Cleavage of GSK‐3β by calpain counteracts the inhibitory effect of Ser9 phosphorylation on GSK‐3β activity induced by H2O2

Glycogen synthase kinase‐3 beta (GSK‐3β) dysfunction may play an essential role in the pathogenesis of psychiatric, metabolic, neurodegenerative diseases, in which oxidative stress exists concurrently. Some studies have shown that GSK‐3β activity is up‐regulated under oxidative stress. This study evaluated how oxidative stress regulates GSK‐3β activity in human embryonic kidney 293 (HEK293)/Tau cells treated with hydrogen peroxide (H₂O₂). Here, we show that H₂O₂ induced an obvious increase of GSK‐3β activity. Surprisingly, H₂O₂ dramatically increased phosphorylation of GSK‐3β at Ser9, an inactive form of GSK‐3β,while there were no changes of phosphorylation of GSK‐3β at Tyr216. Moreover, H₂O₂ led to a transient [Ca²⁺]_i elevation, and simultaneously increased the truncation of GSK‐3β into two fragments of 40 kDa and 30 kDa, whereas inhibition of calpain decreased the truncation and recovered the activity of GSK‐3β. Furthermore, tau was hyperphosphorylated at Ser396, Ser404, and Thr231, three most common GSK‐3β targeted sites after 100 μM H₂O₂ administration in HEK293/Tau cells, whereas inhibition of calpain blocked the tau phosphorylation. In addition, we found that there were no obvious changes of Cyclin‐dependent kinase 5 (CDK5) expression (responsible for tau phosphorylation) and of p35 cleavage, the regulatory subunit of CDK5 in H₂O₂‐treated HEK293/Tau cells. In conclusion, Ca²⁺‐dependent calpain activation leads to GSK‐3β truncation, which counteracts the inhibitory effect of Ser9 phosphorylation, up‐regulates GSK‐3β activity, and phosphorylates tau in H₂O₂‐treated HEK293/Tau cells.     

Role of caspase-3 in tau truncation at D421 is restricted in transgenic mouse models for tauopathies

Truncated tau is widely detected in Alzheimer's disease brain, and caspase-3 has been considered as a major executioner for tau truncation at aspartate421 (D421), according to its capability of cleaving recombinant tau in vitro . Here we investigated the relationship between D421 truncated tau and caspase-3 in two transgenic mouse models for tauopathies. In adult transgenic mice, activated caspase-3 could not be detected in neurons containing truncated tau, with the exception of a few glia-like cells or neurons in postnatal mice. Caspase-3 expression exhibited a dramatic decrease at the early development stage, and kept at constantly low levels during adult stages in both wild type and transgenic mice. On the other hand, co-incubating brain homogenates from adult tau transgenic mice and ethanol-treated postnatal mice promoted tau truncation at D421, which was mildly reduced by caspase inhibitor, but completely suppressed by phosphatase inhibitor, indicating that hyperphosphorylated tau becomes a poor substrate for truncation at D421. Taken together, our study shows that insufficient caspase-3 expression and hyperphosphorylated status of tau in the adult transgenic mouse brain restrict caspase-3 as an efficient enzyme for tau truncation in vivo . Clearly, there is a caspase-3 independent mechanism responsible for tau truncation at D421 in these models.     

Ser422 phosphorylation blocks human Tau cleavage by caspase-3: Biochemical implications to Alzheimer’s Disease

Proteolytic truncation of microtubule associated human (h) Tau protein by caspase-3 at the carboxy (C) terminus has been linked to the pathogenesis of Alzheimer’s Disease (AD). This cleavage likely occurs between Asp⁴²¹↓Ser⁴²² leading to the formation of 421-mer truncated Tau protein which has been found to be present as aggregate in high level after phosphorylation in mortal AD brain tissue compared to normal. At least 50 phosphorylation sites involving Ser, Thr and Tyr residues have been identified or proposed in hTau and a selected number of them have been implicated in hTau aggregation following latter’s proteolytic truncation. Interestingly, it is further noted that Ser⁴²² residue present in the P1′ position of hTau caspase-3 cleavage region is a potential phosphorylation site. So we became interested to examine in vitro the effect of phospho-Ser⁴²² residue on hTau cleavage by caspase-3 which is a crucial upstream event associated with hTau self-assembly leading to AD pathogenesis. The goal of this project is to study in vitro the caspase-3 cleavage site of hTau protein and to examine the kinetics of this cleavage following Ser⁴²² phosphorylation and treatment with caspase-3 inhibitors. This is achieved by designing peptides from the sequence of hTau protein containing the proposed caspase-3 cleavage region. Peptides were designed from 441-mer major human Tau protein sequence that encompasses the proposed caspase-3 cleavage site [Asp⁴²¹↓Ser⁴²²]. Corresponding phospho-, dextro-Ser⁴²² and dextro-Asp⁴²¹ analogs were also designed. Peptides were synthesized by solid phase chemistry, purified and fully characterized by mass spectrometry. These were then incubated with recombinant caspase-3 enzyme under identical condition for digestion and analyzed for cleavage by mass spectrometry and RP-HPLC chromatograms. Our results indicated that while the control peptide is efficiently cleaved by caspase-3 at Asp⁴²¹↓Ser⁴²² site producing the expected N- and C-terminal fragment peptides, the corresponding phospho-Ser⁴²² peptide remained completely resistant to the cleavage. Substitution of Asp⁴²¹ by its dextro isoform also blocks peptide cleavage by caspase-3. However substitution of Ser⁴²² by its dextro isoform in the peptide did not affect the cleavage significantly. The above results were further confirmed by caspase-3 digestion experiment in the presence of varying amounts of caspase-3 inhibitor (Ac-DQVD-aldehyde) which was found to block this cleavage in a highly effective manner. Our results highlighted the crucial significance of Ser⁴²² phosphorylation and suggest that the kinase associated with this Ser-phosphorylation may protect Tau from aggregation. Thus specific promoters/activators of this kinase may find useful therapeutic benefits in arresting Tau truncation by caspase-3 and the progression of AD. In addition our data demonstrated that Tau-peptides where Ser⁴²² or Asp⁴²¹ are substituted by their respective dextro isomers, exhibit different cleavage kinetics by caspase-3 and this may have important implications in therapeutic intervention of Tau aggregation and associated AD.     

Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase

Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H₂O₂ was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H₂O₂ to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H₂O₂ on kinase activity. Similar to the results obtained with H₂O₂-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H₂O₂ are increased. These results demonstrate that physiologically relevant concentrations of H₂O₂ can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status.     

Degradomics Reveals That Cleavage Specificity Profiles of Caspase-2 and Effector Caspases Are Alike

Caspase-2 is considered an initiator caspase because its long prodomain contains a CARD domain that allows its recruitment and activation in several complexes by homotypic death domain-fold interactions. Because little is known about the function and specificity of caspase-2 and its physiological substrates, we compared the cleavage specificity profile of recombinant human caspase-2 with those of caspase-3 and -7 by analyzing cell lysates using N-terminal COmbined FRActional DIagonal Chromatography (COFRADIC). Substrate analysis of the 68 cleavage sites identified in 61 proteins revealed that the protease specificities of human caspases-2, -3, and -7 largely overlap, revealing the DEVD↓G consensus cleavage sequence. We confirmed that Asp⁵⁶³ in eukaryotic translation initiation factor 4B (eIF4B) is a cleavage site preferred by caspase-2 not only in COFRADIC setup but also upon co-expression in HEK 293T cells. These results demonstrate that activated human caspase-2 shares remarkably overlapping protease specificity with the prototype apoptotic executioner caspases-3 and -7, suggesting that caspase-2 could function as a proapoptotic caspase once released from the activating complex.     

Multi-mechanisms are involved in reactive oxygen species regulation of mTORC1 signaling

The mammalian target of rapamycin complex 1(mTORC1) integrates diverse signals to control cell growth, proliferation, survival, and metabolism. Role of reactive oxygen species (ROS) on mTORC1 signaling remains obscure and mechanisms through which ROS modulate mTORC1 are not known. We demonstrate that low doses ROS exposure stimulate mTORC1 while high concentrations or long-term ROS treatment decrease mTORC1 activity in vivo and in a variety of cell lines. The dose/time needed for inhibition or activation are cell type-dependent. In HEK293 cells hydrogen peroxide (H₂O₂) stimulates phosphorylation of AMP-activated kinase (AMPK) (T172) and Raptor (S792), enhances association of activated AMPK with Raptor. Furthermore, AMPK inhibitor compound c inhibits H₂O₂-induced Raptor (S792) phosphorylation and reverses H₂O₂-induced de-phosphorylation of mTORC1 downstream targets p70-S6K1 (T389), S6 (S235/236) and 4E-BP1 (T37/46). H₂O₂ also stimulates association of endogenous protein phosphatase 2A catalytic subunit (PP2Ac) with p70-S6K1. Like compound c, inhibitor of PP2A, okadaic acid partially reverses inactivation of mTORC1 substrates induced by H₂O₂. Moreover, inhibition of PP2A and AMPK partially rescued cells from H₂O₂-induced cell death. High doses of H₂O₂ inhibit while low doses of H₂O₂ activate mTORC1 both in TSC2^?/? P53^?/? and TSC2^+/+ P53^?/? MEFs. These data suggest that PP2A and AMPK-mediated phosphorylation of Raptor mediate H₂O₂-induced inhibition of mTORC1 signaling.     

Cyclophilin A inhibits A549 cell oxidative stress and apoptosis by modulating the PI3K/Akt/mTOR signaling pathway

The excessive and inappropriate production of reactive oxygen species (ROS) can cause oxidative stress and is implicated in the pathogenesis of lung cancer. Cyclophilin A (CypA), a member of the immunophilin family, is secreted in response to ROS. To determine the role of CypA in oxidative stress injury, we investigated the role that CypA plays in human lung carcinoma (A549) cells. Here, we showed the protective effect of human recombinant CypA (hCypA) on hydrogen peroxide (H₂O₂)-induced oxidative damage in A549 cells, which play crucial roles in lung cancer. Our results demonstrated that hCypA substantially promoted cell viability, superoxide dismutase (SOD), glutathione (GSH), and GSH peroxidase (GSH-Px) activities, and attenuated ROS and malondialdehyde (MDA) production in H₂O₂-induced A549 cells. Compared with H₂O₂-induced A549 cells, Caspase-3 activity in hCypA-treated cells was significantly reduced. Using Western blotting, we showed that hCypA facilitated Bcl-2 expression and inhibited Bax, Caspase-3, Caspase-7, and PARP-1 expression. Furthermore, hCypA activates the PI3K/Akt/mTOR pathway in A549 cells in response to H₂O₂ stimulation. Additionally, peptidyl-prolyl isomerase activity was required for PI3K/Akt activation by CypA. The present study showed that CypA protected A549 cells from H₂O₂-induced oxidative injury and apoptosis by activating the PI3K/Akt/mTOR pathway. Thus, CypA might be a potential target for lung cancer therapy.     

Dehydroepiandrosterone ameliorates H2O2-induced Leydig cells oxidation damage and apoptosis through inhibition of ROS production and activation of PI3K/Akt pathways

Dehydroepiandrosterone (DHEA) is widely used as a nutritional supplement, and administration of DHEA produces a number of beneficial effects in the elderly. Many researchers have suggested that DHEA exerts it function after conversion into more biologically active hormones in peripheral target cells. The actions of DHEA in Leydig cells, a major target cell of DHEA biotransformation in males, are not clear. The present study found that DHEA increased cell viability and decreased reactive oxygen species (ROS) and malondialdehyde contents in H₂O₂-induced Leydig cells. DHEA significantly increased the activities of superoxide dismutase, catalase and peroxidase, and decreased the DNA damage in H₂O₂-induced Leydig cells. Apoptosis was significant decreased in H₂O₂-induced Leydig cells after DHEA treatment. DHEA inhibited the loss of mitochondrial membrane potential (ΔΨm) and the upregulation of the caspase-3 protein level induced by H₂O₂ in Leydig cells. DHEA also reversed the decrease in PI3K and p-Akt protein levels induced by H₂O₂. These data showed that DHEA could ameliorate H₂O₂-induced oxidative damage by increasing anti-oxidative enzyme activities, which resulted in reduced ROS content, and decreased apoptosis, mainly by preventing the loss of ΔΨm and inhibiting caspase-3 protein levels via activation of PI3K/Akt signaling pathways. These results increase our understanding of the molecular mechanism of the anti-ageing effect of DHEA.     

Ataxia telangiectasia mutated inhibits oxidative stress-induced apoptosis by regulating heme oxygenase-1 expression

Ataxia telangiectasia (AT) is caused by mutational inactivation of the ataxia telangiectasia mutated (Atm) gene, which is involved in DNA repair. Increased oxidative stress has been shown in human AT cells and neuronal tissues of Atm-deficient mice. Heme oxygenase-1 (HO-1) is an inducible antioxidant enzyme and protects cells against oxidative stress. The purpose of this study is to determine whether ATM induces antioxidant enzyme HO-1 and protects cells from oxidative stress-mediated apoptosis by driving the activation of PKC-δ and NF-κB, by increasing cell viability, and by downregulating DNA fragmentation and apoptotic indicators (apoptosis-inducing factor and cleaved caspase-3). AT fibroblasts stably transfected with human full-length ATM cDNA (YZ5 cells) or the empty vector (MOCK cells) were treated with H₂O₂ as a source of reactive oxygen species (ROS). As a result, transfection with ATM inhibited ROS-induced cell death and DNA fragmentation in MOCK cells. Transfection with ATM induced expression of HO-1 which was mediated by PKC-δ and NF-κB in H₂O₂-treated MOCK cells. ZnPP, an HO-1 inhibitor, and transfection with HO-1 siRNA increased ROS levels and apoptosis, whereas hemin, an HO-1 activator, reduced ROS levels and apoptosis in H₂O₂-treated YZ5 cells. Rottlerin, a PKC-δ inhibitor, inhibited NF-κB activation and HO-1 expression in H₂O₂-treated YZ5 cells. MOCK cells showed increased cell death, DNA fragmentation, and apoptotic indicators compared to YZ5 cells exposed to H₂O₂. In addition, transfection with p65 siRNA increased ROS levels and DNA fragmentation, but decreased HO-1 protein levels in H₂O₂-treated YZ5 cells. In conclusion, ATM induces HO-1 expression via activation of PKC-δ and NF-κB and inhibits oxidative stress-induced apoptosis. A loss of HO-1 induction may explain why AT patients are vulnerable to oxidative stress.     

Inhibition of UVA-induced apoptotic signaling pathway by polypeptide from <Emphasis Type="Italic">Chlamys farreri</Emphasis> in human HaCaT keratinocytes

Chronic UVA irradiation has been reported to induce photoaging and photocarcinogenesis. UVA is a potent inducer of reactive oxygen species (ROS), which can induce various biological processes, including apoptosis. Polypeptide from Chlamys farreri (PCF) is a novel marine active material isolated from the gonochoric Chinese scallop C. farreri. In our previous studies, PCF was found to be an effective antioxidant inhibiting UVA-induced ROS production and a potential inhibitory agent for UVA-induced apoptosis in the human keratinocyte cell line HaCaT. The intracellular mechanisms of how PCF protects HaCaT cells from UVA-induced apoptosis are not understood. Thus, we here investigate the effect of PCF on UVA-induced intracellular signaling of apoptosis. Pretreatment with the ROS scavenger N-acetylcysteine (NAC), the p38 MAPK inhibitor SB203580 or the caspase-3 inhibitor Ac-DEVD-CHO was found to effectively prevent UVA-induced apoptosis, indicating that ROS, p38 MAPK and caspase-3 play important roles in apoptosis. H₂O₂-induced apoptosis was attenuated by PCF, suggesting that PCF plays its anti-apoptotic role through its antioxidant activity. In addition, PCF treatment inhibited UVA-induced p38 MAPK activation and caspase-3 activation, as assayed by Western blot analysis and flow cytometry, respectively. Our results suggest that PCF attenuates UVA-induced apoptosis through a reduction of ROS generation and diminished p38 MAPK and caspase-3 activation.     

Saikosaponin-D Reduces H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced PC12 Cell Apoptosis by Removing ROS and Blocking MAPK-Dependent Oxidative Damage

Neuronal oxidative stress (OS) injury has been proven to be associated with many neurodegenerative diseases, and thus, antioxidation treatment is an effective method for treating these diseases. Saikosaponin-D (SSD) is a sapogenin extracted from Bupleurum falcatum and has been shown to have many pharmacological activities. The main purpose of this study was to investigate whether and how SSD protects PC12 cells from H₂O₂-induced apoptosis. The non-toxic level of SSD significantly mitigated the H₂O₂-induced decrease in cell viability, reduced the apoptosis rate, improved the nuclear morphology, and reduced caspase-3 activation and poly ADP-ribose polymerase (PARP) cleavage. Additionally, exogenous H₂O₂-induced apoptosis by damaging the intracellular antioxidation system. SSD significantly slowed the H₂O₂-induced release of malonic dialdehyde (MDA) and lactate dehydrogenase and increased the activity of superoxide dismutase (SOD) and the total antioxidant capacity, thereby reducing apoptosis. More importantly, SSD effectively blocked H₂O₂-induced phosphorylation of extracellular-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (p38MAPK), and specific inhibitors of ERK, JNK, and p38-reduced OS injury and apoptosis, suggesting that SSD reduces OS injury and apoptosis via MAPK signalling pathways. Finally, we confirmed that SSD significantly reduced H₂O₂-induced reactive oxygen species (ROS) accumulation, and the ROS inhibitor blocked the apoptosis caused by MAPK activation and cellular oxidative damage. In short, our study confirmed that SSD reduces H₂O₂-induced PC12 cell apoptosis by removing ROS and blocking MAPK-dependent oxidative damage.     

Sphingosine 1-phosphate attenuates H2O2-induced apoptosis in endothelial cells

Reactive oxygen species including H₂O₂ lead vascular endothelial cells (EC) to undergo apoptosis. Sphingosine 1-phosphate (S1P) is a platelet-derived sphingolipid mediator that elicits various EC responses. We aimed to explore whether and how S1P modulates EC apoptosis induced by H₂O₂. Treatment of cultured bovine aortic EC (BAEC) with H₂O₂ (750 μM for 6 h) led to DNA fragmentation (ELISA), DNA nick formation (TUNEL staining), and cleavage of caspase-3, key features of EC apoptosis. These responses elicited by H₂O₂ were alike markedly attenuated by pretreatment with S1P (1 μM, 30 min). H₂O₂ induced robust phosphorylation of both p38 and JNK MAP kinases. However, pretreatment with S1P decreased phosphorylation of only p38 MAP kinase, but not that of JNK; conversely, an inhibitor of p38 MAP kinase, but not that of JNK, attenuated H₂O₂-induced caspase-3 activation. Thus S1P attenuates H₂O₂-induced apoptosis of cultured BAEC, involving p38 MAP kinase.     

Cytoprotective effect of dieckol on human endothelial progenitor cells (hEPCs) from oxidative stress-induced apoptosis

Abstract

Although endothelial progenitor cells (EPCs) have been used to promote revascularization after peripheral or myocardial ischemia, excess amounts of reactive oxygen species (ROS) are often involved in senescence and apoptosis of EPCs, thereby causing defective neovascularization and reduced or failed recovery. Here, we examined the cytoprotective effect of Ecklonia cava-derived antioxidant dieckol (DK) on oxidative stress-induced apoptosis in EPCs to improve EPC bioactivity for vessel repair. Although H₂O₂ (10 ^{? 3} M) increased the intracellular ROS level in EPCs, DK (10ug/ml) pretreatment suppressed the H₂O₂-induced ROS increase and drastically reduced the ratios of apoptotic cells. H₂O₂-induced ROS increased the phosphorylation of p38 MAPK and JNK; this was inhibited by DK pretreatment. H₂O₂ treatment increased the phosphorylation of NF-κB, which was blocked by pretreatment with SB 203580, a p38 MAPK inhibitor, or SP 600125, a JNK inhibitor. H₂O₂ decreased the cellular levels of Bcl-2 and c-IAPs, cellular inhibitors of apoptosis proteins, but increased caspase-3 activation. However, all these effects were inhibited by pretreatment with DK. Injection of DK-mixed EPCs (DK + EPCs) into myocardial ischemic sites in vivo induced cellular proliferation and survival of cells at the ischemic sites and, thereby, enhanced the secretion of angiogenic cytokines at the ischemic sites. These results show that DK + EPC exhibit markedly enhanced anti-apoptotic and antioxidative capabilities, unlike that shown by EPCs alone; thus, they contribute to improved repair of ischemic myocardial injury through cell survival and angiogenic cytokine production.     

Using Co-Cultures Expressing Fluorescence Resonance Energy Transfer Based Protein Biosensors to Simultaneously Image Caspase-3 and Ca<Superscript>2+</Superscript> Signaling

Fluorescence resonance energy transfer (FRET)-based protein biosensors allow the spatial and temporal imaging of signaling events in living cells. However, the simultaneous correlation of multiple events of a signaling pathway is hindered by the spectral cross-talk between fluorescent proteins. Here, we show, for signaling pathways that progress synchronously, multiple events can be correlated by using co-cultures expressing different FRET-based protein biosensors. As a demonstration, we investigated the simultaneous caspase-3 and Ca²⁺ signaling events involved in cell death of COS-7 cells induced by 10 mM H₂O₂. Interestingly, this H₂O₂ stimulus induced synchronous caspase-3 activation and Ca²⁺ signaling. In parallel to caspase-3 activation, cytosolic Ca²⁺ concentration, [Ca²⁺]_c, gradually rises to its peak and then slowly drops. As cell shrinkage and rounding ensues, [Ca²⁺]_c again gradually rises to its peak and then reaches a plateau. These observations reveal the relative timing and location of these signaling events in cell death induced by this stimulus of H₂O₂. Finally, our approach offers an exciting opportunity for spatial and temporal imaging of multiple events in a signaling pathway in living cells.     

Metformin protects PC12 cells and hippocampal neurons from H2O 2-induced oxidative damage through activation of AMPK pathway

Metformin, a first line anti type 2 diabetes drug, has recently been shown to extend lifespan in various species, and therefore, became the first antiaging drug in clinical trial. Oxidative stress due to excess reactive oxygen species (ROS) is considered to be an important factor in aging and related disease, such as Alzheimer's disease (AD). However, the antioxidative effects of metformin and its underlying mechanisms in neuronal cells is not known. In the present study, we showed that metformin, in clinically relevant concentrations, protected neuronal PC12 cells from H₂O₂-induced cell death. Metformin significantly ameliorated cell death due to H₂O₂ insult by restoring abnormal changes in nuclear morphology, intracellular ROS, lactate dehydrogenase, and mitochondrial membrane potential induced by H₂O₂. Hoechst staining assay and flow cytometry analysis revealed that metformin significantly reduced the apoptosis in PC12 cells exposed to H₂O₂. Western blot analysis further demonstrated that metformin stimulated the phosphorylation and activation of AMP-activated protein kinase (AMPK) in PC12 cells, while application of AMPK inhibitor compound C, or knockdown of the expression of AMPK by specific small interfering RNA or short hairpin RNA blocked the protective effect of metformin. Similar results were obtained in primary cultured hippocampal neurons. Taken together, these results indicated that metformin is able to protect neuronal cells from oxidative injury, at least in part, via the activation of AMPK. As metformin is comparatively cheaper with much less side effects in clinic, our findings support its potential to be a drug for prevention and treatment of aging and aging-related diseases.     

Tissue transglutaminase 2 promotes apoptosis of rat neonatal cardiomyocytes under oxidative stress

The role of tissue transglutaminase 2 (TG2) in cardiac myocyte apoptosis under oxidative stress induced by ischemic injury remains unclear. Here, we investigated the effects of TG2 on apoptosis of cardiomyocytes under oxidative stress. Ectopic expression of TG2 increased caspase-3 activity and calcium overload in cardiomyocytes. Expression levels of TG2 were significantly increased in H₂O₂-treated cardiomyocytes. Caspase-3 activity assay demonstrated its considerable correlation with TG2 expression, which supported that caspase-3 inhibitor inhibited the apoptosis induced by the ectopic overexpression of TG2. In addition, the other apoptotic signals, such as caspase-8, cytochrome c, and Bax, were increased dependent with TG2 expression in H₂O₂-treated cardiomyocytes. These results indicated that apoptotic signals had a positive correlation with TG2 expression. The decreased expression of phospholipase C (PLC)-δ1 and phospho-PKC in H₂O₂-treated cardiomyocytes were rescued by TG2 silencing. Together, our data strongly suggest that oxidative stress up-regulates TG2 expression in cardiomyocytes, leading to apoptosis.     

Aucubin modulates Bcl-2 family proteins expression and inhibits caspases cascade in H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced PC12 cells

In this study, the effect of aucubin on H₂O₂-induced apoptosis was studied by using a rat pheochromocytoma (PC12) cell line. We have analyzed the apoptosis of H₂O₂-induced PC12 cells, H₂O₂-induced apoptosis appeared to correlate with lower Bcl-2 expression, higher Bax expression and sequential activation of caspase-3 leading to cleavage of poly-ADP-ribose polymerase (PARP). Aucubin not only inhibited lower Bcl-2 expression, high Bax expression, but also modulated caspase-3 activation, PARP cleavage, and eventually protected against H₂O₂-induced apoptosis. These results indicated that aucubin can obstruct H₂O₂-induced apoptosis by regulating of the expression of Bcl-2 and Bax, as well as suppression of caspases cascade activation.     

Activation of cGMP-dependent protein kinase stimulates cardiac ATP-sensitive potassium channels via a ROS/calmodulin/CaMKII signaling cascade

Background

Cyclic GMP (cGMP)-dependent protein kinase (PKG) is recognized as an important signaling component in diverse cell types. PKG may influence the function of cardiac ATP-sensitive potassium (K_ATP) channels, an ion channel critical for stress adaptation in the heart; however, the underlying mechanism remains largely unknown. The present study was designed to address this issue.

Methods and Findings

Single-channel recordings of cardiac K_ATP channels were performed in both cell-attached and inside-out patch configurations using transfected human embryonic kidney (HEK)293 cells and rabbit ventricular cardiomyocytes. We found that Kir6.2/SUR2A (the cardiac-type K_ATP) channels were activated by cGMP-selective phosphodiesterase inhibitor zaprinast in a concentration-dependent manner in cell-attached patches obtained from HEK293 cells, an effect mimicked by the membrane-permeable cGMP analog 8-bromo-cGMP whereas abolished by selective PKG inhibitors. Intriguingly, direct application of PKG moderately reduced rather than augmented Kir6.2/SUR2A single-channel currents in excised, inside-out patches. Moreover, PKG stimulation of Kir6.2/SUR2A channels in intact cells was abrogated by ROS/H₂O₂ scavenging, antagonism of calmodulin, and blockade of calcium/calmodulin-dependent protein kinase II (CaMKII), respectively. Exogenous H₂O₂ also concentration-dependently stimulated Kir6.2/SUR2A channels in intact cells, and its effect was prevented by inhibition of calmodulin or CaMKII. PKG stimulation of K_ATP channels was confirmed in intact ventricular cardiomyocytes, which was ROS- and CaMKII-dependent. Kinetically, PKG appeared to stimulate these channels by destabilizing the longest closed state while stabilizing the long open state and facilitating opening transitions.

Conclusion

The present study provides novel evidence that PKG exerts dual regulation of cardiac K_ATP channels, including marked stimulation resulting from intracellular signaling mediated by ROS (H₂O₂ in particular), calmodulin and CaMKII, alongside of moderate channel suppression likely mediated by direct PKG phosphorylation of the channel or some closely associated proteins. The novel cGMP/PKG/ROS/calmodulin/CaMKII signaling pathway may regulate cardiomyocyte excitability by opening K_ATP channels and contribute to cardiac protection against ischemia-reperfusion injury.