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The structural and functional diversity of plant metabolites is largely created via chemical modification of a basic backbone. However, metabolite modifications in plants have still not been thoroughly investigated by metabolomics approaches. In this study, a widely targeted metabolite modificomics (WTMM) strategy was developed based on ultra-high performance liquid chromatography-quadrupole-linear ion trap (UHPLC-Q-Trap) and UHPLC-Q-Exactive-Orbitrap (UHPLC-QE-Orbitrap), which greatly improved the detection sensitivity and the efficiency of identification of modified metabolites. A metabolite modificomics study was carried out using tomato as a model, and over 34,000 signals with MS2 information were obtained from approximately 232 neutral loss transitions. Unbiased metabolite profiling was also performed by utilizing high-resolution mass spectrometry data to annotate a total of 2,118 metabolites with 125 modification types; of these, 165 modified metabolites were identified in this study. Next, the WTMM database was used to assess diseased tomato tissues and 29 biomarkers were analyzed. In summary, the WTMM strategy is not only capable of large-scale detection and quantitative analysis of plant-modified metabolites in plants, but also can be used for plant biomarker development.
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Solanum lycopersicum , Espectrometria de Massas/métodos , Cromatografia Líquida de Alta Pressão/métodos , Metabolômica/métodosRESUMO
Steroidal glycoalkaloids (SGAs) constitute a characteristic class of antinutritional metabolites that are found in certain Solanum species. Despite the considerable studies on SGA biosynthesis, the mechanisms of crosstalk between hormone signaling pathways that regulate SGA content still remain to be elucidated. Here, we performed a metabolic genome-wide association study (mGWAS) based on the levels of SGA metabolites and identified SlERF.H6 as a negative regulator of bitter-SGA biosynthesis. SlERF.H6 repressed the expression of SGA biosynthetic glycoalkaloid metabolism (GAME) genes and caused a subsequent decrease in the abundance of bitter SGAs. Furthermore, SlERF.H6 were shown to act downstream of GAME9, a regulator of SGA biosynthesis in tomato. We also uncovered the interplay between ethylene and gibberellin (GA) signaling in regulating SGA biosynthesis. SlERF.H6, acting as a downstream component in ethylene signaling, modulated GA content by inhibiting SlGA2ox12 expression. Increasing levels of endogenous GA12 and GA53 in SlERF.H6-OE could inhibit of GA on SGA biosynthesis. Additionally, 1-aminocyclopropane-1-carboxylic acid (ACC) treatment decreased the stability of SlERF.H6, weakening its inhibition on GAME genes and SlGA2ox12, and caused bitter-SGA accumulation. Our findings reveal a key role of SlERF.H6 in the regulation of SGA biosynthesis through the coordinated ethylene-gibberellin signaling.
Assuntos
Solanum lycopersicum , Solanum lycopersicum/genética , Giberelinas , Estudo de Associação Genômica Ampla , Etilenos/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
BACKGROUND: Microglia are key regulators of the inflammatory response in the brain. Adenosine in RNAs can be converted to m6A (N6-methyladenosine), which regulates RNA metabolism and functions as a key epitranscriptomic modification. The m6A modification pattern and m6A-related signatures under pro-inflammatory and anti-inflammatory conditions of microglia remain unclear. METHODS: Primary rat microglia were differentiated into pro-inflammatory M1-like (M1-L), anti-inflammatory M2-like (M2-L), and resting, unstimulated (M0-L) phenotypes. m6A mRNA and lncRNA epitranscriptomic microarray analyses were performed, and pathway analysis was conducted to understand the functional implications of m6A methylation in mRNAs and lncRNAs. The m6A methylation level and gene expression of mRNAs and lncRNAs were subsequently verified by m6A Me-RIP and qRT-PCR. RESULTS: A total of 1588 mRNAs and 340 lncRNAs, 315 mRNAs and 38 lncRNAs, and 521 mRNAs and 244 lncRNAs were differentially m6A methylated between M1-L and M0-L (M1-L/M0-L), M2-L and M0-L (M2-L/M0-L), M2-L and M1-L (M2-L/M1-L), respectively. Furthermore, 4902 mRNAs, 4676 mRNAs, and 5095 mRNAs were identified distinctively expressed in M1-L/M0-L, M2-L/M0-L, and M2-L/M1-L, respectively. Pathway analysis of differentially m6A methylated mRNAs and lncRNAs in M1-L/M0-L identified immune system, signal transduction, and protein degradation processes. In contrast, the distinct m6A methylated mRNAs in M2-L/M0-L were involved in genetic information processing, metabolism, cellular processes, and neurodegenerative disease-related pathways. We validated m6A methylation and the expression levels of five mRNAs and five lncRNAs, which were involved in upregulated pathways in M1-L/M0-L, and five mRNAs involved in upregulated pathways in M2-L/M0-L. CONCLUSIONS: These findings identify a distinct m6A epitranscriptome in microglia, and which may serve as novel and useful regulator during pro-inflammatory and anti-inflammatory response of microglia.
Assuntos
Adenosina/análogos & derivados , Mediadores da Inflamação/metabolismo , Microglia/metabolismo , Adenosina/genética , Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Transcriptoma/fisiologiaRESUMO
Volatile accumulation during tomato ripening greatly affects the fruit flavor. In this study, four accessions from each of the three tomato subgroups (BIG, S. lycopersicum, CER, S. lycopersicumvar. Cerasiforme, and PIM, S. pimpinellifolium) were subjected to a sensory evaluation. The CER subgroup had the highest fruit-flavor score. Using a Headspace solid-phase microextraction/gas chromatography-mass spectrometer (HP-SPME/GC-MS), a volatile database containing 94 volatiles was created. Pentanal accumulated in green fruits and 1-pentanol in red fruits. 1-Octen-3-ol was discovered to underlie the bitterness of green tomatoes, and it was most abundant in PIM green fruits. Phenylethyl alcohol affected the acidity and sweetness of red tomatoes, and it was most abundant in CER red fruits. Branched-chain volatiles were most abundant in PIM and BIG red fruits, while apocarotenoids were most abundant in CER red fruits. These findings suggest that domestication and improvement have influenced volatile content, and apocarotenoids and branched-chain volatiles synergistically mediated aromatic flavors in red fruits. This study provides a metabolic basis for analyses of the molecular mechanisms of fruit-flavor formation.
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Areca catechu L., of the Arecaceae family, is widely distributed in tropical Asia. In A. catechu, the extracts and compounds, including flavonoids, have various pharmacological activities. Although there are many studies of flavonoids, the molecular mechanism of their biosynthesis and regulation remains unclear in A. catechu. In this study, 331 metabolites were identified from the root, stem, and leaf of A. catechu using untargeted metabolomics, including 107 flavonoids, 71 lipids, 44 amino acids and derivatives, and 33 alkaloids. The transcriptome analysis identified 6119 differentially expressed genes, and some were enriched in the flavonoid pathway. To analyze the biosynthetic mechanism of the metabolic differences in A. catechu tissues, 36 genes were identified through combined transcriptomic and metabolomic analysis, in which glycosyltransferase genes Acat_15g017010 and Acat_16g013670 were annotated as being involved in the glycosylation of kaempferol and chrysin by their expression and in vitro activities. Flavonoid biosynthesis could be regulated by the transcription factors, AcMYB5 and AcMYB194. This study laid a foundation for further research on the flavonoid biosynthetic pathway of A. catechu.
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Catequina , Transcriptoma , Areca/química , Flavonoides/metabolismo , Catequina/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de PlantasRESUMO
Ginkgo biloba L. leaf extract (GBE) has been added in many commercial herbal formulations such as EGb 761 and Shuxuening Injection to treat cardiovascular diseases and stroke worldwide. However, the comprehensive effects of GBE on cerebral ischemia remained unclear. Using a novel GBE (nGBE), which consists of all the compounds of traditional (t)GBE and one new compound, pinitol, we investigated its effect on inflammation, white matter integrity, and long-term neurological function in an experimental stroke model. Both transient middle cerebral artery occlusion (MCAO) and distal MCAO were conducted in male C57/BL6 mice. We found that nGBE significantly reduced infarct volume at 1, 3, and 14 days after ischemia. Sensorimotor and cognitive functions were superior in nGBE treated mice after MCAO. nGBE inhibited the release of IL-1ß in the brain, promoted microglial ramification, and regulated the microglial M1 to M2 phenotype shift at 7 days post injury. In vitro analyses showed that nGBE treatment reduced the production of IL-1ß and TNFα in primary microglia. Administration of nGBE also decreased the SMI-32/MBP ratio and enhanced myelin integrity, thus exhibiting improved white matter integrity at 28 days post stroke. These findings demonstrate that nGBE protects against cerebral ischemia by inhibiting microglia-related inflammation and promoting white matter repair, suggesting that nGBE is a promising therapeutic strategy for long-term recovery after stroke.
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Isquemia Encefálica , Acidente Vascular Cerebral , Substância Branca , Camundongos , Masculino , Animais , Ginkgo biloba , Doenças Neuroinflamatórias , Acidente Vascular Cerebral/tratamento farmacológico , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Isquemia Encefálica/tratamento farmacológico , Infarto da Artéria Cerebral Média/tratamento farmacológico , Microglia , Inflamação/tratamento farmacológicoRESUMO
Background: The function of IκB kinase α (IKKα) in the brain is largely unknown. This study examined the effects of IKKα on autophagy after cerebral ischemia. Methods: Permanent distal middle cerebral artery occlusion (dMCAO) was conducted in C57/BL6 mice. Oxygen-glucose deprivation/reperfusion (OGD/R) was performed to mimic ischemia injury in neuro-2A (N2A) cells in vitro. Autophagy activation was assessed by detecting the ratio of microtubule-associated protein 1 light chain 3ß (LC3B)-II/LC3B-I and Cyto-ID autophagic fluorescence. The infarct volume was verified by 2,3,5-triphenyltetrazolium chloride (TTC) staining and magnetic resonance imaging (MRI). Neurological functions were evaluated using the modified Garcia test. Cell death after dMCAO was confirmed with terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay. To determine the role of IKKα, small interfering RNA (siRNA) was transfected into N2A cells or injected intracerebroventricularly. Results: IKKα and LC3B II/I expression levels were increased both in OGD/R treated N2A cells and dMCAO mice. Under the same conditions, IKKß expression was not altered. IKKα siRNA significantly decreased the infarct volume and the apparent diffusion coefficient (ADC) related to brain edema, and promoted the neurological outcomes after dMCAO. Furthermore, inhibition of IKKα attenuated ischemia- induced the conversion of LC3B I to LC3B II both in vitro and in vivo. In addition, IKKα siRNA alleviated the formation of autophagic vacuoles and LC3 positive puncta after cerebral ischemia. Conclusions: These findings indicate that IKKα, but not IKKß, plays a critical role in ischemia-induced autophagy. Inhibition of IKKα protects the brain from ischemia injury and this may have potential benefits in stroke therapy.
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Coconut is a tropical fruit whose flesh has high flavor quality and nutritional value; however, the differences between coconut varieties are still unclear. Here, volatiles and non-volatiles were profiled at three ripening stages by HS-SPME/GC-MS and UHPLC-MS/MS in two coconut varieties (Hainan Tall, HT and Green Dwarf, GD). Four metabolite classes of volatiles were associated with good aroma including hydrocarbons, benzenoids, alcohols and esters, and these volatiles were generally higher in GD, especially at 7 and 9 months of coconut growth. Pathway-based metabolomics revealed that flavonols and their derivatives were significantly enriched in HT, and some of these metabolites were key determinants of HT flesh bitterness, including kaempferol 7-O-glucoside, a known bitter metabolite. Despite the overall accumulation of amino acids, including L-alanine, L-serine and L-methionine in GD, comparative metabolomics revealed that HT flesh provides a higher content of vitamins than GD. This study sheds light on the metabolic pathways and key metabolites differentiating the flesh flavor quality and nutritional value among coconut varieties, and reveals the possible mechanisms of flavor formation and regulation in coconut fruits.
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Background: Dual antiplatelet therapy with aspirin and clopidogrel (ASA + CPG) during the first 21 days has been shown to reduce the risk of major ischemic events in patients with transient ischemic attack (TIA) or minor stroke. However, the mechanisms underlying combination treatment with ASA + CPG in experimental ischemic stroke has not been fully elucidated. Methods: Minor cerebral ischemia was induced in mice by transient distal middle cerebral artery occlusion (tdMCAO). Two doses of ASA + CPG (12 and 24 mg/kg/day) or vehicle were administered by gavage daily. Neurological behaviors were assessed using the modified Garcia scores, Rotarod test, Y maze, and open field test. Platelet function was assessed in vitro by flow cytometry and in vivo by bleeding and clotting time. The neutrophil ratio and the levels of inflammatory cytokines were measured by flow cytometry and the Meso Scale Discovery (MSD) electrochemilunimescence, respectively. Results: Sensorimotor function was partially recovered with ASA + CPG treatment after ischemia. Anxiety levels and cognitive functions showed improvement in the ASA + CPG group at 12 mg/kg/day after 21 days. Both tail bleeding time and flow cytometry showed significantly decreased platelet function after ASA + CPG treatment. Notably, ASA + CPG at 12 mg/kg/day prolonged clotting time at 28 days after injury. Furthermore, the ratio of neutrophils, an indicator of inflammation, was reduced with 12 mg/kg/day ASA + CPG treatment in the bone marrow (BM) at 21 days and in the peripheral blood (PB) at 21 and 28 days after tdMCAO. Both doses of ASA + CPG decreased pro-inflammatory cytokine interleukin (IL)-6 expression 21 days after stroke. Taken together, these results demonstrated that combination treatment with ASA + CPG improved long-term neurological function after stroke and may inhibit platelet-neutrophil interaction by decreasing the concentration of pro-inflammatory cytokine, IL-6. Conclusions: These findings indicate a neuroprotective effect of combination treatment with ASA + CPG for a duration of 21 days in an experimental acute minor stroke model. These findings provide further evidence that dual antiplatelet therapy may be a viable neuroprotective treatment to decrease the recurrence of stroke.
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Neuronal death in the central nervous system contributes to the development of age-related neurodegeneration. The ATR/Chk1 pathway appears to function neuroprotectively to prevent DNA damage induced by cytotoxic agents. Here, we examine the function of Chk1 on cell viability of cortical neurons in the absence of additional DNA damaging stimuli. The Chk1-specific inhibitor, UCN-01, and the ATR inhibitor, Caffeine, cause neuronal apoptosis in differentiated neurons in the absence of additional treatment, whereas inhibition of ATM or Chk2, does not. UCN-01 treatment increased the detection of γ-H2AX phosphorylation, DNA strand breaks, and an activated p53-dependent DNA damage response (DDR), suggesting that Chk1 normally helps to maintain genomic stability. UCN-01 treatment also enhanced the apoptosis seen in neurons treated with DNA damaging agents, such as camptothecin (CPT). Our results indicate that Chk1 is essential for neuronal survival, and perturbation of this pathway increases a cell's sensitivity to naturally accumulating DNA damage.
Assuntos
Apoptose , Córtex Cerebral/citologia , Neurônios/fisiologia , Proteínas Quinases/fisiologia , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Cafeína/farmacologia , Camptotecina/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Diferenciação Celular , Sobrevivência Celular/fisiologia , Células Cultivadas , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Dano ao DNA , Proteínas de Ligação a DNA/antagonistas & inibidores , Feminino , Genes p53 , Instabilidade Genômica , Neurônios/efeitos dos fármacos , Neurônios/enzimologia , Fosforilação/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/fisiologia , Ratos , Ratos Sprague-Dawley , Estaurosporina/análogos & derivados , Estaurosporina/farmacologia , Proteínas Supressoras de Tumor/antagonistas & inibidoresRESUMO
A major phenotype seen in neurodegenerative disorders is the selective loss of neurons due to apoptotic death and evidence suggests that inappropriate re-activation of cell cycle proteins in post-mitotic neurons may be responsible. To investigate whether reactivation of the G1 cell cycle proteins and S phase entry was linked with apoptosis, we examined homocysteine-induced neuronal cell death in a rat cortical neuron tissue culture system. Hyperhomocysteinaemia is a physiological risk factor for a variety of neurodegenerative diseases, including Alzheimer's disease. We found that in response to homocysteine treatment, cyclin D1, and cyclin-dependent kinases 4 and 2 translocated to the nucleus, and p27 levels decreased. Both cyclin-dependent kinases 4 and 2 regained catalytic activity, the G1 gatekeeper retinoblastoma protein was phosphorylated and DNA synthesis was detected, suggesting transit into S phase. Double-labelling immunofluorescence showed a 95% co-localization of anti-bromodeoxyuridine labelling with apoptotic markers, demonstrating that those cells that entered S phase eventually died. Neurons could be protected from homocysteine-induced death by methods that inhibited G1 phase progression, including down-regulation of cyclin D1 expression, inhibition of cyclin-dependent kinases 4 or 2 activity by small molecule inhibitors, or use of the c-Abl kinase inhibitor, Gleevec, which blocked cyclin D and cyclin-dependent kinase 4 nuclear translocation. However, blocking cell cycle progression post G1, using DNA replication inhibitors, did not prevent apoptosis, suggesting that death was not preventable post the G1-S phase checkpoint. While homocysteine treatment caused DNA damage and activated the DNA damage response, its mechanism of action was distinct from that of more traditional DNA damaging agents, such as camptothecin, as it was p53-independent. Likewise, inhibition of the DNA damage sensors, ataxia-telangiectasia mutant and ataxia telangiectasia and Rad3 related proteins, did not rescue apoptosis and in fact exacerbated death, suggesting that the DNA damage response might normally function neuroprotectively to block S phase-dependent apoptosis induction. As cell cycle events appear to be maintained in vivo in affected neurons for weeks to years before apoptosis is observed, activation of the DNA damage response might be able to hold cell cycle-induced death in check.
Assuntos
Apoptose/fisiologia , Homocisteína/metabolismo , Neurônios/fisiologia , Proteínas Quinases/metabolismo , Fase S/fisiologia , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia , Encéfalo/efeitos dos fármacos , Encéfalo/fisiologia , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Proteínas de Ciclo Celular/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Células Cultivadas , Quinase 1 do Ponto de Checagem , Quinase do Ponto de Checagem 2 , Dano ao DNA/fisiologia , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Fase G1/efeitos dos fármacos , Fase G1/fisiologia , Neurônios/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/metabolismo , Ratos , Ratos Sprague-Dawley , Fase S/efeitos dos fármacos , Proteínas Supressoras de Tumor/metabolismoRESUMO
In developing central nervous system, a variety of mechanisms couple cell cycle exit to differentiation during neurogenesis. The cyclin-dependent kinase (CDK) inhibitor p57Kip2 controls the transition from proliferation to differentiation in many tissues, but roles in developing brain remain uncertain. To characterize possible functions, we defined p57Kip2 protein expression in embryonic (E) day 12.5 to 20.5 rat brains using immunohistochemistry combined with markers of proliferation and differentiation. The p57Kip2 was localized primarily in cell nuclei and positive cells formed two distinct patterns including wide dispersion and laminar aggregation that were brain region-specific. From E12.5 to E16.5, p57Kip2 expression was detected mainly in ventricular zone (VZ) and/or mantle zone of hippocampus, septum, basal ganglia, thalamus, hypothalamus, midbrain, and spinal cord. After E18.5, p57Kip2 was detected in select regions undergoing differentiation. The p57Kip2 expression was also compared with regional transcription factors, including Ngn2, Nkx2.1, and Pax6. Time course studies performed in diencephalon showed that p57Kip2 immunoreactivity colocalized with BrdU at 8 hr in nuclei exhibiting the wide dispersion pattern, whereas colocalization in the laminar pattern occurred only later. Moreover, p57Kip2 frequently colocalized with neuronal marker, beta-III tubulin. Finally, we characterized relationships of p57Kip2 to CDK inhibitor p27Kip1: in proliferative regions, p57Kip2 expression preceded p27Kip1 as cells underwent differentiation, though the proteins colocalized in substantial numbers of cells, suggesting potentially related yet distinct functions of Cip/Kip family members during neurogenesis. Our observations that p57Kip2 exhibits nuclear expression as precursors exit the cell cycle and begin expressing neuronal characteristics suggests that the CDK inhibitor contributes to regulating the transition from proliferation to differentiation during brain development.
Assuntos
Encéfalo , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Células-Tronco Embrionárias/fisiologia , Neurônios/fisiologia , Animais , Encéfalo/anatomia & histologia , Encéfalo/embriologia , Encéfalo/metabolismo , Bromodesoxiuridina/metabolismo , Proliferação de Células , Inibidor de Quinase Dependente de Ciclina p57/genética , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurogênese/fisiologia , Gravidez , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
As a member of the CIP/KIP family of cyclin-dependent kinase inhibitors (CKIs), p57Kip2 binds tightly to G1 cyclin/cyclin-dependent kinase complexes to block cell cycle progression. CKIs play critical roles in regulating the transition from proliferation to differentiation in many tissues, including the nervous system. Conversely, CKI dys-regulation contributes to neoplasia and cancer progression. While the combined detection of CKI immunoreactivity and S phase entry using bromodeoxyuridine (BrdU) incorporation may be particularly informative, successful immunostaining may be limited due to "masked" antigen epitopes and acid-induced signal degradation. We now report an improved double immunofluorescent method for detecting p57Kip2 and BrdU in paraformaldehyde-fixed frozen sections of embryonic rat brain. We substituted deoxyribonuclease I (DNAse I) for HCl pre-treatment to expose antigenic sites in frozen sections, and employed a biotinylated tyramide-based system to enhance p57Kip2 visualization. We identified a time- and dose-dependent relationship between DNAse I treatment and double labeling of p57Kip2 and BrdU, increasing both the numbers and intensities of immunopositive nuclei. With excess DNAse I treatment, however, there was signal degradation for both BrdU and total DNA, as reflected by DAPI staining. The use of DNAse I pre-treatment significantly increases the reliability and sensitivity of immunodetection of CKI nuclear factors, and should be useful for both developmental neurobiology studies as well as cancer diagnostic applications.
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Química Encefálica , Encéfalo/embriologia , Bromodesoxiuridina/análise , Inibidor de Quinase Dependente de Ciclina p57/análise , Desoxirribonuclease I/metabolismo , Imuno-Histoquímica/métodos , Animais , Encéfalo/citologia , Bromodesoxiuridina/isolamento & purificação , Inibidor de Quinase Dependente de Ciclina p57/isolamento & purificação , Embrião de Mamíferos , Feminino , Gravidez , Ratos , Ratos Sprague-DawleyRESUMO
Heme plays a central role in oxygen utilization and in the generation of cellular energy. Here we examined the effect of heme and heme deficiency on cell cycle progression and the expression of key regulators in HeLa cells. We found that inhibition of heme synthesis causes cell cycle arrest and induces the expression of molecular markers associated with senescence and apoptosis, such as increased formation of PML nuclear bodies. Our data show that succinyl acetone-induced heme deficiency increases the protein levels of the tumor suppressor gene product p53 and CDK inhibitor p21, and decreases the protein levels of Cdk4, Cdc2, and cyclin D2. Further, we found that heme deficiency diminishes the activation/phosphorylation of Raf, MEK1/2, and ERK1/2-components of the MAP kinase signaling pathway. Our results show that heme is a versatile molecule that can effectively control cell growth and survival by acting on multiple regulators.
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Proteínas de Ciclo Celular/biossíntese , Heme/fisiologia , Proteínas Nucleares , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Regulação da Expressão Gênica , Células HeLa , Heme/antagonistas & inibidores , Heme/biossíntese , Heme/deficiência , Heptanoatos/farmacologia , Humanos , Marcação In Situ das Extremidades Cortadas , Metaloproteinase 3 da Matriz/biossíntese , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Fosforilação , Proteína da Leucemia Promielocítica , Proteínas Proto-Oncogênicas c-raf/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/biossíntese , Proteínas Supressoras de TumorRESUMO
Mitochondria provide cellular energy supply via respiration and are the major sites for the generation of reactive oxygen species (ROS). Mitochondria also play a fundamental role in apoptosis. Heme is a key factor in mitochondrial function. Defective heme synthesis or altered heme metabolism is associated with numerous diseases. Here we investigated the molecular mechanism by which heme promotes HeLa cell growth and survival. We found that heme deficiency-induced apoptosis involves the release of cytochrome c and the activation of caspase 3. However, heme deficiency-induced apoptosis appears to occur by a unique mechanism distinct from those known to mediate mitochondrial-dependent apoptosis. Specifically, our data show that heme deficiency causes apoptosis in a pathway that is independent of ROS generation and the collapse of mitochondrial membrane potential. These results provide insights into how defective heme synthesis or altered heme metabolism causes diseases and how heme may control cell growth and cell death.
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Apoptose/fisiologia , Células HeLa/metabolismo , Heme/deficiência , Espécies Reativas de Oxigênio/metabolismo , Caspase 3 , Caspases/metabolismo , Divisão Celular , Sobrevivência Celular , Citocromos c/metabolismo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Células HeLa/efeitos dos fármacos , Heptanoatos/farmacologia , Humanos , Marcação In Situ das Extremidades Cortadas , Potenciais da Membrana , Mitocôndrias/metabolismoRESUMO
Defective heme synthesis in mammals has been suspected of causing neuropathy associated with porphyrias and lead poisoning. To determine the molecular action of heme in neuronal cells, we examined the effect of the inhibition of heme synthesis on nerve growth factor (NGF) signaling in PC12 cells. We found that the inhibition of heme synthesis by succinyl acetone interferes with NGF-induced neurite outgrowth in PC12 cells. Furthermore, we show that heme deficiency obliterates the activation of the signaling intermediates of the Ras-mitogen-activated protein kinase signaling pathway and its downstream target, the transcription activator cyclic AMP response element-binding protein. Strikingly, microarray expression analysis shows that the inhibition of heme synthesis selectively diminishes the induction of expression of a subset of neuron-specific genes by NGF, such as Ras and neurofilament proteins, whereas NGF induces the expression of several major classes of neuronal genes that encode regulatory and structural proteins at three days after induction. Our data provide insights into how heme deficiency interferes with NGF signaling and abrogates programs of neuronal gene expression, thus ultimately causing defective neuronal functions.