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(Circulation Research. 1997;81:656-663.)
© 1997 American Heart Association, Inc.

Articles

Leukemia Inhibitory Factor, a Potent Cardiac Hypertrophic Cytokine, Activates the JAK/STAT Pathway in Rat Cardiomyocytes

Hiroaki Kodama, Keiichi Fukuda, Jing Pan, Shinji Makino, Akiyasu Baba, Shingo Hori, , Satoshi Ogawa

From the Cardiopulmonary Division (H.K., K.F., J.P., S.M., A.B., S.O.), Department of Internal Medicine, and the Department of Emergency Medicine (S.H.), Keio University, Tokyo, Japan.

Correspondence to Keiichi Fukuda, MD, PhD, Cardiopulmonary Division, Department of Internal Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. E-mail kfukuda{at}mc.med.keio.ac.jp

	Abstract

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Abstract Leukemia inhibitory factor (LIF) is a member of the interleukin-6 family of cytokines, which induces a wide range of responses in a variety of cells. The aim of this study was to investigate whether LIF induces cardiomyocyte hypertrophy and transmits signals through the JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) pathway in primary cultured neonatal rat cardiomyocytes. LIF increased protein content and [³H]phenylalanine uptake in cardiomyocytes in a dose-dependent manner. LIF (10³ U/mL) induced rapid tyrosine phosphorylation of gp130, JAK1, JAK2, STAT1, and STAT3 but not Tyk2 or STAT2. LIF also induced autokinase activity of JAK1 in a time-dependent manner. Gel shift assays for interferon gamma activation site/interferon-stimulated responsive element and sis-inducible element (SIE) revealed that LIF induced dimerization of STAT1 and STAT3 and formation of sis-inducing factor complexes, which subsequently interacted with SIE in the promoter. Preincubation with anti-STAT1 and anti-STAT3 antibodies inhibited the binding of SIF complexes. In conclusion, LIF induces cardiac hypertrophy and directly stimulates the JAK/STAT pathway in cardiomyocytes.

Key Words: signal transduction • JAK kinase • signal transducer(s) and activator(s) of transcription • cardiac hypertrophy • leukemia inhibitory factor

	Introduction

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Cytokines and growth factors interact with their cell surface receptors to activate one or more signaling pathways. These pathways transduce specific responses to the nucleus, culminating in induction of transcription of discrete sets of target genes. Cell surface receptors fall into several large families.^{1 2 3} A number of studies have revealed that stimulation of G protein–coupled seven transmembrane–type receptors, such as ₁ receptors⁴ and angiotensin II receptors, and of tyrosine kinase receptors, such as fibroblast growth factor and platelet-derived growth factor receptors,^{5 6 7} causes myocardial hypertrophy.

Fukada⁸ previously purified a cholinergic neuronal differentiation factor from the supernatant of heart cell culture, and Yamamori et al⁹ showed that this cholinergic neuronal differentiation factor from heart cells is identical to LIF. CT-1 also has recently been found to be a member of the IL-6 cytokine family and to be capable of causing myocardial hypertrophy.^{10 11} These findings suggested that gp130-linked signaling induced by LIF or CT-1 may also play an important role in cardiac hypertrophy. LIF transmits signals via LIF receptors and gp130, a coreceptor of the IL-6 cytokine family (IL-6, IL-11, CNTF, OSM, and LIF).^{12 13 14 15 16} The gp130 and LIF receptor lack a kinase catalytic domain, but they can bind to and activate one or more members of the JAK/STAT tyrosine kinase family.^{17 18 19} The cytokine superfamily uses the JAK/STAT pathway as a major signaling pathway into the nucleus.^{20 21 22 23 24 25 26 27} In contrast to other cytokines that activate only particular combinations of JAK kinases, the IL-6 family can activate all members of the JAK family, although the kinases present vary depending on the cell line examined.²⁸ In spite of using different combinations of JAK family kinases in different cell lines, all members of the IL-6 family of cytokines have been found to stimulate tyrosine phosphorylation of a very similar set of proteins in all sets examined, because the signal-transducing receptor component contains a motif that is specific to the downstream signaling molecules activated by JAK family kinases.²⁹

In the present study, we report (1) that LIF induces cardiomyocyte hypertrophy, (2) that LIF activates the JAK-STAT signal transduction pathway in cardiomyocytes, and (3) the results of a gel mobility shift assay that shows transactivation of the target genes.

	Materials and Methods

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Cell Culture
Primary cultures of cardiomyocytes were prepared from the ventricles of 1-day-old neonatal Wistar rats (Japan Clea) by enzymatic dissociation in 0.03% trypsin, 0.03% collagenase, and 20 µg/mL of DNase I. The cardiomyocytes were collected on the basis of their differential adhesiveness. Cells were seeded at a density of 1x10⁵ cells/cm² on gelatin-coated dishes. The cells were grown in medium 199/DMEM supplemented with 10% FBS and penicillin (50 U/mL)/streptomycin (50 µg/mL) at 37°C in humid air containing 5% CO₂. They were washed two times with serum-free medium 48 hours after plating, incubated in the same medium for 24 hours, and then stimulated with LIF. The nonmyocyte population was 5% to 10%, according to the results of immunofluorescence staining with monoclonal anti–sarcomeric myosin antibody (MF20). In some experiments, cardiac fibroblasts were maintained and subcultured three times, and protein assay was performed.

Protein Content and [³H]Phenylalanine Incorporation
Net protein synthesis was measured in rat neonatal cardiomyocytes and cardiac fibroblasts in gelatin-coated 24-well plates. After 24 hours of serum depletion, primary cultured cardiomyocytes were stimulated with LIF (1 to 1000 U/mL). The cells were then washed with ice-cold PBS three times and lysed with 0.5N NaOH at 24 hours. Protein content was measured by the Lowry method. The results are expressed as micrograms protein per well.

The effects of LIF on [³H]phenylalanine uptake were determined in gelatin-coated 24-well plates. After 24 hours of serum depletion, the cells were stimulated with LIF. [³H]Phenylalanine was added at 0 hours. The cells were then washed three times with ice-cold PBS and incubated for 20 minutes to remove extracellular [³H]phenylalanine, washed three times with 5% trichloroacetic acid to precipitate protein, and then resuspended in 0.5N NaOH. Samples were counted with a scintillation counter. After neutralization with 0.5N HCl, [³H]phenylalanine uptake was measured with a liquid scintillation counter. The results are expressed as disintegrations per minute per well.

Cell-Sizing Protocol
Cells grown on glass coverslips were permeabilized in 1% formaldehyde/PBS for 10 minutes. After blocking with 5% BSA in PBS for 1 hour at room temperature, the cells were incubated with primary antibody (MF20 to sarcomeric myosin). After three washes in PBS for 5 minutes each, the secondary antibody (Texas red–coupled affinity-purified donkey anti-mouse IgG antibody) was applied for 30 minutes. Coverslips were washed three times in PBS and mounted in Permafluor (Lipshaw). Measurement of the size (surface area, perimeter, maximum diameter, and minimal diameter) of the cardiomyocytes was performed using enlarged calibrated fluorescent photomicrographs of the sarcomeric myosin and quantified and validated with a Power Macintosh computer 7600/132 and Epson scanner GT-9000 with Adobe Photoshop version 3.0J and NIH image version 1.56 software.

IP and Western Blot Analysis
Antibodies to JAK1, JAK2, Tyk2, STAT1, and STAT3 were purchased from Santa Cruz Laboratory. Monoclonal antibody to STAT2 was purchased from Transduction Laboratories. Monoclonal antibody for phosphotyrosine (4G10) and polyclonal antibody for gp130 were purchased from Upstate Biotechnology Inc. Recombinant murine LIF was purchased from Genzyme).

After 24 hours of serum depletion, cardiomyocytes were stimulated with LIF at a concentration of 1000 U/mL for 2, 5, 15, 30, and 60 minutes. The cells were then washed with Dulbecco's PBS. To detect phosphotyrosine, cells were lysed in a buffer containing 20 mmol/L Tris-HCl (pH 7.4), 100 mmol/L NaCl, 5 mmol/L EDTA, 1.0% Triton X-100, 10% glycerol, 0.1% SDS, 1.0% deoxycholic acid, 50 mmol/L NaF, 10 mmol/L Na₃P₂O₇, 1 mmol/L Na₃VO₄, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL aprotinin, and 10 µg/mL leupeptin. Cell lysates were precleared by incubation with protein A- or G-Sepharose beads (Sigma Chemical Co). Precipitating antibodies to JAK1, JAK2, Tyk2, STAT1, STAT2, STAT3, and phosphotyrosine were added to the precleared lysates, and the lysates were incubated for 1 hour at 4°C. Immunoprecipitates were pelleted with protein A-Sepharose beads and washed five times with the lysis buffer. The precipitated proteins were dissolved in sample buffer and heated at 95°C for 5 minutes. Proteins were separated on 5.0% to 10.0% SDS-polyacrylamide gel. Fractionated proteins were electrotransferred from the gel to reinforced nitrocellulose membranes (Schleicher & Schuell) in Towbin buffer at a constant current of 400 mA for 2 hours. Nonspecific binding was blocked by incubation in the blocking buffer (5% BSA, 20 mmol/L Tris-HCl [pH 7.4], and 150 mmol/L NaCl) at 4°C overnight. To detect phosphorylated tyrosine, the membranes were incubated with monoclonal antibody 4G10 for 2 hours at room temperature and incubated with peroxidase-conjugated goat anti-mouse IgG. Signals were visualized with an ECL kit (Amersham).

Assay of JAK1 Autokinase Activity
After stimulation with LIF for the indicated periods, cardiomyocytes were lysed in the lysis buffer, and JAK1 was immunoprecipitated with anti-JAK1 antibody. The autophosphorylation activity of JAK1 was assayed by incubating the immunoprecipitates with 10 µCi [-³²P]ATP in 25 mmol/L Tris-HCl (pH 7.4), 140 mmol/L NaCl, 5 mmol/L MnCl2, 10% glycerol, and 20 mmol/L ATP for 20 minutes. Samples were separated by SDS-PAGE, and activity was detected by autoradiography.

Preparation of Nuclear Extracts
After LIF stimulation, cultured neonatal cardiomyocytes were rinsed with PBS at 0°C and scraped into the same buffer. Nuclear extracts were prepared according to standard methods.³⁰ Briefly, harvested cells were resuspended in 5 vol of hypotonic buffer (10 mmol/L HEPES [pH 7.9], 10 mmol/L KCl, 1.5 mmol/L MgCl2, and 0.5 mmol/L dithiothreitol), supplemented with protease and phosphatase inhibitors (0.5 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1 mmol/L Na₃VO₄, and 1 mmol/L NaF), incubated for 10 minutes on ice, and sedimented. Cells were resuspended in 2 vol of the same buffer, Dounce-homogenized, and sedimented at 1000g for 10 minutes, and the pellet (nuclei) was collected. The pelleted nuclei were resuspended with low-salt buffer (20 mmol/L HEPES [pH 7.9], 25% glycerol, 20 mmol/L KCl, 1.5 mmol/L MgCl₂, and 0.2 mmol/L EDTA) supplemented with proteinase and phosphatase inhibitors (see above) and then incubated with high-salt buffer (same as the low-salt buffer except that KCl was 1.2 mol/L) for 30 minutes at 4°C. The nuclear extracts were dialyzed against dialysis buffer (20 mmol/L HEPES [pH 7.9], 20% glycerol, 100 mmol/L KCl, and 0.2 mmol/L EDTA) overnight. The protein concentrations were determined by the Bradford assay,³¹ and the extracts were stored at -80°C.

Gel Mobility Shift Assay
Gel mobility shift assays were performed as described previously with minor modifications.³² Nuclear extracts (5 µg) were incubated with 1 µg of poly(dI-dC)–poly(dI-dC) (Pharmacia Biotech) in 20 µL of 25 mmol/L HEPES (pH 7.9), 50 mmol/L KCl, 0.5 mmol/L EDTA, and 10% glycerol for 20 minutes at 25°C. For supershift analysis, the appropriate antiserum was added to the extracts in the presence of 1 µg poly(dI-dC)/mL for 1 hour before adding the probes. The samples were incubated with 1 or 2 fmol of radiolabeled probes (5000 cpm) for 10 minutes at 25°C. The probes used in the present study were purchased from Santa Cruz Biotechnology, and the sequences have been described (SIE-DNA, 5'-GTGCATTTCCCGTAAATCTTGTCTACA-3'; mutant SIE-DNA, 5'-GTGCATCCACCGTAAATCTTGTCTACA; GAS/ISRE-DNA, 5'-AAGTACTTTCAGTTTCATATTACTCTA-3'; and mutant-GAS/ISRE-DNA, 5'-AAGTACTTTCAGTGGTCTATTACTCTA-3). Binding reactions were resolved by 4% native polyacrylamide gel electrophoresis containing 1x TAE buffer (40 mmol/L Tris-acetate and 1 mmol/L EDTA). Gels were run at 150 V at 4°C for 2 to 3 hours in 1x TAE buffer and dried, and x-ray film was exposed to them for 12 to 24 hours. For supershift assays, nuclear extracts from similarly treated cells were incubated with 2 µg of anti-STAT1 and/or anti-STAT3 antibodies and incubated on ice for 1 hour, and the complexes were resolved in a gel mobility shift assay.

	Results

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Effect of LIF on Protein Content and [³H]Phenylalanine Uptake of Cultured Cardiomyocytes
Fig 1a shows the protein content of cardiomyocytes stimulated with LIF for 24 hours. Protein content increased dose-dependently after LIF stimulation. The maximal increase in protein content was 35.0%. However, LIF did not increase the protein content of the cardiac fibroblasts (data not shown). Fig 1b shows phenylalanine uptake after stimulation with LIF. Phenylalanine uptake was increased dose-dependently by LIF stimulation. The maximal increase in phenylalanine uptake was 16.8%. These results indicate that LIF stimulates protein synthesis and protein accretion in cardiomyocytes.

View larger version (21K): [in this window] [in a new window]   Figure 1. Effect of LIF on cardiomyocyte protein content and [3H]phenylalanine ([3H]-Phe) uptake. a, After 24 hours of serum depletion, primary cultured cardiomyocytes were stimulated with LIF (1 to 1000 U/mL). Protein content was measured after 24 hours of LIF stimulation by the Lowry method. Cardiomyocyte protein content increased dose-dependently after LIF stimulation. b, After 24 hours of serum depletion, the cells were stimulated with LIF, and [3H]-Phe was added. The cells were incubated for 24 hours and then washed three times with ice-cold PBS and three times with 5% trichloroacetic acid to precipitate the protein. Samples were counted with a scintillation counter. Phenylalanine uptake increased dose-dependently after addition of LIF. Each column represents the mean of six separate experiments. *P<.01 vs control.

Effect of LIF on Cell Size and Myofibrillar Assembly in Cardiomyocytes
The Table shows the results of the measurements of the size (surface area, perimeter, and maximal and minimal diameters) of cardiomyocytes exposed to LIF for 48 hours in serum-free cultures. Each value is the mean±SD of 100 cells tested. Four separate experiments yielded similar results. The surface area, perimeter, maximal diameter, and minimal diameter values for cardiomyocytes exposed to LIF were 135.4±4.0%, 143.8±2.9%, 119.4±2.1%, and 118.2±3.1%, respectively, of the control cell values. All of these values were significantly larger than the control values, indicating that LIF enlarged the size of cardiomyocytes.

View this table: [in this window] [in a new window]   Table 1. Effect of LIF on the Size of Cardiomyocytes

Fig 2 shows representative immunofluorescent photographs of the sarcomeric myosin of control cardiomyocytes (Fig 2A) and cardiomyocytes exposed to LIF for 48 hours (Fig 2B) in serum-free cultures. The myofibrillar assemblies were much more abundant in cardiomyocytes exposed to LIF than in the control cells. These results indicate that LIF causes cellular enlargement of cardiomyocytes.

View larger version (89K): [in this window] [in a new window]   Figure 2. Immunofluorescence photograph of the sarcomeric myosin of a cardiomyocyte exposed to LIF. Cardiomyocytes were stained with anti–sarcomeric myosin antibody (MF20). Cell sizing was performed according to the protocol described in "Materials and Methods." A, Control cardiomyocyte cultured without serum for 48 hours. B, Cardiomyocyte exposed to LIF for 48 hours. Bar indicates 10 µm.

Effect of LIF on Tyrosine Phosphorylation of JAK1, JAK2, and Tyk2 in Cardiomyocytes
Members of the IL-6 family of cytokines are known to transactivate JAK family kinases, including JAK1, JAK2, and Tyk2, in other cell types.^{1 14 24 25} To begin elucidating the LIF-induced signal transduction pathway in cardiomyocytes, we performed IP-Western blotting to detect tyrosine phosphorylation of JAK family kinases. Representative blots and densitometric analyses of the four separate experiments are shown in Fig 3. Hardly any JAK1, JAK2, or Tyk2 was phosphorylated in untreated cells. After exposure to LIF, tyrosine-phosphorylated JAK1 and JAK2 increased, peaked at 5 minutes, and decreased gradually thereafter but were still elevated at 30 minutes. The increase in tyrosine phosphorylation of Tyk2 was comparatively small and had returned to the control level at 30 minutes. We also confirmed that almost all JAK1, JAK2, and Tyk2 protein was immunoprecipitated in the individual reactions by Western blotting analysis using anti-JAK1, anti-JAK2, and anti-Tyk2 antibodies. These findings indicated that JAK1 and JAK2 play important roles in LIF signaling in cardiomyocytes. Whether LIF induces phosphorylation of Tyk2 remains controversial, but our findings suggest that Tyk2 phosphorylation is not a critical pathway in cardiomyocytes.

View larger version (21K): [in this window] [in a new window]   Figure 3. a, Representative IP-Western blot showing the effect of LIF on tyrosine phosphorylation of JAK1, JAK2, and Tyk2 in cardiomyocytes. Cardiomyocytes were stimulated with LIF at a concentration of 103 U/mL. Cells were lysed, and IP was performed using each anti-JAK antibody, followed by Western blot analysis with anti-phosphotyrosine and each anti-JAK antibody. Similar results were obtained in three additional experiments. b, The results of densitometric analysis. *P<.01 vs control.

Effect of LIF on Tyrosine Phosphorylation of STAT1, STAT2, and STAT3 in Cardiomyocytes
The results of the IP-Western blotting of the JAK kinases suggested that this pathway plays a critical role in transduction of the LIF-induced signal in cardiomyocytes. In order to demonstrate that STAT is activated by LIF-stimulated JAK family kinases, we performed the IP-Western blotting to detect tyrosine phosphorylation of members of the STAT family (Fig 4). STAT1, STAT2, and STAT3 were unphosphorylated in unstimulated cardiomyocytes. After stimulation with LIF, STAT3 was strongly phosphorylated. Phosphorylation was maximal at 5 minutes and gradually decreased thereafter but remained high at 30 minutes. LIF also caused a moderate increase in STAT1 and STAT1ß phosphorylation at 2 minutes, peaked at 15 minutes, and returned to the control level at 30 minutes. Figure 4b showed the results of densitometric analysis in four separate experiments. STAT2 did not change significantly. We were able to detect Tyr phosphorylation of STAT2 in angiotensin II–treated vascular smooth muscle cells (data not shown). The above findings indicate that LIF uses STAT1 and STAT3, but not STAT2, as a signaling pathway in cardiomyocytes.

View larger version (21K): [in this window] [in a new window]   Figure 4. Effect of LIF on tyrosine phosphorylation of STAT1, STAT2, and STAT3 in cardiomyocytes. a, Cardiomyocytes were stimulated with LIF (103 U/mL). Cells were lysed, and IP was performed using each anti-STAT antibody, followed by Western blot analysis with anti-phosphotyrosine and each anti-STAT antibody. Note that STAT3 is strongly phosphorylated after LIF stimulation. LIF induced rapid tyrosine phosphorylation of STAT1 and STAT3, but not STAT2, in cardiomyocytes. An additional three experiments yielded similar results. b, The results of densitometric analysis are shown. *P<.01 vs control.

Phosphorylation of gp130 in Response to LIF Stimulation
The capacity to bind LIF with high affinity appeared to require coexpression of both the LIF receptor and gp130.^{24 25} A number of studies have revealed that gp130^{33 34} plays a pivotal role in activating JAK family kinases, and a recent study has shown that LIF induces phosphorylation of the tyrosine residue of gp130 in other cells.³⁵ To determine whether LIF induces rapid tyrosine phosphorylation of gp130, cell lysates derived from LIF-stimulated cells were immunoprecipitated with anti-phosphotyrosine antibody, immunoblotted, and detected with anti-gp130 antibody. The results, shown in Fig 5, reveal slight phosphorylation of gp130 at the control level, followed by increasing phosphorylation after the addition of LIF, which peaked at 5 minutes and gradually decreased to the control level thereafter. Densitometric analysis revealed that 5-minute exposure to LIF caused a 4.9±1.1-fold increase (Fig 5b). The significance of the phosphorylation of gp130 is still unknown, but these findings suggest that phosphorylation of gp130 is involved in LIF signaling in cardiomyocytes.

View larger version (26K): [in this window] [in a new window]   Figure 5. Phosphorylation of gp130 in response to LIF stimulation. a, LIF-stimulated cells were immunoprecipitated with anti-phosphotyrosine antibody, and Western blot analysis was performed using anti-gp130 antibodies. b, The results of densitometric analysis in five separate experiments are shown. LIF induced rapid tyrosine phosphorylation of gp130 in cardiomyocytes.

Effect of LIF on the Autokinase Activity of JAK1
Since JAK family kinases phosphorylate and activate themselves after ligand stimulation, we investigated whether LIF induces JAK1 autokinase activity in rat cardiomyocytes. Representative autoradiographs and a summary of the results of densitometric analysis in four separate experiments are shown in Fig 6. The intensity of the 130-kD band corresponding to JAK1 was identified and enhanced after 5 minutes, peaked at 15 to 30 minutes, and decreased thereafter.

View larger version (25K): [in this window] [in a new window]   Figure 6. Effect of LIF on the autokinase activity of JAK1. a, LIF-stimulated cardiomyocytes were immunoprecipitated with anti-JAK1 antibody. Autophosphorylation activity of JAK1 was assayed by incubating the immunoprecipitates with 10 µCi [-32P]ATP. Note that autokinase activity of JAK1 was observed in response to LIF stimulation. An additional three experiments yielded similar results. b, The results of densitometric analysis in four experiments are shown. *P<.01 vs control.

Gel Mobility Shift Assay of GAS/ISRE and SIE in the LIF-Induced Signal Pathways
In order to clarify whether LIF induces SIF-like activity^{36 37 38 39} or ISGF-like activity^{40 41} in neonatal rat cardiomyocytes, nuclear extracts were prepared at various times, ie, 0, 5, 15, and 30 minutes, after LIF stimulation and incubated with ³²P-labeled SIE or GAS/ISRE. The DNA-protein complexes were analyzed in a gel mobility shift assay, and the results are shown in Fig 7. LIF strongly induced the formation of the SIF complex, associated with intranuclear oligonucleotides corresponding to SIE (Fig 7a). This DNA-protein interaction was maximal at 5 minutes and decreased thereafter. Densitometric analysis showed a >100-fold increase in LIF-stimulated SIF activity after 5 minutes of treatment compared with untreated samples. On the other hand, LIF did not induce formation of ISGF complexes capable of associating with GAS/ISRE (Fig 7b). Three complexes, previously designated SIF-A, SIF-B, and SIF-C, have been shown to represent the STAT3 homodimer, STAT1/3 heterodimer, and STAT1 homodimer, respectively. Incubation of the DNA-protein complex with anti-STAT1 antibody inhibited binding of the SIF-C and SIF-B bands, and incubation with anti-STAT3 antibody inhibited binding of SIF-A and decreased binding of the SIF-B band. Incubation with both anti-STAT1 and anti-STAT3 antibodies completely inhibited the binding of all three SIF complexes. The three complexes observed in LIF-treated cardiomyocyte extracts matched the three types of SIF complexes, as determined by inhibition of binding with specific antibodies (Fig 7c). We concluded that LIF induced homodimerization or heterodimerization of STAT1 and STAT3 and formation of SIF complexes, which subsequently interacted with SIE in the promoter of the genes to induce expression.

View larger version (44K): [in this window] [in a new window]   Figure 7. Gel mobility shift assay of SIE and GAS/ISRE in LIF-induced cardiac hypertrophy. a, Gel mobility shift assay of SIE was performed. Mutant SIE was used as a negative control. LIF strongly induced the formation of more than one SIF complex associated with intranuclear oligonucleotides corresponding to SIE. Representative blots from three separate experiments are shown. b, Gel mobility shift assay of GAS/ISRE was performed. LIF did not induce formation of ISGF complexes capable of associating with GAS/ISRE. c, Nuclear extracts from LIF-stimulated cells were preincubated with either anti-STAT1 (lane 4), anti-STAT3 (lane 5), or both antibodies (lane 6), and gel mobility shift assay for SIE was performed. Preincubation with anti-STAT1 antibody inhibited the binding of SIF-C and SIF-B, and preincubation with anti-STAT3 antibody inhibited the binding of SIF-A and decreased the SIF-B band. Preincubation with both antibodies completely inhibited the binding of all SIF complexes. Representative blots from three separate experiments are shown.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we investigated the signal transduction pathway of LIF-induced hypertrophy in primary cultures of neonatal rat cardiomyocytes. The major points of this study are as follows: (1) Protein content and [³H]phenylalanine uptake increased dose-dependently in response to stimulation with LIF. (2) LIF significantly enlarged cell size and increased myofibrillar assemblies in cardiomyocytes. (3) gp130 is tyrosine-phosphorylated after LIF stimulation. (4) LIF-induced rapid tyrosine phosphorylation of JAK1 and JAK2 is a major signaling pathway in cardiomyocytes, but phosphorylation of Tyk2 is not a critical pathway. (5) LIF induced JAK1 autokinase activity. (6) LIF stimulation caused rapid phosphorylation of STAT1 and STAT3 but not STAT2. (7) LIF induced dimerization of STAT and formation of SIF complexes, which subsequently interacted with SIEs in the promoters of genes to induce expression in cardiomyocytes.

Previous studies have revealed that LIF receptors are expressed in various types of cells,^{1 9} such as hemopoietic cells^{42 43 44 45} and nonhemopoietic cells.^{46 47 48 49 50 51} Interestingly, LIF is known to have a variety of effects, including stimulation of cell growth^{44 52 53 54} and differentiation.⁵⁵ LIF also functionally modifies various cell types, causing calcium release from bones, production of acute phase protein by hepatocytes, and elevation of platelet levels. Our findings have also demonstrated that LIF induces cardiomyocyte hypertrophy. Why a single cytokine has different functions in different types of cells is still unknown, but differences in the signal transduction pathway in individual types of cell may be responsible for the differences in response. Therefore, we investigated the role of the signal transduction pathway in LIF-induced hypertrophy of cardiomyocytes.

A recent study has demonstrated that the gene targeting of gp130 leads to failure of the myocardial and hematological system to mature.⁵⁶ Homozygote gp130-targeted mice were embryonically lethal and had hypoplastic left ventricles with septal or trabecular defects or exhibited subcellular ultrastructural disorganization. This indicates that gp130 is necessary for maturation of the heart, which includes cell growth and cell hypertrophy of cardiomyocytes. In contrast, a double transgenic study in the progeny of IL-6–overexpressing mice and IL-6 receptor–overexpressing mice revealed that continuous activation of gp130 causes myocardial hypertrophy.⁵⁷ These findings directly demonstrate that activation of the gp130 signaling pathway plays a critical role in cardiac hypertrophy and that this signaling may be necessary for physiological regulation of the myocardium. In the present study, we demonstrated that gp130-related signaling is involved in LIF-induced cardiac hypertrophy. Our data, together with the results of these studies, indicate that JAK/STAT signaling pathways play an important role in cardiac hypertrophy.

The JAK/STAT family was initially known as a major pathway in cytokine superfamilies.¹ It is generally known that cytokine signaling, including the JAK/STAT pathway, is redundant and that none of the pathways is ligand specific. In other cell types, JAK1 is activated by IL-6, OSM, LIF, CNTF, G-CSF, IFN-, IFN-ß, and IFN-,^{1 11 12 14 15 19 20} whereas JAK2 is activated by IL-3, IL-4, IL-6, OSM, LIF, CNTF, G-CSF, IFN-, erythropoietin, prolactin, and growth hormone.^{1 11 12 15} Tyk2 is activated by IFN-, IFN-ß, IL-6, and OSM.^{1 12 15 23} Although gp130 is believed to play a critical role in cardiac development and cardiomyocyte hypertrophy,^{56 57} it is still unknown which JAK family kinases and STAT transcription factors are important in signaling.

Pennica and colleagues^{10 11} recently cloned a new cytokine, CT-1, from a cDNA library of embryonic stem cells by expression cloning using an embryonic stem cell–based model of cardiogenesis. CT-1 was capable of potently inducing cardiomyocyte hypertrophy in primary cultured neonatal cells, and its hypertrophic effect appeared to be greater than that of any previously known substances, including angiotensin II and endothelin-1. Pennica et al also reported that CT-1 uses the LIF receptor and gp130 complex for binding and signaling, although the precise route of the signaling pathway remains unknown. CT-1 mRNA was expressed not only in heart but in several other organs, such as kidney, muscle, liver, and lung. A recent study has reported that CT-1 induces acute-phase protein production in the liver.⁵⁸ In the future, we must clarify whether CT-1 or LIF plays an important role in cardiac hypertrophy in vivo.

The present study was performed using primary cultured cardiomyocytes. We used bromodeoxyuridine to eliminate contamination by cardiac fibroblasts. Although there is still a possibility that contaminating fibroblasts may have affected the results, the fact that LIF did not increase the protein content of the cardiac fibroblasts indicated that the signals obtained were mainly from cardiomyocytes. Very recently, Kunisada et al⁵⁹ reported that LIF induces cardiac hypertrophy and its related signal in cardiomyocytes. According to their data, LIF activates JAK1, STAT3, and mitogen-activated protein kinases in cardiomyocytes. However, their characterization of LIF signaling in LIF-induced cardiac hypertrophy was incomplete. They did not examine other JAK kinases, such as JAK2 and Tyk2, or other STATs, such as STAT1 and STAT2, and did not describe the results of the gel shift assay. As we have shown in the present study, LIF also activates JAK2 and STAT1 in cardiomyocytes. Gel shift assay and supershift assay also demonstrated that LIF activates SIF complexes, which consist of homodimers or heterodimers of STAT1 and STAT3. These findings suggest that activation of STAT1 is also important in LIF signaling. The complete signal transduction pathway of LIF signaling is still unknown. The signal that is important in mediating cardiac hypertrophy in LIF signaling should be identified in the future.

	Selected Abbreviations and Acronyms

 

CNTF = ciliary neurotrophic factor CT-1 = cardiotrophin-1 GAS/ISRE = IFN- activation site/interferon-stimulated responsive element G-CSF = granulocyte-colony stimulating factor IFN-, -ß, - = interferon alpha, beta, and gamma IL = interleukin IP = immunoprecipitation ISGF = interferon-stimulated gene factor JAK = just another kinase, Janus kinase LIF = leukemia inhibitory factor OSM = oncostatin M SIE = sis-inducible element SIF = sis-inducible factor STAT = signal transducer(s) and activator(s) of transcription

	Acknowledgments

 
This study was supported in part by research grants from the Ministry of Education, Science, and Culture of Japan and the Japan Owner's Association. The authors wish to acknowledge the technical assistance of Yoshiko Kurokawa.

Received April 28, 1997; accepted July 14, 1997.

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