ZAK re-programs atrial natriuretic factor expression and induces hypertrophic growth in H9c2 cardiomyoblast cells

Various intracellular or intercellular stimuli have been associated with the development of cardiac cell hypertrophy. However, the mechanisms underlying this association are not completely understood. In a previous study we determined that ZAK mRNA expression is abundant in heart. ZAK is a mitogen-activated protein kinase kinase kinase (MAP3K) that activates the stress-activated protein kinase/c-jun N-terminal kinase pathway and activates NF-kappaB. We, therefore, investigated the potential involvement of ZAK (which in cultured H9c2 cardiomyoblast cell is a positive mediator of cell hypertrophy). Our results showed that the expression of a wild-type form of ZAK induces the characteristic hypertrophic growth features, including increased cell size, elevated atrial natriuretic factor expression, and increased actin fiber organization.     

Overexpression of calreticulin modulates protein kinase B/Akt signaling to promote apoptosis during cardiac differentiation of cardiomyoblast H9c2 cells

Calreticulin is a Ca(2+)-binding molecular chaperone of the lumen of the endoplasmic reticulum. Calreticulin has been shown to be essential for cardiac and neural development in mice, but the mechanism by which it functions in cell differentiation is not fully understood. To examine the role of calreticulin in cardiac differentiation, the calreticulin gene was introduced into rat cardiomyoblast H9c2 cells, and the effect of calreticulin overexpression on cardiac differentiation was examined. Upon culture in a differentiation medium containing fetal calf serum (1%) and retinoic acid (10 nm), cells transfected with the calreticulin gene were highly susceptible to apoptosis compared with controls. In the gene-transfected cells, protein kinase B/Akt signaling was significantly suppressed during differentiation. Furthermore, protein phosphatase 2A, a Ser/Thr protein phosphatase, was significantly up-regulated, implying suppression of Akt signaling due to dephosphorylation of Akt by the up-regulated protein phosphatase 2A via regulation of Ca(2+) homeostasis. Thus, overexpression of calreticulin promotes differentiation-dependent apoptosis in H9c2 cells by suppressing the Akt signaling pathway. These findings indicate a novel mechanism by which cytoplasmic Akt signaling is modulated to cause apoptosis by a resident protein of the endoplasmic reticulum, calreticulin.     

Knockdown of dishevelled-1 attenuates cyclosporine A-induced apoptosis in H9c2 cardiomyoblast cells

Cyclosporine (CsA) has become a mainstay for immune suppression of organ transplants. It is known that patients receiving CsA manifest increased growth of aggressive cardiotoxicity. We have demonstrated that CsA induces myocardium cell apoptosis in vivo and vitro. Recently, dishevelled-1 (Dvl-1) protein, which is a cytoplasmic mediator of Wnt/β-catenin signaling, was explored in cardiac diseases. However, whether Dvl-1 is involved in CsA-induced apoptosis remains to be determined. The aim of this study was to explore the role of Dvl-1 in CsA-induced apoptosis in H9c2 cardiomyoblast cells and to investigate the role of the Wnt/β-catenin signaling cascade in this progress. H9c2 cells were treated with CsA in dose and time-dependent manners. We found that the appropriate concentrations and time-points of CsA-induced the expression of Dvl-1 and subsequent up-regulation of β-catenin and c-Myc, which is consistent with previously demonstrated concentrations and time-points when H9c2 cells apoptosis occurred. Then, cells were transfected with small interfering RNA (siRNA) against Dvl-1 and stimulated with previously demonstrated concentration of CsA. Dvl-1 down-regulation decreased the apoptotic rate, caspase-3 activity, and the Bax/Bcl-2 ratio in H9c2 cells treated with CsA. Furthermore, knocking down the expression of Dvl-1 partially suppressed the activity of the Wnt/β-catenin pathway. Moreover, we further deleted the downstream member β-catenin by specific siRNA, and found that CsA-induced the Bax/Bcl-2 ratio and the expression of c-Myc, which were attenuated. Our results are the first to unveil this novel aspect of Dvl-1 signaling. In addition, these data provide insight into the pathogenesis and the therapeutic strategies of CsA-induced myocardial injury.     

Protective effect of cholecystokinin octapeptide on angiotensin II-induced apoptosis in H9c2 cardiomyoblast cells

Cholecystokinin (CCK) and its receptors are expressed in mammalian cardiomyocytes and are involved in cardiovascular system regulation; however, the exact effect and underlying mechanism of CCK in cardiomyocyte apoptosis remain to be elucidated. We examined whether sulfated CCK octapeptide (CCK-8) protects H9c2 cardiomyoblast cells against angiotensin II (Ang II)-induced apoptosis. The H9c2 cardiomyoblasts were subjected to Ang II with or without CCK-8 and the viability and apoptotic rate were detected using a Cell Counting Kit-8 assay, Hoechst 33342 staining, terminal deoxyribonucleotide transferase-mediated nick-end labeling assays, and flow cytometry. In addition, specific antiapoptotic mechanisms of CCK-8 were investigated using specific CCK1 (Devazepide) or CCK2 (L365260) receptor antagonists, or the PI3K inhibitor LY294002. The expression of CCK, CCK1 receptor, CCK2 receptor, Akt, p-Akt, Bad, p-Bad, Bax, Bcl-2, and caspase-3 were detected by Western blot analysis and real-time polymerase chain reaction. We found that CCK and its receptor messenger RNA (mRNA) and protein are expressed in H9c2 cardiomyoblasts. Ang II-induced increased levels of CCK mRNA and protein expression and decreased levels of CCK1 receptor protein and mRNA. Pretreatment of CCK-8 attenuated Ang II-induced cell toxicity and apoptosis. In addition, pretreatment of H9c2 cells with CCK-8 markedly induced expression of p-Akt, p-bad, and Bcl-2 and decreased the expression levels of Bax and caspase-3. The protective effects of CCK-8 were partly abolished by Devazepide or LY294002. Our results suggest that CCK-8 protects H9c2 cardiomyoblasts from Ang II-induced apoptosis partly via activation of the CCK1 receptor and the phosphatidyqinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway.     

ZAK induces MMP-2 activity via JNK/p38 signals and reduces MMP-9 activity by increasing TIMP-1/2 expression in H9c2 cardiomyoblast cells

Leucine-zipper and sterile-alpha motif kinase (ZAK) is the key intra-cellular mediator protein in cardiomyocyte hypertrophy induction by transforming growth factor beta 1 (TGF-β1) which has also been identified as a profibrotic cytokine involved in cardiac fibrosis progression. We hypothesized whether ZAK over-expression causes cardiac scar formation due to the extra-cellular matrix (ECM) degraded enzyme regulation in this paper. Using immuno-histochemical analysis of the human cardiovascular tissue array, we found a positively significant association between ZAK over-expression and myocardial scars. ZAK over-expression in H9c2 cardiomyoblast cells increases the metalloproteinase tissue inhibitor 1/2 (TIMP-1/2) protein level, which reduces matria metalloproteinase-9 (MMP-9) activity and also activates c-JNK N-terminal kinase 1/2 (JNK1/2) and p38 signaling, which induces MMP-2, possibly resulting in cardiac fibrosis. Taken together, ZAK activity inhibition may be a good strategy to prevent the cardiac fibrosis progression.     

Insulin protects H9c2 rat cardiomyoblast cells against hydrogen peroxide-induced injury through upregulation of microRNA-210

Abstract

Background. Insulin protects cardiomyocytes from reactive oxygen species (ROS)-induced apoptosis after ischemic/reperfusion injury, but the mechanism is not clear. This study investigated the protective mechanism of insulin in preventing cardiomyocyte apoptosis from ROS injury. Methods. Rat cardiomyoblast H9c2 cells were treated with hydrogen peroxide (H₂O₂) or insulin at various concentrations for various periods of time, or with insulin and H₂O₂ for various periods of time. Cell viability was measured by the methylthiazolydiphenyl-tetrazolium bromide method. Cellular miR-210 levels were quantified using real-time RT-PCR. MiR-210 expression was also manipulated through lentivirus-mediated transfection. LY294002 was used to investigate involvement of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Results. The percentage of viable cells was significantly and inversely associated with H₂O₂ concentration, an effect that was seemingly attenuated by insulin pretreatment. Treatments with H₂O₂ or insulin were associated with a significant increase in miR-210 levels. Manipulation of miR-210 expression by gene transfection showed that miR-210 could attenuate H₂O₂-induced cellular injury. Inhibition of the PI3K/Akt pathway by the Akt inhibitor LY294002 was associated with a decrease in miR-210 expression. Conclusion. Insulin stimulated the expression of miR-210 through the PI3K/Akt pathway, resulting in a protective effect against cardiomyocyte injury that had been induced by H₂O₂/oxygen species. Our results provide novel evidence regarding the mechanism underlying the protective effect of insulin.     

BNIP3 induces IL6 and calcineurin/NFAT3 hypertrophic-related pathways in H9c2 cardiomyoblast cells

Ischemia/reperfusion injury causes cardiomyocyte apoptosis, ventricular remodeling, leading to a dilated heart. Hypoxia is one of the causes involved in ischemia damage, and BNIP3 is a hypoxia-inducible marker and also a sensor to induce mitochondria-dependent apoptosis. Recent reports discussed ablating BNIP3 can restrain cardiomyocytes apoptosis and post-infarction remodeling. BNIP3 is a crucial therapeutic target. However, the BNIP3-induced hypertrophy aspect is rarely investigated. Here, we transiently transfected BNIP3 plasmids into H9c2 cardiomyoblast cells to evaluate the molecular signaling and hypertrophy markers using Western blot. We measured the cell size change using actin staining. We disclose that BNIP3 overexpression induced an increase in cell size, activated the pathological-related hypertrophy signaling pathways, such as IL6-MEK5-ERK5, IL6-JAK2-STAT1/3, calcineurin/NFAT3 and p38β MAPK resulting in the fetal genes, ANP and BNP expressing. Concluding above, BNIP3 acts as a pathological hypertrophy inducer, which might be a potential therapeutic target for heart damage prevention.     

The minor subunit splice variants, H2b and H2c, of the human asialoglycoprotein receptor are present with the major subunit H1 in different hetero-oligomeric receptor complexes

The hepatic asialoglycoprotein receptor (ASGP-R) is an endocytic receptor that mediates the internalization of desialylated glycoproteins and their delivery to lysosomes. The human ASGP-R is a hetero-oligomeric complex composed of H1 and H2 subunits. There are three naturally occurring H2 splice variants, designated H2a, H2b, and H2c, although the expression of the H2c protein had not been reported. Following deglycosylation of purified ASGP-R, we detected the H2b and H2c proteins in HepG2 and HuH-7 hepatoma cells, using an antibody directed against a COOH-terminal peptide common to all H2 isoforms (anti-H2-COOH) and another antibody against a 19-amino acid cytoplasmic insert found only in H2b (anti-H2-Cyto19). H1 and both H2b and H2c were co-purified by affinity chromatography, using asialo-orosomucoid (ASOR)-, anti-H1-, or anti-H2-COOH-Sepharose, whereas only H1 and H2b were immunoprecipitated with anti-H2-Cyto19. These results indicate that H2b and H2c are not present in the same ASGP-R complexes with H1. Similar to the H2b isoform, H2c was also palmitoylated, indicating that the 19-residue cytoplasmic insert does not regulate palmitoylation. Stably transfected SK-Hep-1 cell lines expressing ASGP-R complexes containing H1 and either H2b or H2c had similar binding affinities for ASOR and endocytosed and degraded ASOR at similar rates. The pH dissociation profiles of ASOR.ASGP-R complexes were also identical for complexes containing either H2b or H2c. We conclude that the H2b and H2c isoforms are both functional but are not present with H1 in the same hetero-oligomeric ASGP-R complexes. This structural difference between two functional subpopulations of ASGP-Rs may provide a molecular basis for the existence of two different pathways, designated State 1 and State 2, by which several types of recycling receptors mediate endocytosis.     

Modulation of adipogenesis-related gene expression by estrogen-related receptor gamma during adipocytic differentiation

Estrogen-related receptor gamma (ERRgamma) is an orphan nuclear receptor that regulates cellular energy metabolism by modulating gene expression involved in oxidative metabolism and mitochondrial biogenesis in brown adipose tissue and heart. However, the physiological role of ERRgamma in adipogenesis and the development of white adipose tissue has not been well studied. Here we show that ERRgamma was up-regulated in murine mesenchyme-derived cells, especially in ST2 and C3H10T1/2 cells, at mRNA levels under the adipogenic differentiation condition including the inducer of cAMP, glucocorticoid, and insulin. The up-regulation of ERRgamma mRNA was also observed in inguinal white adipose and brown adipose tissues of mice fed a high-fat diet. Gene knockdown by ERRgamma-specific siRNA results in mRNA down-regulation of adipogenic marker genes including fatty acid binding protein 4, PPARgamma, and PGC-1beta in a preadipocyte cell line 3T3-L1 preadipocytes and mesenchymal ST2 and C3H10T1/2 cells in the adipogenesis medium. In contrast, stable expression of ERRgamma in 3T3-L1 cells resulted in up-regulation of these adipogenic marker genes under the adipogenic condition. These results suggest that ERRgamma positively regulate the adipocyte differentiation with modulating the expression of various adipogenesis-related genes.     

Switch of histamine receptor expression from H2 to H1 during differentiation of monocytes into macrophages

It is known that histamine suppresses gene expression and synthesis of tumor necrosis factor alpha (TNF-alpha) induced by lipopolysaccharide (LPS) in human peripheral blood mononuclear monocytes (HPM) or alveolar macrophages via histamine H2 receptors. We investigated the effect of histamine and differentiation in macrophages on the expression and secretion of TNF-alpha, TNF-alpha-converting enzyme (TACE), and histamine H1 and H2 receptors by use of a leukemia cell line, U937, and HPM. Differentiation of U937 and HPM cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the H1 receptor expression and rather suppressed the H2 receptor, resulting in up-regulation of the histamine-induced expression and secretion of TNF-alpha, modulated via TACE. Therefore, histamine failed to inhibit up-regulated expression of TNF-alpha induced by LPS in macrophages. The switch from H2 to H1 receptors during differentiation in the monocyte/macrophage lineage could participate in the pathogenic processes of atherosclerosis and inflammatory reactions in the arterial wall.     

Galpha protein dependent and independent effects of human RGS1 expression in yeast

Regulators of G-protein Signalling (RGS) regulate the functional lifetime of G-Protein Coupled Receptor (GPCR)-activated heterotrimeric G-protein by serving as GTPase Accelerating Proteins (GAPs) for the G(alpha) subunit. A number of mammalian RGSs can functionally replace the yeast RGS containing SST2 gene and inhibit GPCR signalling. Using yeast strains harbouring a G(betagamma)-responsive FUS1-LacZ reporter gene, we demonstrate that heterologously expressed mammalian RGS1 also serves to decrease basal signalling in the absence of agonist. Although this effect was dependent on the expression of a GPA1-encoded functional G(alpha) protein, the GPCR itself was nevertheless not required. Using the GAL1 inducible promoter to express RGS1, we further demonstrate that in addition to serving as a GAP for Gpa1p in yeast, RGS1 is a dosage-dependent inhibitor of growth. This effect is specific to RGS1 since growth is not altered in cells expressing either mammalian RGS2 or RGS5. We further demonstrate that neither of the two yeast G(alpha) proteins is responsible for mediating this growth inhibitory effect of RGS1. Taken together, our results indicate that RGS1 can function in both G-protein-dependent and -independent manners in yeast.     

Activation of mu-opioid receptors transfers control of Galpha subunits to the regulator of G-protein signaling RGS9-2: role in receptor desensitization

In mouse periaqueductal gray matter (PAG) membranes, the mu-opioid receptor (MOR) coprecipitated the alpha-subunits of the Gi/o/z/q/11 proteins, the Gbeta1/2 subunits, and the regulator of G-protein signaling RGS9-2 and its partner protein Gbeta5. RGS7 and RGS11 present in this neural structure showed no association with MOR. In vivo intracerebroventricular injection of morphine did not alter MOR immunoreactivity, but 30 min and 3 h after administration, the coprecipitation of Galpha subunits with MORs was reduced by up to 50%. Furthermore, the association between Galpha subunits and RGS9-2 proteins was increased. Twenty-four hours after receiving intracerebroventricular morphine, the Galpha subunits left the RGS9-2 proteins and re-associated with the MORs. However, doses of the opioid able to induce tolerance promoted the stable transfer of Galpha subunits to the RGS9-2 control. This was accompanied by Ser phosphorylation of RGS9-2 proteins, which increased their co-precipitation with 14-3-3 proteins. In the PAG membranes of morphine-desensitized mice, the capacity of the opioid to stimulate G-protein-related guanosine 5'-O-(3-[35S]thiotriphosphate) binding as well as low Km GTPase activity was attenuated. The in vivo knockdown of RGS9-2 expression prevented morphine from altering the association between MORs and G-proteins, and tolerance did not develop. In PAG membranes from RGS9-2 knockdown mice, morphine showed full capacity to activate G-proteins. Thus, the tolerance that develops following an adequate dose of morphine is caused by the stabilization and retention of MOR-activated Galpha subunits by RGS9-2 proteins. This multistep process is initiated by the morphine-induced transfer of MOR-associated Galpha subunits to the RGS9-2 proteins, followed by Ser phosphorylation of the latter and their binding to 14-3-3 proteins. This regulatory mechanism probably precedes the loss of MORs from the cell membrane, which has been observed with other opioid agonists.     

RGS9-2 is a negative modulator of mu-opioid receptor function

Regulators of G-protein signaling (RGS) 9-2 is a striatal enriched protein that controls G protein coupled receptor signaling duration by accelerating Galpha subunit guanosine triphosphate hydrolysis. We have previously demonstrated that mice lacking the RGS9 gene show enhanced morphine analgesia and delayed development of tolerance. Here we extend these studies to understand the mechanism via which RGS9-2 modulates opiate actions. Our data suggest that RGS9-2 prevents several events triggered by mu-opioid receptor (MOR) activation. In transiently transfected PC12 cells, RGS9-2 delays agonist induced internalization of epitope HA-tagged mu-opioid receptor. This action of RGS9-2 requires localization of the protein near the cell membrane. Co-immunoprecipitation studies reveal that RGS9-2 interacts with HA-tagged mu-opioid receptor, and that this interaction is enhanced by morphine treatment. In addition, morphine promotes the association of RGS9-2 with another essential component of MOR desensitization, beta-arrestin-2. We also show that over-expression of RGS9-2 prevents opiate-induced extracellular signal-regulated kinase phosphorylation. Our data indicate that RGS9-2 plays an essential role in opiate actions, by negatively modulating MOR downstream signaling as well as the rate of MOR endocytosis.     

Cardiac differentiation promotes mitochondria development and ameliorates oxidative capacity in H9c2 cardiomyoblasts

H9c2 undergoing cardiac differentiation induced by all-trans-retinoic acid were investigated for mitochondria structural features together with the implied functional changes, as a model for the study of mitochondrial development in cardiogenic progenitor cells. As the expression of cardiac markers became detectable, mitochondrial mass increased and mitochondrial morphology and ultrastructure changed. Reticular network organization developed and more bulky mitochondria with greater numbers of closely packed cristae and more electron-dense matrix were detected. Increased expression of PGC-1α proved the occurrence of mitochondrial biogenesis. Improvements in mitochondrial energetic competence were also documented, linked to better assembly between F(0) and F(1) sectors of the F(0)F(1)ATPsynthase enzyme complex.