| Background and Objective:Cytokines related to interleukin-6 (IL-6) comprise a family of substances known to play cytoprotective and growth-promoting roles in different cell types. IL-6 related cytokines potently promote tyrosine-phosphorylation of Janus kinases (JAKs) and cytoplasmic latent transcription factors of the signal transducers and activators (STATs) family. STAT proteins regulate the expression of genes encoding proteins involved in angiogenesis, inflammation, apoptosis, extracellular matrix composition and cellular signaling. The JAK-STAT signaling pathway mediates cytoprotective effects in cardiomyocytes. Eight members have been identified as suppressors of the cytokine signaling (SOCS) family (CIS and SOCS1 to SOCS7), and not only act as direct negative feedback regulators of JAK-STAT signaling, but are also involved in the fine tuning of the myocardial adaptation response and in crosstalk of the complicated cytokine signal network in myocytes.Accumulating evidence indicates that activation and expression of the IL-6-JAK-STAT signaling pathway are facilitated by hypoxia. Among these signaling molecules, STAT3 can be activated by hypoxia/reoxygenation stress and this signaling pathway exerts cardioprotection in the ischemic heart. Blockade of the STAT3 pathway aggravates myocardial injury after infarction. Evidence from cardiomyocyte-restricted ablation of STAT3 mice further indicates that STAT3 protects the heart from ischemic injury by suppressing cardiomyocyte apoptosis, inducing local growth factor production. As a product of the STAT3-inducible gene, SOCS3 is a major feedback regulator of STAT3, and it blocks STAT3 activation by the gp130 receptor. Hypoxia has profound effects on the expression of SOCS3 in pulmonary arterial smooth muscle cell (PASMC). SOCS3 can also be induced and can inhibit signaling by a wide-spectrum of growth factors and cytokines including Toll-like receptor (TLR) agonists (such as lipopolysaccharide and CpG-DNA), IL-10, IL-6 and other gp130 signaling cytokines, leptin and interferon-γ. These properties suggest that SOCS3 may broadly regulate cytokine signaling and might be a chief factor in these classical signal loops. Moreover, it seems that SOCS3 expression determines the character of the response of cardiac myocytes to the IL-6-type cytokines and also impacts on other signaling pathways of other agonists. While many studies have evaluated the protective effects of STAT3 and regulatory effects of SOCS3 for hypoxia/reoxygenation injury of heart, it is unknown whether SOCS3 can be directly induced by hypoxia in cardiac myocytes. Additionally, little is known about the expression, activation and regulation of these signaling molecules in myocytes exposed to chronic low ambient oxygen levels.NF-κB is a ubiquitous transcription factor and its activation involves the regulation of a large variety of genes. NF-κB consists of five Rel-related proteins such as p50, p52 (NF-κB2), p65 (RelA), c-Rel and RelB. The prototypical NF-κB complex is a RelA/p50 heterodimer, which is important for NF-κB-mediated antiapoptotic effects. Recent studies have demonstrated that the amplitude and half-life of nuclear NF-κB are influenced by acetylation of RelA , which requires prior RelA phosphorylation. In particular , endogenous RelA is acetylated in a signal-coupled manner following stimulation. Reversible acetylation of RelA is essential for the duration of NF-κB activity.Signaling pathways that mediate protection from apoptosis involve activation of the transcription factor NF-κB, which in turn induces the expression of proteins that possess cytoprotective roles. NF-κB is induced by hypoxic stimulation and it regulates the secretion of IL-6 in cardiac myocytes, and may be the primary positive regulator of transcriptional activation of the IL-6-JAK-STAT signaling pathway during hypoxia. STAT3 is a transcription factor that can promote oncogenesis, and it is commonly activated in cancer as well as in tumor-associated myeloid cells. STAT3 and NF-κB stimulate a highly overlapping repertoire of pro-survival, proliferative, and pro-angiogenic genes. Crosstalk between STAT3 and NF-κB has been demonstrated at multiple levels, including activation of STAT3 by NF-κB-regulated factors such as IL-6 and Cox-2, possible inhibition of IκB kinase (IKK) activity in normal immune cells by STAT3, and nuclear translocation of unphosphorylated NF-κB by unphosphorylated STAT3. A recent study further demonstrated that activated STAT3 prolongs NF-κB nuclear retention through acetyltransferase p300-mediated RelA acetylation , thereby interfering with NF-κB nuclear export. Therefore, the STAT3/NF-κB interaction is important for tumor cells to adapt to a hypoxic tumor microenvironment and to mediate antiapoptotic effects. The present study was undertaken to test the following hypotheses: in chronic hypoxia cardiac myocytes: 1) the IL-6-STAT3 and NF-κB signaling pathways are sustained and stably activated; 2) STAT3/NF-κB interaction occurs; and 3) expression of SOCS3 is induced by hypoxia, and it suppresses STAT3 phosphorylation and mediates NF-κB activation.We examined the expression and activation of IL-6, STAT3 and NF-κB in the myocardium of infants with cyanotic cardiac defects, as well as in cultured cardiac myocytes subjected to chronic hypoxia. We evaluated the interaction of SOCS3 with STAT3 and NF-κB by transfecting the SOCS3 plasmid to hypoxic cultured H9c2 cells. The purposes of this study were to determine the crosstalk between NF-κB and STAT3 signaling and the effect of SOCS3 on this interaction on myocardial adaptation to chronic hypoxia. Methods:Samples taken from the right ventricular outflow tract were collected from patients with cyanotic (n = 18) or acyanotic (n =22) congenital heart disease. The expression of IL-6, NF-κB p65 (acetyl K310), SOCS3, STAT3, phosphor-Tyr-705-STAT3 and phosphor-Ser-276-NF-κB p65 was examined by immunohistochemistry and western blotting, while IL-6, C-myc and SOCS3 mRNA were tested by Real time RT-PCR. The activity of NF-κB were tested by Gel electrophoretic mobility shift assay (EMSA). Serum levels of IL-6 were measured by ELISA set.To evaluated the effect of chronic hypoxia on expression and activation of IL-6, STAT3 and NF-κB signaling pathway, the cardiomyocytes in embryonic rat-heart-derived H9c2 cells were cultivated and exposed to 1.0% O2, 5.0% CO2 for different durations to establish the chronic hypoxic cell model. Control cells were cultivated in the same conditions except for 21% O2 concentration. After different duration of hypoxic exposure (6, 12, 24, 48, and 72h), cells were collected and subjected to RT-PCR and western blot to detecting the mRNA and protein expression of the two signaling pathways. The expression of NF-κB p65 (acetyl K310), SOCS3, STAT3, phosphor-Tyr-705-STAT3 and phosphor-Ser-276-NF-κB p65 was examined by western blotting, while IL-6, C-myc and SOCS3 mRNA were tested by Real time RT-PCR. IL-6 concentrations in cultured H9c2 cell medium were determined by ELISA. To address whether SOCS3 can inhibit hypoxia-induced STAT3 and NF-κB activation in hypoxic cultured H9c2 cells, we transfected them with the SOCS3 gene and examined changes in activated STAT3 and NF-κB. The expression of NF-κB p65 (acetyl K310), STAT3, phosphor-Tyr-705-STAT3 and phosphor-Ser-276-NF-κB p65 was examined by western blotting, while IL-6 and C-myc mRNA were tested by Real time RT-PCR. IL-6 concentrations in cultured H9c2 cell medium were determined by ELISA, too.To evaluate the effects of SOCS3 overexpression on cell survival , SOCS3 gene-transfected cardiac myocytes and non-transfected cardiac myocytes were submitted to a hypoxic environment for 6, 12, 24, 48, and 72h. Cell viability was assessed by the colorimetric 3-(4, 5-dimethylthia-zol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. LDH levels in the cultured supernatant were measured in duplicate following hypoxia using a commercially available kit. Terminal dUTP Nick End Labeling (TUNEL) and Hoescht staining were used for apoptosis analyses.Results:1. Immunohistochemistry revealed that all of these signaling molecules (IL-6, STAT3, p-STAT3, SOCS3, p-RelA and AC-RelA) were increased in myocardium of patients with cyanotic cardiac defects, in either the cytoplasm or the nuclei. Western blot analysis revealed that the protein levels of these signaling molecules were significantly elevated (p < 0.001) in patients with cyanotic compared to acyanotic congenital heart disease. Also, the expression of IL-6, C-myc and SOCS3 mRNA were markedly increased in cyanotic patients (p < 0.05). While there were no significant differences in serum levels of IL-6 between the acyanotic group and the cyanotic group (p >0.05).2. SOCS3 protein and mRNA were not detected in cardiomyocytes under normoxic conditions.While in hypoxia-exposed cells, the protein expression of STAT3-SOCS3 as well as NF-κB together with the mRNA of IL-6, SOCS3 and C-myc in hypoxia-exposed cells were significantly increased in a duration-dependent manner compared with the normoxia group (p < 0.001).3. The level of p-STAT3, p-RelA and AC-RelA did not increase in SOCS3 gene-transfected cells with prolonging time of hypoxia compared with the level of p-STAT3 in non-transfected cells (p < 0.001). Similarily, the mRNA of IL-6 and C-myc showed the same results(p < 0.001).In contrast to control cells, a greater proportion of SOCS3 gene-transfected cardiac myocytes subjected to hypoxia for 72 h exhibited pyknotic, TUNEL-positive nuclei.The non-transfected group subjected to hypoxia for 72 h showed significantly less hypoxia-mediated apoptosis of myocardial cells compared with the transfected group (p<0.001). Elevated LDH release was also observed in SOCS3 gene-transfected cardiac myocytes after hypoxia at each time interval. SOCS3 overexpression resulted in more LDH leakage from injured myocardium compared with non-transfected cardiac myocytes under hypoxic stress as well as SOCS3 gene-transfected group and control group in normoxia conditions (p < 0.001).Conclusions:1. The IL-6-STAT3-SOCS3 and NF-κB signaling pathways as well as the IL-6, SOCS3 and C-myc mRNA expression increase in cyanotic patients.2. In cardiac myocytes exposed to hypoxia, the activation of Ac-RelA, p-RelA, and p-STAT3 in the nucleus and the expression of SOCS3 and STAT3 in the cytoplasm are significantly increased in a duration-dependent manner compared with the normoxia group.The expression of levels of SOCS3,IL-6 and C-myc mRNA in the cytoplasm also increase in cardiac myocytes exposed to hypoxia.3. Overexpression of SOCS3 shows down-regulation of the IL-6 and C-myc genes. Transfected overexpression of SOCS3 suppresses hypoxia-induced STAT3 phosphorylation and attenuate the activation of Ac-RelA and p-RelA in cardiac myocytes exposed to hypoxia.And overexpression of SOCS3 inhibits the cytoprotective effect of the STAT3 and NF-κB signaling pathways in H9c2 cells exposed to hypoxia. |
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