Here, we demonstrate that the b-AR agonist isoproterenol potently evokes the tyrosine phosphorylation of STAT3 and increases its transcriptional activity in an extracellularly regulated
Trang 1b-adrenergic receptor-induced atrial natriuretic
factor expression in cardiomyocytes
Hui Zhang, Wei Feng, Wenqiang Liao, Xiaowei Ma, Qide Han and Youyi Zhang
Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing, China
b-Adrenergic receptor (b-AR), an archetypal member
of the G-protein-coupled receptor (GPCR)
super-family, has roles in a variety of cardiovascular
patho-logical and physiopatho-logical processes There are three
known subtypes in the heart – b1-AR, b2-AR and
b3-AR Of these, the b1-AR subtype stimulates the
classic Gs–adenylyl cyclase–cAMP–protein kinase A
signaling pathway, whereas b2-AR activates bifurcated
signaling pathways through both Gs and Gi proteins
[1,2] It is well established that stimulation of
myo-cardial b-AR results in cardiac remodeling, which is
characterized by increased cell size and initiation of
the ‘fetal gene’ program, such as atrial natriuretic
factor (ANF) [3] Interestingly, there is increasing
evidence that increased ANF expression may act not
only as a characteristic of cardiac overload and the
resulting myocardial remodeling, but also as a crucial
cardioprotective signal in response to extracellular stress [4,5] Although previous data indicated that b-AR can induce ANF expression via the Akt-GSK3b pathway [6], the precise molecular mechanism
by which b-AR regulates ANF expression is still elusive
As one of the critical cardiac transcriptional factors, signal transducers and activators of transcription 3 (STAT3) is a key mediator of cardiac remodeling in response to many stimuli, such as growth factors, cyto-kines [particularly those of the glycoprotein (gp)130 family, including interleukin-6 (IL-6) and leukemia inhibitory factor (LIF)], and ligands for several mem-bers of the GPCR family, including the type I angio-tensin II receptor [7–9]
A previous study showed that transfection of mutated-type STAT3 cDNA attenuated LIF-stimulated
Keywords
ANF; b-adrenergic receptor; cardiomyocytes;
gp130; STAT3
Correspondence
Y Zhang, Institute of Vascular Medicine,
Peking University Third Hospital, Beijing
100083, China
Fax: +86 10 62361450
Tel: +86 10 82802306
E-mail: zhangyy@bjmu.edu.cn
(Received 31 March 2008, revised 8 May
2008, accepted 13 May 2008)
doi:10.1111/j.1742-4658.2008.06504.x
b-Adrenergic receptor (b-AR)-induced cardiac remodeling is closely linked with the re-expression of the atrial natriuretic factor (ANF) gene How-ever, the exact molecular mechanism of this response remains elusive Here, we demonstrate that the b-AR agonist isoproterenol potently evokes the tyrosine phosphorylation of STAT3 and increases its transcriptional activity in an extracellularly regulated kinase 1⁄ 2 and glycoprotein (gp)130 signaling-dependent manner in rat cardiomyocytes Interestingly, both specific silencing of signal transducers and activators of transcription 3 (STAT3) expression by lentivirus-mediated RNA interference and anta-gonism of gp130 signaling lead to significant inhibition of isoproterenol-stimulated ANF expression Together, these results indicate that gp130⁄ STAT3 signaling has an essential role in ANF expression by b-AR stimulation
Abbreviations
ANF, atrial natriuretic factor; b-AR, b-adrenergic receptor; ERK, extracellularly regulated kinase; GFP, green fluorescent protein; gp,
glycoprotein; GPCR, G-protein-coupled receptor; IL-6, interleukin-6; ISO, isoproterenol; JAK, Janus kinase; JNK, Jun N-terminal kinase; LIF, leukemia inhibitory factor; MAPK, mitogen-activated protein kinase; MOI, multiplicity of infection; NRCM, neonatal rat cardiomyocyte; NS, nonsilencing; qRT-PCR, quantitative real-time RT-PCR; shRNA, short hairpin RNA; siRNA, small interfering RNA; STAT3, signal transducers and activators of transcription 3.
Trang 2ANF expression in cardiomyocytes [10] By contrast,
although STAT3 could associate with the endogenous
ANF gene in type I angiotensin II receptor-activated
cardiomyocytes, no modulation of ANF promoter
activity by STAT3 with angiotensin II stimulation was
observed [11] These reports revealed that regulation of
ANF is a specific, stimulus-dependent process In
addi-tion, except for the report mentioned above, few studies
have investigated the regulatory effects of STAT3 on
ANF expression in the context of GPCR activation
Therefore, in the present study, we investigated whether
STAT3 might play a role in b-AR-induced ANF
expression
Results and Discussion
Activation of b-AR induced delayed STAT3
tyrosine phosphorylation and increased its
transcriptional activity in neonatal rat
cardiomyocytes
We first examined the tyrosine phosphorylation of
STAT3 after stimulation with the b-AR agonist
iso-proterenol (ISO) in neonatal rat cardiomyocytes (NRCMs) ISO at 10 lm markedly induced STAT3 phosphorylation at tyrosine 705 (Fig 1A,B) As com-pared with the rapid LIF-stimulated activation of STAT3 within several minutes [10], ISO-induced STAT3 tyrosine phosphorylation was relatively delayed (it was apparent after 60 min; Fig 1A), indi-cating that b-AR may induce STAT3 activation in an indirect manner In addition, although STAT3 tyro-sine 705 phosphorylation is crucial for its transcrip-tional activity, phosphorylation at serine 727 is also required to achieve full transcriptional activity [12]
We thus investigated whether STAT3 serine 727 was also phosphorylated by b-AR Unlike many other GPCRs [13], b-AR induced only a slight increase in STAT3 serine 727 phosphorylation, and this increase was proven to have no statistical significance (data not shown) These results support the notion that STAT3 activation has high specificity under different stimula-tion condistimula-tions
To investigate whether ISO-induced STAT3 phos-phorylation is a specific action of b-AR, antagonists of b-AR were employed As shown in Fig 1B, the b-AR
Fig 1 Activation of b-AR induced STAT3 tyrosine phosphorylation and increased its transcriptional activity in NRCMs (A) NRCMs were serum-starved for 24 h, and then treated with 10 l M ISO The cell lysates were harvested at the indicated time and analyzed by western blot assays using anti-phospho-tyr705-STAT3 The same membranes were stripped and reprobed with total STAT3 antibody (n = 3) (B, C) Cardiomyocytes were serum-starved, treated with 10 l M ISO for 60 min after pretreatment with 10 l M propranolol, 5 l M CGP 20712A or
5 l M ICI 118551, and then harvested for western blot analysis (n = 3) **P < 0.01 versus control, # P < 0.05 versus ISO, ## P < 0.01 versus ISO (D) Cardiomyocytes were cotransfected with STAT3-driven promoter and renilla luciferase plasmid for 24 h, starved, and then stimu-lated with 10 l M ISO for 8 h with or without pretreatment with 10 l M propranolol The data were converted to relative luciferase activity.
*P < 0.05 versus control, # P < 0.05 versus ISO (n = 3) Prop, propranolol; CGP, CGP 20712A; ICI, ICI 118551.
Trang 3antagonist propranolol entirely abolished the STAT3
tyrosine phosphorylation, confirming the specific
action of b-AR Because ISO can stimulate both
b1-AR and b2-AR, we also investigated which subtype
mediated STAT3 tyrosine phosphorylation
CGP 20712A, a selective b1-AR antagonist, and
ICI 118551, a selective b2-AR antagonist, markedly
reduced the tyrosine phosphorylation of STAT3 by
about 60% and 100%, respectively (Fig 1C), which
indicated that both b1-AR and b2-AR are involved in
this response In addition, given that blockade of
b2-AR completely abolished the STAT3 tyrosine
phos-phorylation, whereas blockade of b1-AR still partially
inhibited the response, the two subtypes may have
synergistic effect in ISO-induced STAT3 activation
The STAT3 transcriptional activity was also
exam-ined by transfection of the STAT3-driven promoter
luciferase plasmid Consistent with the result of STAT3
tyrosine phosphorylation, ISO significantly increased
the transcriptional activity of STAT3, and this increase
was completely inhibited by propranolol, further
con-firming a b-AR-dependent mechanism (Fig 1D)
We previously demonstrated that intraperitoneal
injection of ISO caused delayed phosphorylation of
STAT3 in mouse heart [14] In the present study, we
found that b-AR stimulation in rat cardiomyocytes
per se is able to not only cause phosphorylation of
STAT3 on tyrosine but also promote its transcriptional
activity Despite different internal mechanisms due to
species differences, the two findings indicate an
exten-sive and striking interaction between b-AR and
STAT3 Furthermore, our finding strengthens the
implication, which has not been investigated before,
that STAT3 has an important role in the induction of
the specific cardiac phenotype of b-AR
Extracellularly regulated kinase (ERK)1⁄ 2 but not
p38 or Jun N-terminal kinase (JNK) played an
important role in b-AR-induced STAT3 activation
We further investigated the signaling pathway of
b-AR-induced STAT3 activation It is well known that b-AR
stimulation can activate the mitogen-activated protein
kinase (MAPK) signaling pathway [15,16] On the other
hand, the MAPK pathway has been shown to have an
important role in the regulation of STAT3 signaling
[17,18] We thus examined the potential effects of
MAP-Ks on b-AR-induced STAT3 activation The specific
kinase inhibitors SB203580, U0126 and SP600125 were
used to inhibit p38, ERK1⁄ 2 and JNK, respectively
U0126, an ERK1⁄ 2 inhibitor, significantly inhibited
ISO-induced STAT3 tyrosine phosphorylation as well
as its transcriptional activity, whereas neither p38 nor
JNK inhibition affected these processes (Fig 2A,B) These results indicate an important role of ERK1⁄ 2, but not p38 or JNK, in b-AR-induced STAT3 activa-tion However, as ERK1⁄ 2 is a serine ⁄ threonine kinase, its effect on STAT3 activation, particularly the tyrosine phosphorylation, is probably an indirect action
gp130 family cytokines were involved in b-AR-induced STAT3 activation
Delayed STAT3 tyrosine phosphorylation and the indi-rect action of ERK1⁄ 2 indicate that ISO-stimulated
A
B
Fig 2 ERK1 ⁄ 2, but not p38 or JNK, mediated b-AR-induced STAT3 activation (A) Cardiomyocytes were stimulated with 10 l M ISO for
60 min after pretreatment with 10 l M SB203580, 10 l M SP600125
or 10 l M U0126 for 30 min The cell lysates were harvested and analyzed by western blot analysis **P < 0.01 versus control;
##
P < 0.01 versus ISO; NS, no statistical significance versus ISO (n = 4) (B) Cardiomyocytes were transfected with the STAT3-dri-ven promoter together with renilla luciferase plasmid, starved, and then stimulated with 10 l M ISO after pretreatment with 10 l M
SB203580, 10 l M SP600125 or 10 l M U0126 *P < 0.05 versus control; # P < 0.05 versus ISO; NS, no statistical significance versus ISO (n = 3) SB, SB203580; SP, SP600125.
Trang 4STAT3 activation is a complicated process We
specu-lated that some new secreted cytokines, particularly the
gp130 family cytokines, might contribute to the delayed
STAT3 activation Blockade of the gp130 receptor
using neutralizing antibody to gp130 markedly
inhibited STAT3 tyrosine phosphorylation and its
tran-scriptional activity with ISO stimulation (Fig 3A,B),
confirming that the STAT3 activation is
gp130-depen-dent Consistent with this, inhibition of new RNA
transcription by actinomycin D significantly suppressed
ISO-induced STAT3 tyrosine phosphorylation
(Fig 3C), supporting the requirement for a de novo
transcriptional process In addition, because gp130, as
the coreceptor of gp130 family cytokines, promotes the
recruitment and activation of JAK (Janus kinase)
tyro-sine kinases, thereby activating STAT3 [7], the
poten-tial role of JAK was also determined As shown in
Fig 3D, a specific JAK2 inhibitor, AG490,
dramati-cally attenuated b-AR-induced STAT3 activation
Taken together, these results indicate that autocrine
production of gp130 cytokines is required for STAT3
activation by b-AR
We next explored the possible involvement of IL-6
in STAT3 activation by b-AR, as IL-6 is a typical
gp130 cytokine that activates STAT3 and is stimulated
by many GPCRs, including b-AR [19] The expression
of IL-6 mRNA was monitored by quantitative
real-time RT-PCR (qRT-PCR) Unexpectedly, the IL-6
mRNA levels showed no significant difference with or
without ISO stimulation, at either 1 h or 3 h
(supple-mentary Fig S1) As qRT-PCR could not exclude the
possibility of release of pre-existing IL-6, we further
monitored the secretion of IL-6 protein by ELISA ISO could not increase IL-6 protein production as compared with control within 3 h in the medium of cultured cardiomyocytes (supplementary Fig S2) Therefore, there may be some other, as yet unidenti-fied, cytokines involved in the delayed STAT3 activa-tion by b-AR
With regard to the relationship between the ERK1⁄ 2 and gp130 pathways, given the rapid and transient activation of ERK1⁄ 2 induced by b-AR in NRCMs [20] and the relatively long duration required for cytokine secretion, ERK1⁄ 2 may act upstream of the gp130 signaling pathway in STAT3 activation Consistent with our hypothesis, ERK1⁄ 2 is required for the production of many cytokines in a wide variety
of cells [21–23]
The gp130⁄ STAT3 signaling pathway mediated b-AR-induced ANF expression
b-AR-induced cardiac remodeling is closely linked to ANF expression To determine the role of activated STAT3 in ANF expression resulting from b-AR stimu-lation, we constructed a lentiviral vector derived from HIV-1 to express short hairpin RNA (shRNA) dire-cted against rat STAT3 (rST3 lentivirus) Fluorescent microscopy analysis showed that the transfection effi-ciency was more than 90% in cardiomyocytes, with a multiplicity of infection (MOI) of 150 [The lentiviral infection efficiency was visualized by the expression of green fluorescent protein (GFP), the gene for which is
a marker gene contained within the lentiviral vector.)
A
∗
#
B
C
∗∗
##
D
Fig 3 gp130 family cytokines were
involved in b-AR-induced STAT3 activation.
(A, C) Cardiomyocytes were stimulated with
10 l M ISO for 60 min after pretreatment
with 4 lgÆmL)1gp130 neutralizing antibody
or 6 lgÆmL)1actinomycin D for 30 min The
cell lysates were harvested and analyzed by
western blot analysis (n = 3) (B, D)
Cardio-myocytes were transfected with the
STAT3-driven promoter together with renilla
lucifer-ase plasmid, starved, and then stimulated
with 10 l M ISO after pretreatment with
4 lgÆmL)1gp130 neutralizing antibody or
10 l M AG490 *P < 0.05, **P < 0.01 versus
control, # P < 0.05, ## P < 0.01 versus ISO.
gp130Ab, gp130 neutralizing antibody; ActD,
actinomycin D.
Trang 5The specific and effective silencing of endogenous rat
STAT3 by rST3 lentivirus was also confirmed by
wes-tern blot analysis (Fig 4A) ANF transcription was
examined by cotransfection of the ANF promoter (a
luciferase reporter plasmid containing the ANF
pro-moter region) and lentivirus As compared with
nonsi-lencing (NS) lentivirus, rST3 lentivirus markedly
suppressed the b-AR-promoted ANF promoter
tran-scriptional activity (Fig 4B) In addition, we also
examined the levels of endogenous ANF mRNA using
qRT-PCR The result showed similar trends to that of
the ANF promoter luciferase reporter assay
(supple-mentary Fig S3) On the other hand, we also assessed
the ANF protein expression by ELISA Consistent
with the result of ANF transcription, knock-down of
STAT3 significantly inhibited ISO-induced ANF
pro-tein production in the NRCM culture medium
(Fig 4C)
The involvement of STAT3 in b-AR-induced ANF
expression was further supported by investigation of
the instant upstream signaling of STAT3, in that both
inhibition of gp130 and inhibition of JAK2 markedly
suppressed b-AR-promoted ANF promoter
transcrip-tional activity (Fig 5A,B) It is thus easy to infer that gp130⁄ STAT3 signaling acts virtually as an integral pathway in b-AR-induced ANF expression
Both STAT3 and ANF are important in cardiac remodeling and cardioprotection [4,5,24,25] However, there are only a few reports describing their relation-ships, and few of these are in the context of GPCRs
In the present study, we unequivocally demonstrated that STAT3 activation is required for ANF expression with b-AR stimulation Given that STAT3 can associ-ate with the endogenous ANF gene in vitro [11], its effects on b-AR-induced ANF expression may result from direct transcriptional regulation STAT3 may also act in indirect ways on ANF expression For instance, it may act as a coactivator of other transcrip-tional factors or act by regulating intermediate genes; however, both of these possibilities need further inves-tigation
Conclusion
Taken together, our results provide a new molecular basis for determination of the involvement of the
B
A
C
Fig 4 STAT3-specific lentivirus-mediated RNA interference inhibited b-AR-induced ANF expression (A) Cardiomyocytes were infected with rST3 lentivirus (rST3-lenti or rST3) or NS lentivirus (NS-lenti or NS) at an MOI of 150 for 3 days, and then subjected to fluorescence assays and western blot assays using antibody to STAT3 and antibody to eIF5 (n = 3) The lentiviral infection efficiency was visualized by the expression of a GFP gene, which is a marker gene contained within the lentiviral vector (B) Cardiomyocytes were cotransfected with the ANF promoter (a luciferase reporter plasmid containing the ANF promoter region) and the renilla luciferase plasmid, infected with the rST3 lentivirus or NS lentivirus at an MOI of 150 for 3 days, starved, and then stimulated with 10 l M ISO for 24 h before analysis by the lucifer-ase activity assay *P < 0.05, NS + ISO versus NS; # P < 0.05, rST3 + ISO versus NS + ISO (n = 4) (C) Cardiomyocytes were infected with lentivirus, starved, and then stimulated with 10 l M ISO for 48 h The supernatant concentration of ANF protein was assayed by ELISA.
*P < 0.05, NS + ISO versus NS; # P < 0.05, rST3 + ISO versus NS + ISO (n = 4).
Trang 6gp130⁄ STAT3 signaling pathway in b-AR-stimulated
ANF transcriptional expression
As ANF transcription is often activated in many
types of cardiac hypertrophy and remodeling, the
results of this investigation can be compared with
those studies investigating the regulation of ANF
expression by GPCRs and cytokines On the other
hand, although ANF is a marker of hypertrophy,
sepa-ration of re-emergence of ANF from cardiac growth
has been recently demonstrated by some researchers
[11,26] Thus, understanding how ANF is regulated
may lead to therapeutic strategies that prevent
hyper-trophy while allowing for the beneficial effects of ANF
production Furthermore, this study will further our
understanding of b-AR signaling and provide potential
therapeutic targets for the treatment of heart disease
Experimental procedures
Isolation and culture of rat cardiomyocytes
One-day-old Sprague–Dawley rats were obtained from the
Medical Experimental Animal Center of Peking University
Health Science Center Before their hearts were taken, neo-natal rats were put into a glass beaker containing a cotton mass wetted with ethyl ether After anesthesia and decapi-tation, hearts were taken out immediately and put into ice-cold NaCl⁄ Pi, and then cut into pieces The NRCMs were prepared as previously described [27] Experiments were carried out in accordance with the guidelines laid down by the NIH in the USA
Western blot analysis
Western blot analyses were performed as previously described [27] Antibodies used in this study included anti-STAT3, anti-eIF5 (Santa Cruz Biotechnology, Santa Cruz,
CA, USA), and anti-phospho-tyr705-STAT3 (Cell Signaling Technology, Beverly, MA, USA)
Luciferase reporter assay
NRCMs were transfected with STAT3-driven promoter (2· APRE) or rat ANF promoter firefly luciferase reporter plasmids, as well as the internal control renilla luciferase reporter plasmid (phRL–TK; Promega, Madison, WI, USA) using the fugene 6 reagent (Roche Diagnostics, Mannheim, Germany) in accordance with the manu-facturer’s instructions After 24 h of transfection, cells were serum-starved for 24 h, and then treated with ISO for 8 h (for the STAT3-driven promoter) or 24 h (for the ANF promoter) Cell extracts were prepared and assayed acco-rding to the manufacturer’s instructions (Dual Luciferase Assay System; Promega, Madison, WI, USA) Each mea-sured firefly luciferase activity was normalized by the renilla luciferase activity in the same well To construct the STAT3-driven promoter, the 2· APRE sequence was cloned into the multiple cloning site of the pGL3–TATA plasmid, which encodes a firefly luciferase gene containing
a basic upstream TATA element
Construction of lentiviral vector for silencing of rat STAT3 expression
Small interfering RNAs (siRNAs) targeting the rat STAT3 gene were designed by the Shanghai GeneChem, Co Ltd, China Different siRNAs were screened by cotransfection with a rat STAT3 cDNA plasmid into HEK293T cells with Lipofectamine 2000 (Invitrogen Corporation, Carlsbad,
CA, USA) The optimal sequence of siRNA against rat STAT3 (5¢-CTTCAGACCCGCCAACAAA-3¢) was then cloned into the plasmid pGCL–GFP, which encodes an HIV-derived lentiviral vector containing a multiple cloning site for insertion of shRNA constructs to be driven by an upstream U6 promoter and a downstream cytomegalovirus promoter–GFP fluorescent protein (marker gene) cassette flanked by loxP sites Lentivirus preparations were
A
B
Fig 5 gp130 ⁄ JAK2 signaling pathway was involved in
b-AR-induced ANF expression (A, B) Cardiomyocytes were transfected
with ANF promoter (a luciferase reporter plasmid containing the
ANF promoter region) together with renilla luciferase plasmid,
starved, and then stimulated with 10 l M ISO after pretreatment with
4 lgÆmL)1gp130 neutralizing antibody or 10 l M AG490 *P < 0.05,
**P < 0.01 versus control, # P < 0.05, ## P < 0.01 versus ISO.
Trang 7produced by the Shanghai GeneChem, Co Ltd, China The
resulting lentiviral vector containing rat STAT3 shRNA
was named rST3 lentivirus, and its sequence was confirmed
by PCR and sequencing analysis A negative control
lentivi-ral vector containing NS shRNA was constructed by a
sim-ilar process (NS lentivirus, 5¢-CGTACGCGGAATACTT
CGA-3¢) NRCMs were infected with rST3 lentivirus by
addition of lentivirus into the cell culture at an MOI of
approximately 150 The controls were infected with NS
len-tivirus After 3 days of infection, cells were serum starved
for 24 h and then treated
RNA isolation and qRT-PCR
Total RNA from cardiomyocytes was extracted using
Tri-zol reagent, and first-strand cDNA was generated using the
ImProm-IITM Transcription System (Promega, Madison,
WI, USA) qRT-PCR was performed using the primers of
ANF (5¢-GGGGGTAGGATTGACAGGAT-3¢; 5¢-CTCC
AGGAGGGTATTCACCA-3¢) and
glyceraldehyde-3-phos-phate dehydrogenase (5¢-ATCAAGAAGGTGGTGAAGC
A-3¢; 5¢-AAGGTGGAAGAATGGGAGTTG-3¢)
Amplifi-cations were performed in 35 cycles using an opticon
con-tinuous fluorescence detection system (MJ Research,
Waltham, MA, USA) with SYBR green fluorescence
(Molecular Probes, Eugene, OR, USA) Each cycle
con-sisted of 30 s at 94C, 30 s at 60 C, and 30 s at 72 C All
samples were quantified using the comparative CT method
for relative quantification of gene expression, normalized to
glyceraldehyde-3-phosphate dehydrogenase
ANF protein ELISA assay
ANF protein ELISA assays were performed as previously
described [27]
Statistical analysis
Data are expressed as means ± SE The statistical
signifi-cance of the differences between the means of the groups
was determined by one-way anova or t-tests A P-value
< 0.05 was considered to be significant
Acknowledgements
This work was supported by the National Key
Basic Research Program (NKBRP) of China
(2006CB503806) and the Natural Science Foundation
of China (30470691, 30672466) We thank Dr
Guang-ming Wang (Peking University Health Science Center,
China) for providing the pGL3–TATA, and Prof John
G Edwards (New York Medical College, USA) for
providing rat ANF promoter reporter plasmid
[pANF(-638)-Luc]
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Supplementary material
The following supplementary material is available online:
Fig S1 Relative IL-6 mRNA copy number as a result
of b-AR stimulation
Fig S2 b-AR stimulation of IL-6 production
Fig S3 STAT3-specific lentivirus-mediated RNA inter-ference inhibited b-AR-induced endogenous ANF mRNA expression
This material is available as part of the online article from http://www.blackwell-synergy.com
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