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Jan 17, 2008 - *National Integrative Medicine Centre for Cardiovascular Diseases, China-Japan Friendship Hospital, Beijing 100 029, China, †Department.
Biol. Cell (2009) 101, 263–272 (Printed in Great Britain)

Research article

doi:10.1042/BC20080117

Adiponectin induces interleukin-6 production and activates STAT3 in adult mouse cardiac fibroblasts Wenqiang Liao*, Changan Yu*, Jianyan Wen*, William Jia†, Geng Li*, Yuannan Ke*, Shumin Zhao‡1 and William Campell‡ *National Integrative Medicine Centre for Cardiovascular Diseases, China-Japan Friendship Hospital, Beijing 100 029, China, †Department of Surgery, Brain Research Centre, University of British Columbia, 2211 Wesbrook Mall, Vancouver, Canada V6T 2B5, and ‡Pegasus Pharmaceuticals Group Inc., Unit 110-116, 11800 River Road, Richmond, Canada V6X 1Z7

Background information. APN (adiponectin), an adipocyte-derived cytokine highly presented in serum, which exerts antidiabetic, anti-atherosclerotic and cardioprotective actions, also enhances CFB (cardiac fibroblast) proliferation and protects against cardiac fibrosis. STAT3 (signal transducer and activator of transcription 3), a major mediator in the gp130/JAK2 (Janus kinase 2)/STATs signalling pathway, plays a critical role in cardioprotective events. Almost two-thirds of total myocardial cells are CFBs; however, whether APN regulates STAT3 signalling pathway has not been clarified yet in CFBs. In the present study, we investigated the effect of recombinant globular APN on the STAT3 activity in adult mouse CFBs and explored the possible signalling transduction mechanism. Results. In cultured CFBs, APN (10 μg/ml) can significantly induce delayed STAT3 Tyr705 phosphorylation timedependently, up to 60 min, and mediate STAT3 translocation from cytoplasm to nucleus. Transfection of siRNA (small interfering RNA) specific for AdipoR1 (APN receptor 1), but not AdipoR2, obviously inhibited APN-induced STAT3 Tyr705 phosphorylation, indicating that AdipoR1, not AdipoR2, is required for STAT3 phosphorylation. Both inhibition of gp130 by anti-gp130 neutralizing antibody and JAK2 by AG490 (a specific inhibitor for JAK2) can inhibit APN-induced STAT3 phosphorylation and STAT3 transcription activity detected using 2×pAPRE-Luc (APRE reporter) assay. Furthermore, we found that the IL (interleukin)-6 level in culture medium was significantly increased after stimulation with APN and the IL-6 mRNA level was also markedly increased in CFBs, which can be reversed by siRNA for AdipoR1, but not for AdipoR2, and that anti-IL-6 neutralizing antibody can significantly inhibit APNinduced STAT3 Tyr705 phosphorylation. Conclusions. APN induces IL-6 production mediated by AdipoR1, not AdipoR2, in adult mouse CFBs, which leads to the stimulation of the gp130/JAK signalling pathway, and as a result causes STAT3 activation.

Introduction APN (adiponectin) is a cytokine predominantly secreted from adipose tissues and accounts for 0.01% of the total plasma protein. APN can form a wide range of multimers, from trimers and hexamers to high molecular weight multimers such as dodecamers 1 To

whom correspondence should be addressed (email zhaoshuminxy@ hotmail.com). Key words: adiponectin, cardiac fibroblasts, interleukin-6, signal transducer and activator of transcription 3 (STAT3). Abbreviations used: AdipoR, adiponectin receptor; APN, adiponectin; CFB, cardiac fibroblast; gp130Ab, anti-gp130 neutralizing antibody; IL, interleukin; IL-6Ab, anti-IL-6 neutralizing antibody; JAK, Janus kinase; MCP, monocyte chemotactic protein; NF-κB, nuclear factor-κB; siRNA, short interference RNA; SOCS, suppressor of cytokine signalling; STAT3, signal transducer and activator of transcription 3.

and 18 mers (Shapiro and Scherer, 1998; Kadowaki and Yamauchi, 2005; Ahima and Osei, 2008). APN exists as a full-length protein or a smaller, globular fragment, but almost all APN appears to exist as full-length APN in plasma (Wang and Scherer, 2008; Wang et al., 2008). Two type of APN receptors, AdipoR1 (APN receptor 1) and AdipoR2, have been identified and mediate the beneficial functions of APN (Kadowaki and Yamauchi, 2005). Further studies have revealed that APN is closely associated with metabolic disturbances and cardiac remodelling and exerts antidiabetic, anti-atherosclerotic and cardioprotective actions (Barnett, 2008; Abel et al., 2008). Shibata et al. (2004, 2005, 2007)

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reported that pressure overload in APN-deficient mice resulted in enhanced concentric cardiac hypertrophy and increased mortality, and APN can inhibit hypertrophic signalling in the myocardium. Some other investigators have also reported that APN can obviously diminish infarct size, apoptosis, cardiac hypertrophy and interstitial fibrosis, and significantly increase left ventricular function and coronary flow during myocardial insults, suggesting that APN is a beneficial cytokine and a potential cardioprotective agent (Shibata et al., 2004, 2005, 2007; Gonon et al., 2008). Almost two-thirds of total myocardial cells are CFBs (cardiac fibroblasts). CFBs are responsible for the autocrine/paracrine of many cytokines, such as IL (interleukin)-6, and for the production of some extracellular matrix proteins, such as fibronectin and collagens (Weber and Brilla, 1991), which probably contributes to the process of cardiac remodelling mentioned above. Recently, Fujita et al. (2008) reported that APN can significantly enhance PPARα activity and inhibit angiotensin II-induced cardiac fibrosis in cultured CFBs. A previous report had shown that globular APN isoform can significantly increase MCP (monocyte chemotactic protein)-1 and IL-6 expression in CFBs, and induce collagen synthesis and proliferation in CFBs (Hattori et al., 2007). Therefore we speculated that CFBs, the main player of the autocrine/paracrine system in myocardium, play critical roles in APN-induced cardiac remodelling. It has been established that STAT3 (signal transduction and activator of transcription 3), a key effector protein activated through gp130/JAK (Janus kinase), plays important roles in cardiac remodelling, and exerts cardioprotective actions that are similar to APN (Fischer and Hilfiker-Kleiner, 2007). The IL-6 family of cytokines is the major factor secreted from CFBs and activates STAT3 through the gp130/JAK signalling pathway. Tyr705 phosphorylation of STAT3 is obligatory for its activation. Once activated, phosphorylated STAT3 assembles dimers, translocates from the cytoplasm into the nucleus and as a result initiates gene transcription (Yin et al., 2006; Kurdi and Booz, 2007; Murray, 2007). At the present time there is no information concerning the effects of APN on STAT3 in CFBs. In this study, we examined whether APN can activate STAT3 in CFBs, and the possible signalling pathway involved in this process was investigated.

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Figure 1 APN induces STAT3 phosphorylation time-dependently and its translocation into the nucleus in adult mouse CFBs (A) Adult mice CFBs were incubated in serum-free medium for 24 h, and then treated with 10 μg/ml APN. The whole cell lysate was harvested at the indicated time for Western blot analysis using the specific antibody for STAT3 phosphorylated at Tyr705 (p-tyr STAT3). The same membranes were stripped and re-probed with total STAT3 antibody. These experiments were repeated three times with the same results. (B) CFBs grown on glass coverslips were starved for 24 h and then treated with 10 μg/ml APN for 3 h. The cellular localization of STAT3 was detected using an antibody against STAT3. After washing in PBS, samples were incubated with Cy5-conjugated goat anti-(rabbit IgG) Ig (red) and Hoechst 33342 (blue) and examined by Leica Microscope. STAT3 was predominantly localized in the nucleus upon the treatment of APN. The experiment was repeated two times with the same result.

Results APN induces STAT3 Tyr705 phosphorylation time-dependently and translocation from cytoplasm into nucleus in adult mouse CFBs

We first examined the phosphorylation of STAT3 after treatment with APN (10 μg/ml) in primary cultured adult mouse CFBs. APN can induce phosphorylation of STAT3 at Tyr705 in a time-dependent manner (Figure 1A). Interestingly, the phosphorylation of Tyr705 in STAT3 was delayed and occurred 60 to 120 min after treatment with APN, suggesting that APN-induced STAT3 Tyr705 phosphorylation is not direct but indirect. STAT3 Tyr705 phosphorylation determines the activation of STAT3, and once

Adiponectin activates STAT3 in cardiac fibroblasts

Figure 2 AdipoR1, not AdipoR2, is required for APN-induced STAT3 phosphorylation (A) Adult mouse CFBs were transfected with AdipoR1 siRNA, AdipoR2 siRNA and scramble siRNA, respectively. After transfection, the expression of AdipoR1 and AdipoR2 were measured 36 h later by Western blot with specific antibodies. The filters were reprobed with β-actin antibody. (B) After transfection with siRNA for AdipoR1 and AdipoR2, 36 h later CFBs were deprived of serum and incubated overnight prior to treatment with 10 μg/ml APN for 60 min. The protein levels of STAT3 phosphorylated at Tyr705 were measured by Western blot with anti-phospho-specific antibody. The filters were reprobed with general STAT3 antibody. These experiments were repeated three times with the same results.

Research article gp130/JAK2 signalling pathway is involved in APN-induced STAT3 phosphorylation

As STAT3 is one of the main components in the gp130/JAK2/STATs signalling pathway, we wondered whether gp130 and JAK2 are involved in ANP-induced STAT3 phosphorylation. gp130Ab (anti-gp130 neutralizing antibody) and AG490 (a specific inhibitor of JAK2) were used to inhibit gp130 and JAK2 respectively. Either gp130Ab or AG490 can abolish APN-induced STAT3 Tyr705 phosphorylation (Figures 2A and 2B), which suggested that the gp130/JAK2 signalling pathway is involved in this event. gp130/JAK2 signalling pathway is involved in APN-induced STAT3 transcription activity

Because STAT3 transcriptional activity is dependent upon the STAT3 Tyr705 phosphorylation, the STAT3 transcription activity was detected using 2×pAPRELuc (APRE reporter) assay. APN can significantly increase the transcription activity of STAT3, which can be markedly inhibited either by gp130Ab or AG490 (Figures 3 and 4). These suggested that APN increases STAT3 transcription activity and that gp130/JAK2 signalling is involved in this process. activated, STAT3 translocates from the cytoplasm into the nucleus. As shown in Figure 1(B), STAT3 is predominantly located in the nucleus after treatment with APN for 3 h. AdipoR1, not AdipoR2, is required for APN-induced STAT3 phosphorylation

Two APN receptor subtypes, AdipoR1 and AdipoR2, mediate the biological functions of APN. To examine whether these two receptor subtypes were involved in the STAT3 phosphorylation induced by APN, siRNA (short interference RNA) targeting for these two receptors was used to knockdown the expression of AdipoR1 and AdipoR2. As shown in Figure 2(A), siRNA for AdipoR1 and AdipoR2 inhibits the expression of these two receptors. We further found that AdipoR1 siRNA can significantly inhibit APNinduced STAT3 Tyr705 phosphorylation, and no effect of AdipoR2 siRNA on STAT3 Tyr705 phosphorylation was observed (Figure 2B). These findings indicted that APN-induced STAT3 phosphorylation is through AdipoR1 but not AdipoR2.

IL-6 is induced by APN and is indispensable for APN-induced STAT3 Tyr705 phosphorylation in adult mouse CFBs

IL-6 has been reported to be one of the major cytokines during the autocrine and/or paracrine functions of CFB, and plays a crucial role in the activation of STAT3. APN induces delayed STAT3 Tyr705 phosphorylation (at 60–120 min) and, as gp130/JAK2 signalling pathway is involved in this process, we speculated that this is probably due to IL-6 secretion induced by APN in cultured CFBs. As shown in Figure 5(A), in cultured CFBs, the IL-6 level was significantly increased 3, 6, 12 and 24 h after stimulation with APN (10 μg/ml). Furthermore, we found that the IL-6 mRNA level also obviously increased upon the treatment with APN (10 μg/ml) for 24 h (expressed as the fold increase compared with control, 9.69 + − 2.50, P < 0.05 compared with control, n = 3) (Figure 5B). In addition, the increase both in mRNA level and in protein expression of IL-6 induced by APN were reversed by siRNA of AdipoR1, but not AdipoR2, indicating that AdipoR1, not AdipoR2, mediates APN-induced IL-6 production (Figures 5B

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Figure 3 gp130/JAK2 signalling pathway is involved in APN-induced STAT3 phosphorylation Adult mouse CFBs were incubated in serum-free medium for 24 h, then stimulated with 10 μg/ml APN for 60 min after pretreatment with 4 μg/ml anti-gp130 neutralization antibody (A) or 10 μM AG490 (B) for 60 min respectively. The whole cell lysate was harvested and analysed by Western blot for detection of STAT3 Tyr705 phosphorylation (p-tyr STAT3). The same membranes were re-probed with general STAT3 antibody. These experiments were repeated three times with the same results.

Figure 4 gp130/JAK2 signalling pathway is involved in APN-induced STAT3 transcriptional activity Adult mouse CFBs were starved overnight 24 h after cotransfection with 2×pAPRE-Luc (APRE reporter) and phRL-TK plasmid and then stimulated with 10 μg/ml APN for 24 h prior to treatment with 4 μg/ml gp130 neutralization antibody (A) or 10 μM AG490 (B) for 60 min. The data were converted to relative luciferase activity and expressed as means + − S.E.M. (n = 4), *P < 0.05 compared with control; # P < 0.05 in (A) and # P < 0.01 in (B), compared with APN.

and 5C). To determine whether IL-6 is indispensable for APN-induced STAT3 Tyr705 phosphorylation, IL-6Ab (anti-IL-6 neutralizing antibody) was used to block IL-6 action. As shown in Figure 5(D), IL-6Ab significantly inhibited APN-induced STAT3 phosphorylation (P < 0.01 compared with APN, n = 3).

Discussion In the current study, we demonstrated that globular APN can significantly induce STAT3 Tyr705 phosphorylation and enhance STAT3 transcription activity in cultured mouse CFBs. APN can exist as non-proteolytic (full-length APN) and proteolytic (globular APN) forms in various oligomeric states and different forms of APN exert distinct biological functions mediated by its receptors, AdipoR1 and AdipoR2 (Kadowaki and Yamauchi, 2005). Specific siRNA for AdipoR1, but not siRNA for AdipoR2, obviously abolished STAT3 Tyr705 phosphorylation, which indicated that APN-induced STAT3 Tyr705 phosphorylation is mediated by AdipoR1 and not by AdipoR2. Furthermore, we found that both the

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anti-gp130 neutralizing antibody and AG490 can abolish APN-induced STAT3 Tyr705 phosphorylation and transcriptional activity, indicating that the gp130/JAK2 signalling pathway is involved in this process. The gp130/JAK/STAT signalling pathway has been demonstrated to play important roles in cardiac development, hypertrophy, protection and remodelling in response to physiological and pathophysiological stimuli (Fischer and Hilfiker-Kleiner, 2007, 2008). The IL-6 family of cytokines, which include IL-6, leukemia inhibitory factor and cardiotrophin-1, contribute significantly to cardiac remodelling and appear to be predominant transferable factors in the autocrine/paracrine systems of CFBs (Fredj et al.,

Adiponectin activates STAT3 in cardiac fibroblasts

Research article

Figure 5 APN induces IL-6 production in adult mouse CFBs (A) APN induces IL-6 protein production in adult mouse CFBs. Cells were treated with or without APN (10 μg/ml) for 3, 6, 12 or 24 h. The culture medium was collected at the indicated time and IL-6 concentration was measured by ELISA. The data was expressed as means + − S.E.M. (n = 3), **P < 0.01 compared with control. (B and C) APN induces IL-6 production in cultured adult mouse CFBs. Adult mouse CFBs were transfected with adipoR1 siRNA and adipoR2 siRNA. Cells were starved overnight 36 h after transfection and then treated with 10 μg/ml APN for another 24 h, and the culture medium was collected for the measurement of IL-6 protein production by ELISA and total RNA was extracted for the detection of IL-6 mRNA expression by SYBR green real-time PCR (expressed as fold of control). The values are expressed as # means + − S.E.M. (n = 3), *P < 0.05, **P < 0.01 compared with control; P < 0.01 compared with APN. (D) IL-6 is indispens705 able for APN-induced STAT3 Tyr phosphorylation. Adult mouse CFBs were incubated in serum-free medium for 24 h, then stimulated with 10 μg/ml APN for 60 min after pretreatment with 10 μg/ml IL-6Ab and 10 μg/ml IgG, as a control, for 60 min. The whole cell lysate was prepared for immunoblotting with anti-STAT3 Tyr705 antibody. The same membranes were re-probed with general STAT3 antibody. The graphs indicate results of densitometric analysis. Similar results were ob# tained for three experiments. Values are the means + − S.E.M. *P < 0.05, **P < 0.01 compared with control; P < 0.01 compared with APN.

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2005; Fischer and Hilfiker-Kleiner, 2007; Kurdi and Booz, 2007). Tyr705 phosphorylation is the determinant for STAT3 activation, and once phosphorylated, STAT3 translocates from the cytoplasm into the nucleus and triggers transcription of genes such as Bcl-XL (basal cell lymphoma-extra large), cyclooxygenase-2, vascular endothelial growth factor, manganese superoxide dismutase and atrial natriuretic peptide (Jost et al., 2001; Yamauchi-Takihara, 2002; Hilfiker-Kleiner et al., 2005; Chen and Han, 2008). Notably, STAT3 Tyr705 phosphorylation did not occur instantly after treatment with APN, but was delayed until 60 min, whereas STAT3 Ser727 phosphorylation occurred instantly (results not shown), indicating that APN-induced Tyr705 phosphorylation is indirect. IL-6, an important cytokine in the local secretion system of myocardium, was examined by quantitative real-time PCR and ELISA and, as shown in Figure 5, globular APN significantly enhanced IL-6 mRNA and protein expression in CFBs, which was significantly inhibited by siRNA for AdipoR1, but not for AdipoR2, indicating that APN-induced IL-6 production is mediated by AdipoR1, not by AdipoR2. Furthermore, we found that anti-IL-6 neutralizing antibody completely abolished APN-induced STAT3 Tyr705 phosphorylation. Thus, we speculated that APN-induced STAT3 Tyr705 phosphorylation is due to the activation of the gp130/JAK signalling pathway triggered by IL-6, which probably contributes to the delay in STAT3 Tyr705 phosphorylation. In physiological as well as pathological conditions, functions of cytokines are strictly controlled. Cytokine signalling pathways are negatively regulated by the family of proteins called SOCS (suppressor of cytokine signalling) (Yoshimura et al., 2007; Boyle and Robb, 2008; Dalpke et al., 2008). SOCS-3 is an early response gene, potently induced by various factors, such as leukemia inhibitory factor, IL-1β, IL-6, insulin growth factor-1, erythropoietin and leptin (Patrick et al., 2004; Laubner et al., 2005; Yadav et al., 2005; Wormald et al., 2006; Isomoto et al., 2007; Boyle and Robb, 2008). Subsequently, SOCS3 inhibits JAK/STAT signalling and renders the cell resistant to further stimulation by these gp130 cytokines, thus creating a negative feedback loop that attenuates cytokine signal transduction and maintains homoeostasis. IL-6, via a JAK/STAT-3 pathway, induces expression of SOCS-3, and in turn SOCS-3

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inhibits IL-6 signalling pathway, thereby turning it off in a classic feedback loop (Greenhalgh et al., 2002; Croker et al., 2003; Wormald et al., 2006; Isomoto et al., 2007; Ozawa et al., 2008). In the present study, the IL-6 concentration is elevated in a timedependent manner, reaching its peak at 24 h following APN stimulation, whereas STAT3 phosphorylation by APN declines at 3 h and does not persist to 24 h, which is consistent with a previous report that STAT3 activation in response to IL-6 stimulation is transient as manifested by rapid STAT3-inactivation, and continuous IL-6 stimulation does not lead to sustained STAT3 activation (Patrick et al., 2004). This could be the consequences of a negative feedback mechanism, such as SOCS-3 induction, but the detailed mechanism remains to be investigated. It has been reported that both APN and its receptors, AdipoR1 and AdipoR2, are expressed in cultured ventricular cardiomyocytes, and these constitute a local cardiac-specific APN system in myocardium (Fujioka et al., 2006; Ding et al., 2007; Skurk et al., 2008). Besides cardiomyocytes, AdipoR1 and AdipoR2 were also found expressed in CFBs, and through these receptors, APN significantly induced MCP-1 and IL-6 production, enhanced angiotensin II-induced proliferation and protected against angiotensin II-induced rat cardiac fibrosis (Hattori et al., 2007; Fujita et al., 2008). In this study, we also found that two receptors of APN expressed in cultured adult mouse CFBs and globular APN can substantially induce IL-6 production. Taken together, we believe that cytokines produced from CFBs upon the APN treatment in an autocrine or paracrine manner, and the activation of gp130/JAK/STAT signalling pathway ensued, may enhance the local cardiac APN system and play a crucial role in APN-induced cardioprotection. The present study using siRNA for AdipoR1 and AdipoR2 showed that AdipoR1, but not AdpioR2, mediated APN-induced STAT3 Tyr705 phosphorylation in CFBs. The different responses of AdipoR1 and AdipoR2 are probably due to their different affinity or expression in CFBs. Previous reports showed that AdipoR1 and AdipoR2 have different affinity for full-length and globular APN and the former appears to bind predominantly globular APN with high affinity, whereas the latter binds both full-length and globular APN (Yamauchi et al., 2003; Kadowaki and Yamauchi, 2005). In addition, the expression

Adiponectin activates STAT3 in cardiac fibroblasts

of AdipoR1 and AdipoR2 is rather different in different tissues and cells, AdipoR1 is predominantly found in skeletal muscle, whereas AdipoR2 seems to be preferentially expressed in the liver (Kadowaki and Yamauchi, 2005; Kadowaki et al., 2006). The mRNA expression of AdipoR1 is much higher than that of AdipoR2 in vascular endothelial cells, which might be one of the explanations for the different response of AdipoR1 and AdipoR2 to APN (Kadowaki and Yamauchi, 2005; Hattori et al., 2007). In the present study, the protein levels of AdipoR1 seem to be higher than that of AdipoR2 in CFBs, as shown in Figure 2(A), which probably contributes to the different responses of these two receptors to globular APN. Previous studies have reported that APN can stimulate the release of some cytokines/chemokines and prostaglandins, such as IL-1β, MCP-1, IL-6, IL-10, TNF (tumour necrosis factor)-α, PG (prostaglandin) F2 and PGE2, in macrophage cells, vascular endothelial cells and adipose tissues, which was dependent on the activation of NF-κB (nuclear factorκB) (Martha et al., 2005; Steffens and Mach, 2008; Hattori et al., 2008; Tian et al., 2008). In our study, globular APN can significantly induce IL-6 production in cultured CFBs, but whether NF-κB is involved in this process remains to be clarified. Taking into account all our results, we propose the model shown in Figure 6 to depict APN-induced STAT3 activation in cultured mouse CFBs. APN induces IL-6 production mediated by AdipoR1, which leads to the simulation of the gp130/JAK signalling pathway and as a result causes STAT3 Tyr705 phosphorylation. After this phosphorylation, STAT3 dimerizes and translocates from the cytoplasm into the nucleus where it induces the transcription of target genes leading finally to cardioprotective actions. Although experiments on cultured cells do not necessarily represent the in vivo state, the present results suggest that APN-induced IL-6 production and STAT3 activation most likely make great contributions to the local autocrine/paracrine system in myocardium. Therapeutic approaches that regulate the local myocardium APN system could be useful in preventing or reversing cardiac remodelling.

Materials and methods Materials and reagents

AG490 was purchased from Calbiochem. Anti-phospho-Tyr705 STAT3 antibody was from Cell Signaling Technology (Beverly,

Research article Figure 6 A presumable signalling pathway for APN-induced STAT3 activation in adult mouse CFB Upon APN stimulation, interleukins such as IL-6 are secreted in cultured CFB, which is mediated by AdipoR1, not by AdipoR2. Thereafter, IL-6 cytokine phosphorylates STAT3 at Tyr705 mediated by gp130-JAK2 pathway. Once phosphorylated at Tyr705 , STAT3 translocates from cytoplasm into the nucleus and initiates gene transcription. Phos-STAT3, phosphorylated STAT3; IL-6Ab, anti-IL-6 neutralizing antibody; gp130Ab, anti-gp130 neutralization antibody; AG490, an inhibitor of JAK2.

MA, U.S.A.). Anti-AdipoR1, anti-AdipoR2, anti-β-actin, antiSTAT3 and horseradish peroxidase-conjugated second antibodies were from Santa Cruz Biotechnology. Anti-gp130 neutralizing antibody, anti-IL-6 neutralizing antibody and IgG were from R&D Systems (Minneapolis, MN, U.S.A.). FuGENETM 6 was from Roche Diagnostics and recombinant mouse globular APN [from HEK (human embryonic kidney)-293 cells] were from Alexis Biochemicals (San Diego, CA, U.S.A.). Isolation of adult mouse ventricular fibroblasts

Adult mouse CFBs were isolated from adult male BALB/c mice (20–30 g) by a modification of a previously described protocol (Lee et al., 1995). This work was undertaken as required by the animal ethics committee of China-Japan Friendship Hospital (Beijing, China), and was carried out in accordance with the United States NIH guidelines [Guide for the Care and Use of Laboratory Animals (1985), DHEW Publication no. (NIH) 8523: Office of Science and Health Reports, DRR/NIH, Bethesda, MD, U.S.A.]. Briefly, three to four mice were anaesthetized with pentobarbital i.p. and then killed by decapitation. Hearts were excised and the atria were removed. The ventricles were minced and placed in a solution containing 100 units/ml collagenase I. The ventricles were subjected to periods of digestion at 37◦ C with rotation at 100 rev./min for 15 min; cells from the third to sixth digestion were pooled, centrifuged, and suspended in complete growth medium. The cell suspension was plated in culture dishes (100 mm) for 2 h to allow for the preferential

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attachment of CFBs. CFBs were passaged after confluent within 96 h and all cells used in experiments were from passages 2 to 4. RNA interference for AdipoR

Adult mouse CFBs were plated in 60-mm-diameter plates and cultured overnight in complete growth medium. Cells were transfected with siRNAs (10 nM) in Opti-MEM Medium (Invitrogen) using the CodeBreaker siRNA Transfection Reagent (Promega) according to the manufacturers’ instructions. After 36 h of culture, cells were deprived of serum and incubated overnight before treatment with APN. The sequences of the siRNAs are as follows: AdipoR1, GAGACUGGCAACAUCUGGACAAA; AdipoR2, GCUUAGAGACACCUGUUUGUUAA; scramble siRNA (as a control), AUUUAACUUCUGGUGACGAUACU. Western blot analysis

Adult mouse CFBs were seeded on 60-mm-diameter plates with serum-free medium for 24 h, and then treated with or without APN (10 μg/ml). Cells were washed once with ice-cold PBS, and lysed in 0.10 ml lysis buffer [20 mM Tris/HCl, pH 7.4, 100 mM NaCl, 10 mM sodium pyrophosphate, 5 mM EDTA, 50 mM NaF, 1 mM sodium vandate, 0.1% (w/v) SDS, 10% (w/v) glycerol, 1% (v/v) Triton X-100 and 1% (w/v) sodium deoxycholate] containing 1 μm leupeptin, 0.1 μm aprotinin, 1 mM PMSF and 1 μm pepstatin. Protein concentration was calculated using the BCA (bicinchoninic acid) protein assay kit (Pierce Biotechnology). Protein (60 μg) was loaded onto a SDS/10% polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes (Pall Corporation), analysed with antibodies according to the supplier’s protocol, and visualized with peroxidase and an enhanced chemiluminescence system (ECL kit, Pierce Biotechnology, Inc.). The following antibodies were used in this study: anti-β-actin (1:2000 dilution), antiSTAT3 (1:1000 dilution), anti-AdipoR1, anti-AdipoR1 and anti-phospho-Tyr705 -STAT3 (1:1000 dilution). Immunofluorescence assay

Adult mouse CFBs grown on glass coverslips in six-well dishes were starved for 24 h and then treated with 10 μg/ml APN for 3 h. After washing with 37◦ C PBS, cells were fixed with 4% (w/v) paraformaldehyde, permeabilized with 0.2% (v/v) Triton X-100, incubated with an anti-STAT3 antibody at 4◦ C overnight, and Cy5-conjugated AffiniPure Goat anti-(rabbit IgG) Ig (Jackson Immuno-Research, West Grove, PA, U.S.A.) (1:500) at 37◦ C for 1 h. Cells were counterstained with 5 μg/ml Hoechst 33342 (Sigma–Aldrich) to visualize the nucleus. Microscopic images were acquired using a Leica Microscope.

ELISA

Adult mouse CFBs were seeded on 24-well plates in serumfree medium for 24 h, and then treated with or without APN (10 μg/ml) for 3, 6, 12 and 24 h. The analysis of IL-6 protein concentration in cell culture supernatants was measured using mouse IL-6 Quantikine ELISA Kit (R&D Systems) according to the manufacturer’s instructions. Quantitative estimation of mRNA by real-time RT (reverse transcription)–PCR

To determine whether AdipoR1/2 is involved in APN-induced IL-6 production, siRNA of AdipoR1 and AdipoR2 were used to knockdown the expression of these receptors. Adult mouse CFBs were transfected with 10 nM siRNAs of AdipoR1 or AdipoR2 and, 36 h after transfection, cells were starved overnight, followed by treatment with 10 μg/ml APN for another 24 h, and the culture medium was collected for the measurement of IL-6 protein production by ELISA as described above. Total RNA was extracted using a total RNA isolation system kit (Promega) for the detection of IL-6 mRNA expression by SYBR green real-time PCR. cDNA was synthesized with the Reverse Transcription System kit (Promega). The following primers were used for real-time PCR: IL-6 (amplicon length 141 bp), 5 -GAGGATACCACTCCCAACAGACC-3 and 5 AAGTGCATCATCGTTGTTCATACA-3 ; β-actin (amplicon length 101 bp), 5 -CTTCCTTCTTGGGTATGGAATCC-3 and 5 -CGTAAAGACCTCTATGCCAACACA-3 . cDNA (1 μl) was mixed with 2×SYBR green PCR master mix (Applied Biosystems) and gene-specific primers in a final volume of 20 μl and real-time PCR was performed (ABI PRISM 7000, PE Applied Biosystems): hold for 2 min at 50◦ C and 10 min at 95◦ C for one cycle followed by amplification of cDNA for 40 cycles with melting for 15 s at 95◦ C and annealing and extension for 1 min at 60 ◦ C. The values were normalized using β-actin as an endogenous internal standard. Statistical analysis

Values are expressed as the means + − SEM. The statistical significance of the differences between the means of the groups was determined by one-way ANOVA or two-tailed Student’s t test. A value of P < 0.05 was considered significant.

Funding This research received no specific grant from any funding agency in the public, commercial or notfor-profit sectors.

Luciferase reporter assay

Adult mouse CFBs were transfected with 2×pAPRE-Luc plasmid (APRE reporter) together with phRL-TK using FuGENETM 6 following the manufacturer’s instructions. Luciferase activity in cell lysates was determined by a dual luciferase reporter assay system (Promega) and normalized to the Renilla luciferase activity of co-transfected phRL-TK. After 24 h, cells were serum-starved overnight and then treated with 10 μg/ml APN for 24 h after 1 h pre-incubation with 4 μg/ml anti-gp130 neutralizing antibody or 10 μM AG490. Cell extracts were prepared and assayed according to the manufacturer’s instructions (Dural Luciferase Assay System; Promega).

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Adiponectin activates STAT3 in cardiac fibroblasts

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Received 8 July 2008/2 September 2008; accepted 17 September 2008 Published as Immediate Publication 17 September 2008, doi:10.1042/BC20080117

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