Activated human hydroxy-carboxylic acid receptor-3 signals to MAP kinase cascades via the PLC-dependent PKC and MMP-mediated EGFR pathways.

Abstract

BACKGROUND AND PURPOSE 3-Hydroxy-octanoate, recently identified as a ligand for, the orphan GPCR, HCA(3), is of particular interest given its ability to treat lipid disorders and atherosclerosis. Here we demonstrate the pathway of HCA(3)-mediated activation of ERK1/2. EXPERIMENTAL APPROACH Using CHO-K1 cells stably expressing HCA(3) receptors and A431 cells, a human epidermoid cell line with high levels of endogenous expression of functional HCA(3) receptors, HCA(3)-mediated activation of ERK1/2 was measured by Western blot. KEY RESULTS HCA(3)-mediated activation of ERK1/2 was rapid, peaking at 5 min, and was Pertussis toxin sensitive. Our data, obtained by time course analyses in combination with different kinase inhibitors, demonstrated that on agonist stimulation, HCA(3) receptors evoked ERK1/2 activation via two distinct pathways, the PLC/PKC pathway at early time points (≤ 2 min) and the MMP/ epidermal growth factor receptor (EGFR) transactivation pathway with a maximum response at 5 min. Furthermore, our present results also indicated that the βγ-subunits of the G(i) protein play a critical role in HCA(3)-activated ERK1/2 phosphorylation, whereas β-arrestins and Src were not required for ERK1/2 activation. CONCLUSIONS AND IMPLICATIONS We have described the molecular mechanisms underlying the coupling of human HCA(3) receptors to the ERK1/2 MAP kinase pathway in CHO-K1 and A431 cells, which implicate the G(i) protein-initiated, PLC/PKC -and platelet-derived growth factor receptor/EGFR transactivation-dependent pathways. These observations may provide new insights into the pharmacological effects and the physiological functions modulated by the HCA(3)-mediated activation of ERK1/2.

Full text

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RESEARCH PAPERbph_1875
Activated human
hydroxy-carboxylic acid
receptor-3 signals to MAP
kinase cascades via the
PLC-dependent PKC
and MMP-mediated
EGFR pathways
1756..1773
Q Zhou1*, G Li2*#, XY Deng2, XB He2, LJ Chen2, C Wu2, Y Shi2, KP Wu1,
LJ Mei1,2, JX Lu1and NM Zhou2
1Zhejiang Provincial Key Laboratory of Medical Genetics, School of Laboratory Medicine and Life
Science, Wenzhou Medical College, Wenzhou, Zhejiang, China, and2College of Life Sciences,
Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang, China
Correspondence
Naiming Zhou, College of Life
Sciences, Zhejiang University,
Zijingang Campus, 388 Yuhang
Tang Road, Hangzhou,
310058, China.
E-mail: znm2000@yahoo.com;
Jianxin Lu, Zhejiang Provincial
Key Laboratory of Medical
Genetics, School of Laboratory
Medicine and Life Science,
Wenzhou Medical College,
Wenzhou, Zhejiang, 325035,
China. E-mail: jxlu313@163.com
----------------------------------------------------------------
*Both authors contributed
equally to this work.
#Present address: Institute of
Aging Research, Hangzhou
Normal University, Hangzhou,
Zhejing, China.
----------------------------------------------------------------
Keywords
HCA2; HCA3; cAMP; ERK1/2;
PKC; EGFR; PDGFR; PLC; A431;
b-arrestins
----------------------------------------------------------------
Received
31 May 2011
Revised
13 November 2011
Accepted
13 January 2012
BACKGROUND AND PURPOSE
3-Hydroxy-octanoate, recently identified as a ligand for, the orphan GPCR, HCA3, is of particular interest given its ability to
treat lipid disorders and atherosclerosis. Here we demonstrate the pathway of HCA3-mediated activation of ERK1/2.
EXPERIMENTAL APPROACH
Using CHO-K1 cells stably expressing HCA3 receptors and A431 cells, a human epidermoid cell line with high levels of
endogenous expression of functional HCA3 receptors, HCA3-mediated activation of ERK1/2 was measured by Western blot.
KEY RESULTS
HCA3-mediated activation of ERK1/2 was rapid, peaking at 5 min, and was Pertussis toxin sensitive. Our data, obtained by
time course analyses in combination with different kinase inhibitors, demonstrated that on agonist stimulation, HCA3 receptors
evoked ERK1/2 activation via two distinct pathways, the PLC/PKC pathway at early time points (?2 min) and the MMP/
epidermal growth factor receptor (EGFR) transactivation pathway with a maximum response at 5 min. Furthermore, our
present results also indicated that the bg-subunits of the Gi protein play a critical role in HCA3-activated ERK1/2
phosphorylation, whereas b-arrestins and Src were not required for ERK1/2 activation.
CONCLUSIONS AND IMPLICATIONS
We have described the molecular mechanisms underlying the coupling of human HCA3 receptors to the ERK1/2 MAP kinase
pathway in CHO-K1 and A431 cells, which implicate the Gi protein-initiated, PLC/PKC- and platelet-derived growth factor
receptor/EGFR transactivation-dependent pathways. These observations may provide new insights into the pharmacological
effects and the physiological functions modulated by the HCA3-mediated activation of ERK1/2.
Abbreviations
ADAM, a disintegrin and metalloproteinase; CRE, cAMP response element; EGFR, epidermal growth factor receptor;
ET-18-OCH3, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine; FIPI, 5-fluoro-2-indolyl des-chlorohalopemide ;
Gi, inhibitory G-protein; HCA, hydroxy-carboxylic acid; HDL, high-density lipoproteins; IBC293, 1-(1-methylethyl)-
1H-benzotriazole-5-carboxylic acid; PDGFR, platelet-derived growth factor receptors; PTX, Pertussis toxin; siRNA, small
interfering RNA
BJP
British Journal of
Pharmacology
DOI:10.1111/j.1476-5381.2012.01875.x
www.brjpharmacol.org
1756British Journal of Pharmacology (2012) 166 1756–1773
© 2012 The Authors
British Journal of Pharmacology © 2012 The British Pharmacological Society

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Introduction
The human hydroxy-carboxylic acid HCA3 receptor, also
known as GPR109B or HM74, was first cloned as an orphan
GPCR during a search for novel leukocyte chemoattractant
receptors (Nomura
et al.,1993;
follows Alexander et al., 2011). Amplification of HCA3 from
human spleen cDNA as a template resulted in the discovery
of one close paralogue, termed HCA2also known as GPR109A
or HM74a (Soga et al., 2003). Recently, three research groups
identified HCA2 receptors as the high-affinity receptor for
nicotinic acid, responsible for raising levels of high-density
lipoproteins (HDL) and, thus treating lipid disorders includ-
ing dyslipidaemia and atherosclerosis (Soga et al., 2003;
Tunaru et al., 2003; Wise et al., 2003). Although HCA3recep-
tors share a high degree of similarity with HCA2 receptors,
displaying 96% identity to the HCA2 receptors and with a
24-amino acid extension at its carboxyl terminus (Wise et al.,
2003; Tunaru et al., 2005), they are not simply polymorphic
variants or splice variants, as indicated by their tandem loca-
tion on the human chromosome 12q24, together with GPR81
(Lee et al., 2001). In addition, mRNA expression analyses
have indicated that the tissue distributions of HCA2and HCA3
receptorspartiallyoverlap
(Irukayama-Tomobe et al., 2009).
Despite a high sequence homology with HCA2receptors,
the affinity of nicotinic acid to HCA3 receptors is quite low,
that is, millimolar levels (Wise et al., 2003; Li et al., 2010).
Furthermore, binding assay results have confirmed that the
HCA3receptor does not appreciably bind nicotinic acid (Soga
et al., 2003; Tunaru et al., 2003), which suggests that the
HCA3 receptor has little or no function as a nicotinic acid
receptor in humans. Recently, three aromatic D-amino acids
have been demonstrated to act as specific agonists to activate
HCA3
receptors (Irukayama-Tomobe
3-hydroxylated b-oxidation intermediates, in particular,
3-hydroxy-octanoate has been identified as an endogenous
ligand that decreases the activity of adenylate cyclase
through the activation of Pertussis toxin (PTX)-sensitive
G-proteins (Ahmed et al., 2009). Although the effect of niacin
on the antilipolytic activity is mediated via HCA2 receptors,
HCA3 receptors have also been demonstrated to inhibit
isoprenaline-induced lipolysis in primary human adipocytes
(Semple et al., 2006). Acifran (4,5-dihydro-5- methyl-4-oxo-5-
phenyl-2-furancarboxylic acid), which possesses the same
antilipolytic and triglyceride-lowering effects as nicotinic
acid, activates both HCA2 and HCA3 receptors (Wise et al.,
2003). These data suggest that HCA3receptors are likely to be
involved in modulating lipolysis and, hence, could represent
an interesting target for the treatment of dyslipidemia
(Skinner et al., 2009).
Because the HCA3 receptor is of great interest as a target
for new antidyslipidemic drugs, in the present study, we
aimed to characterize MAPK signalling pathways triggered by
HCA3receptors using the model cell system CHO-K1, which
recombinantly expresses human HCA3 receptors, and A431
cells, a human epidermoid carcinoma cell line that endog-
enously expresses functional human HCA3 receptors (Zhou
et al., 2007). We document here, for the first time, the
molecular mechanisms underlying the coupling of human
HCA3 receptors to the ERK1/2 MAP kinase pathway in
receptornomenclature
andarepartiallydistinct
et al., 2009)and
CHO-K1 and A431 cells, which implicate the Gi protein-
initiated, PLC/PKC- and platelet-derived growth factor recep-
tor (PDGFR)/epidermalgrowth
transactivation-dependent pathways.
factorreceptor(EGFR)
Methods
Molecular cloning and plasmid construction
HCA2and HCA3receptors were cloned as previously described
(Li et al., 2010).
Cell culture and transfection
CHO-K1 cells were grown as monolayers in 50:50 Dulbecco’s
modified Eagle’s medium (DMEM) or Ham’s F-12 medium
containing 10% (v/v) FBS and glutamine (2 mM). Clonal
CHO-K1 lines transfected with HCA2, HCA3receptor or empty
vector were grown in the media mentioned earlier, but with
the addition of G418 (400 mg·L-1). HEK-293 and A431 cells
were grown in DMEM supplemented with 10% (v/v) FBS and
glutamine (2 mM). Clonal HEK-293 lines transfected with
HCA3 were grown in the media mentioned earlier, but with
the addition of G418 (800 mg·L-1). Plasmid constructs were
transfected or co-transfected into CHO-K1 and HEK-293 cells
using Lipofectamine 2000 according to the manufacturer’s
instructions. All cells were incubated at 37°C in a humidified
atmosphere with 5% CO2/95% air.
siRNA synthesis and transfection
siRNAs for b-arrestin1 and 2 were purchased as a SMARTpool
from Dharmacon RNA Technologies (Lafayette, CO). The
transfection protocol for b-arrestin1/2 siRNAs has been pre-
viously reported (Luo et al., 2008; Li et al., 2011). Forty-eight
hours after transfection, cells were split for the indicated
assay on the following day.
cAMP accumulation
After seeding in a 48-well plate overnight, stable CHO-K1
cells co-transfected with HCA3or HCA2receptors and pCRE-
Luc were grown to 90–95% confluence, stimulated with
10 mM forskolin alone or with 10 mM forskolin and different
concentrations of octanoic acid, the selective agonist 1-(1-
methylethyl)-1H-benzotriazole-5-carboxylic
Semple et al., 2006) and acifran in DMEM without FBS, and
incubated for 4 h (37°C). Luciferase activity was detected
using a firefly luciferase kit (Promega, Madison, WI, USA).
When required, cells were treated overnight with or without
PTX (100 ng·mL-1) in serum-free DMEM/F12 before the
experiment.
acid(IBC293;
Intracellular calcium measurement
Calcium mobilization was performed as described previously
with slight modifications (Li et al., 2010). The CHO-HCA3or
CHO-K1 cells were harvested with Cell Stripper (Mediatech,
Herndon, VA, USA), washed twice with PBS and resuspended
to 5 ¥ 106cells·mL-1in Hank’s balanced salt solution (140 mM
NaCl, 5 mM KCl, 10 mM HEPES, pH 7.4, 1 mM CaCl2, 1 mM
MgCl2, 1 mg·mL-1glucose) containing 0.025% BSA. The cells
were then loaded with 3 mM Fura-2 acetoxymethyl ester
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derivative (Fura-2/AM) (Molecular Probes, Eugene, OR, USA)
for 30 min at 37°C. Cells were washed once in Hank’s solu-
tion, resuspended in Hank’s, incubated at room temperature
for 15 min, washed twice in Hank’s solution, and then resus-
pended in Hank’s at a concentration of 3 ¥ 107cells·mL-1.
These cells were then stimulated with 100 mM IBC293.
Calcium flux was measured using excitation at 340 and
380 nm in a Tecan Infinite 200 pro series Microplate Reader
(Tecan, Switzerland). When required, cells were treated over-
night with or without PTX (100 ng·mL-1) in serum-free
DMEM/F12 before the experiment.
Western blot analysis
To analyse the knock-down of siRNA-targeted proteins and
phosphorylation of ERK1/2, siRNA-transfected HEK-293 cells
or agonist-stimulated cells in a six-well plate were washed
twice with ice-cold PBS and lysed with buffer [20 mM HEPES
(pH 7.5), 10 mM EDTA, 150 mM NaCl, 1% Triton X-100, and
one tablet of complete protease inhibitor (Roche, Indianapo-
lis, IN, USA) per 50 mL] on a rocker for 30 min (4°C). The
lysates were centrifuged at 13 500 x g for 15 min (4°C). The
supernatants were separated by electrophoresis (10% SDS-
PAGE), transferred to a PVDF membrane, and immunoblotted
using an anti-b-arrestin1/2 monoclonal antibody (BD Bio-
sciences Pharmingen) or monoclonal anti-phospho-MAPK
E10 antibody (Thr202/Tyr204) (Cell Signaling Technology).
The membrane was then probed with HRP-labelled secondary
antibodies, and chemiluminescence was detected using a
HRP substrate (Cell Signaling Technology). The blots were
stripped and reprobed using an anti-tubulin (1:7500) mono-
clonal antibody as a control for protein loading and anti-total
ERK1/2 (1:2000) as a control for p-ERK1/2.
Measurement of receptor internalization by
confocal imaging
HEK-293 cells stably expressing HCA3-EGFP were transiently
transfected with specific b-arrestin siRNA or a non-specific
control siRNA. After transfection (72 h), cells were stimulated
with 100 mM IBC293 for 40 min. After removal of the agonist,
the cells were fixed with 3% paraformaldehyde for 15 min.
Confocal images were taken on a Zeiss LSM 510 microscope
with an attached Axiovert 200 microscope and LSM5 com-
puter system. Excitation was performed at 488 nm, and fluo-
rescence detection was performed using a 525 ? 25 nm
bandpass filter. Images were collected using QED camera soft-
ware and processed with Adobe Photoshop.
Measurement of receptor internalization
by ELISA
HCA3 receptors on the cell surface were quantitatively
assessed by ELISA as previously described (Li et al., 2010).
Briefly, HEK-293 cells stably expressing Flag-HCA3were tran-
siently transfected with specific b-arrestin siRNA or a non-
specific control siRNA. After transfection (72 h), cells were
stimulated with 100 mM IBC293 for 1 h, the medium was
aspirated, and the cells were washed once with Tris-buffered
saline (TBS). After fixing for 5 min at room temperature with
3.7% formaldehyde in TBS, the cells were washed three times
with TBS and then blocked for 1 h with 1% BSA/TBS. Cells
were then incubated for 1 h with a monoclonal antibody
directed against the Flag epitope (1:2000). The cells were then
washed three times with TBS and incubated for 1 h with a
HRP-labelled secondary antibody. Finally, the cells were
washed three times, and antibody binding was visualized by
adding 0.2 mL of HRP substrate (Sigma). Development was
stopped by transferring 0.1 mL of the substrate to a 96-well
microtiter plate containing 0.1 mL of 1% SDS. The plates
were read at 405 nm in a microplate reader (Bio-Rad, Her-
cules, CA, USA) using Microplate Manager software.
Data analysis
All results are expressed as mean ? SEM from n assays. Data
was analysed using non-linear curve fitting (GraphPad PRISM
version 5.0) to obtain pEC50 values. Statistical significance
was determined using Student’s t-test. Probability values less
than or equal to 0.05 were considered significant.
Materials
Lipofectamine 2000 and G418 were purchased from Invitro-
gen (Carlsbad, CA, USA). Cell culture media and fetal bovine
serum (FBS) were obtained from Hyclone (Beijing, China).
The pEGFP-N1 and pCMV-Flag vectors were purchased from
Clontech Laboratories, Inc. (Palo Alto, CA, USA) and Sigma
(St. Louis, MO, USA) respectively. IBC293, acifran and
5-fluoro-2-indolyl des-chlorohalopemide (FIPI) were pur-
chased from Tocris (Ellisville, MO, USA). RIPA lysis buffer and
EGTA were obtained from Beyotime (Haimen, China). PTX,
Go6983, GF109203X (GFX, bisindolymaleimide), tyrphostin
A9, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine
(ET-18-OCH3), BAPTA-AM, octanoic acid, human recombi-
nant EGF and anti-flag M2 monoclonal antibody were pur-
chased from Sigma. U0126, tyrphostin AG1478, GM6001,
PP2 and wortmannin were from Calbiochem (La Jolla, CA,
USA). Anti-phospho-ERK1/2 (Thr202/Tyr204) and ERK1/2 anti-
bodies and horseradish peroxidase (HRP)-conjugated anti-
rabbit IgG were from Cell Signaling Technology (Danvers,
MA, USA). Anti-b-arrestin1/2 monoclonal antibody was from
BD Biosciences Pharmingen (San Diego, CA, USA).
Results
Functional expression of HCA3 in
CHO-K1 cells
To investigate the HCA3-mediated activation of ERK1/2, we
clonedhuman HCA3
receptors
No.D10923) from human genomic DNA as a template using
PCR and created CHO-K1 cell lines that stably expressed
human HCA3 receptors. We first examined the functional
signalling of HCA3receptors by assaying cAMP accumulation.
As shown in Figure 1A, treatment with the endogenous
ligand octanoic acid (Ahmed et al., 2009), the highly selective
agonist IBC293 (Semple et al., 2006) and the non-selective
agonist acifran, which binds to both HCA2and HCA3recep-
tors (Mahboubi et al., 2006), induced a ligand concentration-
dependent inhibition of forskolin-stimulated cAMP increase
with EC50values of 1.23 mM, 54 nM and 515 nM, respectively,
whereas almost no inhibition of the forskolin-stimulated
cAMP increase was observed in response to niacin in the
range of 0.001–100 mM. The agonist-induced inhibition of
(GenBank Accession
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Figure 1
Characterization of HCA3receptors stably expressed in CHO-K1 cells. A, cAMP accumulation in CHO-K1 cells stably co-transfecting with HCA3and
pCRE-Luc was determined in response to forskolin (FSL) and indicated ligand. Dose-dependent inhibition of forskolin-induced cAMP accumulation
was measured. B and C, CHO-K1 cells stably expressing HCA3 (B) or HCA2 receptors (C) and pCRE-Luc were pretreated with or without
100 ng·mL-1PTX for 12 h, then stimulated with 10 mM forskolin alone or 10 mM forskolin with 100 mM octanoic acid or IBC293 or acifran or niacin
in DMEM/F12 without FBS and incubated for 4 h at 37°C. Luciferase activity was detected by a firefly luciferase kit. (D) CHO-HCA3or CHO-K1
cells were loaded with the calcium probe Fura-2/AM followed by stimulation with 100 mM IBC293. (E) CHO-HCA3cells were loaded with the
calcium probe Fura-2/AM followed by stimulation with 100 mM IBC293 in the presence or absence of PTX, calcium mobilization was assayed by
monitoring the change in Fura-2/AM fluorescence. The data shown are representative of at least three independent experiments. Data were
analysed by Student’s t-test. ***P < 0.001.
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the forskolin-stimulated cAMP increase could be completely
blocked by pretreating with 100 ng·mL-1of PTX for 12 h
(Figure 1B and C). Additionally, octanoic acid (100 mM) or
IBC293 (100 mM) showed no inhibitory effect on the
forskolin-stimulatedcAMP
transfected CHO cells (Figure 1C). In addition, stimulation
with IBC293 (100 mM) elicited a rapid and transient increase
in intracellular Ca2+in CHO-K1 cells expressing HCA3recep-
tors (Figure 1D) and the Ca2+mobilization could be com-
pletely blocked by pretreating with 100 ng·mL-1PTX for 12 h
(Figure 1E). These results suggest that HCA3 receptors in
stably transfected CHO-K1 cells are functional, and octanoic
acid and IBC293 are specific ligands for HCA3receptors.
increase instably HCA2-
HCA3 receptors activate ERK1/2 signalling
via MEK 1/2 following exposure to octanoic
acid, IBC293 and acifran
In CHO-HCA3cells, stimulation with different concentrations
of agonists – octanoic acid, IBC293 and acifran – evoked
ERK1/2 phosphorylation in a dose-dependent manner with
EC50 values of 1.52 mM, 55 nM and 470 nM, respectively
(Figure 2A), whereas almost no ERK1/2 activation was
observed in response to octanoic acid or IBC293 in the range
of 0.1–1000 mM in CHO-HCA2cells (data not shown), which
is consistent with the observation of intracellular cAMP accu-
mulation with no detectable activity up to 1 mM at HCA2
receptors (Semple et al., 2006). In addition, to better charac-
terize the HCA3-mediated ERK1/2 signalling pathway, we also
used the A431 cell line, a human epidermoid cell line with a
high level of endogenous expression of functional HCA3
receptors (Zhou et al., 2007). A431 cells were cultured in
serum-free DMEM media for 24 h followed by stimulation
with various concentrations of IBC293 in fresh serum-free
DMEM for 5 min. There was concentration-dependent acti-
vation of ERK1/2 signalling, with an EC50 of 14.9 mM
(Figure 2B). The HCA3-initiated activation of ERK1/2 was
time-dependent with a maximal activation at 5 min and with
a subsequent reduction to baseline by 30 min in CHO-HCA3
cells (Figure 2C). A similar result was observed during
IBC293-mediatedERK1/2
(Figure 2D).
To investigate whether or not HCA3-induced ERK1/2
phosphorylation is mediated by activation of other signalling
kinases such as MEK1/2, the inhibitor U0126, a highly selec-
tive inhibitor of both MEK1 and MEK2, was used. ERK1/2
activation stimulated by octanoic acid, IBC293 and acifran
were significantly inhibited by preincubation of CHO-
HCA3 cells (Figure 2E) with U0126 (1 mM). A similar result
was observed for IBC293-mediated ERK1/2 activation in
A431 cells (Figure 2F), which indicated that upstream
MEK1/2 activation is required for HCA3-induced ERK1/2
phosphorylation.
activationin A431cells
HCA3 receptors initiate ERK1/2 activation via
the PTX-sensitive Gi protein-dependent
pathway
Previous studies have demonstrated that HCA3 receptors act
via Gi proteins to inhibit adenylyl cyclase (Semple et al.,
2006; Skinner et al., 2007). To assess the role of the Gi
protein in the regulation of HCA3-mediated activation of
ERK1/2, CHO-HCA3 and A431 cells were cultured in the
presence or absence of 100 ng·mL-1PTX in serum-free
DMEM/F-12 or DMEM, respectively, for 24 h, followed by
stimulation with the indicated ligand. As illustrated in
Figure 3A and C, the pretreatment of cells with PTX resulted
in a nearly complete inhibition of ERK1/2 phosphorylation
compared with the agonist alone in both cell lines. A
similar result was observed for octanoic acid or acifran-
mediated ERK1/2 activation in CHO-HCA3 cells (Figure 3B).
Together, these data demonstrate that HCA3receptors signal
to the ERK1/2 pathway via a PTX-sensitive Gi protein-
dependent mechanism.
Involvement of the PLC/PKC pathway in
HCA3-mediated ERK1/2 activation
Next, we investigated whether or not PKC plays a role in
agonist-stimulated ERK1/2 phosphorylation via HCA3 recep-
tors, As shown earlier, in time-course studies, the HCA3-
initiated activation of ERK1/2 revealed a maximal activation
at 5 min and a return to baseline by 30 min (Figure 2C and
D). The CHO-HCA3 (Figure 4A) and A431 cells (Figure 4B)
were pretreated with 10 mM of GFX or 10 mM of Go6983 for
1 h, followed by the agonist IBC293 in a time course. As
shown in Figure 4A and B, both treatment with GFX and
Go6983 resulted in dramatic decreases (>60%) in ERK1/2
activation at an early time point (?2 min), but very little
inhibition was observed at the 5 min time point. Collectively,
these data demonstrate that PKC plays a determinant role in
HCA3-mediated ERK1/2 activation at early time points
(?2 min).
The family of PLCs classically catalyses the hydrolysis of
phosphatidylinositol4,5-bisphosphate
1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). DAG can
directly activate classical types of PKC by interacting with
its lipid-binding domain, and IP3can indirectly activate PKC
by increasing intracellular Ca2+, which interacts with the
PKC Ca2+-binding domain (Chung et al., 1997). Therefore,
we tested the potential involvement of PLC in the activa-
tion of ERK1/2 using the PLC inhibitors U73122 and ET-18-
OCH3. Pretreatment of cells with U73122 (20 mM) led to no
inhibition of HCA3-stimulated ERK1/2 activation in both
CHO-HCA3 and A431 cells (Figure 4C and D), whereas
ET-18-OCH3 significantly blocked HCA3-mediated ERK1/2
phosphorylation in both cell lines (Figure 4C and D). Next,
we examined the role of phospholipase D (PLD) in HCA3-
stimulated ERK1/2 phosphorylation using the PLD inhibitor
FIPI,. As shown in Figure 4C and D, preincubation with FIPI
also did not inhibit ERK1/2 activation in both CHO-
HCA3 and A431 cells. These data suggested that PLC rather
than PLD played a key role in HCA3-mediated ERK1/2
phosphorylation.
Previous studies have shown that octanoic acid causes a
rapid increase of intracellular Ca2+in CHO-K1 cells expressing
HCA3 receptors (Ahmed et al., 2009). Accordingly, we inves-
tigated whether or not intracellular and extracellular Ca2+is
involved in HCA3-stimulated ERK1/2 phosphorylation. Pre-
treatment with the extracellular Ca2+chelator EGTA (5 mM)
or L-type Ca2+channel blocker nifedipine (10 mM) signifi-
cantly inhibited ERK1/2 phosphorylation in CHO-HCA3
cells (Figure 4E). However, these two inhibitors showed no
(PIP2)to inositol
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Figure 2
HCA3receptors activate ERK1/2 signalling via MEK1/2 pathway. (A) Serum-starved CHO-HCA3cells were stimulated with various concentrations
of octanoic acid or IBC293 or acifran for 5 min. (B) Serum-starved A431 cells were stimulated with various concentrations of IBC293 for 5 min.
(C) Serum-starved CHO-HCA3 cells were stimulated with 1 mM IBC293 or 10 mM octanoic acid or 3 mM acifran for indicated time. (D)
Serum-starved A431 cells were stimulated with 100 mM IBC293 for indicated time. (E) Serum-starved CHO-HCA3cells were cultured in serum-free
DMEM/F12 media with or without MEK inhibitor U0126 (1 mM) for 1 h, cells were then stimulated with 1 mM IBC293 or 10 mM octanoic acid or
3 mM acifran for 5 min. (F) Serum-starved A431 cells were cultured in serum-free DMEM media with or without MEK inhibitor U0126 (1 mM) for
1 h, cells were then stimulated with 100 mM IBC293 for 5 min. The data shown are representative of at least three independent experiments. Data
were analysed by Student’s t–test. ***P < 0.001.
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Figure 3
HCA3receptors initiate ERK1/2 activation via PTX-sensitive Giprotein-dependent pathway. CHO-HCA3cells (A) or A431 cells (C) were cultured in
serum-free DMEM/F12 or DMEM media with or without 100 ng·mL-1PTX for 24 h. The next day, media was removed and fresh serum-free
DMEM/F12 or DMEM media with or without 100 ng·mL-1PTX were added for 1 h, cells were then stimulated with 1 mM IBC293 for CHO-HCA3
or 100 mM IBC293 for A431 cells for the indicated time periods. (B) CHO-HCA3cells were cultured in serum-free DMEM/F12 media with or without
100 ng·mL-1PTX for 24 h. The next day, media was removed and fresh serum-free DMEM/F12 media with or without 100 ng·mL-1PTX were
added for 1 h, cells were then stimulated with 1 mM IBC293 or 10 mM octanoic acid or 3 mM acifran for 5 min. The data shown are representative
of at least three independent experiments. Data were analysed by Student’s t–test. ***P < 0.001.
?
Figure 4
Effects of PKC, calcium and PLC on HCA3-stimulated phosphorylation of ERK1/2. Serum-starved CHO-HCA3 cells (A) or A431 cells (B) were
pretreated with DMSO or 10 mM Go6983 or 10 mM GFX for 1 h, and then stimulated with 1 mM IBC293 for CHO-HCA3cells or 100 mM IBC293
for A431 cells for the indicated time periods. Serum-starved CHO-HCA3cells (C) or A431 cells (D) were pretreated with DMSO or 20 mM U73122
or 1 mM FIPI or 100 mM ET-18-OCH3for 1 h, and then stimulated with 1 mM IBC293 for CHO-HCA3cells or 100 mM IBC293 for A431 cells for
2 min. Serum-starved CHO-HCA3cells (E) or A431 cells (F) were cultured in serum-free DMEM/F12 or DMEM media with or without EGTA (5 mM)
or nifedipine (10 mM) or BAPTA-AM(50 mM) or KN62 (1 mM) for 1 h, cells were then stimulated with 1 mM IBC293 for CHO-HCA3or 100 mM
IBC293 for A431 cells for 2 min. The data shown are representative of at least three independent experiments. Data were analysed by Student’s
t–test. ***P < 0.001.
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inhibitory effect on ERK1/2 activation by HCA3 receptors in
A431 cells (Figure 4F). The intracellular Ca2+
BAPTA-AM (100 mM) and Ca2+-calmodulin kinase inhibitor
KN62 (10 mM) also did not impair ERK1/2 activation by HCA3
receptors in both CHO-HCA3 and A431 cells (Figure 4E and
F). Taken together, the results of the present study indicate
that stimulation of HCA3 receptors by agonists leads to
ERK1/2 activation at early time points (?2 min) via the PKC
pathway, consistent with the observation of angiotensin II
receptor (Ahn et al., 2004a) and AT1A receptors (Kim et al.,
2009).
chelators
HCA3-induced ERK1/2 activation is
dependent on a growth factor
receptor-involved transactivation mechanism
It is well known that the transactivation of a growth factor
receptor participates in GPCR-mediated ERK1/2 phosphory-
lation (Pierce et al., 2001). CHO-K1 cells are known to endog-
enously express PDGFR-b (Duckworth and Cantley, 1997)
and lack endogenous EGFR (Shi et al., 2000). Serum-starved
CHO-HCA3cells were preincubated with the PDGFR-selective
receptor tyrosine kinase inhibitor tyrphostin A9 (10 mM) for
1 h, followed by stimulating with 1 mM IBC293 for different
periods of time. As shown in Figure 5A, in the A9 pretreated
cells, there was a > 60% inhibition of ERK1/2 phosphoryla-
tion compared with agonist alone; however, there was no
such effect in the A431 cells (data not shown), which suggests
that PDGFR transactivation is involved in HCA3-induced
ERK1/2 activation in CHO-K1 cells but not in A431 cells.
To assess the role of EGFR transactivation in agonist-
induced ERK1/2 activation in cells endogenously expressing
HCA3 receptors, A431 cells were utilized for further investi-
gations. Serum-starved A431
AG1478, an EGFR-specific tyrosine kinase inhibitor, for 1 h
before exposing them to IBC293. As shown in Figure 5B and
C, AG1478 (100 nM) dramatically inhibited (>70%) IBC293-
induced ERK1/2 phosphorylation. Several studies have
shown that the transactivation of EGFR is sensitive to MMP
inhibitors (Prenzel et al., 1999; Gschwind et al., 2001; Pierce
et al., 2001). To define the mechanism underlying the
IBC293-induced transactivation of EGFR, A431 cells were
treated with the MMP inhibitor GM6001 (10 mM) for 1 h
before exposing them to IBC293 or EGF. GM6001 treatment
led to a significant reduction (>50%) of ERK1/2 activation
induced by IBC293 but not by EGF (Figure 5B and C). These
results demonstrate that HCA3 receptors evoke ERK1/2
activation viathePDGFR
cells weretreated with
transactivationpathwayin
CHO-HCA3 cells and the EGFR transactivation pathway via
the metalloproteinase-dependent shedding of HB-EGF in
A431 cells. Simultaneous inhibition of PLC and PDGFR in
CHO-HCA3 cells and simultaneous inhibition of PLC and
EGFR in A431 cells resulted in a nearly complete inhibition
of ERK1/2 phosphorylation (see Supporting Information
Figure S1), suggesting the involvement of PLC/PKC and
MMP/EGFR or PDGFRin
activation.
theHCA3-mediatedERK1/2
Involvement of the PI3K pathway in
HCA3-mediated ERK1/2 activation
Previous studies have reported that PI3K and Src are involved
in ERK1/2 activation in response to Gi-coupled receptors
(Hawes et al., 1996; Kranenburg et al., 1997; Lopez-Ilasaca
et al., 1997; Luttrell et al., 1999; Ptasznik and Gewirtz, 2000).
Pretreatment with the PI3K inhibitor wortmannin resulted in
decreased IBC293-stimulated ERK1/2 phosphorylation in
both CHO-HCA3 and A431 cells (Figure 6A and B), which
suggests that PI3K plays an important role in HCA3-mediated
ERK1/2 activation. Src activation has been shown to stimu-
late GPCR-mediated MMP induction and EGFR transactiva-
tion (Shah et al., 2003). Therefore, we next examined the role
of Src in HCA3-mediated ERK1/2 activation. As shown in
Figure 6C and D, Src inhibition by the selective Src kinase
inhibitor PP2 did not attenuate IBC293-induced ERK1/2 acti-
vation in both CHO-HCA3and A431 cells. However, pretreat-
ment with PP2 significantly decreased niacin-mediated
ERK1/2 activation in CHO-HCA2cells. These results indicate
that PI3K played an important role in HCA3-mediated ERK1/2
activation, whereas Src kinase was not required for IBC293-
induced EGFR transactivation in A431 cells.
To determine whether PKC and PI3K act upstream or
downstream of the EGFR, we carried out experiments to
examine the effect of PKC and PI3K inhibitors on IBC293-
induced EGFR phosphorylation. As shown in Supporting
Information Figure S2A, in A431 cells pretreated with specific
inhibitors of PKC and PI3K, IBC293-stimulated EGFR phos-
phorylation was significantly reduced. Next, we examined
the effect of PKC and PI3K on EGF-induced ERK1/2 activa-
tion. As shown in Supporting Information Figure S2B and C,
in A431 cells pretreated with PKC and PI3K specific inhibi-
tors, EGF stimulated ERK1/2 activation was also significantly
reduced. These data indicate that PKC and PI3K are more
likely to act downstream of the EGFR, but we cannot rule out
the possibility that PKC and PI3K may also act upstream of
the EGFR.
?
Figure 5
HCA3-induced ERK1/2 activation is dependent on growth factor receptor transactivation. (A) Serum-starved CHO-HCA3cells were pretreated with
DMSO or PDGFR selective receptor tyrosine kinase inhibitor tyrphostin A9 (10 mM) for 1 h, and then stimulated with 1 mM IBC293 for the
indicated time periods. (B) Serum-starved A431 cells were pretreated with DMSO or EGFR selective receptor tyrosine kinase inhibitor tyrphostin
AG1478 (1 mM) or MMP inhibitor GM6001 (20 mM) for 1 h, and then stimulated with 100 mM IBC293 for the indicated time periods. (C)
Serum-starved A431 cells were pretreated with DMSO or AG1478 (1 mM) or GM6001 (20 mM) for 1 h, and then stimulated with 100 mM IBC293
or 10 ng·mL-1EGF for 5 min. (D) Serum-starved A431 cells were stimulated with 100 mM IBC293 for the indicated time periods. (E) Serum-starved
A431 cells were pretreated with DMSO or AG1478 (1 mM) or GM6001 (20 mM) for 1 h, and then stimulated with 100 mM IBC293 for 5 min, cells
were harvested, and equal amounts of total cellular lysate were separated by 10% SDS-PAGE, transferred to a PVDF membrane, and incubated
with anti-p-EGFR (Tyr1173) antibody. Blots were stripped and reprobed for tubulin to control for loading. The data shown are representative of at
least three independent experiments. Data were analysed by Student’s t–test. ***P < 0.001.
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Figure 6
Effects of PI3K and Src on HCA3-mediated ERK1/2 activation. Serum-starved CHO-HCA3cells (A) or A431 cells (B) were pretreated with DMSO or
PI3K selective inhibitor wortmannin (1 mM) for 1 h, and then stimulated with 1 mM IBC293 for CHO-HCA3cells or 100 mM IBC293 for A431 cells
for the indicated time periods. (C) Serum-starved CHO-HCA2or CHO-HCA3cells were pretreated with DMSO or the Src selective inhibitor PP2
(10 mM) for 1 h, and then stimulated with 1 mM niacin or 1 mM IBC293 respectively for 5 min. (D) Serum-starved A431 cells were pretreated with
DMSO or PP2(10 mM) for 1 h, and then stimulated with 100 mM IBC293 for the indicated time periods. The data shown are representative of at
least three independent experiments. Data were analysed by Student’s t–test. **P < 0.01, ***P < 0.001.
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Gbg plays a central role in HCA3-induced
ERK1/2 activation
PLC and PI3K can be activated through a mechanism involv-
ing the Gbg-subunits (Fields and Casey, 1997; Lopez-Ilasaca
et al., 1997). A role for the Gi/o-derived b- and g-subunits was
raised because overnight treatment with 100 ng·mL-1PTX
eliminated HCA3-mediated ERK1/2 activation (Figure 3A–C).
To test for the involvement of the Gbg-subunits in ERK1/2
activation,we transfected
b-adrenoceptor kinase COOH domain (495–689 aa) (bARK1-
CT) or the Gasubunit of transducin, both of which are scav-
engers of the Gbg-subunits. Upon transfection, a significant
inhibition of HCA3-induced ERK1/2 phosphorylation was
observed (Figure 7), which suggests that the Gbg subunit is
likely to play a central role in HCA3-induced ERK1/2 activa-
tion. Simultaneous inhibition of Gbg and PLC or Gbg and
PDGFR in CHO-HCA3cells resulted in enhanced inhibition of
HCA3-mediated ERK1/2 activation, comparing with pre-
treated with only one inhibitor alone (see Supporting Infor-
mation Figure S3), indicating that Gbgsubunits together with
PLC and PDGFR play an important role in HCA3-induced
ERK1/2 activation in CHO-HCA3 cells, although we could
not clearly clarify the detailed mechanism of Gbg subunits-
mediated pathways.
CHO-HCA3
cells withthe
b-arrestin2 is involved in HCA3
internalization, but b-arrestins are
not involved in HCA3-mediated
ERK1/2 activation
To evaluate the role of b-arrestins in the regulation of HCA3
internalization and ERK1/2 activation, we used specific
siRNAs to reduce the expression of b-arrestin1 and b-arrestin2
in HEK-293 cells stably expressing HCA3 receptors. The
endogenous expression of b-arrestins was effectively and spe-
cifically knocked-down by specific siRNA treatment but was
unaffected in cells treated with non-specific or control siRNAs
(Figure 8A). Silencing b-arrestin2 effectively inhibited HCA3
internalization, whereas knock-down of b-arrestin1 had no
effect on the internalization of HCA3receptors, as analysed by
microscopy (Figure 8B) or ELISA (Figure 8C). We further inves-
tigated the effect of knock-down of b-arrestins on ERK1/2
activation, and no difference was observed between control
and knock-down cells (Figure 8D). Taken together, it seems
likely that b-arrestin2 is involved in HCA3receptor internal-
ization, but both b-arrestins are not required for HCA3-
mediated ERK1/2 activation.
Discussion and conclusions
It is generally accepted that HCA3 receptors, which differ
from HCA2receptors by 16 amino acids and in an extended
C-terminal end (Soga et al., 2003; Wise et al., 2003; Tunaru
et al., 2005), are the outcome of a recent gene duplication
because it is only present in higher primates and absent in
rodents and in most other mammals (Zellner et al., 2005).
Although accumulated evidence indicates that the binding
of nicotinic acid to HCA2receptors mediates its antilipolytic
and lipid-lowering effects (Zhang et al., 2005), HCA3 recep-
tors have been shown to have very similar expression pat-
terns to HCA2 receptors (Soga et al., 2003; Wise et al., 2003;
Zellner et al., 2005) and to inhibit isoprenaline-induced
lipolysis in primary human adipocytes (Semple et al., 2006).
The niacin-induced flushing has been shown to be mediated
by HCA2receptors and by PUMA-G (Benyo et al., 2005), and
it suggests that selective activators of HCA3 receptors may
avoid the characteristic and uncomfortable cutaneous flush-
ing response elicited by niacin in humans (Skinner et al.,
2009). However, the exact role of HCA3 receptors in induc-
tion of the flushing side effect is currently not known. More
recently, aromatic
D-amino acids and the endogenous
b-oxidationintermediate3-hydroxy-octanoic
identified as specific agonists that activate HCA3 receptors
withphysiological significance
Irukayama-Tomobe et al., 2009). HCA3, together with HCA2
receptors are of great interest as targets for the development
of new antidyslipidemic drugs. Information about the sig-
nalling pathways linked to activated HCA3 receptors and a
better understanding of their functions are of major signifi-
cance. Analyses of the signalling mechanisms induced by
agonistsof aromatic
D-amino
3-hydroxy-octanoic acid have demonstrated that HCA3
receptors are coupled to PTX-sensitive Gi-proteins, which
results in the inhibition of adenylyl cyclase, a transient rise
of intracellular Ca2+levels and the activation of ERK1/2
acidwere
(Ahmed
et al.,2009;
acidsand endogenous
Figure 7
Effects of Gbgsubunits on HCA3-mediated ERK1/2 Activation. CHO-HCA3cells were transiently transfected with the Gbgscavengers bARK-CT or
Ga-transducin, and the cells were then serum-starved for 24 h and stimulated with various concentrations of IBC293 for 5 min. The data shown
are representative of at least three independent experiments. Data were analysed by using the Student’s t–test. *P < 0.05, **P < 0.01.
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Figure 8
There is no involvement of b-arrestins in HCA3-mediated ERK1/2 activation. (A) HEK-293 cells stably expressing HCA3were transfected with specific
b-arrestin siRNA or a nonspecific control siRNA, 72 h after transfection, cells were harvested, and equal amounts of total cellular lysate were
separated by 10% SDS-PAGE, transferred to a PVDF membrane, and incubated with anti-b-arrestin1/2 antibody. (B) HEK-293 cells stably
expressing HCA3-EGFP were transfected with specific b-arrestin siRNA or a non-specific control siRNA,72 h after transfection, cells were stimulated
with 100 mM IBC293 for 40 min and examined with confocal microscopy as described under ‘Experimental Procedures.’ (C) ELISA determination
of cell surface receptors in Flag-HCA3expressing cells treated with specific b-arrestin siRNA or nonspecific control siRNA. (D) 72 h after transfection
with specific b-arrestin siRNA or non-specific control siRNA, cells were stimulated with 100 mM IBC293 for the indicated time periods and
immunoblotted using monoclonal anti-phospho-MAPK E10 (Thr202/Tyr204), and then the blots were stripped and reprobed for total ERK1/2 to
control for loading. The data and pictures shown are representative of at least three independent experiments. Data were analysed by Student’s
t test **P < 0.01, ***P < 0.001.
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(Ahmed
However, the detailed mechanism of HCA3-mediated ERK1/2
activationviadifferenttemporal
unknown. In the current study, we focused on a detailed
characterization of HCA3-mediated MAPK signalling path-
ways, and we demonstrated that activated HCA3 receptors
signal to ERK1/2 via a PLC-dependent PKC pathway and the
MMP/HB-EGF-dependent EGFR transactivation pathway.
In the present study, we used the CHO-K1 cell line as a
cellular model system for characterizing HCA3-mediated
ERK1/2 activation because it is a commonly used cell line for
investigating GPCR signalling pathways. For better under-
standing of HCA3-induced ERK1/2 phosphorylation, A431
cells, a human epidermoid cell with high levels of endog-
enous expression of functional HCA3 receptors (Zhou et al.,
2007) was also selected for this study. In our preliminary
experiments, we found that although all three agonists of
HCA3receptors triggered significant ERK1/2 phosphorylation
in CHO-HCA3cells, only IBC293 induced significant ERK1/2
phosphorylation in low concentrations in A431 cells,
whereas both octanoic acid and acifran induced a moderate
ERK1/2 phosphorylation in a very high concentration
(>1 mM). This result is in good agreement with our previous
observation with HCA2 receptor (Li et al., 2011); it is likely
that the cell-type specificity and the cell surface expression
level of receptor contributes to the different responses to
agonists between two cell lines. Therefore, we chose the
agonist IBC293 for further study of the ERK1/2 activation
pathway induced by HCA3receptors in A431 cells.
The HCA3receptor is a Giprotein-coupled receptor. Upon
stimulation by agonists, HCA3receptors trigger an inhibitory
effect on adenylate cyclase that leads to a decrease of
intracellular cAMP and, meanwhile, also elicit a transient
rise of intracellular Ca2+levels in a PTX-sensitive manner
(Irukayama-Tomobe et al., 2009). Thus, we explored the effect
of the PLC/PKC pathway in HCA3-mediated ERK1/2 activa-
tion. The inhibitory effect of the PKC inhibitors Go6983 and
GFX suggested a critical role for PKC on HCA3-mediated
ERK1/2 activation at early time points (?2 min). The involve-
ment of PLC as a contributor to HCA3-mediated ERK1/2 acti-
vation was assessed by incubating cells with two PLC
inhibitors, ET-18-OCH3 and U-73122. ET-18-OCH3, but not
U-73122, exhibited significant inhibition of ERK1/2 phos-
phorylation by activated HCA3. U73122 is known to be a
selective inhibitor of PLCb (Ward et al., 2003) and ET-18-
OCH3 to be a PLCg-selective inhibitor (Souttou et al., 2001;
Suzuki et al., 2008). ET-18-OCH3 is also a direct inhibitor of
Raf (Samadder et al., 2003; van der Westhuizen et al., 2007).
Our result with U-73122 is in agreement with observations
that there are receptor-specific differences in the capacity of
U-73122 to inhibit responses (Parker et al., 1998; Morfis et al.,
2008). It is likely that the significant inhibitory effect of
ET-18-OCH3 on HCA3-mediated ERK1/2 activation can be
accounted for suppression of both PLC and Raf in this study.
Furthermore, we also demonstrated that HCA3-induced
ERK1/2 activation was abolished by the depletion of extracel-
lular Ca2+by the chelator EGTA and by nifedipine, an L-type
Ca2+channel blocker in CHO-HCA3cells, suggesting that the
L-type Ca2+channel may play an important part in HCA3-
mediated ERK1/2 activation in CHO-K1 cells. However, Ca2+
was found to play no role in HCA3-mediated ERK1/2 activa-
et al.,2009;Irukayama-Tomobe
et al.,2009).
componentsremains
tion in A431 cells. The discrepancy in the role of Ca2+in
HCA3-mediated ERK1/2 activation between CHO-K1 cells and
A431 cells can be accounted for by cell type specificity, and
our observation is in agreement with previous observation
that AT1 receptor-mediated activation of ERK1/2 by angio-
tensin II was Ca2+–dependent in rat anterior pituitary cells,
but Ca2+–independent in hepatic C9 cells(Suarez et al., 2003)
(Shah and Catt, 2002). Taken together, these data suggest the
involvement of both Ca2+-dependent and -independent PKC
isoforms in HCA3-mediated ERK1/2 activation.
The EGFR tyrosine kinase has emerged as an important
transducer in signalling by GPCRs, a process termed transac-
tivation (Schafer et al., 2004a; Rozengurt, 2007). The role of
EGFR transactivation in ERK1/2 stimulation by GPCR ligands
is cell-specific. COS-7 cells express the EGF receptor (Shah
et al., 2004), but CHO-K1 cells express the PDGFR but lack
endogenous EGFR (Antonelli et al., 2000). Previous studies
have demonstrated that proliferation of adipocytes is regu-
lated by several growth factors, such as EGF, fibroblast growth
factor and insulin-like growth factor (Smith et al., 1988;
Yamasaki et al., 1999; Garcia and Obregon, 2002). Our results
show that HCA3-mediated ERK1/2 activation was potently
inhibited by the PDGFR-selective tyrphostin A9 and the PI3K
inhibitor wortmannin in CHO-K1 cells. However, in A431
cells, HCA3-mediated ERK1/2 activation was impaired by the
EGF receptor-selective inhibitor AG1478 and the MMP
inhibitor GM6001. Our finding that inhibition of metallo-
proteinase activity attenuated the activation of EGFR and
ERK1/2 by HCA3 receptor agonists, but not by EGF, defines
the intermediary action of the MMP-dependent shedding of
HB-EGF in the transactivation of EGFR by HCA3receptors in
A431 cells. HB-EGF is synthesized in the cell as a transmem-
brane precursor that is proteolysed by a MMP of the zinc-
dependent‘disintegrinand
family to form a soluble growth factor that is a potent EGFR
ligand (Riese et al., 1998; Prenzel et al., 1999). Different
members of the ADAM family, including ADAM 10, ADAM 12
and ADAM 17, mediate GPCR-induced EGFR transactivation
in different model systems (Schafer et al., 2004b). The precise
mechanism(s) by which the HCA3receptor stimulates ADAM
activation remains to be further elucidated. Moreover, PI3Ks
(Hawes et al., 1996; Lopez-Ilasaca et al., 1998) and Src family
non-receptor tyrosine kinases (Lin et al., 2008) have each
been proposed as early intermediates in the pathway to
induce EGF receptor transactivation. In the present study, we
observed that PI3K was involved in the PDGFR- or EGFR-
transactivated phosphorylation of ERK1/2, whereas the Src
kinase was not required for HCA3-induced EGFR transactiva-
tion in either CHO or A431 cells. However, more studies are
necessary for the clarification of the exact role of PI3K in
HCA3-induced ERK1/2 activation.
In the current study, our results demonstrate the existence
of two parallel pathways by which ERK1/2 can be activated by
HCA3receptors. One pathway involves PLC and PKC activa-
tion, which results in ERK1/2 phosphorylation at an early
(2 min) time. The other is EGFR transactivation and is medi-
ated by MMP, which leads to activation of ERK1/2 at a later
time point (5 min). This activation via two pathways is abol-
ished by pretreatment with PTX. In addition, we observed
that overexpression of the Gbg subunit scavenger proteins
bARK-CT or Ga-transducin effectively attenuated the ERK1/2
metalloproteinase’ (ADAM)
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activation triggered by HCA3 receptors (Figure 7). These
results indicate that the Gbg subunit acts as an early signal
mediating HCA3-induced PKC activation and EGF receptor
transactivation. The major effects of Gi activation on the
ERK1/2 cascade appear to be mediated via its Gbg subunits
(Crespo et al., 1994; Hawes et al., 1995). Previous studies have
shown that Gi-type GPCRs stimulate Ca2+mobilization
through the binding of the Gbgsubunits to PLC (Dorn et al.,
1997; Dickenson and Hill, 1998). It has also been reported
that the best-understood mechanism whereby the Gbg sub-
units stimulate ERK1/2 is through the ‘transactivation’ of
classical receptor tyrosine kinases, such as the EGF and
PDGFRs (Carpenter, 2000; Gschwind et al., 2001), although
the Gbgsubunit protein effectors that regulate HB-EGF release
remain undefined. Thus, we postulate that upon stimulation
of HCA3 receptors by agonists, activated Gi protein impairs
cAMP production and its released Gbg subunits are able to
trigger the generation of DAG by directly binding to PLC,
which leads to the activation of PKC, followed by a >2 min
early time point peak of ERK1/2 phosphorylation. On the
other hand, the free Gbg subunits also cause activation of a
MMP to cleave HB-EGF (Prenzel et al., 1999) and lead to EGFR
transactivation, which results in a >5 min late time point
peak for ERK1/2 activation. These findings are consistent
with our previous observation of HCA2receptors in CHO-K1
cells and A431 cells (Li et al., 2011), although Src was found
to play no role in HCA3-mediated ERK1/2 activation in
CHO-K1 cells stably expressing HCA3receptors.
b-Arrestins are traditionally recognized as playing a well-
established role in the termination of receptor-G-protein cou-
pling and the initiation of clathrin-dependent internalization
(Luttrell and Lefkowitz, 2002). However, there is a growing
body of evidence that indicates that b-arrestins function as
signal transducers for many GPCRs to mediate ERK1/2 acti-
vation (Lefkowitz and Shenoy, 2005). b-Arrestins are required
for later-phase activation of the ERK1/2 pathway mediated
byangiotensinIIAT1A
receptors
b2-adrenoceptors (Shenoy et al., 2006), vasopressin 2 (Ren
et al., 2005) and parathyroid hormone (Gesty-Palmer et al.,
2006) receptors, whereas, in the dopamine D2and D3recep-
tors (Beom et al., 2004; Quan et al., 2008) and the formyl
peptide receptor (Huet et al., 2007; Gripentrog and Miettinen,
2008), b-arrestins have been found to play no role or only a
minor role in the activation of the ERK1/2 pathway. Our
results using siRNA showed that b-arrestin2 was required for
agonist-mediated internalization of HCA3receptors, whereas
knock-down of b-arrestin2 or b-arrestin1 using siRNA had no
effect on ERK1/2 activation. This result is in good agreement
with our previous observation for the HCA2-mediated activa-
tion of the ERK1/2 pathway (Li et al., 2010).
Our current results have led us to propose a model for the
regulation of HCA3-mediated ERK1/2 activation in CHO cells
that are stably transfected with HCA3receptors and in A431
cells that endogenously express HCA3receptors (Figure 9). In
response to agonists, activated HCA3 receptors induce the
dissociation of Gi proteins from Gbg-subunits, triggering the
(Ahn
et al., 2004b),
Figure 9
Schematic diagram of regulation of HCA3-induced ERK1/2 activation in A431 cells. In response to agonists, activated HCA3 receptors led to
dissociation of Giproteins from Gbg-subunits, triggering the PKC pathway to couple to ERK1/2 phosphorylation at early time points (?2 min), and
the MMP/EGFR transactivation pathway with a maximum response at 5 min.
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1770 British Journal of Pharmacology (2012) 166 1756–1773