Erythropoietin protects cardiac myocytes from hypoxia-induced apoptosis through an Akt-dependent pathway

Apoptosis is a contributing cause of myocyte loss in ischemic heart disease. Recent work has shown that erythropoietin (EPO) offers protection against apoptosis in a wide variety of tissues. We demonstrate that the erythropoietin receptor (EPOR) is expressed in the neonatal rat ventricular myocyte (NRVM). Exposure of NRVMs to hypoxia, with recombinant human EPO, significantly decreased apoptosis as measured by TUNEL, flow cytometry, and caspase 3/7 like activity when compared to hypoxia treatment alone. EPO administered at the initiation of coronary artery occlusion in the rat significantly decreased apoptosis in the myocardial ischemic region. In the NRVM, EPO increased the activity of Akt. The anti-apoptotic effect of EPO was abrogated by co-treatment with LY294002, a specific blocker of phosphatidylinositol 3-kinase (PI3-K). Our study demonstrates that EPO inhibits apoptosis in the NRVM exposed to hypoxia, through an Akt-dependent pathway. EPO also inhibits apoptosis in the in vivo rat model of myocardial ischemia.     

MCP-1 (CCL2) protects human neurons and astrocytes from NMDA or HIV-tat-induced apoptosis

Acquired immunodeficiency syndrome (AIDS)-associated dementia is often characterized by chronic inflammation, with infected macrophage infiltration of the CNS resulting in the production of human immunodeficiency virus type 1 (HIV-1) products, including tat, and neurotoxins that contribute to neuronal loss. In addition to their established role in leukocyte recruitment and activation, we identified an additional role for chemokines in the CNS. Monocyte chemoattractant protein-1 (MCP-1 or CCL2) and regulated upon activation normal T cell expressed and secreted (RANTES) were found to protect mixed cultures of human neurons and astrocytes from tat or NMDA-induced apoptosis. Neuronal and astrocytic apoptosis in these cultures was significantly inhibited by co-treatment with MCP-1 or RANTES but not IP-10. The protective effect of RANTES was blocked by antibodies to MCP-1, indicating that RANTES protection is mediated by the induction of MCP-1. The NMDA blocker, MK801, also abolished the toxic effects of both tat and NMDA. Tat or NMDA treatment of mixed cultures for 24 h resulted in increased extracellular glutamate ([Glu]e) and NMDA receptor 1 (NMDAR1) expression, potential contributors to apoptosis. Co-treatment with MCP-1 inhibited tat and NMDA-induced increases in [Glu]e and NMDAR1, and also reduced the levels and number of neurons containing intracellular tat. These data indicate that MCP-1 may play a novel role as a protective agent against the toxic effects of glutamate and tat.     

Resveratrol protects cardiomyocytes from hypoxia-induced apoptosis through the SIRT1-FoxO1 pathway

Loss of cardiomyocytes through apoptosis has been proposed as a cause of ventricular remodeling and heart failure. Ischemia- and hypoxia-induced apoptosis of cardiomyocytes reportedly plays an important role in many cardiac pathologies. We investigated whether resveratrol (Res) has direct cytoprotective effects against ischemia/hypoxia for cardiomyocytes. Exposure of H9c2 embryonic rat heart-derived cells to hypoxia for 24 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry, while treatment with 20 μM Res greatly decreased hypoxia-induced apoptosis in these cells. Exposure of the cells to Res (20 μM) caused rapid activation of SIRT1, which had a dual effect on FoxO1 function: SIRT1 increased FoxO1’s ability to induce cell cycle arrest, but inhibited FoxO1’s ability to induce cell death. This effect could be reversed by SIRT1 inhibition. Results of our study indicate that Res inhibits hypoxia-induced apoptosis via the SIRT1-FoxO1 pathway in H9c2 cells. This polyphenol may have potential in preventing cardiovascular disease, especially in coronary artery disease (CAD) patients.     

Human lactoferrin exerts bi-directional actions on PC12 cell survival via ERK1/2 pathway

Human lactoferrin (hLF) is a member of the transferrin family and is found in most body fluids of human. Recent study showed that hLF played some roles in the regulation of cell growth. However, the biological function of hLF in the central nervous system and neuronal cells is still unclear. The MTT was used to assay cell viability, ELISA tests were used to assay caspase activities, and TUNEL staining was used to test the cytotoxicity of hLF to the cells. Our result showed that 700 microg/ml hLF significantly reduced the cell viability and increased the caspase 3 and 8 activities in PC12 neuronal cells. TUNEL staining further showed that 700 microg/ml hLF was cytotoxic to the PC12 through apoptosis-mediated pathway. In addition, 700 microg/ml hLF significantly decreased the protein expressions of phosphorylated extracellular-signal-regulated kinase 1/2 (ERK1/2) and Bcl-2 in PC12 cells, whereas 50 microg/ml hLF significantly increased the phosphorylation of ERK1/2 which could be specifically inhibited by PD98059. Furthermore, 50 microg/ml hLF could not only up-regulate the Bcl-2 expression but also protect PC12 cells from FasL-induced apoptosis. In conclusion, hLF plays a crucial role in the regulation of apoptosis and anti-apoptosis in PC12 neuronal cells via ERK1/2 phosphorylation pathway.     

Bcl-2 blocks apoptosis caused by pierisin-1, a guanine-specific ADP-ribosylating toxin from the cabbage butterfly

Pierisin-1, a 98-kDa protein that induces apoptosis in mammalian cell lines, is capable of being incorporated into cells where it ADP-ribosylates guanine residues in DNA. To investigate the apoptotic pathway induced by this unique protein, the bcl-2 gene was transfected into HeLa cells. Cy2-fluorescent pierisin-1 was incorporated into the resultant cells expressing Bcl-2 protein and ADP-ribosylated dG was detected to almost the same extent as in parent cells. However, bcl-2-transfected HeLa cells did not display apoptotic morphological changes, PARP cleavage, and DNA fragmentation, indicating acquisition of resistance. In parent HeLa cells, activation of caspase-9 and release of cytochrome c were observed after 8h treatment with 0.5ng/ml pierisin-1. Caspase substrate assays revealed further cleavage of Ac-DEVD-pNA, Ac-VDVAD-pNA, and Ac-VEID-pNA, suggesting activation of caspase-2, -3, and -6 in pierisin-1-treated HeLa cells. The caspase-3 inhibitor, Ac-DEVD-CHO, was also found to inhibit apoptosis. In contrast, this caspase activation was not observed in bcl-2-transfected HeLa cells. Our results thus indicate that pierisin-1-induced apoptosis is mediated primarily via a mitochondrial pathway involving Bcl-2 and caspases.     

PTPase inhibition restores ERK1/2 phosphorylation and protects mammary epithelial cells from apoptosis

Specific survival signals derived from extracellular matrix (ECM) and growth factors are required for mammary epithelial cell survival. We have previously demonstrated that inhibition of ECM-induced ERK1/2 MAPK pathway with PD98059 leads to apoptosis in primary mouse mammary epithelial cells. In this study, we have further investigated MAPK signal transduction in cell survival of these cells cultured on a laminin rich reconstituted basement membrane. ERK1/2 phosphorylation is activated in the absence of insulin by cell-cell substratum interactions that cause ligand-independent EGFR transactivation. Intact EGFR signal transduction is required for ECM determined cell survival as the EGFR pathway inhibitor, AG1478, induces apoptosis of these cultures. Rescue of AG1478 or PD98059 treated cultures by PTPase inhibition with vanadate restores cellular phospho-ERK1/2 levels and prevents apoptosis. These results emphasize that ERK1/2 phosphorylation and inhibition of PTPase activity are necessary for PMMEC cell survival.     

The kinase domain of MEKK1 induces apoptosis by dysregulation of MAP kinase pathways

MAP kinase pathways comprise a group of parallel protein phosphorylation cascades, which are involved in signaling triggered by a variety of stimuli. Previous findings suggested that the ERK and the JNK pathways have opposing roles in regulating proliferation and survival or apoptosis and that apoptosis can be promoted by inhibiting the ERK pathway or by activation of the JNK pathway. In order to test this hypothesis and explore whether it can be exploited as a strategy for killing human cancer cells, we used gene transfer experiments with a range of cancer cell lines. We expressed the catalytic fragment of human MEKK1 to activate JNK and the Ras-binding domain (RBD) of Raf-1 to inhibit the Ras-ERK pathway. In addition, we designed several RBD-MEKK1 fusion proteins aiming to simultaneously activate the JNK and block the ERK pathway. We found that the MEKK1 proteins as well as the RBD alone could reduce colony formation in all cell lines. The survival time of MEKK1-expressing cells depended on the cell line. In HeLa cells, survival could be prolonged by inhibition of caspases but not by coexpression of the anti-apoptotic protein Bcl-2. Due to a lower kinase activity the RBD-MEKK1 fusion proteins were less effective in apoptosis induction than the MEKK1 kinase domain alone. Using mutant forms of Ras and Raf-1 we could show that the reduced kinase activity of RBD-MEKK1 fusion proteins was caused by binding to the Ras protein. The expression of lethal doses of MEKK1 resulted in a strong activation of all three major MAP kinase families JNK, ERK, and p38. Blocking these pathways either by coexpressing a dominant negative form of MKK4 or with inhibitors of MEK or p38 failed to inhibit apoptosis. This suggests that MEKK1 induces apoptosis by causing a general deregulation of MAP kinase signaling rather than by the activation of a single pathway.     

Crocetin protects against cardiac hypertrophy by blocking MEK-ERK1/2 signalling pathway

Oxidative stress plays a critical role in the progression of pathological cardiac hypertrophy and heart failure. Because crocetin represses oxidative stress in vitro and in vivo , we have suggested that crocetin would repress cardiac hypertrophy by targeting oxidative stress-dependent signalling. We tested this hypothesis using primary cultured cardiac myocytes and fibroblasts and one well-established animal model of cardiac hypertrophy. The results showed that crocetin (1–10 μM) dose-dependently blocked cardiac hypertrophy induced by angiogensin II (Ang II; 1 μM) in vitro . Our data further revealed that crocetin (50 mg/kg/day) both prevented and reversed cardiac hypertrophy induced by aortic banding (AB), as assessed by heart weight/body weight and lung weight/body weight ratios, echocardio-graphic parameters and gene expression of hypertrophic markers. The inhibitory effect of crocetin on cardiac hypertrophy is mediated by blocking the reactive oxygen species (ROS)-dependent mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase-1/2 (MEK/ERK1/2) pathway and GATA binding protein 4 (GATA-4) activation. Further investigation demonstrated that crocetin inhibited inflammation by blocking nuclear factor kappa B (NF-κB) signalling and attenuated fibrosis and collagen synthesis by abrogating MEK-ERK1/2 signalling. Overall, our results indicate that crocetin, which is a potentially safe and inexpensive therapy for clinical use, has protective potential in targeting cardiac hypertrophy and fibrosis by suppression of ROS-dependent signalling pathways.     

Resveratrol- induced apoptosis is mediated by p53-dependent pathway in Hep G2 cells

Resveratrol, a phytoalexin found in many plants, has been reported to possess a wide range of pharmacological properties and is one of the promising chemopreventive agents for cancer. Here, we examined the antiproliferation effect of resveratrol in two human liver cancer cell lines, Hep G2 and Hep 3B. Our results showed that resveratrol inhibited cell growth in p53-positive Hep G2 cells only. This anticancer effect was a result of cellular apoptotic death induced by resveratrol via the p53-dependent pathway. Here we demonstrated that the resveratrol-treated cells were arrested in G1 phase and were associated with the increase of p21 expression. In addition, we also illustrated that the resveratrol-treated cells had enhanced Bax expression but they were not involved in Fas/APO-1 apoptotic signal pathway. In contrast, the p53-negative Hep 3B cells treated with resveratrol did not show the antiproliferation effect neither did they show significant changes in p21 nor Fas/APO-1 levels. In summary, our study demonstrated that the resveratrol effectively inhibited cell growth and induced programmed cell death in Hepatoma cells on a molecular basis. Furthermore, these results implied that resveratrol might also be a new potent chemopreventive drug candidate for liver cancer as it played an important role to trigger p53-mediated molecules involved in the mechanism of p53-dependent apoptotic signal pathway.     

miR-135a inhibition protects A549 cells from LPS-induced apoptosis by targeting Bcl-2

Acute lung injury (ALI) is a severe clinical condition with high morbidity and mortality. Apoptosis is a key pathologic feature of ALI, and Bcl-2 plays an important role during the pathogenesis of ALI via the regulation of apoptosis. However, the regulation of Bcl-2 during ALI, particularly through microRNAs, remains unclear. We hypothesize that certain miRNAs may play deleterious or protective roles in ALI via the regulation of Bcl-2. The LPS stimulation of A549 cells was used to mimic ALI in vitro. First, we confirmed that Bcl-2 is involved in LPS-induced apoptosis in A549 cells. Then, bioinformatic analyses and quantitative real-time polymerase chain reaction assays were performed to screen for miRNAs targeting Bcl-2. We observed that miR-135a was markedly increased in LPS-challenged A549 cells. miR-135a inhibition markedly restored Bcl-2 expression and protected A549 cells from LPS-induced apoptosis. Furthermore, bioinformatic analysis and luciferase activity assays were conducted to confirm that miR-135a binds directly to the 3′-untranslated region of Bcl-2 and suppresses its expression. Interestingly, the inhibition of miR-135a did not attenuate apoptosis under LPS-treated conditions when Bcl-2 was knocked down. Therefore, we suggest that miR-135a regulation of LPS-induced apoptosis in A549 cells may depend in part on the regulation of Bcl-2. The miR-135a/Bcl-2 signaling pathway may be a novel therapeutic target for the prevention of ALI.     

Oxidative stress-induced apoptosis is mediated by ERK1/2 phosphorylation

Oxidative stress is known to induce apoptosis in a wide variety of cell types, apparently by modulating intracellular signaling pathways. High concentrations of H2O2 have been found to induce apoptosis in L929 mouse fibroblast cells. To elucidate the mechanisms of H2O2-mediated apoptosis, ERK1/2, p38-MAPK, and JNK1/2 phosphorylation was examined, and ERK1/2 and JNK1/2 were found to be activated by H2O2. Inhibition of ERK1/2 activation by treatment of L929 cells with PD98059 or dominant-negative ERK2 transfection blocked H2O2-induced apoptosis, while inhibition of JNK1/2 by dominant-negative JNK1 or JNK2 or MKK4 or MKK7 transfection did not affect H2O2-mediated apoptosis. H2O2-mediated ERK1/2 activation was not only Ras-Raf dependent, but also both tyrosine kinase (PDGFbeta receptor and Src) and PKCdelta dependent. H2O2-mediated PKCdelta-dependent and tyrosine kinase-dependent ERK1/2 activations were independent from each other. Based on the above results, we suggest for the first time that oxidative damage-induced apoptosis is mediated by ERK1/2 phosphorylation which is not only Ras-Raf dependent, but also both tyrosine kinase and PKCdelta dependent.     

20(R)-Ginsenoside Rg3 protects SH-SY5Y cells against apoptosis induced by oxygen and glucose deprivation/reperfusion

As shown in our previous studies, 20(R)-ginsenoside Rg3 [20(R)-Rg3] exerts a neuroprotective effect on a rat model of transient focal cerebral ischemia, and the mechanism through which it decreases the mRNA expression of calpain I and caspase-3 has been delineated. However, researchers do not know whether 20(R)-Rg3 exhibits a neuroprotective effect following oxygen-glucose deprivation and reperfusion (OGD/R) injury in vitro. In the present study, 20(R)-Rg3 increased cell viability, decreased the LDH leakage rate, and inhibited the apoptosis rate in a concentration-dependent manner. In addition, 20(R)-Rg3 markedly decreased cleaved caspase-3 protein expression. Furthermore, 20(R)-Rg3 significantly decreased the Bax mRNA and protein levels and increased the levels of Bcl-2 mRNA and protein, subsequently decreasing the Bax/Bcl-2 protein ratio. Based on these findings, 20(R)-Rg3 exerts a neuroprotective effect against OGD/R-induced apoptosis.     

Ischemic but not mechanical preconditioning attenuates ischemia/reperfusion induced myocardial apoptosis in anaesthetized rabbits: The role of Bcl-2 family proteins and ERK1/2

Objective: Recent studies suggest that ischemic preconditioning (IPC) inhibits myocardial apoptosis after ischemia and reperfusion. This study aimed first, to examine whether short mechanical stretch with acute pressure overload (MPC), which has been shown to reduce infarct size after ischemia/reperfusion, mimics IPC in attenuating myocardial apoptosis and second, to evaluate whether induced cardioprotection involves modulation of the expression of the Bcl-2 family proteins and phosphorylation of prosurvival kinases. Methods and Results: A model of anaesthetized rabbit was used and the preconditioning protocol included one cycle of short ischemia/reperfusion, or short mechanical stretch with acute pressure overload. Preconditioning stimuli were equally effective in reducing the infarct size, determined after 4 h reperfusion. However, IPC but not MPC attenuated myocardial apoptosis. IPC restored the decreased expression of Bcl-2 and Bcl-xL observed in hearts subjected to ischemia and reperfusion only. Bax levels were not different among the groups. ERK1/2 were activated during reperfusion in both IPC and MPC groups. Conclusions: The data provide further evidence that apoptosis and necrosis contribute independently to infarct size after ischemia and reperfusion. Inhibition of the myocardial apoptotic processes by IPC may involve modulation of the expression of anti-apoptotic proteins, Bcl-2 and Bcl-xL. ERK1/2 may be involved in the inhibition of both apoptosis and necrosis.     

MAP kinase protects G protein-coupled receptor kinase 2 from proteasomal degradation

The G protein-coupled receptor kinase 2 (GRK2) phosphorylates and shuts down signaling from 7-transmembrane receptors (7TMs). Although, receptor activity controls GRK2 expression levels, the underlying molecular mechanisms are poorly understood. We have previously shown that extracellular signal-regulated kinase (ERK1/2) activation increases GRK2 expression [J. Theilade, J. Lerche Hansen, S. Haunso, S.P. Sheikh, Extracellular signal-regulated kinases control expression of G protein-coupled receptor kinase 2 (GRK2), FEBS Lett. 518 (2002) 195-199]. In the present study, we found that ERK1/2 regulates GRK2 degradation rather than synthesis. ERK1/2 blockade using PD98059 decreased GRK2 cellular levels to 0.25-fold of control in Cos7 cells. This effect was due to enhanced degradation of the GRK2 protein, since proteasome blockade prevented down-regulation of GRK2 protein levels in the presence of PD98059. Further, ERK blockade had no effect on GRK2 synthesis as probed using a reporter construct carrying the GRK2 promoter upstream of the luciferase gene. We predict ERK1/2 mediated GRK2 protection could be a general phenomenon as proteasome inhibition increased GRK2 expression in two other cell lines, HEK293 and NIH3T3.     

INrf2 (Keap1) targets Bcl-2 degradation and controls cellular apoptosis

Cytosolic inhibitor of Nrf2 (INrf2) is an adaptor protein that mediates ubiquitination/degradation of NF-E2-related factor 2 (Nrf2), a master regulator of cytoprotective gene expression. In this paper, we demonstrate that INrf2 degrades endogenous antiapoptotic B-cell CLL/lymphoma 2 (Bcl-2) protein and controls cellular apoptosis. The DGR domain of INrf2 interacts with the BH2 domain of Bcl-2 and facilitates INrf2:Cul3–Rbx1-mediated ubiquitination of Bcl-2 by the conjugation of ubiquitin molecules to lysine17 of Bcl-2. Further studies showed that INrf2 enhanced etoposide-mediated accumulation of Bax, increased release of cytochrome c from mitochondria, activated caspase-3/7, and enhanced DNA fragmentation and apoptosis. Antioxidants antagonized Bcl-2:INrf2 interaction, led to the release and stabilization of Bcl-2, increased Bcl-2:Bax heterodimers and reduced apoptosis. Moreover, dysfunctional/mutant INrf2 in human lung cancer cells failed to degrade Bcl-2, resulting in decreased etoposide and UV/γ radiation-mediated DNA fragmentation. These data provide the first evidence of INrf2 control of Bcl-2 and apoptotic cell death, with implications in antioxidant protection, survival of cancer cells containing dysfunctional INrf2, and drug resistance.     

Baicalein protects against cardiac hypertrophy through blocking MEK‐ERK1/2 signaling

Baicalein, a flavonoid present in the root of Scutellaria baicalensis, is well known for its antibacterial, antiviral, anti‐inflammatory, antithrombotic, and antioxidant effects. Here we show that baicalein also attenuates cardiac hypertrophy. Aortic banding (AB) was performed to induce cardiac hypertrophy secondary to pressure overload in mice. Mouse chow containing 0.05% baicalein (dose: 100 mg/kg/day baicalein) was begun 1 week prior to surgery and continued for 8 weeks after surgery. Our data demonstrated that baicalein prevented cardiac hypertrophy and fibrosis induced by AB, as assessed by echocardiographic and hemodynamic parameters and by pathological and molecular analysis. The inhibitory action of baicalein on cardiac hypertrophy was mediated by effects on mitogen‐activated protein kinase kinase (MEK)‐extracellular signal‐regulated kinases (ERK1/2) signaling and GATA‐4 activation. In vitro studies performed in rat cardiac H9c2 cells confirmed that baicalein attenuated cardiomyocyte hypertrophy induced by angiotensin II, which was associated with inhibiting MEK‐ERK1/2 signaling. In conclusion, our results suggest that baicalein has protective potential for targeting cardiac hypertrophy and fibrosis through suppression of MEK‐ERK1/2 signaling. Baicalein warrants further research as a potential antihypertrophic agent that might be clinically useful to treat cardiac hypertrophy and heart failure. J. Cell. Biochem. 114: 1058–1065, 2013. © 2012 Wiley Periodicals, Inc.     

MEKK1-induced apoptosis is mediated by Smac/Diablo release from the mitochondria

During apoptotic stimulation, the serine threonine kinase, MEKK1, is cleaved into an activated 91 kDa kinase fragment. This cleavage is mediated by caspase 3 and leads to further caspase 3 activation and apoptosis. Forced expression of the 91 kDa kinase fragment induces apoptosis through changes in membrane potential of the mitochondria mediated by permeability transition pore opening. MEKK1 activation, however, fails to release cytochrome c from the mitochondria. Herein, we determined that overexpression of MEKK1 causes mitochondrial Smac/Diablo release correlating with MEKK1-induced apoptosis. Furthermore, using siRNA that lowers Smac/Diablo expression, MEKK1-induced apoptosis was significantly reduced. Mouse embryonic fibroblast cells lacking MEKK1 expression are also resistant to etoposide-induced mitochondrial Smac/Diablo release. In contrast, etoposide-induced mitochondrial cytochrome c release was not inhibited. MEKK1 also activates the MAP kinase JNK, but MEKK1-induced mitochondrial Smac/Diablo release and apoptosis are independent of MEKK1 mediated JNK activation. Taken together, release of Smac/Diablo from the mitochondria plays a role in MEKK1-induced apoptosis.