NDP kinase 2 interacts with two oxidative stress-activated MAPKs to regulate cellular redox state and enhances multiple stress tolerance in transgenic plants

NDP kinases (NDPKs) are multifunctional proteins that regulate a variety of eukaryotic cellular activities, including cell proliferation, development, and differentiation. However, much less is known about the functional significance of NDPKs in plants. We show here that NDPK is associated with H(2)O(2)-mediated mitogen-activated protein kinase signaling in plants. H(2)O(2) stress strongly induces the expression of the NDPK2 gene in Arabidopsis thaliana (AtNDPK2). Proteins from transgenic plants overexpressing AtNDPK2 showed high levels of autophosphorylation and NDPK activity, and they have lower levels of reactive oxygen species (ROS) than wild-type plants. Mutants lacking AtNDPK2 had higher levels of ROS than wild type. H(2)O(2) treatment induced the phosphorylation of two endogenous proteins whose molecular weights suggested they are AtMPK3 and AtMPK6, two H(2)O(2)-activated A. thaliana mitogen-activated protein kinases. In the absence of H(2)O(2) treatment, phosphorylation of these proteins was slightly elevated in plants overexpressing AtNDPK2 but markedly decreased in the AtNDPK2 deletion mutant. Yeast two-hybrid and in vitro protein pull-down assays revealed that AtNDPK2 specifically interacts with AtMPK3 and AtMPK6. Furthermore, AtNDPK2 also enhances the myelin basic protein phosphorylation activity of AtMPK3 in vitro. Finally, constitutive overexpression of AtNDPK2 in Arabidopsis plants conferred an enhanced tolerance to multiple environmental stresses that elicit ROS accumulation in situ. Thus, AtNDPK2 appears to play a previously uncharacterized regulatory role in H(2)O(2)-mediated MAPK signaling in plants.     

EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana

Shortly after the release of singlet oxygen ((1)O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of (1)O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of (1)O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of (1)O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all (1)O2-responsive genes. Retrograde control of (1)O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.     

白念珠菌CDR1/CDR2和MDR1基因高表达对氧化应激的影响

目的探索白念珠菌CDR1/CDR2和MDR1基因高表达对氧化应激的影响。方法以白念珠菌CDR1/CDR2和MDR1基因高表达株及其亲本株为研究对象,测定过氧化氢(H2O2)对菌株生长的影响。以H2O2建立氧化应激模型,比较各菌株细胞内活性氧(Reactive oxygen species,ROS)、线粒体膜电位(Mitochondrial membrane potential,ΔΨm)、氧化应激相关基因(CAP1和GRP2)和ROS清除相关基因(SOD2和SOD5)转录水平的差异。结果各菌株均在5mmol/L H2O2作用时出现100%生长抑制;H2O2能升高细胞内ROS和降低ΔΨm(P0.05),其中高表达株中两者的变化幅度较亲本株低(P0.05);同时高表达株中CAP1和GRP2基因在转录水平的表达上调以及SOD2和SOD5基因的下调也较亲本株少(P0.05)。结论 CDR1/CDR2和MDR1基因高表达能够降低白念珠菌的氧化应激反应,增强其对氧化应激的适应性。     

NF-κB and ERK-signaling pathways contribute to the gene expression induced by cag PAI-positive- Helicobacterpy/oriinfection

AIM: To elucidate the sequential gene expression profile in AGS cells co-cultured with wild-type Helicobacter pylori (H pylori) as a model of H pylori-infected gastric epithelium, and to further examine the contribution of cag-pathogenicity islands (cagPAI)-coding type IV secretion system and the two pathways, nuclear factor kappa B (NF-kappaB) and extracellular signal-regulated kinases (ERK) on wild-type H pylori-induced gene expression. METHODS: Gene expression profiles induced by H pylori were evaluated in AGS gastric epithelial cells using cDNA microarray, which were present in the 4 600 independent clones picked up from the human gastric tissue. We also analyzed the contribution of NF-kappaB and ERK signaling on H pylori-induced gene expression by using inhibitors of specific signal pathways. The isogenic mutant with disrupted cagE (Delta cagE) was used to elucidate the role of cagPAI-encoding type IV secretion system in the gene expression profile. RESULTS: According to the expression profile, the genes were classified into four clusters. Among them, the clusters characterized by continuous upregulation were most conspicuous, and it contained many signal transducer activity-associated genes. The role of cagPAI on cultured cells was also investigated using isogenic mutant cagE, which carries non-functional cagPAI. Then the upregulation of more than 80% of the induced genes (476/566) was found to depend on cagPAI. Signal transducer pathway through NF-kappaB or ERK are the major pathways which are known to be activated by cagPAI-positive H pylori. The role of these pathways in the whole signal activation by cagPAI-positive H pylori was analyzed. The specific inhibitors against NF-kappaB or ERK pathway blocked the activation of gene expression in 65% (367/566) or 76% (429/566) of the genes whose activation appealed to depend on cagPAI. CONCLUSION: These results suggest that more than half of the genes induced by cagPAI-positive H pylori depend on NF-kappaB and ERK signaling activation, and these pathways may play a role in the gene expression induced by host-bacterial interaction which may associate with H pylori-related gastro-duodenal diseases.     

Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants

Despite the recognition of H(2)O(2) as a central signaling molecule in stress and wounding responses, pathogen defense, and regulation of cell cycle and cell death, little is known about how the H(2)O(2) signal is perceived and transduced in plant cells. We report here that H(2)O(2) is a potent activator of mitogen-activated protein kinases (MAPKs) in Arabidopsis leaf cells. Using epitope tagging and a protoplast transient expression assay, we show that H(2)O(2) can activate a specific Arabidopsis mitogen-activated protein kinase kinase kinase, ANP1, which initiates a phosphorylation cascade involving two stress MAPKs, AtMPK3 and AtMPK6. Constitutively active ANP1 mimics the H(2)O(2) effect and initiates the MAPK cascade that induces specific stress-responsive genes, but it blocks the action of auxin, a plant mitogen and growth hormone. The latter observation provides a molecular link between oxidative stress and auxin signal transduction. Finally, we show that transgenic tobacco plants that express a constitutively active tobacco ANP1 orthologue, NPK1, display enhanced tolerance to multiple environmental stress conditions without activating previously described drought, cold, and abscisic acid signaling pathways. Thus, manipulation of key regulators of an oxidative stress signaling pathway, such as ANP1/NPK1, provides a strategy for engineering multiple stress tolerance that may greatly benefit agriculture.     

Oscillatory shear stress upregulation of endothelial nitric oxide synthase requires intracellular hydrogen peroxide and CaMKII

We have previously shown that hydrogen peroxide (H(2)O(2)) upregulates endothelial nitric oxide synthase (eNOS) expression via a calcium/calmodulin-dependent protein kinase II (CaMKII)-mediated mechanism whereas it also acutely activates eNOS enzyme. We hypothesized that oscillatory shear stress (OSS), which stimulates endogenous H(2)O(2), would have effects on eNOS expression and function similar to that of exogenous H(2)O(2). Exposure of bovine aortic endothelial cells to OSS (+/-15 dynes/cm(2)) increased eNOS mRNA expression by 3-fold. Pretreatment with either polyethylene glycol-catalase (PEG-CAT, a scavenger of H(2)O(2)) or KN93, an inhibitor of CaMKII, abolished this response. OSS activated CaMKII in an H(2)O(2)-dependent fashion whereas unidirectional laminar shear stress (LSS) inhibited CaMKII phosphorylation. Inhibition of c-Src (essential for LSS upregulation of eNOS) had no effect on OSS upregulation of eNOS. Additionally, OSS stimulated NO* production acutely. Scavenging of H(2)O(2) by PEG-CAT attenuated OSS stimulation of NO* by 50% whereas it had no effect on LSS regulation of NO* production. These data suggest that intracellular H(2)O(2) and CaMKII mediate OSS upregulation of eNOS. The acute activation of eNOS by OSS also partially requires H(2)O(2). As OSS has been shown previously to stimulate sustained production of superoxide (O(2)*-) which would inactivate NO*, these responses may represent attempted compensation to restore NO* bioavailability in areas exposed to OSS. Simultaneous stimulation of O(2)*- and NO* by this mechanism, however, could facilitate peroxynitrite formation and protein nitration, which may enhance atherosclerotic lesion formation. Both OSS and LSS upregulate eNOS expression but via different signaling mechanisms.     

NF-κB and ERK-signaling pathways contribute to the gene expression induced by cag PAI-positive- Helicobacter pylori infection

AIM: To elucidate the sequential gene expression profile in AGS cells co-cultured with wild-type Helicobacter pylori(H pylori) as a model of H pylori-infected gastric epithelium,and to further examine the contribution of cag-pathogenicity islands (cagPAI)-coding type Ⅳ secretion system and the two pathways, nuclear factor kappa B (NF-κB) and extracellular signal-regulated kinases (ERK) on wild-type H pylori-induced gene expression.METHODS: Gene expression profiles induced by H pylori were evaluated in AGS gastric epithelial cells using cDNA microarray, which were present in the 4 600 independent clones picked up from the human gastric tissue. We also analyzed the contribution of NF-κB and ERK signaling on H pylori-induced gene expression by using inhibitors of specific signal pathways. The isogenic mutant with disrupted cagE (△cagE) was used to elucidate the role of cagPAI-encoding type Ⅳ secretion system in the gene expression profile.RESULTS: According to the expression profile, the genes were classified into four clusters. Among them, the clusters characterized by continuous upregulation were most conspicuous, and it contained many signal transducer activity-associated genes. The role of cagPAI on cultured cells was also investigated using isogenic mutant cagE,which carries non-functional cagPAI. Then the upregulation of more than 80% of the induced genes (476/566) was found to depend on cagPAI. Signal transducer pathway through NF-κB or ERK are the major pathways which are known to be activated by cagPAI-positive H pylori. The role of these pathways in the whole signal activation by cagPAIpositive H pylori was analyzed. The specific inhibitors against NF-κB or ERK pathway blocked the activation of gene expression in 65% (367/566) or 76% (429/566) of the genes whose activation appealed to depend on cagPAI.CONCLUSION: These results suggest that more than half of the genes induced by cagPAI-positive H pylori depend on NF-κB and ERK signaling activation, and these pathways may play a role in the gene expression induced by hostbacterial interaction which may associate with H pylorirelated gastro-duodenal diseases.     

Extranuclear protection of chromosomal DNA from oxidative stress

Eukaryotic organisms evolved under aerobic conditions subjecting nuclear DNA to damage provoked by reactive oxygen species (ROS). Although ROS are thought to be a major cause of DNA damage, little is known about the molecular mechanisms protecting nuclear DNA from oxidative stress. Here we show that protection of nuclear DNA in plants requires a coordinated function of ROS-scavenging pathways residing in the cytosol and peroxisomes, demonstrating that nuclear ROS scavengers such as peroxiredoxin and glutathione are insufficient to safeguard DNA integrity. Both catalase (CAT2) and cytosolic ascorbate peroxidase (APX1) play a key role in protecting the plant genome against photorespiratory-dependent H(2)O(2)-induced DNA damage. In apx1/cat2 double-mutant plants, a DNA damage response is activated, suppressing growth via a WEE1 kinase-dependent cell-cycle checkpoint. This response is correlated with enhanced tolerance to oxidative stress, DNA stress-causing agents, and inhibited programmed cell death.     

Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart

G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart.     

Nitric oxide inhibits H2O2-induced transferrin receptor-dependent apoptosis in endothelial cells: Role of ubiquitin-proteasome pathway

We investigated here the mechanism of cytoprotection of nitric oxide (*NO) in bovine aortic endothelial cells treated with H2O2. NONOates were used as *NO donors that released *NO slowly at a well defined rate in the extracellular and intracellular milieus. H2O2-mediated intracellular dichlorofluorescein fluorescence and apoptosis were enhanced by the transferrin receptor (TfR)-mediated iron uptake. *NO inhibited the TfR-mediated iron uptake, dichlorofluorescein fluorescence, and apoptosis in H2O2-treated cells. *NO increased the proteasomal activity and degradation of nitrated TfR via ubiquitination. Nomega-nitro-L-arginine methyl ester, a nonspecific inhibitor of endogenous *NO biosynthesis, decreased the trypsin-like activity of 26S proteasome. *NO, by activating proteolysis, mitigates TfR-dependent iron uptake, dichlorodihydrofluorescein oxidation, and apoptosis in H2O2-treated bovine aortic endothelial cells. The relevance of biological nitration on redox signaling is discussed.     

Melatonin induction and its role in high light stress tolerance in Arabidopsis thaliana

In plants, melatonin is a potent bioactive molecule involved in the response against various biotic and abiotic stresses. However, little is known of its defensive role against high light (HL ) stress. In this study, we found that melatonin was transiently induced in response to HL stress in Arabidopsis thaliana with a simultaneous increase in the expression of melatonin biosynthetic genes, including serotonin N ‐acetyltransferase1 (SNAT 1 ). Transient induction of melatonin was also observed in the flu mutant, a singlet oxygen (¹O₂)‐producing mutant, upon light exposure, suggestive of melatonin induction by chloroplastidic ¹O₂ against HL stress. An Arabidopsis snat1 mutant was devoid of melatonin induction upon HL stress, resulting in high susceptibility to HL stress. Exogenous melatonin treatment mitigated damage caused by HL stress in the snat1 mutant by reducing O₂^? production and increasing the expression of various ROS ‐responsive genes. In analogy, an Arabidopsis SNAT 1 ‐overexpressing line showed increased tolerance of HL stress concomitant with a reduction in malondialdehyde and ion leakage. A complementation line expressing an Arabidopsis SNAT 1 genomic fragment in the snat1 mutant completely restored HL stress susceptibility in the snat1 mutant to levels comparable to that of wild‐type Col‐0 plants. The results of the analysis of several Arabidopsis genetic lines reveal for the first time at the genetic level that melatonin is involved in conferring HL stress tolerance in plants.     

Annexin-like protein from Arabidopsis thaliana rescues delta oxyR mutant of Escherichia coli from H2O2 stress.

Reactive oxygen species are common causes of cellular damages in all aerobic organisms. In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H2O2 stress. To identify genes involved in counteracting oxidative stress in plants, we transformed a delta oxyR mutant of E. coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the delta oxyR mutant to grow in the presence of H2O2. Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family. The complemented mutant showed higher catalase activity. mRNA expression of the annexin gene in A. thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H2O2 stress or salicylic acid. Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H2O2 stress.     

Inhibition of endothelial nitric oxide synthase activity by proline-rich tyrosine kinase 2 in response to fluid shear stress and insulin

In native and primary cultures of endothelial cells, fluid shear stress elicits the tyrosine phosphorylation of the endothelial NO synthase (eNOS), however, the consequences of this modification on enzyme activity are unclear. We found that fluid shear stress induces the association of eNOS with the proline-rich tyrosine kinase 2 (PYK2) in endothelial cells and that the eNOS immunoprecipitated from eNOS- and PYK2-overexpressing HEK293 cells was tyrosine-phosphorylated on Tyr657. In mouse carotid arteries, the overexpression of wild-type PYK2, but not a dominant-negative PYK2, decreased eNOS activity (approximately 50%), whereas in murine lung endothelial cells, the downregulation of PYK2 (small interfering RNA) increased ionomycin-induced NO production. Mutation of Tyr657 to the phosphomimetic residues aspartate (D) or glutamate (E) abolished enzyme activity, whereas a nonphosphorylatable mutant (phenylalanine [F]) showed activity comparable to the wild-type enzyme. Moreover, normal flow-induced vasodilatation was apparent in carotid arteries from eNOS(-/-) mice overexpressing either the wild-type eNOS or the Y657F mutant, whereas no flow-induced vasodilatation was apparent in arteries expressing the Y657E eNOS mutant. Insulin also activated PYK2 and stimulated eNOS in endothelial cells expressing the Y657F mutant but not wild-type eNOS. These data indicate that PYK2 mediates the tyrosine phosphorylation of eNOS on Tyr657 in response to fluid shear stress and insulin stimulation and that this modification attenuates the activity of the enzyme. The PYK2-dependent inhibition of NO production may serve to keep eNOS activity low and limit the detrimental consequences of maintained high NO output, ie, the generation of peroxynitrite.