Keiko Yamauchi-Takihara

Activation of gp130 Transduces Hypertrophic Signals via STAT3 in Cardiac Myocytes

BACKGROUNDngp130, a signal transducer of the IL-6-related cytokines, is expressed ubiquitously, including in the heart. The activation of gp130 in cardiac myocytes was reported to induce myocardial hypertrophy. The downstream side of gp130 consists of two distinct pathways in cardiac myocytes, one a Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway, the other a mitogen-activated protein kinase (MAPK) pathway. In the present study, we examined whether the JAK/STAT pathway, especially the STAT3-mediated pathway, plays a critical role in gp130-dependent myocardial hypertrophy by transfecting wild-type and mutated-type STAT3 cDNA to cardiac myocytes.nnnMETHODS AND RESULTSnWe constructed three kinds of replication-defective adenovirus vectors carrying wild-type (AD/WT) or mutated-type (AD/DN) STAT3 cDNA or adenovirus vector itself (AD). Cultured murine cardiac myocytes infected with adenovirus were stimulated with leukemia inhibitory factor (LIF), and the expression of c-fos and atrial natriuretic factor (ANF) mRNAs and [3H]leucine incorporation were examined. There were no significant differences in MAPK activity among the three groups. Compared with AD-transfected cardiac myocytes, induction of c-fos and ANF mRNAs and protein synthesis after LIF stimulation were significantly augmented in AD/WT-transfected cells. In contrast, induction of c-fos and ANF mRNA expression and protein synthesis were attenuated after LIF stimulation in cardiac myocytes transfected with AD/DN.nnnCONCLUSIONSnThese results suggest that the STAT3-dependent signaling pathway downstream of gp 130 promotes cardiac myocyte hypertrophy under stimulation with LIF.

Glycoprotein 130 Regulates Cardiac Myocyte Survival in Doxorubicin-Induced Apoptosis Through Phosphatidylinositol 3-Kinase/Akt Phosphorylation and Bcl-xL/Caspase-3 Interaction

Background —We recently reported that the activation of glycoprotein (gp) 130 by leukemia inhibitory factor (LIF) upregulates Bcl-xL and exerts antiapoptotic effects in cardiac myocytes. In addition, LIF induces activation of phosphatidylinositol (PI) 3-kinase and Akt, which are known to be required for cell survival. However, their regulatory roles in cell death remain unknown. Methods and Results —We investigated the fate of these proteins and the cytoprotective effects of LIF on doxorubicin (DOX)-induced apoptosis in cultured neonatal rat cardiac myocytes. Myocyte apoptosis increased significantly in DOX-treated cells but was significantly reduced by LIF pretreatment. The kinase activities of PI 3-kinase and Akt declined below basal levels but were partially recovered with LIF. Moreover, DOX-induced caspase-3 activation and decrease in Bcl-xL abundance are completely inhibited by LIF and caspase inhibitor. LIF phosphorylates Bad through PI 3-kinase and reduces the heterodimerization of Bad with Bcl-xL. Adenovirus transfer of the constitutively active form of Akt to cardiac myocytes restored cardiac myocyte survival after DOX treatment. Conversely, the dominant-negative form of Akt inhibited LIF-induced increase in cell viability and suppression of caspase-9 activation. Conclusions —Activation of gp130 inhibits DOX-induced cell death in cardiac myocytes, resulting in the restoration of PI 3-kinase/Akt activities and in the inactivation of caspase-3, leading to facilitation of the protective function of Bcl-xL.

Activation of JAK/STAT pathway transduces cytoprotective signal in rat acute myocardial infarction

OBJECTIVESnWe reported that the activation of gp130 transduced hypertrophic and cytoprotective signals via Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway in cardiac myocytes. Recent in vivo experiments have demonstrated that the JAK/STAT pathway is activated in acute pressure overload hearts. The present study was designed to examine whether the JAK/STAT pathway is also activated in acute myocardial infarction (AMI) and to determine its pathophysiological roles in ischemic heart disease.nnnMETHODS AND RESULTSnAMI model was generated by the ligation of proximal left anterior descending coronary artery of male Wistar rat. They were sacrificed at various time points ranging from 1 to 24 h after coronary ligation and their hearts were examined. Tyrosine phosphorylation of STAT3 was observed in the myocardium obtained from both the ischemic area and the healthy border area adjacent to the infarcted area. The AMI rats were next randomly assigned to two groups, one with coronary ligation only (group M), and the other with coronary ligation with AG-490 treatment (1 mg/kg i.v., every 4 h), a specific JAK2 inhibitor (group A). In group A, phosphorylation of STAT3 was significantly suppressed and caspase-3 activity and Bax expression were significantly increased in the myocardium after AMI. In group M, few apoptotic myocytes were identified in the border area by means of TUNEL assay. However, a significant increase in apoptotic cells was observed in group A.nnnCONCLUSIONSnAdministration of JAK2 inhibitor resulted in deterioration of myocardial viability in AMI hearts. The JAK/STAT pathway is activated in AMI myocardium and plays a pivotal role in cytoprotective signaling.

Activation of gp130 Transduces Hypertrophic Signal Through Interaction of Scaffolding/Docking Protein Gab1 With Tyrosine Phosphatase SHP2 in Cardiomyocytes

Abstract— Grb2-associated binder-1 (Gab1) is a scaffolding/docking protein and contains a Pleckstrin homology domain and potential binding sites for Src homology (SH) 2 and SH3 domains. Gab1 is tyrosine phosphorylated and associates with protein tyrosine phosphatase SHP2 and p85 phosphatidylinositol 3-kinase on stimulation with various cytokines and growth factors, including interleukin-6. We previously demonstrated that interleukin-6–related cytokine, leukemia inhibitory factor (LIF), induced cardiac hypertrophy through gp130. In this study, we report the role of Gab1 in gp130-mediated cardiac hypertrophy. Stimulation with LIF induced tyrosine phosphorylation of Gab1, and phosphorylated Gab1 interacted with SHP2 and p85 in cultured cardiomyocytes. We constructed three kinds of adenovirus vectors, those carrying wild-type Gab1 (AdGab1WT), mutated Gab1 lacking SHP2 binding site (AdGab1F627/659), and &bgr;-galactosidase (Ad&bgr;-gal). Compared with cardiomyocytes infected with Ad&bgr;-gal, longitudinal elongation of cardiomyocytes induced by LIF was enhanced in cardiomyocytes infected with AdGab1WT but inhibited in cardiomyocytes infected with AdGab1F627/659. Upregulation of BNP mRNA expression by LIF was evoked in cardiomyocytes infected with Ad&bgr;-gal and AdGab1WT but not in cardiomyocytes infected with AdGab1F627/659. In contrast, Gab1 repressed skeletal &agr;-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 cardiomyocytes infected with Ad&bgr;-gal but repressed in cardiomyocytes infected with AdGab1F627/659. Coinfection of AdGab1WT with adenovirus vector carrying dominant-negative ERK5 abrogated longitudinal elongation of cardiomyocytes induced by LIF. Taken together, these findings indicate that Gab1-SHP2 interaction plays a crucial role in gp130-dependent longitudinal elongation of cardiomyoctes through activation of ERK5.

Cytokines and their receptors in cardiovascular diseases — role of gp130 signalling pathway in cardiac myocyte growth and maintenance

Interleukin (IL)‐6‐related cytokines share gp130 as the signal‐transducing protein. Cardiac myocytes produce various kinds of cytokines including IL‐6 and cardiotrophin‐1. Activation of gp130 transduces hypertrophic and cytoprotective signals in cardiac myocytes via JAK/STAT, MAP kinase and PI‐3 kinase pathways. Besides various well‐established mechanisms by which cardiac growth and myocardial remodeling are regulated, gp130 signalling may be a newly discovered mechanism that regulates these events in association with cytoprotective effect in myocardial diseases.

The Polycomb-group gene Rae28 sustains Nkx2.5/Csx expression and is essential for cardiac morphogenesis

The Polycomb-group (PcG) gene Rae28 is a mammalian homologue of the Drosophila gene polyhomeotic. PcG genes are known to maintain transcription states, once initiated, probably by regulating chromatin structure. Since homozygous Rae28-deficient (Rae28(-/-)) mice displayed cardiac anomalies similar to congenital heart diseases in humans, we examined the role of Rae28 in cardiac morphogenesis at the molecular level. In Rae28(-/-) embryos, expression of the cardiac selector gene Nkx2.5/Csx (Nkx2.5) was initiated properly but was not sufficiently sustained later in development. This impaired expression of Nkx2.5 in the maintenance phase proved to have a crucial effect on cardiac morphogenesis, as demonstrated by the results of a genetic complementation experiment in which the cardiac anomalies were suppressed by overexpression of human NKX2.5/CSX1 in Rae28(-/-) embryos. Ubiquitous expression of exogenous Rae28 likewise restored the impaired Nkx2.5 expression in Rae28(-/-) embryos, further supporting the notion that Rae28 sustains Nkx2.5 expression in cardiomyocytes. Thus, our data show that a mammalian PcG gene can play a key role in organogenesis by helping to maintain the expression of a selector gene.

Smad1 Protects Cardiomyocytes From Ischemia-Reperfusion Injury

Background—We previously reported that bone morphogenetic protein 2 (BMP2) protected against apoptosis of serum-deprived cardiomyocytes via induction of Bcl-xL through the Smad1 pathway. To investigate whether Smad1 signaling promotes cell survival in the adult heart, we subjected transgenic mice with cardiac-specific overexpression of smad1 gene (Smad1TG) to ischemia-reperfusion (I/R) injury. Methods and Results—The effects of BMP2 or adenovirus-mediated transfection of smad1 on cardiomyocyte survival in hypoxia-reoxygenation were examined using rat neonatal cardiomyocytes. BMP2 and Smad1 each significantly promoted survival and diminished apoptotic death of cardiomyocytes during hypoxia-reoxygenation. Interestingly, Smad1 was found to be activated during I/R in normal mouse heart. To examine physiological and pathological roles of Smad1 in I/R, we generated Smad1TG using the α-myosin heavy chain gene promoter. Phosphorylation of Smad1 was found in all smad1 transgene–positive mouse hearts. To examine whether Smad1 prevents injury of cardiomyocytes in vivo, we subjected Smad1TG and age-matched wild-type mice (WT) to I/R injury induced by 1 hour of ligation of the left coronary artery and 1 hour of reperfusion. TUNEL and DNA ladder analyses showed that Smad1TG had significantly smaller myocardial infarctions and fewer apoptotic deaths of cardiomyocytes than did WT. Interestingly, increased expression of Bcl-xL and β-catenin was more remarkable whereas caspase3 was less activated in Smad1TG heart than in that of WT. Conclusions—These findings suggest that the Smad1 signaling pathway plays a role in cardioprotection against I/R injury.

Bcl-xl reduces doxorubicin-induced myocardial damage but fails to control cardiac gene downregulation

OBJECTIVEnWe recently reported that doxorubicin (Dox), an effective anti-cancer drug, induces apoptosis in cardiac myocytes in association with reduction of Bcl-xl expression. In the present study, we further examined whether overexpression of Bcl-xl ameliorates Dox-induced cardiac myocyte damage.nnnMETHODS AND RESULTSnOverexpression of the Bcl-xl gene by adenovirus vector resulted in an 11-fold increase in Bcl-xl protein in neonatal rat cardiac myocytes (BCL) compared to that in cells with beta-galactosidase gene transfection (CTL). Although Dox treatment generated similar amounts of reactive oxygen species (ROS) in BCL and CTL, cell viability was maintained and the number of apoptotic cardiac myocytes was significantly decreased in BCL. Cytochrome c release and enhanced caspase-3 activity after Dox treatment were significantly suppressed and Bax expression level was decreased in BCL. Cardiac-specific gene expression is known to be inhibited by Dox. The expression of cardiac alpha-actin and sarcoplasmic reticulum Ca(2+)-ATPase 2a mRNA was equally inhibited in BCL and CTL after Dox treatment.nnnCONCLUSIONSnOverexpression of Bcl-xl in cardiac myocytes failed to regulate Dox-induced ROS generation and cardiac-specific gene downregulation but inhibited apoptosis accompanied by reduction of Bax protein.

Expressional Patterns of Cytokines in a Murine Model of Acute Myocarditis: Early Expression of Cardiotrophin-1

To determine the role of cytokines in acute myocarditis, we examined expressional patterns of cardiotrophin-1 (CT-1), TNF-α, and IL-1α in a murine model of acute myocarditis. Ten-day-old Institute of Cancer Research mice were injected with Coxsackievirus B3 and killed on Days 1, 2, 3, 4, 5, 7, 10, 14, and 28 of injection. TNF-α and IL-1α expressions were investigated on histological sections from each heart, and mRNA expression of TNF-α, IL-1α, and CT-1 in the heart was examined by reverse transcription-polymerase chain reaction and RNase protection assay. To determine myocardial regeneration, cardiomyocytic DNA synthesis was investigated using bromodeoxyuridine on Days 3, 5, 7, and 10, and the labeling index was calculated in each heart. Age-matched uninfected mice were used as controls. TNFα and IL-1α expression was first detected in the cardiomyocytes on Day 3 and reached the maximum level on Day 7, when inflammatory changes were most prominent. Although an increased expression of TNFα and IL-1α mRNA was also detected on Day 3, CT-1 mRNA expression was distinctly augmented on Day 2. The labeling indices in the hearts with myocarditis were significantly higher than in those of the controls in all of the time points examined. CT-1 expression preceded TNF-α and IL-1α expressions and active DNA synthesis in a murine model of acute myocarditis. All CVB3-infected mice with anti-glycoprotein-130 antibody treatment died within 6 days. CT-1 may exert a protective role by modulating cytokine production and by inducing cardiomyocytic proliferation in CVB3-infected murine hearts.

Ral GDP Dissociation Stimulator and Ral GTPase Are Involved in Myocardial Hypertrophy

Abstract—Ras-related GTPase (Ral) is converted to the GTP-bound form by Ral GDP dissociation stimulator (Ral-GDS), a putative effector protein of Ras. Although a number of studies indicate that Ras induces cardiac hypertrophy, the functional role of Ral-GDS/Ral signaling pathway is as yet unknown in cardiac myocytes. We investigated the role of the Ral-GDS/Ral pathway in cardiac hypertrophy. Transfection of Ral-GDS and constitutively active mutant of Ral (RalG23V) in cultured rat neonatal myocytes stimulated promoter activity of c-fos (5.4-fold and 2.6-fold, P <0.01), &agr;-skeletal actin (2.7-fold and 2.1-fold, P <0.01), and &bgr;-myosin heavy chain-luciferase (2.8-fold and 2.3-fold, P <0.01). Ral-GDS–induced or RalG23V-induced promoter activation was increased synergistically with activated Ras (RasG12V). Dominant-negative mutant of Ral (RalS28N) partially inhibited RasG12V induced promoter activation. Cardiac myocytes transfected with RalG23V showed increased cell size compared with nontransfected or vector-transfected cells (2.1-fold, P <0.01). Cardiotrophin-1 (CT-1) upregulated Ral-GDS mRNA expression and induced Ral activation. CT-1–induced Ral-GDS mRNA expression was inhibited by overexpression of the dominant-negative mutant of STAT3. Moreover, Ral activity was elevated in hypertrophied hearts (2.1-fold, P <0.01) by mechanical stress in association with increased CT-1 expression and signal transducer and activator of transcription 3 (STAT3) phosphorylation in the rat aortic banding model. Ral-GDS/Ral pathway is involved in a wide range of gene expressions and is activated by hypertrophic stimuli in vitro and in vivo. SATA3 may play a key role in Ral-GDS expression and Ral activation. Our data provide evidence that the Ral-GDS/Ral signaling pathway is a link to the process of cardiac hypertrophy.