Original Paper
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Acta Biochim Biophys
Sin 2008, 40: 311-318
doi:10.1111/j.1745-7270.2008.00403.x
Apelin-13 induces ERK1/2 but
not p38 MAPK activation through coupling of the human apelin receptor to the Gi2
pathway
Bo Bai1#*,
Jiyou Tang2#*, Haiqing Liu1,
Jing Chen1, Yalin Li1, and Wengang Song1
1 Department of Neurobiology, Taishan Medical
College, Taian 271016, China
2 Department of Neurology,
Qianfoshan Hospital of Shandong, Jinan 250014, China
Received: November
11, 2007
Accepted: February
21, 2008
This work was supported
by grants from the Natural Science Foundation of Shandong Province (No.
Y2005C47), the Department of Science and Technology of Shandong Province (No.
2006GG2202015), and the Natural Science Foundation of Taishan Medical College
(No. 2007QNZR022)
#These authors
contributed equally to this work
*Corresponding
authors:
Bo Bai: Tel,
86-538-6229908; Fax, 86-538-6229953; E-mail, [email protected]
Jiyou Tang: Tel,
86-531-89268106; Fax, 86-531-82963647; E-mail, [email protected]
Apelin
signaling to the family of mitogen-activated protein kinases (MAPKs), such as
extracellular-regulated kinases 1/2 (ERK1/2) and p38 MAPK, through the coupling
of apelin receptor (APJ) to G-protein, mediates important pathophysiological
responses. Although apelin fragments have been reported to induce ERK1/2
activation through Gi-protein, the
intracellular pathways by which APJ activates these MAPKs are only partially
understood. Here, using stably transfected human embryonic kidney 293 (HEK293)
cells overexpressing human APJ (HEK293-apelinR), we showed that apelin-13
signaling leads to ERK1/2 and p38 MAPK pathways through APJ activation. It was
found in HEK293-apelinR cells that ERK1/2 activation was initiated by apelin-13
at 5 min, with the peak of activation occurring at 15 min, and a return to the
basal level within 60 min. The activation of ERK1/2 appeared to be
dose-dependent with a significant activation being observed at 10 nM apelin-13
and maximal activation at 100 nM. However, phosphorylated-p38 MAPK was not detected
in HEK293-apelinR cells treated with apelin-13. We also shown that the
apelin-13-induced ERK1/2 activation requires a coupling with pertussis
toxin-sensitive G-protein, and that overexpression of dominant-negative Gi2 completely inhibits the apelin-13-induced ERK1/2
activation. In addition, treatment with apelin-13 resulted in a
concentration-dependent reduction of forskolin-stimulated cAMP production. It
is therefore suggested that apelin-13 activates ERK1/2 but not p38 MAPK, which
involves the coupling of APJ to the Gi2 cascade. In
conclusion, the ERK1/2, but not p38 MAPK pathway is activated by apelin-13
through coupling of human APJ to Gi2-protein,
which contributes to cellular responses.
Keywords apelin receptor; apelin-13; ERK1/2; p38 MAPK; G-protein
Apelin, a novel endogenous peptide from bovine stomach tissue
extracts discovered in 1998, orchestrates its functions by binding and activating
the 7-transmembrane G-protein-coupled receptor, apelin receptor (APJ)
[1,2]. Apelin and its receptor have been shown to be expressed not only in the
central nervous system, but also in peripheral tissues and cells, such as
stomach, gastrointestinal tract, heart, kidney, adipose, lung, and endothelial
cells [3–6]. Apelin is originally synthesized as a 77 amino acid
prepropeptide that is cleaved into biologically active C-terminal fragments of
various sizes [5]. Apelin peptides containing 13 (65–77), 17 (61–77), and 36 (42–77) amino acids
(carboxyl-terminal peptides) have been isolated in vivo, and all
of them bind to APJ and activate the second messenger signaling cascades [1].
Apelin is highly conserved among different species, and the C-terminal 13 amino
acids (65–77) are completely conserved across all species studied [5],
indicating the paramount biological importance of this peptide, apelin-13 [7].
Apelin-36 and shorter C-terminal sequences have different potencies and
efficacies in regulating these functions. Shorter sequences, especially
apelin-13, are potent regulators of cardiovascular function, whereas longer
peptides such as apelin-36 are more effective in inhibiting HIV infection by
blocking the HIV co-receptor APJ. In addition to the control of water and food
intake, apelin plays an important role in the central and peripheral regulation
of the cardiovascular system, the release of hormones and neuropeptides, as
well as in the modulation of immune function [3,8–10]. Recent studies have shown
that the endogenous peptide apelin is crucial to maintaining
cardiac contractility in pressure overload and aging [11].Recently, studies have indicated that apelin is able to trigger
intracellular signaling cascades through APJ activation in transfected Chinese
hamster ovary (CHO) cells expressing APJ, and in neural cells [2,12,13]. One of
the central signaling molecules activated by APJ is the family of
mitogen-activated protein kinases (MAPKs). Activation of MAPKs, in particular
extracellular-regulated kinases (ERK1/2), has been implicated in mediating
important pathophysiological processes, such as control of endothelial cell
proliferation [13]. However, little is known about the activation of p38 MAPK
by apelin, which also participates in the regulation of cell proliferation and
differentiation as well as in apoptosis. Further studies have shown that apelin-36 and apelin-13 activate
ERK1/2 and inhibit forskolin-stimulated cAMP production through a pertussis
toxin (PTX)-sensitive G-protein in CHO cells expressing APJ [12], suggesting
that APJ might preferentially couple to Gi-protein.
Furthermore, apelin-induced ERK1/2 activation is not mediated by the Gbg subunits and is protein kinase C (PKC)-dependent and
Ras-independent. However, the intracellular transduction pathways that provide
the molecular link between activation of APJ by apelin fragments and their
biological responses at the cellular level remain to be clarified. In this study, we focused on the coupling of human APJ to G-protein
leading to ERK1/2 and p38 MAPK activation in human embryonic kidney 293
(HEK293) cells overexpressing human APJ (HEK293-apelinR). Our results revealed
that apelin-13 can activate the ERK1/2 pathway and inhibit forskolin-induced
intracellular cAMP production through a PTX-sensitive G-protein, but does not
activate the p38 MAPK pathway. The properties of apelin-13 in the activation of
MAPKs might be involved in the modulation of different biological responses.
Materials and Methods
Materials
Human apelin-13 was obtained from Phoenix Pharmaceuticals (Belmont,
USA). The amino acid sequence of apelin-13 is
pGlu-Arg-Pro-Arg-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe. Lipofectamine 2000 was
obtained from Invitrogen (Grand Island, USA). Human G-protein a i2
[dominant-negative (Q205L/D273N) Gi2] was obtained from the
UMR cDNA Resource Centre, University of Missouri-Rolla (Rolla, USA). Forskolin,
3-isobutyl-1-methylxanthine, and PTX were obtained from Sigma (St. Louis, USA).
Polyvinylidene difluoride membranes and enhanced chemiluminescence (ECL) plus
Western blotting detection reagents were purchased from Amersham Biosciences
(Little Chalfont, UK). APJ antibody was purchased from Abcam (Cambridge, UK).
Anti-phospho-ERK1/2 (Thr202/Tyr204) antibody, anti-ERK1/2 antibody,
anti-phospho-p38 MAPK (Thr180/Tyr182) antibody, and anti-p38 MAPK antibody were
purchased from Cell Signaling Technology (Danvers, USA).
Total RNA isolation and RT-PCR
To amplify the full-length human APJ cDNA, total RNA was extracted
from human brain using the SV total RNA isolation kit (Promega, Madison, USA)
and reverse-transcribed into cDNA. First-strand cDNA synthesis was carried out
using Moloney murine leukemia virus reverse transcriptase and random hexamers
as primers. The set of primers for the amplification of APJ was: sense, 5-CCGGAATTCATGGAGGAAGGTGGTGATTTTG-3;
and antisense, 5-CCGCTCGAGCTAGTCAACCACAAGGGTCTC-3 (GenBank accession
No. NM005161). The 5-ends of the APJ sense and antisense primers were designed
with the cleavage sites by the restriction enzymes of EcoRI and XhoI
(Invitrogen), respectively. PCR was carried out using a Taq DNA
polymerase kit (MBI Fermentas, Hanover, USA). The reaction mixture (10 PCR
buffer, 10 mM dNTP, 25 mM MgCl2, 200 ng/ml specific
primer, cDNA, and Taq DNA polymerase) was placed in a thermal cycler to
start PCR at 95 ?C (45 s), 56 ?C (45 s), and 72 ?C (1.5 min) for a total of 40
cycles, with a final extension step at 72 ?C for 10 min. The PCR product was
purified using QIAquick gel extraction kit (Qiagen, Crawley, UK) for cloning.
Construction of human APJ with
pcDNA3.1(-)
To create the recombinant pcDNA3.1(+)-apelinR plasmid, the PCR
product and pcDNA3.1(+) plasmid were digested with EcoRI and XhoI,
followed by purification using agarose gel and the QIAquick gel extraction kit.
The APJ cDNA was inserted between the EcoRI and XhoI sites of
pcDNA3.1(+), and subsequently cloned as described previously [14]. In order to
identify the construct, the recombinant plasmid was digested with two
restriction enzymes and sequenced. The sequence data were analyzed using Blast
Nucleic Acid Database searches from the National Centre for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/).
To create the recombinant pcDNA3.1(+)-apelinR plasmid, the PCR
product and pcDNA3.1(+) plasmid were digested with EcoRI and XhoI,
followed by purification using agarose gel and the QIAquick gel extraction kit.
The APJ cDNA was inserted between the EcoRI and XhoI sites of
pcDNA3.1(+), and subsequently cloned as described previously [14]. In order to
identify the construct, the recombinant plasmid was digested with two
restriction enzymes and sequenced. The sequence data were analyzed using Blast
Nucleic Acid Database searches from the National Centre for Biotechnology
Information (http://www.ncbi.nlm.nih.gov/).
Stable transfection of HEK293
cells
HEK293 cells were grown in culture medium containing high-glucose
Dulbecco’s modified Eagle’s) medium (DMEM; Gibco BRL, Paisley, UK), 10% heat
inactivated fetal calf serum, 200 U/ml penicillin, and 200 mg/ml
streptomycin incubated at 37 ?C in 5% CO2. The
cells were seeded in 25 cm2 flasks until 50%–70% confluence
and washed with fresh medium to remove any antibiotics. Ten micrograms of
pcDNA3.1(+)-apelinR plasmid with 10 ml Lipofectamine 2000 reagent in Opti-MEM
medium containing GlutaMax (Invitrogen) was added to HEK293 cells and incubated
overnight at 37 ?C. After incubation for 18 h, the transfection mixture was
replaced by DMEM. For generation of cell lines stably expressing APJ (HEK293-apelinR),
the transfected cells were cultured in DMEM in the presence of G418 (0.5 mg/ml)
(Gibco BRL). The surviving cells were subcultured in the selective process for
8 weeks and used for all subsequent experiments.
Confocal microscopy for APJ
expression in HEK293-apelinR cells
HEK293-apelinR cells, grown on poly-L-lysine pretreated coverslips
until they reached 70%–80% confluence, were fixed in 4% paraformaldehyde in
phosphate-buffered saline (PBS) for 30 min at room temperature. Non-specific
binding was blocked by incubating cells with 3% bovine serum albumin in
PBS-Triton X-100 (0.01%) for 1 h at room temperature. Cells were washed three
times with PBS-Triton X-100 (0.01%) then incubated overnight with anti-APJ
monoclonal antibody (1:100) in PBS-Triton X-100 (0.01%) at 4 ?C. Cells were
washed as before with PBS-Triton X-100 (0.01%), subsequently incubated in the
dark for 1 h at room temperature with a 1:400 dilution of Alexa Fluor
633-conjugated goat anti-mouse IgG (Molecular Probes, Eugene, USA). After three
washes for 5 min in PBS-Triton X-100 (0.01%), cells were mounted in Vectashield
mounting medium (Vector Laboratories, Burlingame, USA) with
4-6-diamidino-2-phenylindole (Vector Laboratories, Peterborough, UK) on
microscope slides and observed under an oil immersion objective (63) using a
Leica model DMRE laser-scanning confocal microscope (Leica, Heidelberg,
Germany).
Measurement of intracellular
cAMP production
HEK293-apelinR cells cultured in 12-well plates coated with 10 mg/ml
poly-L-lysine were pre-incubated with stimulation buffer including DMEM, 500 mM
3-isobutyl-1-methylxanthine, and 10 mM MgCl2 at 37
?C for 20 min. Cells were then stimulated with stimulation buffer containing
forskolin (10 mM) in the absence or presence of various concentrations of apelin-13
(0.01–1000 nM) at 37 ?C for 15 min. The cAMP level was determined using a
cyclic AMP assay kit (R&D Systems, Minneapolis, USA) according to the
manufacturer’s instructions. Determination of optical density at 405 nm for
each treatment was carried out immediately on a microplate reader with
wavelength correction set to 570 nm.
SDS-PAGE and immunoblotting
Ten micrograms of cell lysate was separated by 10% SDS-PAGE, and then
transferred onto polyvinylidene difluoride membranes at 100 V for 1 h.
Non-specific binding was blocked by incubating membranes for 1 h in blocking
buffer [5% (W/V) non-fat milk in 200 mM NaCl, 50 mM Tris-HCl (pH
7.4), and 0.1% (V/V) Tris-buffered saline Tween-20 (TBST)] at
room temperature. The membranes were incubated overnight with a 1:1000 dilution
of phospho-ERK1/2 and p38 MAPK antibodies in 5% (W/V) bovine
serum albumin and TBST (0.1%) at 4 ?C. Phospho-ERK1/2 and phospho-p38 MAPK were
detected using rabbit polyclonal antibodies. Membranes were subsequently washed
four times in 1?BST for 10 min, then incubated with a 1:2000 dilution of horseradish
peroxidase-conjugated goat anti-rabbit immunoglobulins in 5% (W/V)
non-fat milk in TBST for 1 h at room temperature, followed by washing four
times with 1?BST as
previously described. To detect activation of ERK1/2 and p38 MAPK, ECL reagents
(Amersham Biosciences) were applied to generate light when the ECL system
reacted with the horseradish peroxidase. After immersion into ECL solution for
5 min, the membranes were drained of excess solution and exposed to X-ray film
in the dark room. The film was scanned and the bands were analyzed by
densitometry using Scion Image (http://www.scioncorp.com).
For standardization, the same membranes were submerged in stripping buffer
[final concentration: 2% SDS, 62.5 mM Tris-HCl (pH 6.8), and 100 mM b-mercaptoethanol]
and incubated at 50 ?C for 30 min, followed by two washes with TBST at room
temperature. The membranes were blocked with 5% non-fat dried milk in TBST for
1 h at room temperature then reprobed with total ERK1/2 and p38 MAPK antibodies
as described above.
Statistical analysis
All quantitative data are presented as the mean±SEM. Statistical
significance was evaluated using Student’s t-test. In the case of
multiple group comparisons, ANOVA
was adopted. P<0.05 was used for consideration of significance of the findings.
Results
Amplification and cloning of human APJ
To obtain the human full-length APJ cDNA, total RNA from human brain
was reverse-transcribed and amplified with specific primers for human APJ using
RT-PCR. As shown in Fig. 1(A), the band between 1 kb and 1.5 kb (marker)
was shown to correspond to the size of 1143 bp APJ. The cloned recombinant cDNA
was digested with two restriction enzymes, EcoRI and XhoI. As
shown in Fig. 1(B), two bands were observed from the digested product in
1% agarose gel electrophoresis, one of them between 5.0 kb and 6.0 kb
representing pcDNA3.1(+) plasmid, the other corresponding to the size of
full-length APJ (1143 bp). The sequence data for APJ appeared to be identical
to that of NM005161 (GenBank) in the Blast Nucleic Acid Database.
Localization of APJ in
HEK293-apelinR cells
As shown in Fig. 2, immunofluorescence confocal microscopy
indicated that the heterologous protein expression of APJ in HEK293-apelinR
cells was exclusively localized to the cellular membrane (red). However, APJ
was not detected in non-transfected HEK293 cells, nor in cells transfected with
empty pcDNA3.1(+) plasmid (data not shown). As expected, APJ is indeed a
membrane protein properly expressed and localized on the surface of HEK293-apelinR
cells.
Characteristics of
apelin-13-induced ERK1/2 activation in HEK293-apelinR cells
In order to characterize ERK1/2 activation involved in human APJ,
HEK293-apelinR cells were treated with 100 nM apelin-13 for 5–60 min and stimulated
with different concentrations of apelin-13 (0.01–1000 nM) for 15 min. The
results showed that a significant activation of ERK1/2 by apelin-13 was seen
after 5 min, with maximal activation at 15 min, and a return to the basal level
after 60 min [Fig. 3(A)]. We also observed that treatment of
HEK293-apelinR cells with different concentrations of apelin-13 for 15 min led
to a dose-dependent increase of ERK1/2 activation, with a significant response
to apelin-13 from a concentration of 10 nM, reaching maximal activation at 100
nM, with an EC50 value of 2.8 nM [Fig. 3(B)]. It is
therefore suggested that the ERK1/2 pathway can be rapidly activated by
apelin-13, and that ERK1/2 activation tends to decline at concentrations higher
than 1000 nM.
Apelin-13 does not induce p38
MAPK activation in HEK293-apelinR cells
Given that apelin-13 can activate ERK1/2, it is interesting to
determine whether apelin-13 could activate the p38 MAPK pathway. The results
indicated that p38 MAPK was not activated by treatment with 100 nM apelin-13
within 60 min in HEK293-apelinR cells, although treatment with thrombin (0.1
U/ml) for 30 min resulted in remarkable p38 MAPK activation in the same cell
line [Fig. 4(A)]. After stimulation with different concentrations of
apelin-13, as indicated in Fig. 4(B), for 10 min, the activation of p38
MAPK was not observed in HEK293-apelinR cells, suggesting that apelin-13 does
not induce activation of the p38 MAPK pathway through APJ in the HEK293 cell
line.
Apelin-13 inhibits
forskolin-stimulated cAMP production through Gi-protein
To determine the extent of adenylyl cyclase inhibition elicited by
the different concentrations of apelin-13, we observed the effect of apelin-13
on forskolin-induced cAMP production in HEK293-apelinR cells. As shown in Fig.
5, the forskolin-induced cAMP production was significantly reduced when
apelin-13 was added at 1 nM concentration. In addition, it was found that the
inhibition of forskolin-stimulated cAMP production appeared to be
dose-dependent, with saturation and maximum at 100 nM apelin-13 (EC50=3.43 nM), indicating that the inhibition of adenylyl cyclase might
require the coupling of APJ to Gi.
Apelin-13 induces ERK1/2
activation through coupling of APJ to Gi2
In view of these results, we next asked whether apelin-13 activates
ERK1/2 activation through coupling of APJ to Gi2
identical to that of inhibiting adenylyl cyclase. First, using
dominant-negative techniques, we observed that the apelin-13-induced activation
of ERK1/2 was completely inhibited by dominant-negative Gi2 in HEK293-apelinR cells [Fig. 6(A)], consistent with the
inhibition of forskolin-stimulated cAMP production by apelin-13. Second,
pretreatment of HEK293-apelinR cells with PTX for 6 h or 12 h, which
selectively deactivated Gi/o-protein through ADP
ribosylation to prevent its interaction with the receptor, mostly abrogated the
apelin-13-induced ERK1/2 activation [Fig. 6(B)], indicating that APJ
preferentially couples to a PTX-sensitive G-protein. Taken together, it was
suggested that the Gi2 pathway plays a predominant role in
apelin-13-induced ERK1/2 activation.
Discussion
In the present study, we cloned the human APJ gene and
established a stable HEK293 cell line (HEK293-apelinR) expressing human APJ. It
was found that human APJ is intensely expressed at the plasma membrane of
HEK293-apelinR cells. Thus, this cell line can be used to investigate the
profiles of intracellular signal transduction of APJ-mediated MAPK activation.
We showed that apelin-13 is able to induce, in a dose-dependent manner, the
activation of ERK1/2, but not p38 MAPK, through human APJ coupling to the Gi2 pathway in HEK293-apelinR cells. This is the first study to provide
evidence that the p38 MAPK pathway might not be activated by apelin-13 while
the peak of ERK1/2 activation by apelin-13 occurs. As activation of ERK1/2 is
related to cell survival and p38 MAPK is associated with induction of apoptosis
[15,16], we speculate that the ERK1/2 and p38 MAPK pathways might be regulated
differently by APJ, which contributes to different physiological responses to
apelin during the cycle of cellular processes.Previous studies have shown that in CHO cells expressing the murine
APJ, ERK1/2 can be activated by apelin-13 and apelin-36 through Gi1 and Gi2 proteins [13]. In order to further shed
light on the intracellular signal cascade from apelin-13 to MAPKs, the human
G-protein dominant-negative technique was used in this study. Dominant-negative
Gi2, which derives from the Q205L and D273N double mutations of human
G-protein alpha i2 subunit, is conferred a preference for xanthine but not
guanine nucleotide binding. It was reported that GoaQ205L/D273N was regulated
by xanthine, not by guanine nucleotides, and bound xanthine diphosphate and
xanthine triphosphate instead of guanine diphosphate or guanine triphosphate
[17]. It has been shown that GoaQ205L/D273N protein retained the receptor binding specificity of the
wild-type Goa and was able to interact with Go-coupled receptors in transfected
COS-7 cells. However, in the absence of xanthine triphosphate, GoaQ205L/D273N did
not dissociate from the receptors and thus inhibited their activities. Because
cells lack xanthine nucleotides, Gi2Q205L/D273N might act as
a dominant-negative mutant to disrupt Gi2-coupled
receptor signal transduction pathways. Our results showed that the
apelin-13-induced ERK1/2 activation is completely blocked by dominant-negative
Gi2 in HEK293 cells expressing human APJ. It is further supported by
our study that the activation of ERK1/2 is abrogated by PTX, and that apelin-13
attenuates forskolin-stimulated cAMP production through inhibition of adenylyl
cyclase by the coupling of APJ to Gi. These findings suggest
that the Gi2 pathway plays a crucial role in signal transduction from the
stimulation of APJ to the intracellular kinase cascades. Similar to the d-opioid receptor
[18], APJ-mediated ERK1/2 activation requires Gi-protein
and does not involve Gbg and Ras in CHO cells [12], although other G-protein-coupled
receptors, like a2A-adrenergic receptor coupling to Gi,
mediate ERK1/2 activation through the Gbg/Src/Ras cascade in HEK293
cells [19]. However, apelin-13-induced ERK1/2 activation is reduced by the PKC
inhibitor [12], suggesting the involvement of PKC. In light of these observations,
it is tempting to speculate that the apelin-13 signal to ERK1/2 through APJ
might be tissue-specific and involves multiple signaling cascades through
coupling to Ga subunits.In addition to the APJ-mediated ERK1/2 activation, we also observed that
apelin-13 slightly, but not significantly, decreased basal cAMP levels without
forskolin at a concentration of 0.01–1000 nM in HEK293-apelinR cells. This prompted
us to guess whether the activation of ERK1/2 by apelin-13 is affected by the
change of cAMP. cAMP has already been reported to have either a stimulatory
effect through B-Raf or an inhibitory effect through c-Raf-1 on ERK1/2
activation, depending on the cell types [20]. It is therefore possible that in
HEK293 cells expressing both B-Raf and c-Raf-1, activation of ERK1/2 is
modulated by the mechanisms of stimulation and inhibition of cAMP [20,21].
Additionally, in CHO cells expressing the human dopaminergic D2 receptor, the Goa-induced ERK1/2 activation through Ras-independent and PKC- and
phosphatidylinositol-3 kinase-dependent activation of B-Raf pathways is greatly
enhanced by the Gi/Gbg/Ras/Raf-1 cascade [22]. These speculations
that the apelin signal from Ga-protein subunits to the ERK1/2 cascade is mediated through
activation of B-Raf or/and c-Raf-1 remains to be clarified in our future study.Interestingly, apelin-13 at 100 nM can induce maximal activation of
ERK1/2, whereas the activation of p38 MAPK was not seen at the same time points
in response to 100 nM apelin-13 in HEK293-apelinR cells. It is in agreement
with the study in human osteoblasts that apelin-13 does not result in
activation of p38 MAPK within 60 min [23]. This raises the possibility that the
coupling of APJ to Gi2 does not activate the upstream effectors of
p38 MAPK pathway, although APJ can mediate ERK1/2 activation via Gi2 cascade. Previous studies have shown that p38 MAPK can be activated
by muscarinic acetylcholine receptors and b-adrenergic receptor
through coupling to Gq, Gi, and Gbg subunits in
HEK293 cells [24,25], and by adenosine A2B receptor
through coupling to the Gs/AC/cAMP/PKA pathway in CHO
cells [26], which is dependent on the expression of receptor and cells.
Therefore, we speculate that the coupling of APJ to Gi2, but
not to Gbg, Gq, or Gs, does
not target the signaling molecules to p38 MAPK activation. It is suggested that
the activation of different signaling pathways is tissue- or/and
receptor-specific, and that the regulation of APJ for MAPKs might contribute to
different physiological responses.In conclusion, we showed that apelin-13
induces ERK1/2 activation through human APJ in a time-dependent and
dose-dependent manner, and involves the Gi2-dependent
pathway. However, p38 MAPK is not activated upon stimulation by apelin-13 when
a maximal activation of ERK1/2 occurs. These properties of apelin-13 for ERK1/2
and p38 MAPK activation could be useful for designing specific pharmacological
tools to treat the pathological dysfunctions caused by lack of apelin
signaling.
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