the gp130/stat3 signaling pathway mediates β-adrenergic receptor-induced atrial natriuretic factor...

The gp130 ⁄STAT3 signaling pathway mediatesb-adrenergic receptor-induced atrial natriureticfactor expression in cardiomyocytesHui 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 physiological 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: [email protected]

(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.

3590 FEBS Journal 275 (2008) 3590–3597 ª 2008 The Authors Journal compilation ª 2008 FEBS

ANF 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 conditions.

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

A B

C D

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 lM 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 lM ISO for 60 min after pretreatment with 10 lM propranolol, 5 lM CGP 20712A or

5 lM 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 lM ISO for 8 h with or without pretreatment with 10 lM 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.

H. Zhang et al. gp130 ⁄ STAT3 mediates ANF expression by b-AR

FEBS Journal 275 (2008) 3590–3597 ª 2008 The Authors Journal compilation ª 2008 FEBS 3591

antagonist 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 lM ISO for

60 min after pretreatment with 10 lM SB203580, 10 lM SP600125

or 10 lM 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 lM ISO after pretreatment with 10 lM

SB203580, 10 lM SP600125 or 10 lM U0126. *P < 0.05 versus

control; #P < 0.05 versus ISO; NS, no statistical significance versus

ISO (n = 3). SB, SB203580; SP, SP600125.

gp130 ⁄ STAT3 mediates ANF expression by b-AR H. Zhang et al.


STAT3 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 lM ISO for 60 min after pretreatment

with 4 lgÆmL)1 gp130 neutralizing antibody

or 6 lgÆmL)1 actinomycin 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 lM ISO after pretreatment with

4 lgÆmL)1 gp130 neutralizing antibody or

10 lM AG490. *P < 0.05, **P < 0.01 versus

control, #P < 0.05, ##P < 0.01 versus ISO.

gp130Ab, gp130 neutralizing antibody; ActD,

actinomycin D.



The 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 lM 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 lM 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).



gp130 ⁄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 lM ISO after pretreatment with

4 lgÆmL)1 gp130 neutralizing antibody or 10 lM AG490. *P < 0.05,

**P < 0.01 versus control, #P < 0.05, ##P < 0.01 versus ISO.



produced 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¢-CTCCAGGAGGGTATTCACCA-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 94 �C, 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

Please note: Blackwell Publishing are not responsible

for the content or functionality of any supplementary

materials supplied by the authors. Any queries (other

than missing material) should be directed to the corre-

sponding author for the article.



the gp130/stat3 signaling pathway mediates β-adrenergic receptor-induced atrial natriuretic factor...

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