The gp130 ⁄ STAT3 signaling pathway mediates b-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 physiological processes. There are three known subtypes in the heart – b 1 -AR, b 2 -AR and b 3 -AR. Of these, the b 1 -AR subtype stimulates the classic Gs–adenylyl cyclase–cAMP–protein kinase A signaling pathway, whereas b 2 -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. 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 CD 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 lM propranolol, 5 lM 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 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 b 1 -AR and b 2 -AR, we also investigated which subtype mediated STAT3 tyrosine phosphorylation. CGP 20712A, a selective b 1 -AR antagonist, and ICI 118551, a selective b 2 -AR antagonist, markedly reduced the tyrosine phosphorylation of STAT3 by about 60% and 100%, respectively (Fig. 1C), which indicated that both b 1 -AR and b 2 -AR are involved in this response. In addition, given that blockade of b 2 -AR completely abolished the STAT3 tyrosine phos- phorylation, whereas blockade of b 1 -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 lM 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 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. 3592 FEBS Journal 275 (2008) 3590–3597 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 l M 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 l M ISO after pretreatment with 4 lgÆmL )1 gp130 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. 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 3593 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 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). gp130 ⁄ STAT3 mediates ANF expression by b-AR H. Zhang et al. 3594 FEBS Journal 275 (2008) 3590–3597 ª 2008 The Authors Journal compilation ª 2008 FEBS 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 ⁄ P i , 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 )1 gp130 neutralizing antibody or 10 lM AG490. *P < 0.05, **P < 0.01 versus control, # P < 0.05, ## P < 0.01 versus ISO. 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 3595 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-II TM 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 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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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. 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 3597 . The gp130 ⁄ STAT3 signaling pathway mediates b-adrenergic receptor-induced atrial natriuretic factor expression in cardiomyocytes Hui. 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