Interleukin-6 Family of Cytokines Mediates Isoproterenol-induced Delayed STAT3 Activation in Mouse Heart

This study was aimed to determine whether β-adrenergic receptor (β-AR) stimulated by isoproterenol (ISO) activates signal transducers and activators of transcription (STAT) in mouse heart and, if so, to examine the underlying mechanism. We found that treatment of adult male mice by ISO (15 mg/kg body weight, intraperitoneal) caused a delayed STAT3 activation (at 60–120 min), which was fully abolished by β-AR antagonist, propranolol. ISO-induced phosphorylation of STAT3 was markedly enhanced by phosphodiesterase inhibitor amrinone, indicating that cAMP is critically involved in β-AR-mediated STAT3 activation. In addition, β-AR stimulation significantly increased gene expression of interleukin-6 (IL-6) family of cytokines (IL-6, leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin-1). IL-6 protein levels in serum and mouse myocardium were also significantly increased in response to ISO treatment. In cultured cardiac fibroblasts, IL-6 level was enhanced significantly after ISO (10-6 mol/liter) stimulation for 2 h and then peaked at 12 h, whereas the response of IL-6 in cultured cardiomyocytes to ISO stimulation was not significant, suggesting that ISO-induced increase in IL-6 is primarily from cardiac fibroblasts rather than cardiomyocytes. Most importantly, IL-6 could activate STAT3 in a time-dependent manner in cultured cardiomyocytes, and inhibition of IL-6 level by anti-IL-6-neutralizing antibody clearly attenuated ISO-induced phosphorylation of STAT3 in myocardium. Taken together, these results indicate that β-AR stimulation leads to a delayed STAT3 activation via an IL-6 family of cytokine-mediated pathway and that cardiac fibroblasts, but not cardiomyocytes, is probably the predominant source of IL-6 in response to ISO stimulation in mouse myocardium. This study was aimed to determine whether β-adrenergic receptor (β-AR) stimulated by isoproterenol (ISO) activates signal transducers and activators of transcription (STAT) in mouse heart and, if so, to examine the underlying mechanism. We found that treatment of adult male mice by ISO (15 mg/kg body weight, intraperitoneal) caused a delayed STAT3 activation (at 60–120 min), which was fully abolished by β-AR antagonist, propranolol. ISO-induced phosphorylation of STAT3 was markedly enhanced by phosphodiesterase inhibitor amrinone, indicating that cAMP is critically involved in β-AR-mediated STAT3 activation. In addition, β-AR stimulation significantly increased gene expression of interleukin-6 (IL-6) family of cytokines (IL-6, leukemia inhibitory factor, ciliary neurotrophic factor, and cardiotrophin-1). IL-6 protein levels in serum and mouse myocardium were also significantly increased in response to ISO treatment. In cultured cardiac fibroblasts, IL-6 level was enhanced significantly after ISO (10-6 mol/liter) stimulation for 2 h and then peaked at 12 h, whereas the response of IL-6 in cultured cardiomyocytes to ISO stimulation was not significant, suggesting that ISO-induced increase in IL-6 is primarily from cardiac fibroblasts rather than cardiomyocytes. Most importantly, IL-6 could activate STAT3 in a time-dependent manner in cultured cardiomyocytes, and inhibition of IL-6 level by anti-IL-6-neutralizing antibody clearly attenuated ISO-induced phosphorylation of STAT3 in myocardium. Taken together, these results indicate that β-AR stimulation leads to a delayed STAT3 activation via an IL-6 family of cytokine-mediated pathway and that cardiac fibroblasts, but not cardiomyocytes, is probably the predominant source of IL-6 in response to ISO stimulation in mouse myocardium. It is widely accepted that when the heart is subject to neurohumoral factors or mechanical pressure overload, cardiomyocytes exhibit hypertrophic response, which is a leading predictor of heart failure (1Molkentin J.D. Wdorn G. Annu. Rev. Physiol. 2001; 63: 391-426Crossref PubMed Scopus (578) Google Scholar). Increasing evidence has demonstrated that cardiomyocyte hypertrophy is initiated by endocrine, paracrine, or autocrine factors that activate a variety of intracellular signaling pathways and ultimately modulate transcription factors and gene expression (2Sadoshima J. Xu Y. Slayter H.S. Izumo S. Cell. 1993; 75: 977-984Abstract Full Text PDF PubMed Scopus (1163) Google Scholar, 3Ito H. Hirata Y. Adachi S. Tanaka M. Tsujino M. Koike A. Nogami A. Murumo F. Hiroe M. J. Clin. Invest. 1993; 92: 398-403Crossref PubMed Scopus (526) Google Scholar, 4Long C.S. Ordahl C.P. Simpson P.C. J. Clin. Invest. 1989; 83: 1078-1082Crossref PubMed Scopus (108) Google Scholar, 5Pennica D. King K.L. 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For instance, angiotensin II, endothelin-1, catecholamines, and the IL-6 1The abbreviations used are: IL, interleukin; STAT, signal transducers and activators of transcription; Jak, Janus kinase; LIF, leukemia inhibitory factor; ELISA, enzyme-linked immunosorbent assay; CNTF, ciliary neurotrophic factor; CT, cardiotrophin-1; gp, glycoprotein; ISO, isoproterenol; ANOVA, analysis of variance; PDE, phosphodiesterase; β-AR, β-adrenergic receptor. family of cytokines that regulate proliferation in cancer cells or immune cells instead trigger hypertrophic growth in cardiomyocytes. Both in vivo and in vitro studies have shown that stimulation of myocardium β-ARs results in cardiac remodeling characterized by increased cell size, reexpression of the "fetal gene" program (atrial natriuretic factor and skeletal α-actin), and organization of actin cytoskeleton (6Dzimiri N. Pharmacol. Rev. 1999; 51: 465-501PubMed Google Scholar, 7Morisco C. Zebrowski D. Condorelli G. Tsichlis P. Vatner S.F. Sadoshima J. J. Biol. Chem. 2000; 275: 14466-14475Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). The mechanism underlying β-AR-mediated cardiac remodeling in vivo, however, remains largely unclear. Recently, it has been reported that Janus kinase/signal transducers and activators of transcription (Jak/STAT), a newly discovered intracellular signal transduction pathway, may play an important role in the process of cardiac remodeling. In vivo studies have demonstrated that Jak1, Jak2, and Tyk2 kinases as well as STATs are activated in the rat heart during acute pressure overloading (8Pan J. Fukuda K. Kodama H. Makino S. Takahashi T. Sano M. Hori S. Ogawa S. Circ. Res. 1997; 81: 611-617Crossref PubMed Scopus (126) Google Scholar). Jak/STAT also responds to many cytokines and growth factors (9Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5028) Google Scholar, 10Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1266) Google Scholar). The binding of ligands to receptor leads to the activation of the Jak tyrosine kinase family, and subsequently, the activated receptor-kinase complexes recruit and activate members of the STAT family by phosphorylation. As a result, the phosphorylated STAT proteins dimerize, translocate into the nucleus, and bind to response elements in the promoters of target genes, thereby regulating gene expression. Up to date, seven mammalian STAT proteins have been identified. Among these proteins, STAT3 is highlighted as a critical mediator of cardiac remodeling and survival of cardiomyocytes (11Yamauchi-Takihara K. Kishimoto T. Trends Cardiovasc. Med. 2000; 10: 298-303Crossref PubMed Scopus (62) Google Scholar, 12Negoro S. Kunisada K. Tone E. Funamoto M. Oh H. Kishimoto T. Yamauchi-Takihara K. Cardiovasc. Res. 2000; 47: 797-805Crossref PubMed Scopus (189) Google Scholar, 13Sano M. Fukuda K. Kodama H. Pan J. Saito M. Matsuzaki J. Takahashi T. Makino S. Kato T. Ogawa S. J. Biol. Chem. 2000; 275: 29717-29723Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar). Accumulating evidence has suggested that IL-6 family of cytokines plays an important role in the activation of STAT3 (11Yamauchi-Takihara K. Kishimoto T. Trends Cardiovasc. Med. 2000; 10: 298-303Crossref PubMed Scopus (62) Google Scholar). The IL-6 family of cytokines including IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M, ciliary neurotrophic factor (CNTF), and cardiotrophin-1 (CT-1) interacts with membrane-bound receptors, which consist of a common signaltransducing subunit, gp130, and various ligand-binding sub-units. Propagation of these cytokine signals requires gp130, which activates STAT3 by binding to its SH2 domain via phosphotyrosine residues in the gp130 cytoplasmic domain. We hypothesized that Jak/STAT pathway could be activated by β-AR stimulation and then involved in β-AR-induced cardiac remodeling. To test this hypothesis, the effect of isoproterenol, a β-AR agonist, on activation of STAT3 in mouse heart was studied. Materials and Animals—Materials were obtained from the following sources. Isoproterenol (ISO), propranolol, atenolol, amrinone, and goat IgG were from (Sigma). Anti-phospho-STAT3 was from New England Biolabs (Beverly, MA). Anti-STAT3 and horseradish peroxidase-conjugated anti-rabbit antibody was from Santa Cruz Biotechnology (Santa Cruz, CA). Murine IL-6 ELISA kit was from Diaclone (Besancon, France). Recombinant murine IL-6 was from PeproTech EC Ltd. (London, United Kingdom). Goat anti-murine IL-6 polyclonal antibody was from R&D Systems (Minneapolis, MN). Male 8-week-old BALB/c mice (weighing 18–20 g) and 1-day-old Balb/c mice were obtained from the Medical Experimental Animal Center of Peking University. Experiments were approved by the Committee on the Ethical Aspects of Research Involving Animals of the Peking University Health Science Center. Western Blot Analysis—Mice were treated with ISO (15 mg/kg body weight) or vehicle by intraperitoneal injection. The left ventricular myocardium was excised at various time points and homogenized by using a Polytron homogenizer with ice-cold lysis buffer containing 20 mmol/liter Tris-HCl (pH 7.4), 150 mmol/liter NaCl, 2.5 mmol/liter EDTA, 50 mmol/liter NaF, 0.1 mmol/liter Na4P2O7, 1 mmol/liter Na3VO4, 1% Triton X-100, 10% glycerol, 0.1% SDS, 1% deoxycholic acid, 1 mmol/liter phenylmethylsulfonyl fluoride, and 1 μg/ml aprotinin. The protein concentration was determined with the BCA protein assay kit (Pierce) using the manufacturer's instructions, and the extracts were stored at -70 °C before use. For Western blot analysis, samples (30 μg each) were separated by electrophoresis on 10% SDS-polyacrylamide gel and transferred onto polyvinylidene difluoride membranes. The membranes were probed with antibodies to phospho-STAT3, and horseradish peroxidase-conjugated anti-rabbit antibodies were used as secondary antibodies. The peroxidase reaction products were visualized by LumiGLO® chemiluminescent substrate (New England Biolabs). The same membrane was then stripped and reprobed with anti-STAT3 antibody to determine the total protein abundance using a similar procedure. Immunohistochemistry—Paraffin sections (6 μm) were deparaffinized, hydrated, and pretreated by boiling in 0.01 mol/liter phosphatebuffered saline (pH 6.0) for 10 min. After treatment for 30 min with 5% normal rabbit serum, samples were treated with polyclonal STAT3 antibody overnight at 4 °C, rinsed with phosphate-buffered saline, and treated for 2 h with horseradish peroxidase-conjugated anti-rabbit antibodies (1:200 dilution) and the peroxidase activity was visualized by using diamine benzidine, resulting in a brown precipitate. RNA Extraction and Reverse Transcriptase-PCR Analysis—Total RNA was extracted from cultured cells and mouse hearts using TRIzol reagent (Invitrogen). The samples were treated with DNase I and then subjected to first-strand synthesis using oligo(dT) primer and reverse transcriptase (Superscript II). Mouse IL-6, LIF, CNTF, CT-1, and β-actin mRNA were amplified by PCR using the following primers (14Ito Y. Yamamoto M. Li M. Doyu M. Tanaka F. Mutch T. Mitsuma T. Sobue G. Brain Res. 1998; 793: 321-327Crossref PubMed Scopus (88) Google Scholar, 15Funamoto M. Hishinuma S. Fujio Y. Matsuda Y. Kunisada K. Oh H. Negoro S. Tone E. Kishimoto T. Yamauchi-Takihara K. J. Mol. Cell Cardiol. 2000; 32: 1275-1284Abstract Full Text PDF PubMed Scopus (45) Google Scholar): mouse IL-6, 5′-GGA GAC TTC ACA GAG GAT ACC-3′ and 5′-CAA GAT GAA TTG GAT GGT CTT-3′ (483-bp product size); mouse LIF, 5′-ATG CCA CGG CAA CCT CAT GAA-3′ and 5′-GAC TTG CTT GTA TGT CCC CAG-3′ (466 bp); mouse CNTF, 5′-TGG CTA GCA AGG AAG ATT CGT T-3′ and 5′-CCC ATA ATG GCT CTC ATG TGC-3′ (519 bp); mouse CT-1, 5′-GAG ACA GTG CTG GCC GCG CTG-3′ and 5′-AGA GGA GAG CAG AAG AGA GAG A-3′ (345 bp); and mouse β-actin, 5′-GTG GGG CGC CCC AGG CAC CA-3′ and 5′-CTT CCT TAA TGT CAC GCA CGA TTT C-3′ (540 bp). The PCR mixture was incubated on a DNA Thermal Cycler (Stratagene) using various cycles. The reaction conditions involved denaturation at 95 °C for 1 min, annealing at 60 °C for 1 min, and extension at 72 °C for 1 min, 20 s with an initial denaturation at 95 °C for 5 min and a final extension step at 72 °C for 7 min, 30 s. After amplification, products were analyzed by electrophoresis on a 2% agarose gel. Cell Culture—Primary ventricular myocytes were prepared as described previously (16Gray M.O. Long C.S. Kalinyak J.E. Li H.T. Karliner J.S. Cardiovasc. Res. 1998; 40: 352-363Crossref PubMed Scopus (356) Google Scholar). Ventricles from 1-day-old BALB/c mice were minced, and cells were isolated by multiple rounds of 8-min-long tissue dissociation with 0.01% trypsin. After each incubation with trypsin, the supernatant was added to an equal volume of Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and all of the supernatants were combined. The cardiomyocytes were collected by differential adhesiveness. Cardiomyocyte-enriched suspensions were removed from the culture dishes and plated at a density of 150–200 cells/mm2 after overnight incubation. Cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 0.1 mm bromodeoxyuridine to prevent fibroblast proliferation. Cardiac fibroblasts obtained during the preplating step of the myocytes isolation procedure were maintained in complete culture medium, allowed to proliferate, and then trypsinized and passaged once at 1:3 dilution. Cardiac fibroblasts in the third passages were used. By immunostaining and by examination of the morphology of the cells, it has been indicated that the cultured cells were pure cardiomyocytes and cardiac fibroblasts. Enzyme-linked Immunosorbent Assay—IL-6 levels in serum, myocardium homogenates, or cell culture medium were measured by ELISA using commercially available kit (Diaclone) according to the manufacturer's protocol. As reported by the manufacturer, this kit is a solidphase sandwich ELISA. A specific anti-IL-6 antibody was coated onto the wells of the microtiter strips. Standards of known IL-6 content, control specimens, and unknown samples were placed into the wells by pipette followed by the addition of biotinylated secondary antibody. After a first incubation and the removal of excess secondary antibody, streptavidin peroxidase was added, which bound to the biotinylated antibody to complete the 4-member sandwich. After a second incubation and washing to remove all of the unbound enzyme, a substrate (tetra-methyl benzidine) solution was then added to produce color. The intensity of this colored product is directly proportional to the concentration of IL-6 present in the sample. Absorbance was read with a microtiter plate reader at 450 nm. In Vivo Antibodies Treatment—Goat anti-murine IL-6 polyclonal antibody was obtained from R&D Systems (<10 ng of endotoxin/mg of polyclonal antibody). Goat IgG was used as an isotype control antibody. Four male 8-week-old BALB/c mice were given 200 μg of anti-murine IL-6-neutralizing antibody intraperitoneally 30 min before ISO treatment (15 mg/kg body weight, intraperitoneal). Control mice received intraperitoneal injection of goat IgG (200 μg each, n = 4). At the conclusion of the study, all of the mice were sacrificed and the blood and tissue samples were collected for further analysis. Statistical Analysis—Data are expressed as the mean ± S.E. The statistical significance of the differences between the means of the groups was determined by one-way ANOVA or unpaired two-tailed Student's t tests. A value of p < 0.05 was considered significant. ISO Activates Jak/STAT Pathway in Mouse Myocardium— Western blot analysis revealed that treatment of mice with ISO (15 mg/kg body weight, intraperitoneal) for 1 or 2 h markedly increased tyrosine phosphorylation of STAT3 in myocardium without altering the protein abundance of STAT3 (Fig. 1A). In contrast, the phosphorylation status of STAT1, STAT5, and STAT6 was unaffected by β-AR stimulation (data not shown). Using immunohistochemical staining, we examined whether the increase in STAT3 tyrosine phosphorylation after ISO treatment was correlated with its changes in subcellular localization. Mouse myocardium treated with ISO for 2 h was processed for immunohistochemical staining with specific rabbit antibody to STAT3 (upper panel). As shown in Fig. 1B, although nontreated myocardium showed little nuclear staining for STAT3, clear nuclear staining was observed in mouse myocardium treated with ISO for 2 h. To further explore whether the increased accumulation of STAT3 in the nucleus was tyrosine-phosphorylated STAT3, we also performed the immunohistochemical staining with rabbit anti-pSTAT3 antibody (lower panel) and the results showed a similar staining pattern. These findings indicate that ISO causes delayed tyrosine phosphorylation of STAT3 and mediates its translocation from the cytoplasm to the nucleus. cAMP-mediate β-AR-induced STAT3 Activation—Fig. 2A shows that phosphorylation of STAT3 was completely inhibited by pretreatment with a nonselective β-AR antagonist, propranolol (30 mg/kg, intraperitoneal), but only partially inhibited by atenolol, a selective β1-AR antagonist, indicating that ISO-induced STAT3 activation was probably mediated by both β1-AR and β2-AR. Because β-AR stimulation increases intracellular cAMP formation, cAMP signaling in mammalian cells is terminated by phosphodiesterases (PDEs), which could catalyze the hydrolysis of cyclic nucleotides to 5′-nucleotide monophosphates so that inhibition of the breakdown of cAMP by PDE inhibitors would produce a sustained increase in the intracellular level of cAMP (6Dzimiri N. Pharmacol. Rev. 1999; 51: 465-501PubMed Google Scholar). Therefore, we next evaluated the possible role of this important second messenger in ISO-induced STAT3 activation by using amrinone, a widely used PDE3 inhibitor. As shown in Fig. 2B, ISO-induced phosphorylation of STAT3 was markedly enhanced by pretreatment with amrinone in a dose-dependent manner. These results strongly suggest that ISO-induced STAT3 activation is mainly mediated by β-AR-cAMP signaling cascade. β-AR Stimulation Up-regulates Gene Expression of IL-6 Family Cytokines and IL-6 Secretion—In contrast to the present in vivo study, our preliminary in vitro studies have shown that ISO could not increase the phosphorylation level of STAT3 in either cultured cardiomyocytes or cardiac fibroblasts (data not shown). This discrepancy suggests that there are some key factors missing in cultured myocytes or fibroblasts. Because IL-6 family of cytokines has been implicated in the regulation of STAT3 activation, we examined the potential effect of ISO on IL-6 family of cytokines gene expression in mouse myocardium. Indeed, the expression of IL-6 family of cytokines including IL-6, LIF, and CNTF mRNA was increased after ISO injection and peaked at 60–120 min (Fig. 3), which was temporally correlated with the delayed phosphorylation of STAT3 (Fig. 1A). Interestingly, although substantial CT-1 mRNA expression was observed in mouse myocardium, the expression was significantly increased at 15 min after ISO injection and had declined to its basal level by 60 min. To further investigate alterations in protein levels of IL-6, we examined their levels in serum and myocardium homogenates after ISO stimulation. The mean IL-6 serum level determined before ISO injection was 120.3 ± 33.7 pg/ml, which increased at 1 h (195.4 ± 28.4 pg/ml), and was further elevated significantly at 2 h (448.0 ± 46.1 pg/ml, p < 0.01) (Fig. 4A). We also observed that in myocardium homogenate samples, the mean IL-6 level was 632.1 ± 38.7 pg/mg in control group and then reached the plateau at 1 h after ISO injection (824.2 ± 38.9 pg/mg, p < 0.05) (Fig. 4B). These data strongly suggest that autocrine/paracrine-secreted IL-6 family of cytokines may be involved in ISO-induced STAT3 activation. Cardiac Fibroblast Is the Main Source of IL-6 Secretion in Myocardium—To clarify the source of increased IL-6 in mouse myocardium, cardiomyocytes and cardiac fibroblasts were prepared from 1-day-old BALB/c mice. Cells were serum-deprived for 24 h prior to ISO (10-6 mol/liter), and the supernatants were collected at different time points for the measurement of IL-6 by ELISA. In unstimulated cardiac fibroblasts, low levels of IL-6 were detected in culture medium (67.4 ± 1.3 pg/ml). IL-6 level was enhanced significantly at 2 h after ISO stimulation (502.1 ± 146.7 pg/ml, p < 0.05) and then peaked at 12 h (773.7 ± 161.9 pg/ml, p < 0.01). Interestingly, despite similar IL-6 levels between unstimulated cardiomyocytes and cardiac fibroblasts, the response of IL-6 measured in the supernatants from cultured cardiomyocytes to ISO stimulation was not significant (Fig. 5A). Our data indicate that cardiac fibroblast but not cardiomyocytes is probably a predominant source of IL-6 in response to ISO stimulation in mouse myocardium. Effect of IL-6 on Tyrosine Phosphorylation of STAT3 in Cardiomyocytes—To demonstrate that IL-6 could activate STAT3 in cultured cardiomyocytes, we analyzed the tyrosine phosphorylation of STAT3 by Western blot analysis. After 24 h of serum depletion, primary cultured cardiomyocytes were stimulated with recombinant murine IL-6 (10 ng/ml) for 30 min. As illustrated in Fig. 5B, tyrosine phosphorylation of STAT3 was observed at 5 min after ISO stimulation and then peaked at 30 min, and it was still sustained at 60 min. These findings suggest that in cultured cardiomyocytes, IL-6 could activate STAT3 in a time-dependent manner. Effect of Anti-IL-6-neutralizing Antibody on Tyrosine Phosphorylation of STAT3 in Vivo—Mice were treated with either anti-murine IL-6-neutralizing antibody or goat IgG (200 μg, intraperitoneal) for 30 min and then received intraperitoneal injection of ISO (15 mg/kg body weight). Blood and myocardium samples were taken at 2 h after ISO injection. In ISO-treated mice, serum IL-6 levels were elevated 4-fold above sham group (p < 0.01). Treatment with an anti-murine IL-6 polyclonal antibody completely inhibited the elevated IL-6 levels (94.4 ± 14.9% versus control, n = 4, p > 0.05). (Fig. 6A) Based on the potential of anti-IL-6 antibody treatment to neutralize endogenous IL-6 in our model, further studies examined the effects of blocking IL-6 on ISO-induced myocardium STAT3 activation in vivo. As shown in Fig. 6B, treatment with anti-murine IL-6-neutralizing antibody significantly inhibited ISO-induced STAT3 phosphorylation, which was consistent with the complete inhibition of IL-6 level. These data clearly indicate that IL-6 plays a pivotal role in ISO-induced STAT3 activation in mouse myocardium. The manifestation of cardiac remodeling is almost always associated with hypertension and left ventricular pressure/volume overload disease. In addition to heart muscle diseases, chronic pressure overload and the associated cardiac remodeling are considered to be not only very common causes but also predictors for the development of chronic heart failure. Previous studies have been focused on the functional roles of β-AR stimulation in left ventricular hypertrophy (6Dzimiri N. Pharmacol. Rev. 1999; 51: 465-501PubMed Google Scholar). More recently, the Jak/STAT pathway, which is activated by a multitude of cytokines and tyrosine kinase receptors (9Darnell J.E. Kerr I.M. Stark G.R. Science. 1994; 264: 1415-1421Crossref PubMed Scopus (5028) Google Scholar, 10Ihle J.N. Cell. 1996; 84: 331-334Abstract Full Text Full Text PDF PubMed Scopus (1266) Google Scholar), has been also implicated in cardiac hypertrophy. However, there is no information available linking these two cardiac hypertrophy pathways. The major finding of this study is that β-AR stimulation leads to delayed phosphorylation of STAT3 via an IL-6-dependent mechanism in addition to the well established cAMP/protein kinase A pathway. Moreover, the present study provides direct evidence that cardiac fibroblast but not cardiomyocytes is probably the predominant source of IL-6 in response to ISO stimulation in mouse myocardium. The coexistence of β1-AR and β2-AR has been demonstrated with the radioligand binding assay in the hearts of rat, mouse, cat, guinea pig, dog, and rabbit (17Brodde O.E. Pharmacol. Rev. 1991; 43: 203-243PubMed Google Scholar). Stimulation of these β-ARs activates the classic guanine nucleotide-binding proteins (G proteins), adenylate cyclases, and cAMP-protein kinase A cascade, which regulates multiple effects. In this study, we have demonstrated that ISO injection could induce tyrosine phosphorylation of STAT3 in myocardium, simultaneously mediating its translocation from the cytoplasm to the nucleus. Furthermore, our findings indicate that ISO-induced STAT3 activation is mediated by both β1-AR and β2-AR via increasing intracellular cAMP. Interestingly, we have found that ISO robustly increases mouse myocardium STAT3 phosphorylation in vivo but has no such effect in cultured cardiomyocytes or cardiac fibroblasts. This discrepancy reminds us that some key factors might be missing in cultured cells. Recent studies have demonstrated that autocrine/paracrine-secreted IL-6 family of cytokines plays a critical role in STAT activation in response to mechanical stretch and angiotensin II (18Taga T. Kishimoto T. Annu. Rev. Immunol. 1997; 15: 797-819Crossref PubMed Scopus (1298) Google Scholar, 19Pan J. Fukuda K. Sato T. Matsuzaki J. Kodama H. Sano M. Takahashi T. Kato T. Ogawa S. Circ. Res. 1999; 84: 1127-1136Crossref PubMed Scopus (250) Google Scholar). Moreover, the levels of angiotensin II, plasma renin activity, and norepinephrine are proportionally correlated with the level of IL-6, suggesting a link between neurohormones and cytokine activation (20Orus J. Roig E. Perez-Villa F. Pare C. Azqueta M. Filella X. Heras M. Sanz G. J. Heart Lung Transplant. 2000; 19: 419-425Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). In this study, we have observed that the gene expression of IL-6 family of cytokines including IL-6, LIF, and CNTF are increased in response to β-AR stimulation by ISO and the time course is comparable with STAT3 activation. The ELISA was used to determine whether the increases in the expression level of IL-6 mRNA was associated with increases in the extracellular secretion of IL-6. Our results indicate that ISO significantly increased IL-6 levels in serum and myocardium homogenates in a pattern similar to that of IL-6 mRNA expression in mouse myocardium. Thus, we have for the first time demonstrated that autocrine/paracrine-secreted IL-6 family of cytokines may be critically involved in β-AR-induced STAT3 activation. It should be noted that the elevation of IL-6 level in mouse myocardium is slightly premature than those in serum. The myocardium is believed to be a major source of IL-6 in patients with acute myocardial infarction and heart failure (21Kucharz E.J. Wilk T. Eur. J. Intern. Med. 2000; 11: 253-256Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar). It is well accepted that the heart is composed of multiple cell types including cardiomyocytes and nonmyocytes (mostly cardiac fibroblasts). Accumulating evidence has established that cardiac fibroblasts similar to cardiomyocytes synthesize and secrete a local peptide hormone and cytokines that may modulate myocardial structure and function (16Gray M.O. Long C.S. Kalinyak J.E. Li H.T. Karliner J.S. Cardiovasc. Res. 1998; 40: 352-363Crossref PubMed Scopus (356) Google Scholar, 22Wan S. DeSmet J.M. Barvais L. Goldstein M. Vincent J.L. LeClerc J.L. J. Thorac. Cardiovasc. Surg. 1996; 112: 806-811Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). 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Besch-Williford C.L. Hook R.R. Riley L.K. J. Med. Microbiol. 2000; 49: 171-176Crossref PubMed Scopus (11) Google Scholar). In agreement with those of previous reports (34Van Andel R.A. Franklin C.L. Besch-Williford C.L. Hook R.R. Riley L.K. J. Med. Microbiol. 2000; 49: 171-176Crossref PubMed Scopus (11) Google Scholar), our results here indicate that polyclonal antibody is capable of depleting serum IL-6. Most importantly, the inhibition of IL-6 level by anti-IL-6-neutralizing antibody significantly attenuated ISO-induced phosphorylation of STAT3 in mouse myocardium. Thus, these direct evidences confirm the critical role of IL-6 in STAT3 activation after ISO injection. Moreover, based on the present finding, (I) expression of other IL-6 families of cytokines including LIF, CNTF, and CT-1 mRNA were also increased after ISO treatment and (II) anti-IL-6-neutralizing antibody at a high dose (200 μg each) failed to completely inhibit STAT3 activation. Despite its great capability in depleting endogenous IL-6, we supposed that other IL-6-related cytokines (LIF, CNTF, and CT-1) might be also partially involved in this activation, although their contribution should be weaker than that of IL-6. In summary, β-AR stimulation induces the Jak/STAT pathway activation in mouse heart and autocrine/paracrine-secreted IL-6 family of cytokines plays a pivotal role in ISO-induced delayed STAT3 activation. Moreover, the present findings suggest that cardiac fibroblasts but not cardiomyocytes is probably the predominant source of IL-6 in response to ISO stimulation in mouse myocardium. We thank Dr. Rui-Ping Xiao for the critical reading of the paper.