
Whether this scenario is particular to 2B-AR in LLC-PK1
or whether it can be extended to other cell types and/or other
G-protein-coupled receptors remains to be established.
View larger version (19K):
[in a new window]
Fig. 9.
Signal transduction pathway involved
in ERK1/2 activation in response to
2B-AR stimulation.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: INSERM Unit 388, Institut Louis Bugnard, CHU Rangueil, Bat. L3, 31403 Toulouse Cedex 4, France. Tel.: 33-561-32-30-90; Fax: 33-562-17-25-54; E-mail:
paris@toulouse.inserm.fr.
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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