Cardiotrophin-1 (7. Cardiotrophin-1 Signalling)

Gp130 is required for CT-1 signaling in cardiomyocytes, by demonstrating that a monoclonal anti-gp130 antibody completely inhibits c-fos induction by CT-1. Similarly, a leukemia inhibitory factor receptor subunit beta (LIFRbeta) antagonist effectively blocks the CT-1 induction of c-fos, indicating a requirement for LIFRbeta in the hypertrophic response, as well. Upon stimulation with CT-1, both gpl30 and the LIFRbeta are tyrosine-phosphorylated, providing further evidence that CT-1 signals through the gp130/LIFRbeta heterodimer in cardiomyocytes ^Ref . CT-1 promotes cardiac myocyte survival via the activation of an antiapoptotic signaling pathway that requires Map kinases, whereas the hypertrophy induced by CT-1 may be mediated by alternative pathways, e.g. Janus kinase/STAT or MEK kinase/c-Jun NH2-terminal protein kinase ^Ref . It was proposed that CT-1 may therefore be of use as a novel cardio-protective agent, particularly if its hypertrophic effect can be specifically inhibited ^Ref . A more recent study concluded that STAT3 transduces not only a hypertrophic signal but also a protective signal against doxorubicine-induced cardiomyopathy by inhibiting reduction of cardiac contractile genes and inducing cardiac protective factors ^Ref . In another study, CT-1 simultaneously activated phosphorylation of STAT3, ERK1/2, and PI3-K in rat cardiomyocytes. Parthenolide, an inhibitor of STAT, suppressed CT-1-induced [3H]leucine incorporation by 88.3 % and protein-to-DNA ratio by 75.0 %. U0126, an MEK1/2 inhibitor, increased CT-1-induced the phosphorylation of STAT3 in a dose-dependent manner and, consistently, augmented CT-1-induced increase in [3H]leucine incorporation and cellular protein-to-DNA ratio by 17.6 % and 16.3 %, respectively. Wortmannin, a PI3-K inhibitor, did not influence CT-1-induced [3H]leucine incorporation and cellular protein-toDNA ratio. The authors concluded that the hypertrophic effect of CT-1 was essentially mediated by STAT3, independent of PI3-K, and negatively regulated by ERK1/2 via inhibiting the phosphorylation of STAT3. Taken together, the interaction between STAT3 and ERK1/2 in CT-1-induced signaling contributes to development of cardiac hypertrophy ^Ref . Inhibition of ERK1/2 by U0126 resulted in an increase of CT-1-induced tyrosine phosphorylation of STAT3 and, consequently, the protein-to-DNA ratio and [(3)H]-leucine incorporation. Transient transfection of the cells with STAT3S727A had no significant effect on CT-1-induced tyrosine phosphorylation of STAT3. It was concuded that STAT3 is activated by CT-1 in rat cardiomyocytes, but full activation is mitigated by the simultaneous activation of ERK1/2. The inhibition of ERK1/2 increases the activity of STAT3, which, in turn, enhances the hypertrophic effect of CT-1. The crosstalk between ERK1/2 and STAT3 is independent of the phosphorylation of the S727 in STAT3. Such crosstalk may contribute to the development of adequate cardiac hypertrophy ^Ref . Cultured cardiomyocytes were infected with adenoviral vectors harboring a dominant-negative STAT3 mutant or one of two endogenous negative regulators of cytokine signaling via the Janus kinase (JAK)-signal transducer and activator of transcription (STAT) pathways [suppressor of cytokine signaling (SOCS) 1 and 3] and then examined their effects on three indexes of CT-1-induced cell hypertrophy: protein synthesis, secretion of brain natriuretic peptide and changes in cell surface area. In control cells, CT-1-induced both STAT3 phosphorylation and cell hypertrophy. Overexpression of dominant-negative STAT3 mutant suppressed CT-1-induced STAT3 phosphorylation, but did not affect cell hypertrophy. On the other hand overexpression of SOCS1 or SOCS3 inhibited both CT-1-induced STAT3 phosphorylation and cell hypertrophy. CT-1 also induced phosphorylations of ERK1/2 and ERK5 in cardiomyocytes, and those, too, were suppressed by overexpression of SOCSs. CT-1-induced cell hypertrophy was suppressed by overexpression of a dominant-negative MEK5 mutant, and not by overexpression of a dominant-negative MEK1 mutant. These findings indicate that the major pathway responsible for the hypertrophic responses to CT-1 is not JAK-STAT3 pathway nor MEK1-ERK1/2 pathway, but MEK5-ERK5 pathway ^Ref . Intravenous injection of 20 microgram/kg body weight of CT-1 induced a transient, marked increase in STAT3 activation in various tissues, including heart and lung, and subsequent upregulation of 2 members of the CIS (cytokine-inducible SH2 protein) family, JAK-binding protein (JAB)/SOCS-1/SSI-1 and CIS3/SOCS-3/SSI-3 (cytokine-inducible negative regulators of cytokine signaling via Janus kinase (JAK)-STAT pathways) , in the same tissues. It was also observed that CIS3 was directly associated with JAK2 in vivo. Pretreatment with the same dose of CT-1 60 minutes before significantly attenuated the STAT3 activation induced by a second injection of CT-1. A similar phenomenon was previously reported regarding CT-1 and NO-synthase: intravenous injection of CT-1 results in the nitric oxide (NO)-dependent hypotension accompanied by the induction of inducible NO synthase mRNA. In rats pretreated with CT-1, the induction of inducible NO synthase mRNA or hypotension by subsequent CT-1 injection was not observed. Forced expression of JAB or CIS3, but not other CISs, directly blocked CT-1-induced STAT3 activation in 293 cells. These results suggest that JAB and CIS3 serve as endogenous inhibitors of CT-1-mediated JAK-STAT signaling in the cardiovascular system in vivo ^Ref . It was found that CT-1 phosphorylated and activated Akt, and the effect was blocked by the PI3K inhibitors LY294002 and wortmannin. CT-1 also phosphorylated the pro-apoptotic factor, BAD, and the BAD phosphorylation was inhibited by LY294002, suggesting that phosphorylation of BAD is one of the key events by which the PI3K/Akt pathway mediates CT-1-induced survival signaling. Further, CT-1 PI3K-dependently prolonged the survival of serum-starved ventricular myocytes by preventing apoptosis. In summary, these findings show that PI3K-dependent survival signals contribute to CT-1-mediated ventricular myocyte survival. In vivo, the death of ventricular myocytes leads to heart failure, and downregulation of survival signals and/or augmentation of proapoptotic signals are likely to be important components of disease processes. Thus, the extent to which CT-1 and the PI3K/Akt pathway mitigate such pathological processes, in vivo, is an important question for the future ^Ref . Dominant negative gene based inhibitors of MEK1/2, PI3-kinase and Akt inhibited CT-1 mediated cardioprotection in re-oxygenation as did chemical inhibitors of the PI3-kinase pathway. Hence the PI3-kinase/Akt pathway is required in addition to MEK1/2 to mediate CT-1 cardioprotection in ischemia reoxygenisation ^Ref . CT-1 signals through p38, ERK, and Akt in a parallel manner to activate NF-kappa B and that NF-kappa B is required for CT-1 to mediate its full cytoprotective effects in cardiac myocytes ^Ref . Treatment of embryoid bodies grown from pluripotent murine embryonic stem (ES) cells with CT-1 significantly stimulated cardiomyogenesis and increased nuclear expression of the proliferation marker Ki-67. The increase in Ki-67 expression was inhibited upon pretreatment with the free radical scavenger vitamin E, indicating a role for reactive oxygen species (ROS) in the signaling cascade. CT-1 treatment of cardiac cells raised intracellular ROS in ES cell-derived cardiomyocytes. ROS were presumably generated by an NADPH-oxidase since ROS generation was down-regulated upon preincubation with the NADPH-oxidase inhibitor diphenylen iodonium chloride (DPI) and LY294002, which inhibits phosphatidyli­nositol 3 kinase (PI3-kinase). CT-1 activated nuclear factorkappaB (NF-kappaB) and induced phosphorylation of the Janus kinase signal transducer-2 (Jak-2), the signal transducer and activator of transcription3 (STAT-3) as well as the extracellular signal-regulated kinase 1,2 (ERK1/2). STAT-3 and ERK1/2 phosphorylation as well as NF-kappaB activation were inhibited by pretreatment with the Jak-2 antagonist aG490, the ERK1/2 inhibitor pd98059, the free radical scavenger vitamin E, the NADPH-oxidase inhibitor DPI, as well as by LY294002. PD98059 failed to inhibit Jak-2 phosphorylation, indicating that the ERK and the JAK/STAT signaling cascade interact on a level downstream of Jak-2. It is concluded that CT-1 stimulates the proliferation of ES cell-derived cardiomyocytes by signaling pathways that involve ROS as signaling molecules in the signal transduction cascade ^Ref .