Activation of gp130 Transduces Hypertrophic Signal Through ...

Activation of gp130 Transduces Hypertrophic SignalThrough Interaction of Scaffolding/Docking Protein Gab1

With Tyrosine Phosphatase SHP2 in CardiomyocytesYoshikazu Nakaoka, Keigo Nishida, Yasushi Fujio, Masahiro Izumi, Kazuo Terai,

Yuichi Oshima, Shoko Sugiyama, Satoshi Matsuda, Shigeo Koyasu, Keiko Yamauchi-Takihara,Toshio Hirano, Ichiro Kawase, Hisao Hirota

Abstract—Grb2-associated binder-1 (Gab1) is a scaffolding/docking protein and contains a Pleckstrin homology domainand potential binding sites for Src homology (SH) 2 and SH3 domains. Gab1 is tyrosine phosphorylated and associateswith protein tyrosine phosphatase SHP2 and p85 phosphatidylinositol 3-kinase on stimulation with various cytokinesand growth factors, including interleukin-6. We previously demonstrated that interleukin-6–related cytokine, leukemiainhibitory factor (LIF), induced cardiac hypertrophy through gp130. In this study, we report the role of Gab1 ingp130-mediated cardiac hypertrophy. Stimulation with LIF induced tyrosine phosphorylation of Gab1, and phosphor-ylated Gab1 interacted with SHP2 and p85 in cultured cardiomyocytes. We constructed three kinds of adenovirusvectors, those carrying wild-type Gab1 (AdGab1WT), mutated Gab1 lacking SHP2 binding site (AdGab1F627/659), and�-galactosidase (Ad�-gal). Compared with cardiomyocytes infected with Ad�-gal, longitudinal elongation of cardio-myocytes induced by LIF was enhanced in cardiomyocytes infected with AdGab1WT but inhibited in cardiomyocytesinfected with AdGab1F627/659. Upregulation of BNP mRNA expression by LIF was evoked in cardiomyocytes infectedwith Ad�-gal and AdGab1WT but not in cardiomyocytes infected with AdGab1F627/659. In contrast, Gab1 repressedskeletal �-actin mRNA expression through interaction with SHP2. Furthermore, activation of extracellular signal–regulated kinase 5 (ERK5) was enhanced in cardiomyocytes infected with AdGab1WT compared with cardiomyocytesinfected with Ad�-gal but repressed in cardiomyocytes infected with AdGab1F627/659. Coinfection of AdGab1WT withadenovirus vector carrying dominant-negative ERK5 abrogated longitudinal elongation of cardiomyocytes induced byLIF. Taken together, these findings indicate that Gab1-SHP2 interaction plays a crucial role in gp130-dependentlongitudinal elongation of cardiomyoctes through activation of ERK5. (Circ Res. 2003;93:221-229.)

Key Words: hypertrophy � Gab1 � SHP2 � gp130 � ERK5

Grb2-associated binder-1 (Gab1) is a member of the Gabfamily of scaffolding/docking proteins (Gab1, Gab2,

and Gab3).1,2 Gab1 contains a Pleckstrin homology (PH)domain in the amino-terminal region, as well as tyrosine-based motifs and proline-rich sequences, which are potentialbinding sites for various Src homology (SH) 2 domains andSH3 domains, respectively.3 Gab1 is tyrosine phosphorylatedon stimulation with various growth factors, cytokines, and Gprotein–coupled receptor (GPCR) agonists.3–7

Gab1 interacts with multiple signaling molecules, such asprotein tyrosine phosphatase SHP2, p85 phosphatidylinositol3-kinase, phospholipase C-�, and Grb2.3–6 Among these, the

major binding partner of Gab1 in cells stimulated with growthfactors and cytokines is SHP2, a ubiquitously expressedprotein tyrosine phosphatase with two SH2 domains.8 Twotyrosine residues located in the most C-terminal ends of theGab family proteins have been reported to fall within con-sensus binding motifs (YXXV/I/L) for SHP2 on tyrosinephosphorylation.9–12 The functional significance of Gab1-SHP2 interaction has been extensively studied using mutantsof Gab1 unable to bind SHP2 in vitro and in vivo. The Gab1mutant unable to bind SHP2 is defective in delivering signalsfor Met-dependent morphogenesis and for epidermal growthfactor (EGF)-dependent epidermal proliferation and also

Original received February 10, 2003; revision received June 11, 2003; accepted June 26, 2003.From the Departments of Molecular Medicine (Y.N., M.I., K.T., Y.O., S.S., K.Y.-T., I.K., H.H.) and Molecular Oncology (T.H.), Osaka University

Graduate School of Medicine, Osaka, Japan; Laboratory for Cytokine Signaling (K.N., T.H.), RIKEN Research Center for Allergy and Immunology,Kanagawa, Japan; Department of Clinical Evaluation of Medicines and Therapeutics (Y.F.), Osaka University Graduate School of PharmaceuticalSciences, Osaka, Japan; Department of Microbiology and Immunology (S.M., S.K.), Keio University School of Medicine, Tokyo, Japan; Core Researchfor Evolutional Science and Technology (CREST) (S.M., S.K.), Japan Science and Technology Corporation, Saitama, Japan; and Laboratory forDevelopmental Immunology (T.H.), Osaka University Graduate School of Frontier Bioscience, Osaka, Japan.

Presented in part at the 75th Scientific Sessions of the American Heart Association, Chicago, Ill, November 17–20, 2002, and published in abstractform (Circulation. 2002;106[suppl]:II-259).

Correspondence to Hisao Hirota, MD, PhD, Assistant Professor, Department of Molecular Medicine, Osaka University Graduate School of Medicine,2-2, Yamadaoka, Suita City, Osaka, 565-0871, Japan. E-mail [email protected]

© 2003 American Heart Association, Inc.

Circulation Research is available at http://www.circresaha.org DOI: 10.1161/01.RES.0000085562.48906.4A

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blocks extracellular signal-regulated kinase 1/2 (ERK1/2)activation by EGF and lysophosphatidic acid.9–14 Thesefindings underscore the importance of Gab1-SHP2 interac-tion and strongly suggest that the primary role of Gab1 mightbe to recruit SHP2.

To reveal the functional role of Gab1 in vivo, we andothers generated mice lacking Gab1 by gene targeting.15,16

Gab1-deficient mice died in utero and displayed developmen-tal defects in the heart, placenta, liver, and skin. Gab1 washighly expressed in embryonic heart from E10.5 to E13.5.The ventricular chamber displayed dilatation, and the ven-tricular wall was extremely thin in all of the Gab1�/� embryosthat survived past E13.5.15 These findings indicate that Gab1is necessary for development of the heart.

Leukemia inhibitory factor (LIF) and cardiotrophin-1(CT-1) are interleukin-6 (IL-6)-related cytokines and bind toa heterodimer of gp130 and LIF receptor �.17,18 LIF and CT-1are potent inducers of cardiomyocyte hypertrophy and alsoserve as myocyte survival factors in vitro and in vivo.19–24

The hypertrophic response in cardiomyocytes induced by LIFand CT-1 is distinct from the hypertrophic response observedafter GPCR stimulation.20 Adrenergic agonists, endothelin-1(ET-1), and angiotensin II (Ang II) binding to GPCR inducea rather uniform increase in cardiomyocyte size, resultingfrom the addition of myofibrils in parallel.25–27 In contrast,LIF and CT-1 induce a predominant increase in cell lengthwith the addition of new sarcomeric units in series.20 Inter-estingly, recent reports have shown that the mitogen-activatedprotein kinase (MAPK) kinase 5 (MEK5)-MAPK extracellu-lar signal-regulated kinase 5 (ERK5) pathway plays a criticalrole in gp130-mediated eccentric cardiac hypertrophy in vitroand in vivo.28

In this study, we found that Gab1 was tyrosine phosphor-ylated and associated with SHP2 after stimulation with LIF incardiomyocytes. It was also revealed that Gab1 plays acritical role in elongation of cardiomyocytes induced by LIFthrough interaction with SHP2, using adenovirus vectorsexpressing wild-type Gab1 and mutated Gab1, which couldnot bind SHP2. In addition, we found that the interaction ofGab1 with SHP2 is involved not only in the regulation ofbrain natriuretic polypeptide (BNP) and skeletal �-actin(SKA) gene expression but also in the activation of ERK5after stimulation with LIF in cardiomyocytes. Furthermore,dual infection of adenovirus vectors carrying wild-type Gab1and dominant-negative ERK5 abrogated elongation of car-diomyocytes induced by LIF, suggesting that ERK5 may bean essential component of gp130-dependent cardiomyocytehypertrophy through Gab1-SHP2 interaction.

Materials and MethodsConstruction of Recombinant AdenovirusAccording to a previous study,9,12 the most C-terminal 2 tyrosineresidues (Tyr-627 and Tyr-659) of Gab1 are required for binding toSHP2. Substitution of these tyrosine residues by phenylalaninerenders the molecule incapable of binding to SHP2. The wild-typeand mutated human Gab1 cDNAs are designated as Gab1WT andGab1F627/659, respectively. A dominant-negative form of murineERK5 (ERK5AEF) was created by mutating dual phosphorylation site(Thr-219 and Tyr-221 with alanine and phenylalanine, respective-ly).29 The adenovirus vectors expressing Gab1WT (AdGab1WT),

Gab1F627/659 (AdGab1F627/659), and ERK5AEF (AdERK5AEF) were gen-erated according to a protocol described elsewhere.30

Cell Culture and Protocol forAdenovirus InfectionPrimary cultures of neonatal rat cardiomyocytes were prepared fromventricles of 1-day-old Wistar rats (Kiwa Jikken Dobutsu) asdescribed previously.31 At 16 hours after plating, cardiomyocyteswere infected with adenovirus diluted in Medium-199 with 2% FBSat a multiplicity of infection (moi) of 20 and incubated for 8 hours.In the dual infection of adenovirus vectors, cardiomyocytes wereinfected with each virus at an moi of 10. After removal of viralsuspension, cardiomyocytes were serum starved for 16 hours andstimulated with reagents. Infection efficiency, determined by lacZgene expression in cultured cardiomyocytes, is consistently �90%with this method. Adenovirus vector expressing �-galactosidase(Ad�-gal) was used as a control.

Immunoprecipitation and ImmunoblottingThe methods of immunoprecipitation were essentially as describedpreviously.5 After stimulation, cells were immediately lysed in lysisbuffer (20 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, 1%Nonidet P-40, 500 �mol/L sodium vanadate, 1 mmol/L dithiothrei-tol, 5 �g/mL aprotinin, 5 �g/mL leupeptin, and 1 mmol/L phenyl-methylsulfonyl fluoride). The cleared lysates were incubated with 2�L of anti-Gab1 serum or 4 �L of anti-SHP2 antibody and 20 �L ofprotein A-Sepharose for 12 hours at 4°C. Collected immune com-plexes were eluted with 20 �L of 2�Laemmli’s SDS loading buffer,separated on a 4% to 20% gradient polyacrylamide gel (Dai-ichiKagaku), electrotransferred to a polyvinylidene difluoride membrane(Immobilon-P; Millipore), and processed for immunoblotting anal-ysis essentially as described previously.5 The ECL system was usedfor detection.

Northern Blot AnalysisNorthern blot analysis was performed as previously described.21

Total cellular RNA (8 �g) was loaded in each lane and sizefractionated by 1% formaldehyde-agarose gel electrophoresis. Theprobes for BNP, SKA, and GAPDH were kindly donated by Dr K.R.Chien (University of California, San Diego, Calif).

ImmunofluorescenceFor immunofluorescence, cardiomyocytes were grown on glasscoverslips coated with gelatin. Cells were incubated in the presenceor absence of LIF 1�103 U/mL for 24 hours. Cells were fixed with2% formaldehyde and permeabilized with 0.1% Triton X-100. Cellswere incubated with monoclonal anti-sarcomeric �-actinin antibody,followed by incubation with fluorescein-conjugated goat anti-mousesecondary antibody. Cardiomyocytes stained against sarcomeric�-actinin were viewed by fluorescence microscopy. Cell size wasestimated by measuring the area over which individual sarcomeric�-actinin–positive cells had attached (planimetry), and cell lengthand cell width were determined as described previously.20

StatisticsStatistical analysis was performed with Student’s t test. Values ofP�0.05 were considered significant.

An expanded Materials and Methods section can be found in theonline data supplement available at http://www.circresaha.org.

ResultsGab1 and SHP2 Are Tyrosine Phosphorylated byLIF, and Phosphorylated Gab1 Interacts WithSHP2 and p85 in CardiomyocytesTo examine which ligand induces tyrosine phosphorylation ofGab1 in cardiomyocytes, cells were incubated with LIF,norepinephrine (NE), ET-1, and Ang II for 5 minutes. Among

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these, LIF exclusively induced tyrosine phosphorylation ofGab1 (Figure 1A). Furthermore, SHP2, the major bindingpartner of Gab1, was also tyrosine phosphorylated only byLIF (Figure 1B). Therefore, we focused on the gp130-dependent signaling pathway through Gab1 incardiomyocytes.

Gab1 was tyrosine phosphorylated by LIF in a time-dependent and dose-dependent manner (Figures 2A and 2B).

SHP2 was also tyrosine phosphorylated by LIF in a time-dependent and dose-dependent manner (Figures 2C and 2D).To elucidate the association of Gab1 with other SH2-containing molecules, cell lysates from cardiomyocytestreated with LIF were immunoprecipitated with anti-Gab1serum and subjected to immunoblotting with anti-SHP2 andanti-p85 antibodies. SHP2 and p85 were coprecipitated withGab1 in response to LIF (Figure 2E). Gab1 was also copre-cipitated with SHP2 by the immunoprecipitation with anti-SHP2 antibody (Figure 2F). These results demonstrated thatLIF induced tyrosine phosphorylation of Gab1, leading toassociation of Gab1 with SHP2 and p85 in cardiomyocytes.

Tyrosine Phosphorylation of Gab1 and Associationof Gab1 With SHP2 in AdGab1WT orAdGab1F627/659-Treated CardiomyocytesWe constructed recombinant adenovirus vectors expressingGab1WT and Gab1F627/659. Figure 3A shows a schematic repre-sentation of these adenovirus vectors, which are namedAdGab1WT and AdGab1F627/659. We examined tyrosine phos-phorylation of Gab1 in Ad�-gal–treated, AdGab1WT–treated,or AdGab1F627/659-treated cardiomyocytes. As shown in Figure3B, tyrosine phosphorylation of Gab1 and the amount ofcoprecipitated SHP2 with Gab1 were increased in AdGab1WT-treated cardiomyocytes after LIF stimulation, compared withAd�-gal–treated cardiomyocytes. In AdGab1F627/659-treatedcardiomyocytes, tyrosine phosphorylation of Gab1 was ob-served in the same manner as in AdGab1WT-treated cardio-myocytes, but SHP2 was not coprecipitated with Gab1. Asshown in Figure 3C, Gab1 was not coprecipitated with SHP2in AdGab1F627/659-treated cardiomyocytes. These results indi-cate that Gab1WT and Gab1F627/659, which were overexpressed

Figure 1. LIF induces tyrosine phosphorylation of Gab1 andSHP2 in cardiomyocytes. A, Serum-deprived neonatal rat car-diomyocytes were treated with 1�103 U/mL LIF, 2 �g/mL NE,100 nmol/L ET-1, and 100 nmol/L Ang II for 5 minutes. Celllysates were immunoprecipitated with anti-Gab1 serum followedby immunoblotting with anti-phosphotyrosine antibody (PY99)(top). Blots were reprobed with anti-Gab1 antibody (bottom). B,Cell lysates were immunoprecipitated with anti-SHP2 antibodyfollowed by immunoblotting with PY99 (top). Blots werereprobed with anti-SHP2 antibody (bottom).

Figure 2. Tyrosine phosphorylation ofGab1 and SHP2 and complex formationof Gab1 with SHP2 and p85 after stimu-lation with LIF in cardiomyocytes. A andC, Cardiomyocytes were stimulated with1�103 U/mL LIF for the indicated peri-ods of time. Cell lysates were immuno-precipitated with anti-Gab1 serum (A) oranti-SHP2 antibody (C) followed by im-munoblotting with PY99. B and D, Cardi-omyocytes were stimulated with the indi-cated concentrations of LIF for 5minutes. Cell lysates were treated asabove. E and F, Cardiomyocytes werestimulated with 1�103 U/mL LIF for 5minutes. Tyrosine phosphorylation wasdetected by immunoprecipitation withanti-Gab1 serum (E) or anti-SHP2 anti-body (F) followed by immunoblotting withPY99 (top). Blots were reprobed withanti-SHP2 (E and F), Gab1 (E and F), andp85 antibodies (E) (bottom).

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through adenovirus-mediated gene transfer, could functioneffectively in cardiomyocytes.

Elongation of Cardiomyocytes Induced byLIF Is Enhanced in AdGab1WT-TreatedCardiomyocytes but Suppressed inAdGab1F627/659-Treated CardiomyocytesTo elucidate the biological roles of Gab1 in gp130-signalingpathway in cardiomyocytes, we examined the morphological

effects of Gab1WT and Gab1F627/659 on cardiomyocyte hyper-trophy in response to LIF. As shown in Figures 4A and 4D,LIF induced longitudinal elongation in Ad�-gal–treated car-diomyocytes. In AdGab1WT-treated cardiomyocytes, elonga-tion of cardiomyocytes induced by LIF was enhanced com-pared with Ad�-gal–treated cardiomyocytes (Figures 4Band 4E). On the contrary, in cardiomyocytes expressingGab1F627/659, this morphological change was significantly in-hibited (Figures 4C and 4F).

We quantified the cell surface area of cardiomyocytesinfected with these adenovirus vectors. As shown in Figure4G, LIF increased cell surface area by 70% in Ad�-gal–treated or AdGab1WT-treated cardiomyocytes. However, inAdGab1F627/659-treated cardiomyocytes, the increase in cellsurface area induced by LIF was almost abolished. Tocharacterize the hypertrophic phenotype of these cardio-myocytes, we measured cell length and cell width accord-ing to a previously reported method.20 As shown in Figure4H, cell length was significantly increased in AdGab1WT-treated cardiomyocytes, compared with that in Ad�-gal–treated cardiomyocytes. In contrast, increase in cell lengthwas significantly suppressed in AdGab1F627/659-treated car-diomyocytes. Cell width was significantly decreased inresponse to LIF in AdGab1WT-treated cardiomyocytes butnot altered in Ad�-gal–treated or AdGab1F627/659-treatedcardiomyocytes (Figure 4I). Compared with Ad�-gal–treated cardiomyocytes, the cell length to width ratio wassignificantly increased in AdGab1WT-treated cardiomyo-cytes but suppressed significantly in AdGab1F627/659-treatedcardiomyocytes (Figure 4J). These findings indicate thatelongation of cardiomyocytes in response to LIF is en-hanced by overexpression of Gab1WT but suppressed bythat of Gab1F627/659, suggesting that the interaction of Gab1with SHP2 plays a crucial role in longitudinal elongationof cardiomyocytes in response to LIF.

Gab1 Regulates LIF-Induced Embryonic GeneExpression Through Interaction With SHP2Reactivation of embryonic phenotype genes, such as atrialnatriuretic factor (ANF), BNP, and SKA, is known to beassociated with hypertrophic responses in cardiomyocytes.We examined how the interaction of Gab1 with SHP2contributes to the induction of BNP and SKA mRNAexpression after LIF stimulation. BNP mRNA was upregu-lated by LIF in AdGab1WT-treated cardiomyocytes to thesame extent as in Ad�-gal–treated cardiomyocytes. On thecontrary, induction of BNP mRNA was abrogated inAdGab1F627/659-treated cardiomyocytes (Figures 5A and5B). In contrast, SKA mRNA expression was slightlyincreased in response to LIF in Ad�-gal–treated cardio-myocytes but was almost completely suppressed inAdGab1WT-treated cardiomyocytes both in basal level andafter LIF stimulation. In AdGab1F627/659-treated cardiomyo-cytes, the expression of SKA mRNA was restored to thesame extent as in Ad�-gal–treated cardiomyocytes (Fig-ures 5A and 5C). These results indicate that the interactionof Gab1 with SHP2 is involved in the regulation ofembryonic gene expression after stimulation with LIF.

Figure 3. Generation of adenovirus vectors expressing Gab1WT

and Gab1F627/659. A, Structures of adenovirus vectors expressingwild-type Gab1 (AdGab1WT) and mutated Gab1, which could notbind SHP2 (AdGab1F627/659). Substitution of Tyr-627 and Tyr-659each by phenylalanine renders the molecule incapable of bind-ing to SHP2 on tyrosine phosphorylation. The transgene isdriven under the control of the CMV promoter. Adenovirus vec-tor expressing Ad�-gal was used as a control. MBD indicatesc-Met binding domain. B and C, Tyrosine phosphorylation ofGab1 or SHP2 in cardiomyocytes infected with adenovirus vec-tors and association of Gab1 with SHP2. Cardiomyocytes wereinfected at an moi of 20 with AdGab1WT, AdGab1F627/659, or Ad�-gal. Cardiomyocytes were treated with 1�103 U/mL LIF for 5minutes. Tyrosine phosphorylation was detected by immunopre-cipitation with anti-Gab1 serum (B) or anti-SHP2 antibody (C)followed by immunoblotting with PY99 (top). Blots werereprobed with anti-Gab1 and SHP2 antibodies.



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Interaction of Gab1 With SHP2 Plays aCrucial Role in Activation of ERK5 by LIFin CardiomyocytesTo elucidate a potential mechanism in which Gab1-SHP2interaction plays a role in gp130-mediated longitudinalelongation of cardiomyocytes, we examined the effects ofGab1WT and Gab1F627/659 on LIF-induced activation of MAPkinases (ERK5 and ERK1/2), AKT, and signal transducerand activator of transcription 3 (STAT3), which are knownto mediate biological functions through gp130.21,28,31–33

These signaling molecules were rapidly activated by LIF inAd�-gal–treated cardiomyocytes. Activation of ERK5 byLIF was augmented in AdGab1WT-treated cardiomyocytescompared with Ad�-gal–treated cardiomyocytes. On theother hand, activation of ERK5 was reduced in AdGab1F627/659-treated cardiomyocytes (Figure 6B). ERK1/2 was activated tothe same extent in AdGab1WT-treated cardiomyocytes as inAd�-gal–treated cardiomyocytes. On the contrary, activationof ERK1/2 was reduced in AdGab1F627/659-treated cardiomyo-

cytes compared with Ad�-gal–treated cardiomyocytes (Fig-ure 6C). Activation of AKT was enhanced in AdGab1WT-treated or AdGab1F627/659-treated cardiomyocytes comparedwith Ad�-gal–treated cardiomyocytes (Figure 6D). Activa-tion of STAT3 was not altered in cardiomyocytes infectedwith Ad�-gal, AdGab1WT, or AdGab1F627/659. These resultsindicate that Gab1 plays a critical role in activation of ERK5and ERK1/2 by LIF through interaction with SHP2 incardiomyocytes. Based on a previous report,28 the presentfinding suggests that the interaction of Gab1 with SHP2might be involved in LIF-induced elongation of cardiomyo-cytes through activation of ERK5.

Overexpression of the Dominant-Negative Form ofERK5 Abrogates the Effect of Gab1WT onLongitudinal Elongation of CardiomyocytesInduced by LIFTo determine whether ERK5 might participate in the LIF-ac-tivated signaling pathway that mediates longitudinal elonga-

Figure 4. Gab1 mediates the longitudinalelongation of cardiomyocytes inresponse to LIF. Cardiomyocytes wereinfected at an moi of 20 with Ad�-gal (Aand D), AdGab1WT (B and E), orAdGab1F627/659 (C and F) for 8 hours.Cells were serum deprived for 16 hoursand treated without (A through C) or with1�103 U/mL LIF (D through F) for anadditional 24 hours before fixation andimmunostaining with anti-sarcomeric�-actinin antibody. Representative dataare shown. G through J, Cardiomyocytesstained for sarcomeric �-actinin wereviewed by fluorescence microscopy. Atotal of 150 sarcomeric �-actinin–positivecells were examined for each measure-ment. Cell size was examined by mea-suring the area to which individual sarco-meric �-actinin–positive cells attached(planimetry). Cell surface areas, length,and width were determined using Mac-Scope software as a relative ratio tothose of control Ad�-gal–treated cardio-myocytes without LIF stimulation. Foreach cardiomyocyte measured, celllength to width ratio was also calculated(J). Values are mean�SD; *P�0.001 vsLIF(�); †P�0.001 vs Ad�-gal; #P�0.001vs AdGab1WT. Experiments wererepeated 3 times with similar results.



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tion of cardiomyocytes induced by LIF, we constructedrecombinant adenovirus vector expressing dominant-negativeform of ERK5 (AdERK5AEF). As shown in Figure 7A,overexpression of ERK5AEF almost abrogated LIF-inducedlongitudinal elongation of cardiomyocytes.

To test whether overexpression of ERK5AEF abrogatesLIF-induced longitudinal elongation of cardiomyocytes over-expressing Gab1WT, cardiomyocytes were dual infected withAdGab1WT and Ad�-gal or with AdGab1WT and AdERK5AEF.LIF induced elongation of cardiomyocytes infected withAdGab1WT and Ad�-gal. On the contrary, this morphologicalchange was significantly inhibited in cardiomyocytes infectedwith AdGab1WT and AdERK5AEF (Figure 7B). We quantifiedthe cell surface area, cell length, and cell width of thesecardiomyocytes. As shown in Figures 7C and 7D, LIFincreased cell surface area by 60% and cell length by 84% incardiomyocytes infected with AdGab1WT and Ad�-gal. How-ever, in cardiomyocytes infected with AdGab1WT and

AdERK5AEF, the increases in cell surface area and cell lengthinduced by LIF were almost abrogated. Cell width wassignificantly decreased in response to LIF in cardiomyocytesinfected with AdGab1WT and Ad�-gal but not in cardiomyo-cytes infected with AdGab1WT and AdERK5AEF (Figure 7E).The cell length to width ratio was significantly increased byLIF in cardiomyocytes infected with AdGab1WT and Ad�-galbut not in cardiomyocytes infected with AdGab1WT andAdERK5AEF (Figure 7F). Therefore, it seems that ERK5might be an essential component of LIF-activated signaling

Figure 5. Gab1 regulates embryonic gene expression throughinteraction with SHP2 in cardiomyocytes. A, Cardiomyocyteswere infected with Ad�-gal, AdGab1WT, or AdGab1F627/659 at anmoi of 20 for 8 hours and serum deprived. At 24 hours afterinfection, cells were treated without or with 1�103 U/mL LIF foran additional 24 hours. Total RNA was isolated and subjected toNorthern blot analysis (8 �g/lane) using BNP and SKA cDNAprobes. Equal loading and transfer conditions were confirmedby GAPDH hybridization. B and C, Relative intensity of thebands for BNP or SKA was assessed as the ratio to the inten-sity of GAPDH. The results were expressed as relative intensityover Ad�-gal–treated cells without LIF stimulation. Values areshown as mean�SD. *P�0.05 vs LIF(�); †P�0.05 vs Ad�-gal(n�3).

Figure 6. Gab1 is involved in activation of ERK5 by LIF throughinteraction with SHP2 in cardiomyocytes. A, Cardiomyocyteswere infected at an moi of 20 with Ad�-gal, AdGab1WT, orAdGab1F627/659 and serum deprived. At 24 hours after infection,cells were stimulated with 3�102 U/mL LIF for indicated periodsof time. Total cell extracts were prepared and blotted with anti-phospho ERK5 (p-ERK5), anti-ERK5, anti-phospho ERK1/2(p-ERK1/2), anti-ERK1/2, anti-phospho AKT (pAKT), anti-AKT,anti-phospho STAT3 (p-STAT3), and anti-STAT3 antibodies.Gab1 expression was confirmed with anti-Gab1 antibody. Alldata shown are one representative result from 3 independentexperiments that had a similar result. B through D, Phosphoryla-tion of ERK5, ERK1/2, and AKT was normalized to total ERK5,ERK1/2, and AKT using FluorChem-8000 (Alpha-Innotech).



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pathway, leading to elongated morphology of cardiomyocytesthrough Gab1-SHP2 interaction.

DiscussionThe present study is the first to reveal the role of Gab1 ingp130-mediated hypertrophic signaling in cardiomyocytes invitro. Gab1 is tyrosine phosphorylated and interacts with SHP2and p85 after stimulation with LIF. Overexpression of Gab1WT

enhances elongation of cardiomyocytes induced by LIF. Con-sistent with potential involvement of Gab1 in gp130-signalingpathway, overexpression of Gab1F627/659, which could not asso-ciate with SHP2, blocks morphological change and induction ofBNP mRNA in response to LIF in cardiomyocytes. Gab1 is alsoinvolved in regulation of SKA gene expression through interac-tion with SHP2. Moreover, Gab1 regulates LIF-induced activa-tion of ERK5 through interaction with SHP2, leading to gp130-dependent elongation of cardiomyocytes.

Among several hypertrophic factors, we found that LIFinduced remarkable tyrosine phosphorylation of Gab1 andSHP2. On the other hand, GPCR agonists, such as NE, ET-1,

and Ang II, did not induce tyrosine phosphorylation of Gab1 andSHP2 in cardiomyocytes. LIF and CT-1 induce cardiomyocytehypertrophy,20,21 which is distinct from the hypertrophic pheno-type observed after stimulation of GPCR agonists, both on amorphological and a molecular level.20 GPCR agonists induce arelatively uniform increase in myocyte size and the addition ofnew myofibrils in parallel.25–27 In contrast, LIF and CT-1 inducea predominant increase in cell length with the addition of newsarcomeric units in series but no concomitant increase in cellwidth.20 In the present study, we showed that Gab1 enhancedelongation of cardiomyocytes induced by LIF and that overex-pression of Gab1F627/659 inhibited increase in cell size and celllength of cardiomyocytes after LIF stimulation. On the otherhand, our data showed that overexpression of both Gab1WT andGab1F627/659 did not affect the morphological change after stim-ulation with GPCR agonist ET-1 in cardiomyocytes (data notshown). These findings suggest that the interaction of Gab1 withSHP2 specifically contributes to longitudinal elongation ofcardiomyocytes induced by stimulation of gp130. However, theLIF-induced increase of cell length was not completely abol-

Figure 7. Overexpression of ERK5AEF

abrogates the effect of Gab1WT on longi-tudinal elongation of cardiomyocytesinduced by LIF. A, Cardiomyocytes wereinfected with AdERK5AEF. Cells weretreated as described in Figure 4. Repre-sentative data are shown. B, Cardiomyo-cytes were dual infected with AdGab1WT

and Ad�-gal or with AdGab1WT andAdERK5AEF. Cells were treated asdescribed in Figure 4. Representativedata are shown. C through F, A total of150 sarcomeric �-actinin–positive cellswere examined for each measurement.Cell surface area, cell length, and cellwidth were examined as described inFigure 4. Values are mean�SD;*P�0.001 vs LIF(�); †P�0.001 vs Ad�-gal. Experiments were repeated 3 timeswith similar results.



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ished in AdGab1F627/659-treated cardiomyocytes (Figure 4H). Thisincrease may be related to augmented activation of AKT inAdGab1F627/659-treated cardiomyocytes, as shown in Figures 6Aand 6D.

To additionally investigate the molecular mechanisms ofGab1-mediated longitudinal elongation of cardiomyocytes, weexamined the downstream signaling pathway of gp130. Interest-ingly, activation of ERK5 by LIF was enhanced by overexpres-sion of Gab1WT but suppressed by that of Gab1F627/659. Further-more, in cardiomyocytes dual infected with AdGab1WT andAdERK5AEF, the increases in cell surface area and cell lengthwere almost abrogated. These data indicate that Gab1-SHP2interaction plays a critical role in gp130-dependent elongation ofcardiomyocytes through activation of ERK5. Although it hasbeen reported that the interaction of Gab1 with SHP2 regulatesactivation of ERK1/2,9–12,14 the present study is the first dem-onstration that the interaction of Gab1 with SHP2 also regulatesactivation of ERK5. Consistent with our results, Nicol et al28

recently reported that activation of ERK5 is necessary andsufficient for elongation of cardiomyocytes induced by LIF,providing the causality between Gab1-mediated ERK5 activa-tion and elongation of cardiomyocytes. Nicol et al28 also dem-onstrated that the gp130-MEK5-ERK5 pathway has a spe-cific role in inhibition of parallel assembly of sarcomeresusing adenovirus vectors expressing constitutive activeand dominant-negative MEK5.28 Our data showing thatcell width was decreased after LIF stimulation inAdGab1WT-treated cardiomyocytes suggest that Gab1might enhance gp130-MEK5-ERK5 signaling pathway toinhibit parallel assembly of sarcomeres.

On the other hand, ERK5 was also shown to be activated byGPCR agonist phenylephrine in cardiomyocytes.28 Based onthese findings, we could hypothesize that tyrosine phosphoryla-tion of Gab1 and subsequent complex formation of Gab1 andSHP2 are primarily responsible for the specification of gp130-mediated cardiac hypertrophy (Figure 8). However, additionalinvestigation is needed to elucidate the functional role of SHP2in cardiac hypertrophy.

In addition to morphological change, stimulation of cardio-myocytes with LIF and CT-1 induced ANF and BNP mRNAexpression but not SKA mRNA expression.20,34 In contrast,GPCR agonists induced ANF, BNP, and SKA mRNA expres-sion in a coordinate fashion.25–27,35 Although overexpression ofGab1WT or Gab1F627/659 did not alter the upregulation of BNPmRNA after stimulation with GPCR agonist ET-1 in cardiomyo-cytes (data not shown), our results showed that Gab1-SHP2interaction mediated LIF-induced BNP and SKA mRNA indifferent directions. Accordingly, these findings suggest thatGab1-SHP2 interaction contributes to the unique pattern ofembryonic gene expression in gp130-mediated cardiac hypertro-phy. However, additional investigation is needed to reveal themolecular mechanism underlying Gab1-mediated gene regula-tion in gp130-mediated cardiac hypertrophy.

Finally, it is very important to investigate the concernedpathological conditions in which gp130-Gab1 pathway is in-volved in human clinical case or animal models. To ourknowledge, one previous study has demonstrated the distinctgene expression pattern in the hearts with pressure overload(PO) and volume overload (VO) in rat model.36 In this report,

mRNA levels were quantified in the left ventricular myocardiumfrom rats with cardiac hypertrophy attributable to PO or VOcaused by suprarenal aortic constriction or an abdominal aorto-caval fistula, respectively. Although PO and VO caused com-parable increases in LV weight and prepro-ANF mRNA, PO butnot VO increased mRNA levels of SKA. This pattern of geneexpression induced by VO in vivo is reminiscent of thatobserved in cultured cardiomyocytes after LIF stimulation.Additionally, recent reports have shown that the signalingpathway through gp130-dependent pathway is profoundly al-tered in patients with end-stage heart failure attributable todilated and ischemic cardiomyopathy.37 Although little is knownregarding involvement of Gab1 in human clinical case or animalmodels, these findings and our findings suggest that Gab1 mayplay a role in the left ventricular remodeling in volume-overloaded hearts, providing novel insights into a therapeuticstrategy for heart failure by manipulating Gab1-SHP2interaction.

AcknowledgmentsThis study was supported by a Grant-in-Aid for Scientific Researchfrom the Ministry of Education, Science, and Culture of Japan and

Figure 8. Schematic model of the roles of Gab1-SHP2 interactionin the control of cardiac hypertrophy by stimulation of gp130. IL-6–related cytokines, such as LIF and CT-1, induce tyrosine phos-phorylation of Gab1 and SHP2, leading to the association of Gab1with SHP2. Gab1-SHP2 interaction plays a critical role in activationof ERK5 in cardiomyocytes. Furthermore, this interaction plays arole in the formation of eccentric hypertrophy of cardiomyocytes,characterized not only by morphological change but also by regu-lation of embryonic gene expression.



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grants from the Ministry of Health and Welfare of Japan. The authorsthank J. Hironaka for secretarial assistance, M. Yoshida and Y.Takemura for expert technical assistance, and N. Mochizuki (Na-tional Cardiovascular Center Research Institute, Osaka, Japan) forhelpful discussion.

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Hirano, Ichiro Kawase and Hisao HirotaToshioOshima, Shoko Sugiyama, Satoshi Matsuda, Shigeo Koyasu, Keiko Yamauchi-Takihara,

Yoshikazu Nakaoka, Keigo Nishida, Yasushi Fujio, Masahiro Izumi, Kazuo Terai, YuichiScaffolding/Docking Protein Gab1 With Tyrosine Phosphatase SHP2 in Cardiomyocytes

Activation of gp130 Transduces Hypertrophic Signal Through Interaction of

Print ISSN: 0009-7330. Online ISSN: 1524-4571 Copyright © 2003 American Heart Association, Inc. All rights reserved.is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Circulation Research

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MS #5369/ R1

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Activation of gp130 Transduces Hypertrophic Signal through

Interaction of Scaffolding/Docking Protein Gab1 with

Tyrosine Phosphatase SHP2 in Cardiomyocytes (Short Title; Gab1 in gp130-dependent cardiac hypertrophy)

Online Supplemental information

Materials

Murine leukemia inhibitory factor was purchased from Chemicon International Inc.

Norepinephrine was supplied by Sankyo. Endothelin -1 was from Peptide Institute Inc.

Medium-199 was from GibcoBRL. Angiotensin II and monoclonal

anti-sarcomeric α-actinin (clone EA-53) antibody were obtained from Sigma.

Anti-phosphotyrosine monoclonal antibody (PY99), anti-signal transducer and activator of

transcription 3 (STAT3), anti-ERK1, anti-ERK2, and anti-SHP2 antibodies were from Santa

Cruz Biotechnology. Anti-p85 antibody and anti-Gab1 antibody for immunoblotting and

immunocytochemistry were from Upstate Biotechnology. Anti-Gab1 serum for

immunoprecipitation was described previously. 1 Anti-phospho STAT3, anti-phospho

ERK1/2, anti-phospho ERK5, anti-phospho AKT, and anti-AKT antibodies were purchased

from Cell Signaling Technology. Anti-ERK5 antibody was from Calbiochem.

MS #5369/ R1

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Fluorescein-conjugated goat anti-mouse antibody and Cy3-conjugated goat anti-rabbit

antibody were from Jackson Immunoresearch Laboratories. Protein A-sepharose and the

ECL system were from Amersham Bioscience. All other chemicals were reagents of

molecular biology grade and were obtained from standard commercial sources.

Reference

1. Takahashi-Tezuka M, Yoshida Y, Fukada T, Ohtani T, Yamanaka Y, Nishida K,

Nakajima K, Hibi M, Hirano T. Gab1 acts as an adapter molecule linking the cytokine

receptor gp130 to ERK mitogen-activated protein kinase. Mol Cell Biol.

1998;18:4109-17.

MS #5369/ R1

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Online Figure Legends

Online Figure 1. LIF induces surface translocation of Gab1 in cardiomyocytes.

Cardiomyocytes were infected with AdGab1WT at an m.o.i. of 20 for 8 hours and

serum-deprived. At 24 hours post-infection, cells were treated without or with 1×103 U/ml

LIF for 5 minutes. Cells were fixed and dual-immunostained with anti-Gab1 antibody

(Online Figure 1A, 1C) and anti-sarcomeric α-actinin antibody (Online Figure 1B, 1D).

Gab1 and sarcomeric α-actinin were detected by Cy3-conjugated secondary goat anti-rabbit

antibody and fluorescein-conjugated secondary goat anti-mouse antibody, respectively.

Fluorescence microscopy revealed diffuse cytoplasmic distribution of Gab1 in

vehicle -treated cells (Online Figure 1A) and translocation of Gab1 to the cell periphery in

LIF-treated cells (Online Figure 1C). Representative data are shown. Experiments were

repeated three times with similar results. In addition, LIF induced surface translocation of

Gab1 in cardiomyocytes overexpressing Gab1F627/659 (data not shown).

Online Figure 2. Sarcomeric organaization of cardiomyocytes infected with Adβ-gal,

AdGab1WT , or AdGab1F627/659 .

Cardiomyocytes were infected with Adβ-gal, AdGab1WT , or AdGab1F627/659 at an m.o.i. of 20

MS #5369/ R1

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for 8 hours and serum-deprived. At 24 hours post-infection, cells were treated with 1×103

U/ml LIF for additional 24 hours. Cells were dual-labeled with monoclonal

anti-sarcomeric α-actinin antibody (Online Figure 2A, 2B, 2C) and rhodamine phalloidine

(Online Figure 2D, 2E, 2F), to allow a simultaneous assessment of Z-band and thin filament

(F-actin) assembly. Images were obtained by fluorescence microscopy. In cardiomyocytes

infected with Adβ-gal and AdGab1WT , LIF induced high degree of sarcomeric organaization

in which myofibrils were oriented along the longitudinal cell axis and extended into the

cytoplasmic projections. On the other hand, LIF could not induce longitudinal elongation of

cardiomyocytes infected with AdGab1F627/659, in which myofibrils were not oriented along

the longitudinal cell axis.

Online Figure 1.

LIF(-)

LIF(+)

Gab1 sarcomeric α-actinin

D

A B

C

Online Figure 2.

sarcomericα-actinin

phalloidine

Adβ-gal AdGab1F627/659AdGab1WT

A B C

D E F

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