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In hypoxic and pseudohypoxic rodent models of pulmonary hypertension (PH), hypoxia-inducible factor (HIF) inhibition attenuates disease initiation. However, HIF activation alone, due to genetic alterations or use of inhibitors of prolyl hydroxylase domain (PHD) enzymes, has not been definitively shown to cause PH in humans, indicating the involvement of other mechanisms. Given the association between endothelial cell dysfunction and PH, the effects of pseudohypoxia and its underlying pathways were investigated in primary human lung endothelial cells. PHD2 silencing or inhibition, while activating HIF2α, induced apoptosis-resistance and IFN/STAT activation in endothelial cells, independent of HIF signaling. Mechanistically, PHD2 deficiency activated AKT and ERK, inhibited JNK, and reduced AIP1 (ASK1-interacting protein 1), all independent of HIF2α. Like PHD2, AIP1 silencing affected these same kinase pathways and produced a similar dysfunctional endothelial cell phenotype, which were partially reversed by AKT inhibition. Consistent with these in vitro findings, AIP1 protein levels in lung endothelial cells were decreased in Tie2-Cre/Phd2 knockout mice compared to wild-type controls. Lung vascular endothelial cells from patients with pulmonary arterial hypertension (PAH) showed IFN/STAT activation. Lung tissue from both SU5416/hypoxia PAH rats and PAH patients all showed AKT activation and dysregulated AIP1 expression. In conclusion, PHD2 deficiency in lung vascular endothelial cells drives an apoptosis-resistant and inflammatory phenotype, mediated by AKT activation and AIP1 loss independent of HIF signaling. Targeting these pathways, including PHD2, AKT, and AIP1, holds potential for developing new treatments for endothelial dysfunction in PH.
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Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, activates NOTCH1 signaling and plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in PAECs activated AKT and decreased DLL4 expression. DLL4 loss was also seen in lungs of patients with IPAH and HPAH. Over-expression of DLL4 in PAECs induced BMPR2 promoter activity and exogenous DLL4 increased BMPR2 mRNA through NOTCH1 activation. Furthermore, DLL4/NOTCH1 signaling blocked AKT activation, decreased proliferation and reversed EndoMT in BMPR2-silenced PAECs and ECs from IPAH patients. PPARγ, suppressed by BMPR2 loss, was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH PAECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Finally, leniolisib, a well-tolerated oral PI3Kδ/AKT inhibitor, decreased cell proliferation, induced apoptosis and reversed markers of EndoMT in BMPR2-silenced PAECs. Restoring DLL4/NOTCH1/PPARγ signaling and/or suppressing AKT activation may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.
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Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by pathologic vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in pulmonary artery endothelial cells (PAECs) activated AKT and suppressed the expression of DLL4. Consistent with these in vitro findings, increased AKT activation and reduced DLL4 expression was found in the small pulmonary arteries of patients with PAH. Increased NOTCH1 activation through exogenous DLL4 blocked AKT activation, decreased proliferation and reversed EndoMT. Exogenous and overexpression of DLL4 induced BMPR2 and PPRE promoter activity, and BMPR2 and PPARG mRNA in idiopathic PAH (IPAH) ECs. PPARγ, a nuclear receptor associated with EC homeostasis, suppressed by BMPR2 loss was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH ECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Directly blocking AKT or restoring DLL4/NOTCH1/PPARγ signaling may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II , Células Endoteliais , PPAR gama , Proteínas Proto-Oncogênicas c-akt , Artéria Pulmonar , Receptor Notch1 , Transdução de Sinais , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , PPAR gama/metabolismo , PPAR gama/genética , Receptor Notch1/metabolismo , Receptor Notch1/genética , Artéria Pulmonar/metabolismo , Artéria Pulmonar/patologia , Células Endoteliais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/patologia , Masculino , Proliferação de Células , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/patologia , Feminino , Células CultivadasRESUMO
Lymphocyte depletion is a distinctive feature of Ebola virus (EBOV) disease. The ectodomain of EBOV glycoprotein (GP) is cleaved off the surface of infected cells into circulation as shed GP. To test the hypothesis that shed GP induces lymphocyte death, we cultured primary human B, NK, or T cells with shed GP in vitro. We found that shed GP dependably decreased B, NK, and T cell viability across donors. B and NK cells exhibited higher susceptibility than T cells. Continuous monitoring revealed shed GP began to kill B and NK cells by 4 h and T cells by 5 h. We also demonstrated that shed GP-induced lymphocyte death can be both caspase dependent and caspase independent. Our data are evidence that the cytotoxic effect of shed GP on lymphocytes may contribute to EBOV disease and highlight the need for further research to clarify mechanisms of shed GP-induced death.
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NR2F2 is expressed in endothelial cells (ECs) and Nr2f2 knockout produces lethal cardiovascular defects. In humans, reduced NR2F2 expression is associated with cardiovascular diseases including congenital heart disease and atherosclerosis. Here, NR2F2 silencing in human primary ECs led to inflammation, endothelial-to-mesenchymal transition (EndMT), proliferation, hypermigration, apoptosis-resistance, and increased production of reactive oxygen species. These changes were associated with STAT and AKT activation along with increased production of DKK1. Co-silencing DKK1 and NR2F2 prevented NR2F2-loss-induced STAT and AKT activation and reversed EndMT. Serum DKK1 concentrations were elevated in patients with pulmonary arterial hypertension (PAH) and DKK1 was secreted by ECs in response to in vitro loss of either BMPR2 or CAV1, which are genetic defects associated with the development of PAH. In human primary ECs, NR2F2 suppressed DKK1, whereas its loss conversely induced DKK1 and disrupted endothelial homeostasis, promoting phenotypic abnormalities associated with pathologic vascular remodeling. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating chronic vascular diseases associated with EC dysfunction.NEW & NOTEWORTHY NR2F2 loss in the endothelial lining of blood vessels is associated with cardiovascular disease. Here, NR2F2-silenced human endothelial cells were inflammatory, proliferative, hypermigratory, and apoptosis-resistant with increased oxidant stress and endothelial-to-mesenchymal transition. DKK1 was induced in NR2F2-silenced endothelial cells, while co-silencing NR2F2 and DKK1 prevented NR2F2-loss-associated abnormalities in endothelial signaling and phenotype. Activating NR2F2 or blocking DKK1 may be useful therapeutic targets for treating vascular diseases associated with endothelial dysfunction.
Assuntos
Hipertensão Arterial Pulmonar , Doenças Vasculares , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Endoteliais/metabolismo , Doenças Vasculares/metabolismo , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Pulmonar Primária Familiar/metabolismo , Inflamação/patologia , Fator II de Transcrição COUP/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismoRESUMO
Interferonopathies, interferon (IFN)-α/ß therapy, and caveolin-1 (CAV1) loss-of-function have all been associated with pulmonary arterial hypertension (PAH). Here, CAV1-silenced primary human pulmonary artery endothelial cells (PAECs) were proliferative and hypermigratory, with reduced cytoskeletal stress fibers. Signal transducers and activators of transcription (STAT) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) were both constitutively activated in these cells, resulting in a type I IFN-biased inflammatory signature. Cav1-/- mice that spontaneously develop pulmonary hypertension were found to have STAT1 and AKT activation in lung homogenates and increased circulating levels of CXCL10, a hallmark of IFN-mediated inflammation. PAH patients with CAV1 mutations also had elevated serum CXCL10 levels and their fibroblasts mirrored phenotypic and molecular features of CAV1-deficient PAECs. Moreover, immunofluorescence staining revealed endothelial CAV1 loss and STAT1 activation in the pulmonary arterioles of patients with idiopathic PAH, suggesting that this paradigm might not be limited to rare CAV1 frameshift mutations. While blocking JAK/STAT or AKT rescued aspects of CAV1 loss, only AKT inhibitors suppressed activation of both signaling pathways simultaneously. Silencing endothelial nitric oxide synthase (NOS3) prevented STAT1 and AKT activation induced by CAV1 loss, implicating CAV1/NOS3 uncoupling and NOS3 dysregulation in the inflammatory phenotype. Exogenous IFN reduced CAV1 expression, activated STAT1 and AKT, and altered the cytoskeleton of PAECs, implicating these mechanisms in PAH associated with autoimmune and autoinflammatory diseases, as well as IFN therapy. CAV1 insufficiency elicits an IFN inflammatory response that results in a dysfunctional endothelial cell phenotype and targeting this pathway may reduce pathologic vascular remodeling in PAH.
Assuntos
Caveolina 1/genética , Endotélio Vascular/metabolismo , Hipertensão Pulmonar/metabolismo , Interferon Tipo I/metabolismo , Animais , Células Cultivadas , Endotélio Vascular/enzimologia , Endotélio Vascular/fisiopatologia , Inativação Gênica , Humanos , Hipertensão Pulmonar/fisiopatologia , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/genética , Fator de Transcrição STAT1/metabolismo , Transdução de SinaisRESUMO
Aims: Spironolactone (SPL) improves endothelial dysfunction and survival in heart failure. Immune modulation, including poorly understood mineralocorticoid receptor (MR)-independent effects of SPL might contribute to these benefits and possibly be useful in other inflammatory cardiovascular diseases such as pulmonary arterial hypertension. Methods and results: Using human embryonic kidney cells (HEK 293) expressing specific nuclear receptors, SPL suppressed NF-κB and AP-1 reporter activity independent of MR and other recognized nuclear receptor partners. NF-κB and AP-1 DNA binding were not affected by SPL and protein synthesis blockade did not interfere with SPL-induced suppression of inflammatory signalling. In contrast, proteasome blockade to inhibit degradation of xeroderma pigmentosum group B complementing protein (XPB), a subunit of the general transcription factor TFIIH, or XPB overexpression both prevented SPL-mediated suppression of inflammation. Similar to HEK 293 cells, a proteasome inhibitor blocked XPB loss and SPL suppression of AP-1 induced target genes in human pulmonary artery endothelial cells (PAECs). Unlike SPL, eplerenone (EPL) did not cause XPB degradation and failed to similarly suppress inflammatory signalling. SPL combined with siRNA XPB knockdown further reduced XPB protein levels and had the greatest effect on PAEC inflammatory gene transcription. Using chromatin-immunoprecipitation, PAEC target gene susceptibility to SPL was associated with low basal RNA polymerase II (RNAPII) occupancy and TNFα-induced RNAPII and XPB recruitment. XP patient-derived fibroblasts carrying an N-terminal but not C-terminal XPB mutations were insensitive to both SPL-mediated XPB degradation and TNFα-induced target gene suppression. Importantly, SPL treatment decreased whole lung XPB protein levels in a monocrotaline rat model of pulmonary hypertension and reduced inflammatory markers in an observational cohort of PAH patients. Conclusion: SPL has important anti-inflammatory effects independent of aldosterone and MR, not shared with EPL. Drug-induced, proteasome-dependent XPB degradation may be a useful therapeutic approach in cardiovascular diseases driven by inflammation.
Assuntos
Anti-Inflamatórios/farmacologia , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Endoteliais/efeitos dos fármacos , Hipertensão Pulmonar/tratamento farmacológico , Mediadores da Inflamação/metabolismo , Antagonistas de Receptores de Mineralocorticoides/farmacologia , NF-kappa B/metabolismo , Artéria Pulmonar/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Espironolactona/farmacologia , Fator de Transcrição AP-1/metabolismo , Fator de Transcrição TFIIH/metabolismo , Animais , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Eplerenona/farmacologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Células HEK293 , Humanos , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/fisiopatologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Mutação , NF-kappa B/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , RNA Polimerase II/metabolismo , Ratos Sprague-Dawley , Estudos Retrospectivos , Fator de Transcrição AP-1/genética , Fator de Transcrição TFIIH/genéticaRESUMO
The proliferative endothelial and smooth muscle cell phenotype, inflammation, and pulmonary vascular remodeling are prominent features of pulmonary arterial hypertension (PAH). Mutations in bone morphogenetic protein type 2 receptor (BMPR2) have been identified as the most common genetic cause of PAH and females with BMPR2 mutations are 2.5 times as likely to develop heritable forms of PAH than males. Higher levels of estrogen have also been observed in males with PAH, implicating sex hormones in PAH pathogenesis. Recently, the estrogen metabolite 16α-OHE1 (hydroxyestrone) was implicated in the regulation of miR29, a microRNA involved in modulating energy metabolism. In females, decreased miR96 enhances serotonin's effect by upregulating the 5-hydroxytryptamine 1B (5HT1B) receptor. Because PAH is characterized as a quasi-malignant disease, likely due to BMPR2 loss of function, altered signaling pathways that sustain this cancer-like phenotype are being explored. Extracellular signal-regulated kinases 1 and 2 and p38 mitogen-activated protein kinases (MAPKs) play a critical role in proliferation and cell motility, and dysregulated MAPK signaling is observed in various experimental models of PAH. Wnt signaling pathways preserve pulmonary vascular homeostasis, and dysregulation of this pathway could contribute to limited vascular regeneration in response to injury. In this review, we take a closer look at sex, sex hormones, and the interplay between sex hormones and microRNA regulation. We also focus on MAPK and Wnt signaling pathways in the emergence of a proproliferative, antiapoptotic endothelial phenotype, which then orchestrates an angioproliferative process of vascular remodeling, with the hope of developing novel therapies that could reverse the phenotype.
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A proliferative endothelial cell phenotype, inflammation, and pulmonary vascular remodeling are prominent features of pulmonary arterial hypertension (PAH). Bone morphogenetic protein type II receptor (BMPR2) loss-of-function is the most common cause of heritable PAH and has been closely linked to the formation of pathological plexiform lesions. Although some BMPR2 mutations leave ligand-dependent responses intact, the disruption of ligand-independent, noncanonical functions are universal among PAH-associated BMPR2 genotypes, but incompletely understood. This study examined the noncanonical signaling consequences of BMPR2 silencing in human pulmonary artery endothelial cells to identify potential therapeutic targets. BMPR2 siRNA silencing resulted in a proliferative, promigratory pulmonary artery endothelial cell phenotype and disruption of cytoskeletal architecture. Expression profiling closely reflected these phenotypic changes. Gene set enrichment and promoter analyses, as well as the differential expression of pathway components identified Ras/Raf/ERK signaling as an important consequence of BMPR2 silencing. Raf family members and ERK1/2 were constitutively activated after BMPR2 knockdown. Two Raf inhibitors, sorafenib and AZ628, and low-dose nintedanib, a triple receptor tyrosine kinase inhibitor upstream from Ras, reversed the abnormal proliferation and hypermotility of BMPR2 deficiency. Inhibition of dysregulated Ras/Raf/ERK signaling may be useful in reversing vascular remodeling in PAH.
Assuntos
Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Proliferação de Células , Células Endoteliais/citologia , Hipertensão Pulmonar/metabolismo , Pulmão/metabolismo , Artéria Pulmonar/metabolismo , Adulto , Idoso , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hipertensão Pulmonar Primária Familiar/metabolismo , Feminino , Inativação Gênica , Humanos , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Mutação/genética , Fenótipo , RNA Interferente Pequeno/genética , Transdução de Sinais/fisiologia , Remodelação Vascular/genética , Adulto Jovem , Quinases raf/metabolismoRESUMO
Peroxisome proliferator-activated receptor γ (PPARγ) ligands have been widely used to treat type 2 diabetes mellitus. However, knowledge of PPARγ signaling remains incomplete. In addition to PPARγ, these drugs also activate G protein-coupled receptor 40 (GPR40), a Gαq-coupled free fatty acid receptor linked to MAPK networks and glucose homeostasis. Notably, p38 MAPK activation has been implicated in PPARγ signaling. Here, rosiglitazone (RGZ) activation of GPR40 and p38 MAPK was found to boost PPARγ-induced gene transcription in human endothelium. Inhibition or knockdown of p38 MAPK or expression of a dominant negative (DN) p38 MAPK mutant blunted RGZ-induced PPARγ DNA binding and reporter activity in EA.hy926 human endothelial cells. GPR40 inhibition or knockdown, or expression of a DN-Gαq mutant likewise blocked activation of both p38 MAPK and PPARγ reporters. Importantly, RGZ induction of PPARγ target genes in primary human pulmonary artery endothelial cells (PAECs) was suppressed by knockdown of either p38 MAPK or GPR40. GPR40/PPARγ signal transduction was dependent on p38 MAPK activation and induction of PPARγ co-activator-1 (PGC1α). Silencing of p38 MAPK or GPR40 abolished the ability of RGZ to induce phosphorylation and expression of PGC1α in PAECs. Knockdown of PGC1α, its essential activator SIRT1, or its binding partner/co-activator EP300 inhibited RGZ induction of PPARγ-regulated genes in PAECs. RGZ/GPR40/p38 MAPK signaling also led to EP300 phosphorylation, an event that enhances PPARγ target gene transcription. Thus, GPR40 and PPARγ can function as an integrated two-receptor signal transduction pathway, a finding with implications for rational drug development.
Assuntos
Células Endoteliais/metabolismo , PPAR gama/metabolismo , Receptor Cross-Talk , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Linhagem Celular , DNA/genética , DNA/metabolismo , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Regulação da Expressão Gênica , Genes Reporter , Humanos , Hipoglicemiantes/farmacologia , Ligantes , Luciferases/genética , Luciferases/metabolismo , PPAR gama/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Pioglitazona , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores Acoplados a Proteínas G/genética , Rosiglitazona , Sirtuína 1/genética , Sirtuína 1/metabolismo , Tiazolidinedionas/farmacologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
It is unclear whether Mediator complex in yeast is necessary for all RNA polymerase II (Pol II) transcription or if it is limited to genes activated by environmental stress. In mammals, amino acid limitation induces SNAT2 transcription through ATF4 binding at an amino acid response element. ATF4 is the functional counterpart to the yeast amino acid-dependent regulator GCN4 and GCN4 recruits Mediator during transcriptional activation. Consistent with enhanced SNAT2 transcription activity, the present data demonstrate that amino acid limitation increased SNAT2 promoter association of the general transcription factors that make up the preinitiation complex, including Pol II, but there was no increase in Mediator recruitment. Furthermore, siRNA knockdown of eight Mediator subunits caused no significant decrease in SNAT2 transcription. The estrogen-dependent pS2 gene was used as a positive control for both the ChIP and the siRNA approaches and the data demonstrated the requirement for Mediator recruitment. These results document that activation of the SNAT2 gene by the mammalian amino acid response pathway occurs independently of enhanced Mediator recruitment.
Assuntos
Sistema A de Transporte de Aminoácidos/genética , Aminoácidos/metabolismo , Elementos de Resposta , Fatores Genéricos de Transcrição/metabolismo , Ativação Transcricional , Linhagem Celular Tumoral , Histonas/metabolismo , Humanos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/genética , Interferência de RNA , Fatores Genéricos de Transcrição/antagonistas & inibidores , Fatores Genéricos de Transcrição/genéticaRESUMO
Persistent infection with oncogenic human papillomaviruses (HPVs) is the most important factor in the induction of uterine cervical cancer, a leading cause of cancer mortality in women worldwide. Upon cell transformation, continual expression of the viral oncogenes is required to maintain the transformed phenotype. The viral E6 protein forms a ternary complex with the cellular E6-AP protein and p53 protein which promotes the rapid degradation of p53. Recent studies have revealed that lignans from the creosote bush (3'-O-methyl-nordihydroguaiaretic acid) can repress the viral promoter responsible for E6 gene expression. Work reported here shows that the lignan can subvert viral oncogene function resulting in stabilized p53 protein within treated HPV-containing tumor cells. The stabilized p53 is transcriptionally active as demonstrated by a luciferase reporter vector and induction of genes for Bax and PUMA proteins. Apoptosis is detected by annexin V binding to treated cells as analyzed by flow cytometry. Programmed cell death is confirmed by the induction of active caspases and TUNEL assay. Initiator caspase-9 is activated first, followed later by the effector caspase-3 enzyme. The stabilization and induced apoptosis are not observed within treated HPV-negative cervical tumor cells. Quantitative real time RT-PCR analysis of endogenous E6 gene transcription from the integrated HPV 16 promoter shows at least a fivefold repression of expression as compared to untreated cells. These results indicate that the loss of E6 protein in treated cells could be, in part, responsible for the stabilization of p53 within the lignan treated cells.
Assuntos
Apoptose/efeitos dos fármacos , Masoprocol/análogos & derivados , Proteínas Oncogênicas Virais/metabolismo , Proteínas Repressoras/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológico , Proteínas Reguladoras de Apoptose/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Luciferases , Masoprocol/uso terapêutico , Plantas/química , Proteínas Proto-Oncogênicas/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologia , Proteína X Associada a bcl-2/metabolismoRESUMO
Evidence from human amyotrophic lateral sclerosis (ALS) patients and ALS-linked Cu/Zn superoxide dismutase (Cu/Zn-SOD) transgenic mice bearing the mutation of glycine to alanine at position 93 (G93A) suggests that the pro-apoptotic protein prostate apoptosis response-4 (Par-4) might be a critical link in the chain of events leading to motor neuron degeneration. We now report that Par-4 is enriched in synaptosomes and post-synaptic density from the ventral horn of the spinal cord. Levels of Par-4 in synaptic compartments increased significantly during rapid and slow declining stages of muscle strength in hSOD1 G93A mutant mice. In the pre-muscle weakness stage, hSOD1 G93A mutation sensitized synaptosomes from the ventral horn of the spinal cord to increased levels of Par-4 expression following excitotoxic and apoptotic insults. In ventral spinal synaptosomes, Par-4-mediated production of pro-apoptotic cytosolic factor(s) was significantly enhanced by the hSOD1 G93A mutation. RNA interference (RNAi) knockdown of Par-4 inhibited mitochondrial dysfunction and caspase-3 activation induced by G93A mutation in synaptosomes from the ventral horn of the spinal cord, and protected spinal motor neurons from apoptosis. These results identify the synapse as a crucial cellular site for the cell death promoting actions of Par-4 in motor neurons, and suggest that targeted inhibition of Par-4 by RNAi may prove to be a neuroprotective strategy for motor neuron degeneration.