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(Circulation. 1996;94:2626-2632.)
© 1996 American Heart Association, Inc.

Articles

Activation of JAK-STAT and MAP Kinases by Leukemia Inhibitory Factor Through gp130 in Cardiac Myocytes

Keita Kunisada, MD; Hisao Hirota, MD; Yasushi Fujio, MD; Hideo Matsui, MD; Yoshihiko Tani, MD; Keiko Yamauchi-Takihara, MD; Tadamitsu Kishimoto, MD

Department of Medicine III, Osaka University Medical School, Japan.

Correspondence to Keiko Yamauchi-Takihara, MD, PhD, Department of Medicine III, Osaka University Medical School, 2-2 Yamadaoka, Suita, Osaka 565, Japan. E-mail takihara@imed3.med.osaka-u.ac.jp.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Interleukin (IL)-6–related cytokines share gp130 as the signal-transducing protein. Downstream of gp130, two signal-transducing pathways have been recognized, the Janus kinase–signal transducer and activator of transcription (JAK-STAT) pathway and the Ras–mitogen-activated protein kinase (MAPK) pathway. To determine whether these two signaling pathways through gp130 are present in cardiac myocytes, we examined their activation by using leukemia inhibitory factor (LIF), which is a member of the IL-6 cytokine family.

Methods and Results Lysates from neonatal rat cardiac myocytes were immunoprecipitated with anti-gp130, anti-JAK1, or anti-STAT3 antibody and blotted with anti-phosphotyrosine antibody. Tyrosine phosphorylation of gp130, JAK1, and STAT3 was observed after LIF stimulation in cardiac myocytes. MAPKs were maximally activated 5 minutes after LIF stimulation. Furthermore, anti-gp130 antibody significantly inhibited the LIF-induced activation of JAK1, STAT3, and MAPKs. To examine whether these signaling pathways were also activated in the adult heart in vivo, LIF was injected intravenously into a 6-week-old mouse, and the heart was examined subsequently. gp130, STAT3, and MAPKs were activated in the heart after LIF treatment.

Conclusions These results demonstrate for the first time that a JAK-STAT pathway and a MAPK pathway are present downstream of gp130 in cardiac myocytes and are rapidly activated by LIF both in vitro and in vivo. Activation of gp130 constitutes a novel signaling pathway in cardiac myocytes.

Key Words: signal transduction • hypertrophy • interleukins • gp130

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Cardiac hypertrophy is reported to be stimulated by ET-1,^{1 2} NE,^{3 4} and Ang II.⁵ Recently, culture medium from mouse embryoid bodies was reported to exert a potent hypertrophic action on cultured cardiac myocytes in vitro.⁶ The protein responsible for this activity was characterized by use of an expression cloning approach, leading to the isolation and cloning of a 21.5-kD protein named CT-1. The amino acid sequence of CT-1 is highly homologous with those of LIF, IL-11, and CNTF, which are members of the IL-6 cytokine family.⁶

This cytokine family is known to share a common ß-receptor, known as gp130, as a signal-transducing receptor. In the IL-6 receptor system, a complex of IL-6 and IL-6 receptor associates with gp130 to induce homodimerization of gp130. Formation of the gp130 homodimer activates some kinases and transduces signals to the nucleus.⁷ To investigate the physiological functions of gp130 in vivo, Hirota and colleagues⁸ produced and examined transgenic mice with continuously activated gp130 protein. These double-transgenic mice that overexpressed IL-6 and IL-6 receptors demonstrated severe ventricular hypertrophy at 20 weeks of age.⁸

Two major signaling pathways from gp130 to the nucleus have been identified: an MAPK pathway and a JAK-STAT pathway.⁹ In cardiac myocytes, the MAPK pathway is activated by many factors (eg, ET-1,¹⁰ FGF,¹¹ NE,^{10 12} and mechanical loading¹³ ) is thought to be important in the induction of hypertrophy. But the importance of the STAT pathway in cardiac myocytes has not been examined yet.

The present study was designed to assess whether a JAK-STAT pathway and an MAPK pathway are present downstream of gp130 in cardiac myocytes and to demonstrate whether these pathways are activated in cardiac myocytes after phosphorylation of gp130 by LIF.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Materials
Medium-199 (Flow Laboratories, Inc), newborn calf serum (GIBCO), bovine pancreas insulin, human transferrin, bromodeoxyuridine, trypsin, and collagenase (Sigma Chemical Co) were used for myocardial cell culture. Murine recombinant LIF (AMRAD Co, Ltd), human recombinant IL-6 (donated by Ajinomoto), and norepinephrine (Sankyo Co) were used in the present study.

Rabbit anti-STAT3, anti-JAK1, anti-gp130, anti-ERK1, and anti-ERK2 polyclonal antibodies were purchased from Santa Cruz Biotechnology, Inc. Mouse anti-phosphotyrosine monoclonal antibody (4G10) was purchased from Upsate Biotechnology, Inc. Murine anti-gp130 functional blocking antibody (RX435) was kindly provided by Dr T. Taga (Osaka University). MAPK assay was performed by use of a BIOTRAK MAPK assay kit, currently available from Amersham International, plc. An enhanced chemiluminescence detection system was also obtained from Amersham.

Cell Culture
Primary cultures of neonatal cardiac myocytes were prepared from the ventricles of 1-day-old Sprague-Dawley rats (Nippon Dobutsu, Japan) as described previously.¹⁴ Cultures were enriched with myocardial cells by preplating them for 60 minutes to deplete the population of NMCs. Nonattached cells were then suspended in medium-199 (adjusted to pH 7.4 with 20 mmol/L sodium bicarbonate, M-199), supplemented with 10% newborn calf serum and 0.1 mmol/L bromodeoxyuridine, plated 3 mL onto 60-mm plastic culture dishes at a concentration of 2x10⁵ cells/mL, and cultured for 24 hours at 37°C in 95% air/5% CO₂. More than 90% of the cells displayed spontaneous contractile activity. Attached cells during preplating were cultured as NMCs and maintained for 4 days with a change of medium every 2 days. NMCs were passaged by treatment with 0.1% trypsin/0.02% EDTA at 37°C for 10 minutes and replated onto new dishes. NMC cultures were composed of >95% fibroblasts. Cells were passaged twice and seeded at a density of 1.0x10⁵ cells/mL onto 60-mm dishes. The culture medium was changed to M-199 24 hours before the experiments. Neonatal mouse ventricular myocytes were isolated from 1- to 2-day-old DDY mice following the protocol outlined above. One litter (8 to 12 pups) yielded 4x10⁵ cells.

Animal Preparation
Male DDY mice (6 weeks old) weighing between 45 and 55 g were obtained (Nippon Dobutsu) for this study. They were anesthetized with diethylether, and LIF (6x10⁴ U/kg), IL-6 (2x10³ U/kg), or NE (1x10² µg/kg) was injected into the tail vein. The mice were killed by cervical dislocation, and the hearts and livers were removed and used in the subsequent experiments.

Immunoprecipitation
At the end of the experiments, cells were rinsed rapidly with ice-cold PBS containing 1 mmol/L sodium orthovanadate, harvested with lysis buffer (20 mmol/L Tris-HCl, pH 7.4; 1% NP40; 0.1% SDS; 150 mmol/L NaCl; 1 mmol/L EDTA; 10 µg/mL aprotinin; 1 mmol/L sodium orthovanadate; and 0.5 mmol/L PMSF), and homogenized in a Teflon-glass homogenizer at 4°C. (Aprotinin, PMSF, and sodium orthovanadate were added just before homogenization.) The homogenates were centrifuged at 100 000g for 30 minutes at 4°C. Supernatants were collected and then incubated with either anti-STAT3 antibody, anti-JAK1 antibody, or anti-gp130 antibody and protein A–sepharose (Pharmacia) for 4 hours at 4°C. The immunoprecipitates were washed three times with TBS buffer (50 mmol/L Tris, pH 7.4; 150 mmol/L NaCl; 0.5 mmol/L PMSF; and 1 mmol/L sodium orthovanadate), eluted with 25 µL of sample buffer (62.5 mmol/L Tris, pH 6.8; 2% SDS; 5% 2-mercaptoethanol; and 10% glycerol), and boiled for 10 minutes. Thereafter, the samples were centrifuged at 5000 rpm for 1 minute, and the supernatant was collected and stored at -80°C until assay.

The hearts and livers were homogenized with lysis buffer using 15 strokes in a motor-driven Teflon-glass homogenizer at 4°C. These lysates were treated as described above.

Western Blot Analysis
The samples were separated in a 7.5% SDS-PAGE, and the resolved proteins were electrophoretically transferred onto a polyvinylidene difluoride membrane (Immobilion-P, Millipore Co) with a transfer buffer (25 mmol/L Tris, 190 mmol/L glycine, and 20% methanol). Membranes were blocked with 5% skim milk (DIFCO Laboratories) and probed with anti-phosphotyrosine antibody at a 1:1000 dilution for 1 hour to detect phosphorylated gp130, JAK1, or STAT3. The immune complexes were visualized with Kodak X-OMAT-AR film (Eastman Kodak Co) by use of the enhanced chemiluminescence system according to the manufacturer's instructions. The filters were then stripped and reprobed with anti-STAT3, anti-JAK1, or anti-gp130 Ab.

MAPK Assay
Protein kinase activity was measured with the P42/P44 MAPK assay system as previously described.¹⁵ The cardiac myocytes and NMCs were lysed at 4°C with a lysis buffer containing 20 mmol/L Tris-HCl, pH 7.4; 20 mmol/L ß-glycerophosphate; 1 mmol/L sodium orthovanadate; 2 mmol/L EGTA; 0.5 mmol/L PMSF; 10 µg/mL aprotinin, and 20 mmol/L NaF. The lysates were centrifuged at 100 000g for 30 minutes at 4°C. The supernatant containing MAPKs was collected, and the protein concentration was measured with a protein assay system (Bio-Rad Laboratories). The supernatant was diluted with lysis buffer to equalize the protein concentration and then applied to an assay tube. A phosphorylation assay of the synthetic peptide substrate was performed by use of [³²P]-ATP. The peptide used in this assay contains the phosphorylation sequence PLS/TP as a site for phosphorylation.¹⁶ This synthetic peptide is phosphorylated more specifically by MAPK than myelin basic protein, which is commonly used to detect MAPK activity. The reaction was initiated by the addition of ATP to the mixture, which was then incubated for 30 minutes at 30°C. The reaction was terminated by the addition of a stop buffer. The phosphorylated synthetic peptide was isolated by application of the reaction mixture to phosphocellulose paper. The papers were then washed twice with 50 mmol/L H₃PO₄ and placed in scintillation vials with a 10-mL liquid scintillation cocktail. Radioactivity was determined with a liquid scintillation counter. MAPK activity was adjusted by the protein content of each sample.

Kinase Assay of Anti-ERK1 and ERK2 Immunoprecipitates in Myelin Basic Protein–Containing Gels After SDS-PAGE
MAPKs were immunoprecipitated with anti-ERK1 or anti-ERK2 antibody and treated as described above. The immunoprecipitates were electrophoresed on a 10% SDS-PAGE gel containing 0.5 mg/mL myelin basic protein. After the gel was washed in 20% 2-propanol and 50 mmol/L Tris-HCl, pH 8.0, MAPKs were denatured by 6 mol/L guanidine and 50 mmol/L Tris-HCl for 1 hour and renatured in 50 mmol/L Tris-HCl containing 0.04% Tween 40 and 5 mmol/L 2-mercaptoethanol for 16 hours at 4°C. A phosphorylation of myelin basic protein was assayed by incubation of a gel with 25 µCi [³²P]-ATP; 40 mmol/L HEPES, pH 7.5; 0.1 mmol/L EGTA; 20 mmol/L MgCl₂; and 2 mmol/L dithiothreitol for 30 minutes. Thereafter, the gel was washed extensively, dried, and subjected to autoradiography.

Statistical Analysis
Statistical analysis was performed by use of Student's t test. A value of P<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
LIF Induces Tyrosine Phosphorylation of gp130 in Cardiac Myocytes
To determine whether LIF transduces signals in cardiac myocytes, we initially examined the tyrosine phosphorylation of gp130 after LIF stimulation. Cardiac myocytes were cultured with M-199 with the addition of LIF (1x10³ U/mL) for 2, 5, or 15 minutes. Lysates of cardiac myocytes were immunoprecipitated with anti-gp130 antibody; then Western blots of the precipitated proteins were probed with anti-phosphotyrosine antibody. gp130 was rapidly tyrosine phosphorylated within 2 minutes of LIF stimulation and maximally phosphorylated after 5 minutes (Fig 1, top). gp130 was gradually dephosphorylated by 15 minutes. The amounts of precipitated gp130 were the same for each period (Fig 1, bottom).

View larger version (47K): [in this window] [in a new window]   Figure 1. Effect of LIF on the activation of gp130 in cardiac myocytes. Lysates from cardiac myocytes stimulated with 1x103 U/mL LIF were prepared at the times indicated and incubated with anti-gp130 antibody for 4 hours at 4°C. The immunoprecipitated proteins were separated by a 7.5% SDS-PAGE and transferred onto an immobilion-P membrane. The blot was probed with anti-phosphotyrosine (pTy) antibody 4G10 (top). The blot was reprobed with anti-gp130 antibody (bottom). Molecular weight protein markers indicate 200 and 97 kD. Ig indicates immunoglobulin.

LIF Induces Tyrosine Phosphorylation of JAK1 in Cardiac Myocytes
Next, we examined whether LIF tyrosine phosphorylated JAK1 in cardiac myocytes after gp130 activation. JAK1 is recognized to bind constitutively to the cytoplasmic domain of gp130.^{17 18} Cardiac myocytes were stimulated with LIF (1x10³ U/mL), and the lysates were immunoprecipitated with anti-JAK1 antibody. JAK1 was phosphorylated within 5 minutes and continued to be phosphorylated after 15 minutes (Fig 2, top). JAK2 was also activated by LIF with a similar time course (data not shown). The amounts of precipitated JAK1 were the same for each period (Fig 2, bottom).

View larger version (50K): [in this window] [in a new window]   Figure 2. Effect of LIF on the activation of JAK1 in cardiac myocytes. Lysates from cardiac myocytes stimulated with 1x103 U/mL LIF were prepared at the times indicated and incubated with anti-JAK1 antibody for 4 hours at 4°C. The immunoprecipitated proteins were separated by a 7.5% SDS-PAGE and transferred onto an immobilion-P membrane. The blot was probed with anti-phosphotyrosine (pTy) antibody 4G10 (top). The blot was reprobed with anti-JAK1 antibody (bottom). Molecular weight protein markers indicate 97 and 69 kD. Ig indicates immunoglobulin.

STAT3 is Tyrosine Phosphorylated With LIF in Cardiac Myocytes
The JAK family consists of non–receptor-type protein tyrosine kinases that phosphorylate STATs on one particular tyrosine residue. We next examined whether LIF activates STAT3 in cardiac myocytes. Fig 3 shows the time course of LIF-stimulated tyrosine phosphorylation of STAT3 in cardiac myocytes and NMCs 5, 15, 30, and 60 minutes after the addition of 1x10³ U/mL LIF. STAT3 was tyrosine phosphorylated within 5 minutes of LIF stimulation in cardiac myocytes and gradually dephosphorylated by 30 minutes. The extent of STAT3 phosphorylation returned to baseline by 60 minutes after LIF stimulation (Fig 3A, top). LIF also phosphorylated STAT3 in NMCs according to a similar time course. However, the extent of phosphorylation of STAT3 was lower in NMCs than in cardiac myocytes (Fig 3B, top). The amounts of precipitated STAT3 were the same for each period (Fig 3A and 3B, bottom).

View larger version (34K): [in this window] [in a new window]   Figure 3. Effect of LIF on the activation of STAT3 in cardiac myocytes and NMCs. A, Cardiac myocytes stimulated with 1x103 U/mL LIF. B, NMCs stimulated with 1x103 U/mL LIF. The cell lysates prepared at the time indicated were incubated with anti-STAT3 antibody for 4 hours at 4°C. The immunoprecipitated proteins were separated by 7.5% SDS-PAGE and transferred onto an immobilion-P membrane. The blot was probed with anti-phosphotyrosine (pTy) antibody 4G10 (top). The blot was reprobed with anti-STAT3 antibody (top). Molecular weight protein markers indicate 97 and 69 kD.

Fig 4 shows the dose-dependent effect of LIF on tyrosine phosphorylation of STAT3 in cardiac myocytes. The myocytes were incubated with the indicated doses of LIF (0, 1x10, 1x10², or 1x10³ U/mL) for 15 minutes. Although 1x10 U/mL LIF did not phosphorylate STAT3, a significant increase in the phosphorylation of STAT3 was observed with 1x10³ U/mL LIF. A dose-dependent effect of LIF on tyrosine phosphorylation of STAT3 was observed in cardiac myocytes (Fig 4, top). The amounts of precipitated protein were the same for each period (Fig 4, bottom).

View larger version (56K): [in this window] [in a new window]   Figure 4. Dose-dependent effect of LIF on the activation of STAT3 in cardiac myocytes. Lysates from cardiac myocytes incubated with indicated doses of LIF (0, 1x10, 1x102, or 1x103 U/mL) for 15 minutes were treated as described in Fig 3. The blot was probed with anti-phosphotyrosine (pTy) antibody 4G10 (top). The blot was reprobed with anti-STAT3 antibody (bottom). Molecular weight protein markers indicate 97 and 69 kD. Ig indicates immunoglobulin.

Anti-gp130 Antibody Inhibits LIF-Induced Activation of JAK1 and STAT3 in Cardiac Myocytes
To determine whether LIF activates JAK1 and STAT3 through gp130, cardiac myocytes were incubated with anti-gp130 blocking antibody (RX435) for 2 hours preceding the LIF stimulation. This antibody has been shown specifically to block the actions of IL-6, LIF, oncostatin M, and IL-11 on a mouse myeloid leukemic M1 cell line (Drs T. Taga and Kishimoto, unpublished data, 1994) and inhibits the binding of CT-1 to M1 cells.¹⁹ In addition, RX435 is reported to abolish the induction of c-fos by CT-1 and LIF in cardiac myocytes.²⁰ Because RX435 does not cross-react with rat gp130, we used neonatal murine cardiac myocytes only in this set of experiments.

As Fig 5 shows, anti-gp130 blocking antibody significantly inhibited the tyrosine phosphorylation of JAK1 (Fig 5A, top) and STAT3 (Fig 5B, top) induced by LIF stimulation in cardiac myocytes. The amounts of precipitated proteins were the same for each period (Fig 5A and 5B, bottom). These results indicate that LIF also evokes gp130 to activate the downstream JAK-STAT signaling pathway in cardiac myocytes, as observed in other cell types.

View larger version (29K): [in this window] [in a new window]   Figure 5. Effect of anti-gp130 blocking antibody on LIF-induced activation of JAK1 and STAT3. Neonatal murine cardiac myocytes were incubated with or without anti-gp130 blocking antibody RX435 for 2 hours preceding LIF (1x103 U/mL) stimulation. A, Lysates from cardiac myocytes stimulated with LIF for 5 minutes were treated as described in Fig 2. The blot was probed with anti-phosphotyrosine (pTy) antibody 4G10 (top). The blot was reprobed with anti-JAK1 antibody (bottom). Molecular weight protein markers indicate 200 and 97 kD. B, Lysates from cardiac myocytes stimulated with LIF for 15 minutes were treated as described in Fig 3. The blot was probed with anti-phosphotyrosine antibody 4G10 (top). The blot was reprobed with anti-STAT3 antibody (bottom). Molecular weight protein markers indicate 97 and 69 kD. These experiments were repeated three times with identical results.

LIF Activates MAPK in Cardiac Myocytes
Another signaling pathway downstream of gp130 is the MAPK pathway. MAPK activity was measured 0, 2, 5, 15, 30, and 60 minutes after LIF (1x10³ U/mL) or NE (1x10^-5 mol/L) stimulation in cardiac myocytes. The results were expressed as a percentage of the baseline MAPK activity value. MAPK was activated in cardiac myocytes as early as 2 minutes after LIF stimulation and maximally augmented to 460% after 5 minutes (Fig 6A). This augmented activity declined 15 minutes after LIF stimulation and returned to the control level by 30 minutes. As Fig 6A shows, NE also activated MAPK in cardiac myocytes, with maximal augmentation at 5 minutes. The maximal MAPK activity produced by NE stimulation was 2.5 times higher than that caused by LIF stimulation. Furthermore, the augmented MAPK activity observed after NE stimulation was sustained even after 60 minutes. LIF also activated MAPK in NMCs, and the time course was similar to that observed in cardiac myocytes. However, the maximal activity of MAPK in NMCs was half that observed in cardiac myocytes (data not shown).

View larger version (78K): [in this window] [in a new window]   Figure 6. MAPK activity in cardiac myocytes after LIF stimulation. A, Lysates from cardiac myocytes stimulated with LIF (1x103 U/mL) or NE (1x10-5 mol/L) were prepared at the times indicated (0, 2, 5, 15, 30, and 60 minutes). The lysates were assayed for MAPK activity with a BIOTRAK MAPK assay kit, as described in "Methods." Results are expressed as a percentage of the baseline MAPK activity value. *P<.05 vs control. B, Cardiac myocytes were stimulated with LIF (1x103 U/mL) for the times indicated. The immunoprecipitates with anti-ERK1 (top) or anti-ERK2 (bottom) antibody were subjected to MAPK assays in a myelin basic protein–containing gel as described in "Methods." Molecular weight markers indicate 46 and 30 kD. These experiments were repeated three times with identical results. C, Neonatal murine cardiac myocytes were stimulated with LIF (1x103 U/mL) for 5 minutes with or without anti-gp130 blocking antibody RX435. Results are expressed as a percentage of the baseline MAPK activity value. Data are mean±SD from three samples. *P<.05 vs control.

Furthermore, LIF-induced MAPK activation was also examined by kinase assay with a myelin basic protein–containing gel. As Fig 6B shows, stimulation with LIF (1x10³ U/mL) evoked activation of both ERK1 (top) and ERK2 (bottom) in cardiac myocytes, with a similar time course as that observed in Fig 6A. To investigate whether LIF activates MAPK through gp130, cardiac myocytes were incubated with the anti-gp130 blocking antibody RX435 for 2 hours preceding the LIF stimulation. As Fig 6C shows, anti-gp130 blocking antibody completely inhibited the increase in MAPK activity induced by LIF.

These results indicate that LIF requires gp130 to activate the downstream signaling pathway that leads to the activation of MAPKs (ERK1 and ERK2) in cardiac myocytes.

STAT3 in the Adult Heart Is Phosphorylated by LIF But Not by IL-6 or NE
To investigate whether LIF also transduces signals in the adult heart in vivo, we examined the activation of gp130 and STAT3 in the murine heart after LIF stimulation. Heart lysates obtained from 6-week-old mice that had been intravenously injected with LIF were immunoprecipitated with either anti-gp130 or anti-STAT3 antibody. Thereafter, they were probed with anti-phosphotyrosine antibody.

gp130 in the adult heart was tyrosine phosphorylated within 5 minutes of treatment with 6x10⁴ U/kg LIF (Fig 7), as observed in the cultured cardiac myocytes (Fig 1). STAT3 in the heart was also tyrosine phosphorylated within 5 minutes of treatment with LIF, maximally activated at 15 minutes, and gradually declined to near baseline by 60 minutes (Fig 8A, top left). The amounts of precipitated STAT3 were the same for each period (Fig 8A, bottom left). Because STAT3 was isolated and cloned from the liver,²¹ the livers from the same mice were also examined. The time course of phosphorylation of STAT3 in the liver was similar to that observed in the heart (Fig 8A, right). STAT3 was also maximally phosphorylated 15 minutes after LIF treatment in the liver. Furthermore, the time course of STAT3 phosphorylation observed in this study was similar to that observed in nuclear extract obtained from murine liver.²¹

View larger version (48K): [in this window] [in a new window]   Figure 7. LIF activates gp130 in the adult heart. Heart lysates were prepared from 6-week-old mice 0, 5, and 15 minutes after LIF (6x104 U/kg IV) injection. The lysates were immunoprecipitated with anti-gp130 antibody and treated as described in Fig 1. The blot was probed with anti-phosphotyrosine (pTy) antibody 4G10 (top). The blot was reprobed with anti-gp130 antibody (bottom). Molecular weight protein markers indicate 97 and 69 kD. Ig indicates immunoglobulin.

View larger version (39K): [in this window] [in a new window]   Figure 8. Effects of LIF, IL-6, and NE on the activation of STAT3 in the adult heart. A, Effect of LIF on the activation of STAT3 in the heart (left). Heart lysates were prepared from LIF (6x104 U/kg)-injected mice at the times indicated (0, 5, 15, 30, and 60 minutes). Effect of LIF on the activation of STAT3 in the liver (right). Hepatic lysates were prepared from the same mice as described above. B, Effect of IL-6 on the activation of STAT3 in the heart. Heart lysates were prepared from IL-6 (2x103 U/kg)–injected mice at the times indicated (0, 5, 15, and 30 minutes). C, Effect of NE on the activation of STAT3 in the heart. Heart lysates were prepared from NE (1x102 µg/kg)-injected mice at the times indicated (0, 5, 15, and 30 minutes). The heart and liver lysates were immunoprecipitated with anti-STAT3 antibody and blotted with anti-phosphotyrosine (pTy) antibody 4G10 (top) or anti-STAT3 antibody (bottom). Molecular weight protein markers indicate 97 and 69 kD.

To examine whether other cytokines that share gp130 as a receptor also stimulate tyrosine phosphorylation of STAT3 in the heart, we administered IL-6 (2x10³ U/kg) intravenously in mice and then examined the hearts. Although LIF significantly activated STAT3 in the heart, IL-6 did not induce STAT3 phosphorylation in the heart (Fig 8B).

Because the activation of gp130 is reported to produce myocardial hypertrophy,⁸ we next examined whether NE stimulates tyrosine phosphorylation of STAT3 in the heart. However, intravenous administration of NE (1x10² µg/kg) did not activate STAT3 within the first 30 minutes (Fig 8C).

LIF Activates MAPK in Murine Hearts
The hearts from LIF-treated mice also were examined to investigate whether LIF activates MAPK in vivo. MAPK in murine hearts was measured 0, 5, 15, and 30 minutes after administration of 6x10⁴ U/kg LIF. The results were expressed as a percentage of the baseline MAPK activity value. As Fig 9 shows, LIF treatment significantly augmented MAPK activity by 270% at 5 minutes. NE (1x10² µg/kg) treatment also augmented MAPK activity by 300% at 5 minutes in vivo (data not shown).

View larger version (15K): [in this window] [in a new window]   Figure 9. Effect of LIF on the MAPK activity in the adult heart. The heart lysates were prepared 0, 5, 15, and 30 minutes after LIF (6x104 U/kg) administration. The lysates were assayed for MAPK activity as described in Fig 6A. Results are expressed as a percentage of the baseline MAPK activity value. Data are mean±SD from four samples. *P<.05 vs control.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In this study, we showed that JAK-STAT and MAPK pathways are present in cardiac myocytes and demonstrated for the first time the activation of JAK1 and STAT3 after tyrosine phosphorylation of gp130 in cardiac myocytes. gp130 was rapidly phosphorylated within 2 minutes of LIF stimulation. Thereafter, JAK1 and STAT3 were activated within 5 minutes and declined by 60 minutes. These activations of JAK1 and STAT3 induced by LIF stimulation were significantly inhibited with the addition of anti-gp130 blocking antibody. This rapid activation of the STAT pathway is considered to be a direct effect of exogenous LIF but not of cytokines subsequently induced by LIF stimulation.

The JAK-STAT pathway was found as a result of extensive studies on transcriptional regulation induced by interferon-. The components of interferon-stimulated gene factor 3 have been identified as two novel proteins, STAT1 (91 kD) and STAT2 (113 kD),^{22 23 24} which are members of the STAT family and activate gene expression. As far as we know, six members of the STAT family have been discovered.^{21 23 24 25 26 27 28} STAT3, which was found and cloned by Akira and colleagues,²¹ binds to IL-6 response elements identified in the promoter region of various acute-phase protein genes. In a manner similar to interferon-stimulated gene factor 3, STAT3 is tyrosine phosphorylated and translocates to the nucleus in response to IL-6. Tyrosine phosphorylation of STAT3 was also observed in response to other cytokines, such as LIF or CNTF, whose receptors also share gp130.²¹

CT-1, a member of the IL-6 cytokine family, was recently reported to induce heterodimerization of gp130 and LIF receptor during signal processing.¹⁹ LIF is also reported to induce this heterodimerization.²⁹ We observed that the LIF receptor is expressed in the murine heart throughout life and that LIF itself has a strong potential to induce hypertrophy in cultured neonatal rat cardiac myocytes.³⁰ IL-6 also activates STAT3 in various cells through gp130 after binding to the IL-6 receptor.^{21 25} However, few IL-6 receptors are reported to be expressed in cardiac myocytes.³¹ The concentration of IL-6 used in this study did not allow induction of STAT3 phosphorylation in the heart. Although the downstream region of activated gp130 in cardiac myocytes has not been studied fully yet, at least a JAK-STAT pathway and a MAPK pathway have been observed to be activated in cardiac myocytes, as reported in other cell lines.^{21 25 32}

Transgenic mice overexpressing IL-6 have been reported to show mesangial proliferative glomerulonephritis, rheumatoid arthritis, and plasmacytoma.³³ Recently, Hirota and colleagues⁸ constructed double-transgenic mice carrying the IL-6 and IL-6 receptor genes in which gp130 is also continuously activated in the heart. One of the remarkable phenotypic differences between these two types of transgenic mice involved the heart. Although hearts from IL-6 transgenic mice did not show hypertrophy, the double-transgenic mice had hypertrophied ventricles associated with the same abnormality as observed in the IL-6 transgenic mice.^{8 33} These results suggested that a gp130-dependent signaling pathway may be coupled to a hypertrophic response in cardiac myocytes.

Many studies have revealed that ET-1, FGF, NE, and mechanical loading activate an MAPK pathway through G-protein binding receptors and protein kinase C and promote a hypertrophic response in cultured cardiac myocytes.^{10 11 12 13} Previous studies revealed that MAPK activation by G-protein–coupled receptors is not necessarily due to protein kinase C activation. Intracellular Ca²⁺ rather than protein kinase C seems to be critical for Ang II–induced activation of MAPKs in cardiac myocytes.³⁴ LIF activates MAPK maximally at 5 minutes, which is as rapid as the STAT3 activation seen in cardiac myocytes. This rapid activation of MAPKs observed in cardiac myocytes after LIF stimulation resembles that observed in cultured cardiac myocytes treated with ET-1.¹¹

The significance of a STAT pathway for myocardial hypertrophy has not been studied yet. Yoshida and colleagues³⁵ recently reported that a mouse with a disrupted gene for gp130 had an immature left ventricle that resembled dilated cardiomyopathy and proved to be lethal. The gp130 signaling pathway is considered to play an important role in the proliferation and differentiation of cardiac myocytes, and the STAT pathway might be related to this phenomenon. The target genes of STAT3 and the role of the STAT pathway in cardiac myocytes should be explored further.

In summary, this study demonstrated that a JAK-STAT pathway is present downstream of gp130 in cardiac myocytes and is rapidly activated by LIF. An MAPK pathway is also activated by LIF. These signaling pathways are also activated in the heart by LIF administration in vivo. Phosphorylation of gp130 constitutes a novel signaling pathway in the progression of myocardial hypertrophy.

	Selected Abbreviations and Acronyms

 

Ang = angiotensin CNTF = ciliary neurotrophic factor CT-1 = cardiotrophin-1 ERK = extracellular signal-regulated kinase ET = endothelin FGF = fibroblast growth factor IL = interleukin JAK = Janus kinase LIF = leukemia inhibitory factor MAPK = mitogen-activated protein kinase NE = norepinephrine NMC = nonmyocardial cell PMSF = phenylmethyl sulfonyl fluoride STAT = signal transducer and activator of transcription

	Acknowledgments

 
This study was supported by a grant-in-aid for Scientific Research of Priority Areas from the Japanese Ministry of Education, Science, and Culture and a grant from the Study Group of Molecular Cardiology. We thank Y. Yamaguchi for excellent secretarial assistance.

	Footnotes

 
Presented in part at the 68th Scientific Sessions of the American Heart Association, Anaheim, Calif, November 13-16, 1995, and published in abstract form (Circulation. 1995;92[suppl I]:I-570).

Received April 18, 1996; revision received August 1, 1996; accepted August 7, 1996.

	References

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