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1.
Korean J Parasitol ; 58(4): 393-402, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32871633

RESUMO

Toxoplasma gondii is an intracellular parasite that causes severe disease when the infection occurs during pregnancy. Adenosine is a purine nucleoside involved in numerous physiological processes; however, the role of adenosine receptors in T. gondii-induced trophoblast cell function has not been investigated until now. The goal of the present study was to evaluate the intracellular signaling pathways regulated by adenosine receptors using a HTR-8/SVneo trophoblast cell model of T. gondii infection. HTR8/SVneo human extravillous trophoblast cells were infected with or without T. gondii and then evaluated for cell morphology, intracellular proliferation of the parasite, adenosine receptor expression, TNF-α production and mitogen-activated protein (MAP) kinase signaling pathways triggered by adenosine A3 receptor (A3AR). HTR8/SVneo cells infected with T. gondii exhibited an altered cytoskeletal changes, an increased infection rate and reduced viability in an infection time-dependent manner. T. gondii significantly promoted increased TNF-α production, A3AR protein levels and p38, ERK1/2 and JNK phosphorylation compared to those observed in uninfected control cells. Moreover, the inhibition of A3AR by A3AR siRNA transfection apparently suppressed the T. gondii infection-mediated upregulation of TNF-α, A3AR production and MAPK activation. In addition, T. gondii-promoted TNF-α secretion was dramatically attenuated by pretreatment with PD098059 or SP600125. These results indicate that A3AR-mediated activation of ERK1/2 and JNK positively regulates TNF-α secretion in T. gondii-infected HTR8/SVneo cells.


Assuntos
MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Receptor A3 de Adenosina/fisiologia , Toxoplasmose/metabolismo , Trofoblastos/metabolismo , Trofoblastos/parasitologia , Fator de Necrose Tumoral alfa/metabolismo , Células Cultivadas , Humanos
2.
PLoS Biol ; 18(8): e3000774, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32745097

RESUMO

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.


Assuntos
Dictyostelium/genética , MAP Quinase Quinase Quinase 3/genética , Proteínas de Protozoários/genética , Pseudópodes/metabolismo , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Animais , Sistemas CRISPR-Cas , Adesão Celular , Linhagem Celular Tumoral , Quimiotaxia/genética , Dictyostelium/metabolismo , Dictyostelium/ultraestrutura , Edição de Genes/métodos , Regulação da Expressão Gênica , MAP Quinase Quinase Quinase 3/metabolismo , Melanócitos/metabolismo , Melanócitos/ultraestrutura , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Mutação , Células NIH 3T3 , Fenótipo , Fosforilação , Ploidias , Proteínas de Protozoários/metabolismo , Pseudópodes/genética , Pseudópodes/ultraestrutura , Família de Proteínas da Síndrome de Wiskott-Aldrich/metabolismo
3.
PLoS One ; 15(8): e0237098, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32745124

RESUMO

The EGFR-targeting cancer therapies are commonly facing drug resistance, mostly due to mutations. Gene therapy with artificial microRNA targeting EGFR conserved sequence may avoid such problem. In this study, we constructed a recombinant adenovirus expressing EGFR-targeting artificial microRNA and active revCASP3 (Ad-EC), under the control of tumor-specific SLPI promoter, and evaluated its inhibitory effect on HEP-2 cancer cells both in vitro and in vivo. MTT assay showed that cell growth inhibition rate at 72h was 44.0% in Ad-EC group at MOI 50, while the rate was 7.7% in the control virus Ad-GFP group and 3.6% in Cetuximab (500 µg/ml) group respectively. Flow cytometry analysis revealed the late apoptotic cells rate was 36.1% in Ad-EC group, significantly higher than 6.5% of Ad-GFP group (p < 0.001). When Ad-EC (MOI 50) was combined with CDDP (0.25 µg/ml), late apoptotic cells rate increased to 61.2%, significantly higher than each monotherapy group (P < 0.001). The real-time xCELLigence system recorded an effective cell growth inhibition in Ad-EC and CDDP groups, and more enhanced effect in Ad-EC plus CDDP group. Western blot revealed that Ad-EC could inhibit the activation of AKT pathway and ERK1/2 pathway, while Cetuximab had the AKT pathway over-activated. In vivo experiments with HEP-2 xenograft in nude mice confirmed the tumor inhibition in Ad-EC, CDDP and Ad-EC plus CDDP groups compared with PBS group (P < 0.01). Collectively, these data support the effective inhibition of cancer cells by this novel gene therapy strategy.


Assuntos
Caspase 3/metabolismo , Receptores ErbB/genética , MicroRNAs/genética , Neoplasias Experimentais/terapia , Terapêutica com RNAi/métodos , Adenoviridae/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptose , Caspase 3/genética , Linhagem Celular Tumoral , Proliferação de Células , Cetuximab/administração & dosagem , Cetuximab/uso terapêutico , Cisplatino/administração & dosagem , Cisplatino/uso terapêutico , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo
4.
PLoS One ; 15(8): e0237017, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32756588

RESUMO

Procyandin A2 (PCA2) is a polyphenolic compound which is isolated from grape seeds. It has been reported that PCA2 exhibits antioxidative and anti-inflammatory effects, but its molecular mechanism is still poorly understood. This study tests the hypothesis that PCA2 suppresses lipopolysaccharide (LPS)-induced inflammation and oxidative stress through targeting the nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and NF-E2-related factor 2 (Nrf2) pathways in RAW264.7 cells. PCA2 (20, 40, 80 µM) exhibited no significant cytotoxicity in RAW264.7 cells and showed an inhibitory effect on an LPS-induced nitrite level. Pro-inflammatory cytokines like tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), prostaglandin E2 (PGE2), nitric oxide (NO), and reactive oxygen species (ROS) were suppressed by PCA2 with a concentration range of 0-80 µM. The mRNA levels of TNF-α and IL-6 were inhibited by PCA2 (80 µM). The hallmark-protein expression of the NF-κB (p-IKKα/ß, p-IκBα, and p-p65) and MAPK (p-p38, p-JNK, and p-ERK) pathways were decreased by PCA2 in LPS-stimulated RAW264.7 cells. In addition, immunofluorescence results indicated that PCA2 (80 µM) promoted the translocation of NF-κB/p65 from the cytoplasm into the nucleus. PCA2 upregulated the expressions of Nrf2 and HO-1 and downregulated the expression of Keap-1. Simultaneously, PCA2 (80 µM) reversed LPS-induced Nrf2 translocation from the nucleus into the cytoplasm. Collectively, PCA2 protect cells against the damage from inflammation and oxidative injury, which suggest a potential therapeutic strategy for inflammatory and oxidative stress through targeting NF-κB, MAPK, and Nrf2 pathways in RAW264.7 cells.


Assuntos
Catequina/metabolismo , Catequina/farmacologia , Inflamação/tratamento farmacológico , Proantocianidinas/metabolismo , Proantocianidinas/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Citocinas/metabolismo , Dinoprostona/metabolismo , Heme Oxigenase-1/metabolismo , Lipopolissacarídeos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Inibidor de NF-kappaB alfa/metabolismo , NF-kappa B/efeitos dos fármacos , NF-kappa B/metabolismo , Óxido Nítrico/metabolismo , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacos
5.
Mol Cell ; 78(6): 1178-1191.e6, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32485148

RESUMO

The RAS-ERK/MAPK (RAS-extracellular signal-regulated kinase/mitogen-activated protein kinase) pathway integrates growth-promoting signals to stimulate cell growth and proliferation, at least in part, through alterations in metabolic gene expression. However, examples of direct and rapid regulation of the metabolic pathways by the RAS-ERK pathway remain elusive. We find that physiological and oncogenic ERK signaling activation leads to acute metabolic flux stimulation through the de novo purine synthesis pathway, thereby increasing building block availability for RNA and DNA synthesis, which is required for cell growth and proliferation. We demonstrate that ERK2, but not ERK1, phosphorylates the purine synthesis enzyme PFAS (phosphoribosylformylglycinamidine synthase) at T619 in cells to stimulate de novo purine synthesis. The expression of nonphosphorylatable PFAS (T619A) decreases purine synthesis, RAS-dependent cancer cell-colony formation, and tumor growth. Thus, ERK2-mediated PFAS phosphorylation facilitates the increase in nucleic acid synthesis required for anabolic cell growth and proliferation.


Assuntos
Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Purinas/biossíntese , Células A549 , Animais , Carbono-Nitrogênio Ligases com Glutamina como Doadora de N-Amida/genética , Ciclo Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HeLa , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Fosforilação , Purinas/metabolismo , Transdução de Sinais/fisiologia , Proteínas ras/metabolismo
6.
Proc Natl Acad Sci U S A ; 117(25): 14139-14149, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32503917

RESUMO

Agonist-activated G protein-coupled receptors (GPCRs) must correctly select from hundreds of potential downstream signaling cascades and effectors. To accomplish this, GPCRs first bind to an intermediary signaling protein, such as G protein or arrestin. These intermediaries initiate signaling cascades that promote the activity of different effectors, including several protein kinases. The relative roles of G proteins versus arrestins in initiating and directing signaling is hotly debated, and it remains unclear how the correct final signaling pathway is chosen given the ready availability of protein partners. Here, we begin to deconvolute the process of signal bias from the dopamine D1 receptor (D1R) by exploring factors that promote the activation of ERK1/2 or Src, the kinases that lead to cell growth and proliferation. We found that ERK1/2 activation involves both arrestin and Gαs, while Src activation depends solely on arrestin. Interestingly, we found that the phosphorylation pattern influences both arrestin and Gαs coupling, suggesting an additional way the cells regulate G protein signaling. The phosphorylation sites in the D1R intracellular loop 3 are particularly important for directing the binding of G protein versus arrestin and for selecting between the activation of ERK1/2 and Src. Collectively, these studies correlate functional outcomes with a physical basis for signaling bias and provide fundamental information on how GPCR signaling is directed.


Assuntos
Receptores de Dopamina D1/metabolismo , Transdução de Sinais , Arrestina/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Domínios Proteicos , Receptores de Dopamina D1/química , Quinases da Família src/metabolismo
7.
Mol Pharmacol ; 98(1): 49-60, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32358164

RESUMO

Negative allosteric modulation of the metabotropic glutamate 5 (mGlu5) receptor has emerged as a potential strategy for the treatment of neurologic disorders. Despite the success in preclinical studies, many mGlu5 negative allosteric modulators (NAMs) that have reached clinical trials failed due to lack of efficacy. In this study, we provide a detailed in vitro pharmacological characterization of nine clinically and preclinically tested NAMs. We evaluated inhibition of l-glutamate-induced signaling with Ca2+ mobilization, inositol monophosphate (IP1) accumulation, extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation, and real-time receptor internalization assays on rat mGlu5 expressed in HEK293A cells. Moreover, we determined association rates (kon) and dissociation rates (koff), as well as NAM affinities with [3H]methoxy-PEPy binding experiments. kon and koff values varied greatly between the nine NAMs (34- and 139-fold, respectively) resulting in long receptor residence times (>400 min) for basimglurant and mavoglurant, medium residence times (10-30 min) for AZD2066, remeglurant, and (RS)-remeglurant, and low residence times (<10 mins) for dipraglurant, F169521, F1699611, and STX107. We found that all NAMs inhibited l-glutamate-induced mGlu5 receptor internalization, generally with a similar potency to IP1 accumulation and ERK1/2 phosphorylation, whereas Ca2+ mobilization was less potently inhibited. Operational model of allosterism analyses revealed that dipraglurant and (RS)-remeglurant were biased toward (affinity) receptor internalization and away (cooperativity) from the ERK1/2 phosphorylation pathway, respectively. Our study is the first to measure mGlu5 NAM binding kinetics and negative allosteric modulation of mGlu5 receptor internalization and adds significant new knowledge about the molecular pharmacology of a diverse range of clinically relevant NAMs. SIGNIFICANCE STATEMENT: The metabotropic glutamate 5 (mGlu5) receptor is important in many brain functions and implicated in several neurological pathologies. Negative allosteric modulators (NAMs) have shown promising results in preclinical models but have so far failed in human clinical trials. Here we provide the most comprehensive and comparative molecular pharmacological study to date of nine preclinically/clinically tested NAMs at the mGlu5 receptor, which is also the first study to measure ligand binding kinetics and negative allosteric modulation of mGlu5 receptor internalization.


Assuntos
Imidazóis/farmacologia , Indóis/farmacologia , Isoxazóis/farmacologia , Piridinas/farmacologia , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Triazóis/farmacologia , Regulação Alostérica/efeitos dos fármacos , Animais , Cálcio/metabolismo , Células HEK293 , Humanos , Imidazóis/química , Indóis/química , Fosfatos de Inositol/metabolismo , Isoxazóis/química , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Estrutura Molecular , Fosforilação/efeitos dos fármacos , Piridinas/química , Ratos , Fatores de Tempo , Triazóis/química
8.
Am J Physiol Endocrinol Metab ; 319(1): E110-E116, 2020 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-32421368

RESUMO

Statins lower cholesterol and risk of cardiovascular disease. Statins can increase blood glucose and risk of new-onset diabetes. It is unclear why statins can have opposing effects on lipids versus glucose. Statins have cholesterol-independent pleiotropic effects that influence both insulin and glucose control. Statin lowering of isoprenoids required for protein prenylation promotes pancreatic ß-cell dysfunction and adipose tissue insulin resistance. Protein prenylation influences immune function and statin-mediated adipose tissue insulin resistance involves the NLR family pyrin domain-containing 3 (NLRP3) inflammasome and IL-1ß. However, the intracellular cues that statins engage to activate the NLRP3 inflammasome and those responsible for IL-1ß-mediated insulin resistance in adipose tissue have not been identified. We hypothesized that stress kinases or components of the insulin signaling pathway mediated statin-induced insulin resistance. We tested the associations of p38, ERK, JNK, phosphatase, and tensin homolog (PTEN), and mTOR in statin-exposed adipose tissue from WT and IL-1ß-/- mice. We found that statins increased phosphorylation of p38 in WT and IL-1ß-/- mice. Statin activation of p38 upstream of IL-1ß led to priming of this NLRP3 inflammasome effector in macrophages. We found that mTORC1 inhibition with low doses of rapamycin (2 or 20 nM) lowered macrophage priming of IL-1ß mRNA and secretion of IL-1ß caused by multiple statins. Rapamycin (20 nM) or the rapalog everolimus (20 nM) prevented atorvastatin-induced lowering of insulin-mediated phosphorylation of Akt in mouse adipose tissue. These results position p38 and mTOR as mediators of statin-induced insulin resistance in adipose tissue and highlight rapalogs as candidates to mitigate the insulin resistance and glycemic side effects of statins.


Assuntos
Atorvastatina/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inflamassomos/efeitos dos fármacos , Resistência à Insulina , Insulina/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/efeitos dos fármacos , Serina-Treonina Quinases TOR/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Inflamassomos/metabolismo , Interleucina-1beta/genética , MAP Quinase Quinase 4/efeitos dos fármacos , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , PTEN Fosfo-Hidrolase/efeitos dos fármacos , PTEN Fosfo-Hidrolase/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
PLoS One ; 15(4): e0222969, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32352958

RESUMO

In inflammatory skin conditions, such as psoriasis, vascular enlargement is associated with endothelial cell proliferation, release of cytokines and adhesion molecule expression. Interleukin (IL)-17A is a pro-inflammatory cytokine mainly secreted by T helper-17 cells that is critically involved in psoriasis pathogenesis. IL-36α, IL-36ß and IL-36γ are also inflammatory cytokines up-regulated in psoriasis and induced by various stimuli, including IL-17A. In this study, we found that human keratinocytes are the main source of IL-36, in particular of IL-36γ. This cytokine was strongly induced by IL-17A and, together with IL-17A, efficiently activated human dermal microvascular endothelial cells (HDMECs), which expressed both IL-17 and IL-36 receptors. Both IL-36γ and IL-17A induced cell proliferation through specific molecular cascades involving ERK1/2 only or ERK1/2, STAT3 and NF-κB, respectively. We highlighted the intense IL-17A- and IL-36γ -dependent interplay between keratinocytes and HDMECs, likely active in the psoriatic lesions and leading to the establishment of a cytokine network responsible for the development and maintenance of the inflamed state. IL-17A or IL-36γ showed in HDMECs a synergic activity with TNF-α by potently inducing inflammatory cytokine/chemokine release and ICAM-1 expression. We also investigated the involvement of IL-36γ and VEGF-A, substantially reduced in lesional skin of psoriatic patients pharmacologically treated with the anti-IL-17A antibody Secukinumab. Importantly, keratinocyte-derived IL-36γ represented an additional pro-angiogenic mediator of IL-17A. We observed that keratinocyte-derived VEGF-A influenced proliferation but did not act on expression of adhesion molecules in HDMECs. On the other hand, inhibition of IL-36γ released by IL-17A-treated keratinocytes impaired either proliferation or ICAM-1 expression both in HDMECs and in an in vivo murine model of psoriasis. Taken together, our data demonstrated that IL-17A and IL-36γ are highly involved in endothelial cells/keratinocytes crosstalk in inflammatory skin conditions.


Assuntos
Comunicação Celular , Células Endoteliais/metabolismo , Interleucina-17/metabolismo , Interleucina-1/metabolismo , Queratinócitos/metabolismo , Psoríase/metabolismo , Células Cultivadas , Humanos , Molécula 1 de Adesão Intercelular/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fator de Transcrição STAT3/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
10.
Virus Res ; 283: 197961, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32283129

RESUMO

Porcine deltacoronavirus (PDCoV) is a newly emerged swine coronavirus that causes acute enteritis in neonatal piglets. To date, little is known about the host factors or cellular signaling mechanisms associated with PDCoV replication. Since the Raf/MEK/ERK pathway is involved in modulation of various important cellular functions, numerous DNA and RNA viruses coopt this pathway for efficient propagation. In the present study, we found that PDCoV induces the activation of ERK1/2 and its downstream substrate Elk-1 early in infection irrespective of viral biosynthesis. Chemical inhibition or knockdown of ERK1/2 significantly suppressed viral replication, whereas treatment with an ERK activator increased viral yields. Direct pharmacological inhibition of ERK activation had no effect on the viral entry process but sequentially affected the post-entry steps of the virus life cycle. In addition, pharmacological sequestration of cellular or viral cholesterol downregulated PDCoV-induced ERK signaling, highlighting the significance of the cholesterol contents in ERK activation. However, ERK inhibition had no effect on PDCoV-triggered apoptosis through activation of the cytochrome c-mediated intrinsic mitochondrial pathway, suggesting the irrelevance of ERK activation to the apoptosis pathway during PDCoV infection. Altogether, our findings indicate that the ERK signaling pathway plays a pivotal role in viral biosynthesis to facilitate the optimal replication of PDCoV.


Assuntos
Infecções por Coronavirus/metabolismo , Coronavirus/fisiologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Replicação Viral , Transporte Ativo do Núcleo Celular , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Colesterol/metabolismo , Infecções por Coronavirus/virologia , Interações Hospedeiro-Patógeno , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Fosforilação , Transdução de Sinais , Suínos , Proteínas Elk-1 do Domínio ets/metabolismo
11.
Nat Commun ; 11(1): 1943, 2020 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-32327648

RESUMO

Kidney fibrosis is a highly deleterious process and a final manifestation of chronic kidney disease. Alpha-(α)-synuclein (SNCA) is an actin-binding neuronal protein with various functions within the brain; however, its role in other tissues is unknown. Here, we describe the expression of SNCA in renal epithelial cells and demonstrate its decrease in renal tubules of murine and human fibrotic kidneys, as well as its downregulation in renal proximal tubular epithelial cells (RPTECs) after TGF-ß1 treatment. shRNA-mediated knockdown of SNCA in RPTECs results in de novo expression of vimentin and α-SMA, while SNCA overexpression represses TGF-ß1-induced mesenchymal markers. Conditional gene silencing of SNCA in RPTECs leads to an exacerbated tubulointerstitial fibrosis (TIF) in two unrelated in vivo fibrotic models, which is associated with an increased activation of MAPK-p38 and PI3K-Akt pathways. Our study provides an evidence that disruption of SNCA signaling in RPTECs contributes to the pathogenesis of renal TIF by facilitating partial epithelial-to-mesenchymal transition and extracellular matrix accumulation.


Assuntos
Nefropatias/patologia , Rim/patologia , alfa-Sinucleína/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Fibrose , Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Rim/metabolismo , Nefropatias/genética , Nefropatias/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Crescimento Transformador beta1/farmacologia , Obstrução Ureteral/genética , Obstrução Ureteral/metabolismo , Obstrução Ureteral/patologia , Vimentina/genética , Vimentina/metabolismo , alfa-Sinucleína/genética , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
Biol Res ; 53(1): 14, 2020 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293550

RESUMO

BACKGROUND: Previous studies have shown that long noncoding RNA (lncRNA) LINC00483 was aberrantly expressed in human cancers, including gastric cancer. However, the regulatory mechanism of this lncRNA in gastric cancer remains largely unknown. The present study aimed to investigate the effect of LINC00483 on gastric cancer development and explore the potential regulatory network of LINC00483/microRNA (miR)-490-3p/mitogen-activated protein kinase 1 (MAPK1). METHODS: Thirty patients with gastric cancer were recruited for tissues collection. The expression levels of LINC00483, miR-490-3p and MAPK1 were detected by quantitative real-time polymerase chain reaction or western blot. Cell viability, apoptosis, migration and invasion were determined by MTT, flow cytometry, transwell assays and western blot, respectively. The target association between miR-490-3p and LINC00483 or MAPK1 was confirmed by luciferase reporter assay. Xenograft model was established to assess the function of LINC00483 in vivo. RESULTS: LINC00483 and MAPK1 levels were increased in gastric cancer tissues and cells. Knockdown of LINC00483 or MAPK1 inhibited cells viability, migration and invasion but promoted apoptosis in gastric cancer cells. Moreover, MAPK1 overexpression attenuated the effect of LINC00483 knockdown on gastric cancer development. LINC00483 could increase MAPK1 expression by competitively sponging miR-490-3p. miR-490-3p overexpression suppressed gastric cancer development, which was abated by introduction of LINC00483. Besides, inhibition of LINC00483 decreased xenograft tumor growth by regulating miR-490-3p/MAPK1 axis. CONCLUSION: Knockdown of LINC00483 inhibited gastric cancer development in vitro and in vivo by increasing miR-490-3p and decreasing MAPK1, elucidating a novel mechanism for understanding the development of gastric cancer.


Assuntos
MicroRNAs/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , RNA Longo não Codificante/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Apoptose , Carcinogênese/metabolismo , Linhagem Celular Tumoral/metabolismo , Movimento Celular , Sobrevivência Celular , Células Epiteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Luciferases/metabolismo , Masculino , Camundongos Endogâmicos BALB C , MicroRNAs/genética , RNA Longo não Codificante/genética , Neoplasias Gástricas/genética , Ensaios Antitumorais Modelo de Xenoenxerto
13.
Acta Diabetol ; 57(8): 947-958, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32130518

RESUMO

AIMS: Excessive glucose serum concentration, endothelial dysfunction and microangiopathy are key features of diabetes mellitus, being both diagnostic parameters and pathogenetic mechanisms. Vascular endothelial growth factor (VEGF) is importantly implicated in the physiology and pathology of blood vessels, including diabetic vascular damage. METHODS: These factors certainly affect endothelial cells, and to evaluate mechanisms involved, we took advantage of telomerase-immortalized human microvascular endothelial (TIME) cells. TIME cells were exposed to different glucose concentrations and to VEGF treatments. Culture conditions also included the use of basement membrane extract, as an in vitro differentiation model. Cell morphology was then evaluated in the different conditions, and cellular proteins were extracted to analyze specific protein products by Western blot. RESULTS: High glucose concentrations and VEGF did substantially affect neither morphology nor growth of cultured TIME cells, while both considerably increased differentiation into "capillary-like" structures when cells were cultured on basement membrane extract. CONCLUSIONS: Under these conditions, high glucose concentration and VEGF also produced a short-term increase in pERK1/2 and p85 proteins, while total and phosphorylated AKT were not affected. These data suggest a direct angiogenetic effect of glucose, affecting intracellular transduction mechanisms with an action similar to that of VEGF. This effect on endothelial cell proliferation and differentiation could be part of pathogenetic mechanisms producing diabetic microvascular alterations.


Assuntos
Indutores da Angiogênese/metabolismo , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Glucose/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fator A de Crescimento do Endotélio Vascular/farmacologia , Indutores da Angiogênese/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Fosforilação/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos
14.
Mol Carcinog ; 59(5): 520-532, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32134157

RESUMO

Glioblastoma (GBM) is the most common and malignant brain tumor in adults. Recently, programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) checkpoint blockades have been applied for GBM treatment. However, the mechanism of PD-L1 upregulation in GBM is still unclear. COP9 signalosome 6 (CSN6) is crucial for maintaining the protein stabilization in cancer cells. In this study, we applied human GBM specimens and cell lines to investigate whether the EGFR-ERK pathway regulates CSN6 for PD-L1 upregulation. Data from The Cancer Genome Atlas dataset showed that high expression of EGFR, CSN6, and PD-L1 in patients with glioma was associated with poor prognosis. In 47 human GBM specimens, high expression of PD-L1 was associated with low amount of CD8+ T cell infiltration as well as the poor prognosis of patients. CSN6 was positively correlated with EGFR and PD-L1 expression in human GBM specimens. We treated two GBM cell lines (U87 and U251) with epidermal growth factor (EGF) in vitro, and found EGF-upregulated p-EGFR, p-ERK, CSN6, and PD-L1 expression in GBM cells. PD98059, the ERK blocker, inhibited upregulations of CSN6 and PD-L1 in EGF-treated cells. Inhibition of CSN6 by small interfering RNA decreased PD-L1 expression but also increased CHIP expression in GBM cells. When the cells were treated with EGF and cycloheximide (CHX), a protein synthesis inhibitor, EGF-reduced CHX-induced CSN6 and PD-L1 turnover in GBM cells. Furthermore, CSN6-mediated downregulation of PD-L1 was inhibited by MG132, a proteasome inhibitor in U87 cells. Thus, these results suggest that the EGFR-ERK pathway may upregulate CSN6, which may inhibit PD-L1 degradation and subsequently maintain PD-L1 stability in GBM.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígeno B7-H1/metabolismo , Biomarcadores Tumorais/metabolismo , Complexo do Signalossomo COP9/metabolismo , Glioblastoma/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Idoso , Apoptose , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Movimento Celular , Proliferação de Células , Receptores ErbB/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Humanos , Masculino , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
15.
J Oleo Sci ; 69(3): 255-260, 2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32051357

RESUMO

Lysophosphatidylcholine (lysoPtdCho) is produced by the phospholipase A2-mediated hydrolysis of phosphatidylcholine and can stimulate proliferation and apoptosis of vascular smooth muscle cells. We examined the influence of fetal bovine serum (FBS) concentration in the culture medium on lysoPtdCho-mediated apoptosis and proliferation of human aortic smooth muscle cells (HASMCs) as well as on the activation of extracellular signal-regulated kinases (ERK)1/2. In the presence of 1% FBS, HASMC viability increased after lysoPtdCho treatment at 1 and 10 µM but decreased at 25 and 50 µM. However, lysoPtdCho increased HASMC viability in a dose-dependent manner in the presence of 10% FBS. The activity of caspase 3/7 in HASMCs was increased by 25 µM lysoPtdCho in the presence of 1% FBS, but not 10% FBS. Furthermore, lysoPtdCho at 1 and 10 µM triggered ERK1/2 phosphorylation in the presence of 1% FBS, but not at 10% FBS. Thus, lysoPtdCho-mediated HASMC apoptosis, proliferation, and ERK1/2 activation are dependent on the concentration of FBS.


Assuntos
Aorta/citologia , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Lisofosfatidilcolinas/farmacologia , Músculo Liso Vascular/citologia , Soro/fisiologia , Animais , Bovinos , Células Cultivadas , Ativação Enzimática , Humanos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo
16.
Sci Adv ; 6(4): eaay9819, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-32010791

RESUMO

Disassembly of intercellular junctions is a hallmark of epithelial-mesenchymal transition (EMT). However, how the junctions disassemble remains largely unknown. Here, we report that E3 ubiquitin ligase Smurf1 targets p120-catenin, a core component of adherens junction (AJ) complex, for monoubiquitination during transforming growth factor ß (TGFß)-induced EMT, thereby leading to AJ dissociation. Upon TGFß treatment, activated extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylates T900 of p120-catenin to promote its interaction with Smurf1 and subsequent monoubiquitination. Inhibition of T900 phosphorylation or ubiquitination of p120-catenin abrogates TGFß-induced AJ dissociation and consequent tight junction (TJ) dissociation and cytoskeleton rearrangement, hence markedly blocking lung metastasis of murine breast cancer. Moreover, the T900 phosphorylation level of p120-catenin is positively correlated with malignancy of human breast cancer. Hence, our study reveals the underlying mechanism by which TGFß induces dissociation of AJs during EMT and provides a potential strategy to block tumor metastasis.


Assuntos
Cateninas/metabolismo , Transição Epitelial-Mesenquimal , Neoplasias/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Junções Aderentes , Animais , Neoplasias da Mama/etiologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Células Cultivadas , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neoplasias/etiologia , Neoplasias/patologia , Fosforilação , Fator de Crescimento Transformador beta/farmacologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
17.
Mol Cell Biochem ; 466(1-2): 55-63, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32052247

RESUMO

Rap2B, belonging to the Ras superfamily of small guanosine triphosphate-binding proteins, is upregulated and contributes to the progression of several tumors by acting as an oncogene, including hepatocellular carcinoma (HCC). However, the mechanism underlying the functional roles of Rap2B in HCC remains unclear. In this study, the evaluation of Rap2B expression in HCC cells and tissues was achieved by qRT-PCR and western blot assays. The effects of Rap2B on the malignant biological behaviors in HCC were explored by means of MTT assay, flow cytometry analysis, and Transwell invasion assay, respectively. Protein levels of Ki67, matrix metalloproteinase (MMP)-2, MMP-9, and cleaved caspase-3, together with the alternations of the ERK1/2 and PTEN/PI3K/Akt pathways were qualified by western blot assay. Further verification of the Rap2B function on HCC tumorigenesis was attained by performing in vivo assays. We found that Rap2B levels were upregulated in HCC tissues and cells. Rap2B silencing led to a reduction of cell-proliferative and invasive abilities, and an increase of apoptosis in HCC cells. In addition, xenograft tumor assay demonstrated that Rap2B silencing repressed HCC xenograft tumor growth in vivo. In addition, we found that Rap2B knockdown significantly inhibited the ERK1/2 and PTEN/PI3K/Akt cascades in HCC cells and xenograft tumor tissues. Together, Rap2B knockdown inhibited HCC-malignant progression, which was involved in inhibiting the ERK1/2 and PTEN/PI3K/Akt pathways. Our findings contribute to understanding of the molecular mechanism of Rap2B in HCC progression.


Assuntos
Carcinoma Hepatocelular/metabolismo , Técnicas de Silenciamento de Genes , Neoplasias Hepáticas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína rab2 de Ligação ao GTP/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Proteínas Proto-Oncogênicas c-akt , Proteína rab2 de Ligação ao GTP/genética
18.
EBioMedicine ; 52: 102635, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32028069

RESUMO

BACKGROUND: The ovulatory dysfunction mechanisms underlying polycystic ovary syndrome (PCOS) are not completely understood. There is no effective therapy for PCOS so far. METHODS: We measured the expression of four and a half LIM domain 2 (FHL2) and other related-genes in human granulosa cells (hGCs) from patients with and without PCOS. To minimise the heterogeneity of patients with PCOS, we only included PCOS patients meeting all three criteria according to the revised Rotterdam consensus. The in vitro effects of FHL2 on ovulatory genes and the underlying mechanisms were examined in KGN cells. The role of FHL2 in ovulation was investigated in vivo by overexpressing FHL2 in rat ovaries via intrabursal lentivirus injection. FINDINGS: Increased FHL2 and androgen receptor (AR) expression and decreased CCAAT/enhancer-binding protein ß (C/EBPß) expression were observed in hGCs from patients with PCOS. FHL2 inhibited the expression of ovulation-related genes, including phosphorylated ERK1/2, C/EBPß, COX2 and HAS2 in KGN cells. It was partially by interacting with AR to act as its co-regulator to inhibit C/EBPß expression and by binding to ERK1/2 to inhibit its phosphorylation. Moreover, FHL2 abundance in hGCs was positively correlated with the basal serum testosterone concentration of patients with PCOS, and dihydrotestosterone (DHT)-induced FHL2 upregulation was mediated by AR signalling in KGN cells. Additionally, lentiviral-mediated functional FHL2 overexpression in rat ovaries for 1 week contributed to an impaired superovulatory response, displaying decreased numbers of retrieved oocytes and a lower MII oocyte rate. 3-week FHL2 overexpression rat models without superovulation led to acyclicity and polycystic ovary morphology. INTERPRETATION: Our findings provide novel insights into the mechanisms underlying the pathogenesis of PCOS, suggesting that FHL2 could be a potential treatment target for ovulatory obstacles in PCOS. FUND: National Key Research and Development Program of China, National Natural Science Foundation, National Institutes of Health project and Shanghai Commission of Science and Technology.


Assuntos
Regulação da Expressão Gênica , Proteínas com Homeodomínio LIM/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas Musculares/genética , Ovulação/genética , Síndrome do Ovário Policístico/etiologia , Síndrome do Ovário Policístico/metabolismo , Receptores Androgênicos/metabolismo , Fatores de Transcrição/genética , Animais , Biomarcadores , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Modelos Animais de Doenças , Feminino , Imunofluorescência , Humanos , Proteínas com Homeodomínio LIM/metabolismo , Sistema de Sinalização das MAP Quinases , Proteínas Musculares/metabolismo , Ligação Proteica , Ratos , Receptores Androgênicos/genética , Fatores de Transcrição/metabolismo
19.
Mol Biol Rep ; 47(3): 2023-2034, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32030599

RESUMO

The purpose of the current study was to examine the neuroprotective effect of rutin against colistin-induced neurotoxicity in rats. Thirty-five male Sprague Dawley rats were randomly divided into 5 groups. The control group (orally received physiological saline), the rutin group (orally administered 100 mg/kg body weight), the colistin group (i.p. administered 15 mg/kg body weight), the Col + Rut 50 group (i.p. administered 15 mg/kg body weight of colistin, and orally received 50 mg/kg body weight of rutin), the Col + Rut 100 group (i.p. administered 15 mg/kg body weight of colistin, and orally received 100 mg/kg body weight of rutin). Administration of colistin increased levels of glial fibrillary acidic protein and brain-derived neurotrophic factor and acetylcholinesterase and butyrylcholinesterase activities while decreasing level of cyclic AMP response element binding protein and extracellular signal regulated kinases 1 and 2 (ERK1/2) expressions. Colistin increased oxidative impairments as evidenced by a decrease in level of nuclear factor erythroid 2-related factor 2 (Nrf-2), glutathione, superoxide dismutase, glutathione peroxidase and catalase activities, and increased malondialdehyde content. Colistin also increased the levels of the apoptotic and inflammatoric parameters such as cysteine aspartate specific protease-3 (caspase-3), p53, B-cell lymphoma-2 (Bcl-2), nuclear factor kappa B (NF-κB), Bcl-2 associated X protein (Bax), tumor necrosis factor-α (TNF-α) and neuronal nitric oxide synthase (nNOS). Rutin treatment restored the brain function by attenuating colistin-induced oxidative stress, apoptosis, inflammation, histopathological and immunohistochemical alteration suggesting that rutin supplementation mitigated colistin-induced neurotoxicity in male rats.


Assuntos
Apoptose/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/genética , Colistina/farmacologia , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Inflamação/etiologia , Inflamação/metabolismo , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Rutina/farmacologia , Animais , Biomarcadores , Encéfalo/metabolismo , Encéfalo/patologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Colistina/efeitos adversos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Imuno-Histoquímica , Inflamação/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ratos
20.
Cardiovasc Drugs Ther ; 34(1): 3-14, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32103377

RESUMO

PURPOSE: We investigated whether increased expression of activated mitogen-activated protein kinase (MAPK) kinases 1 (MEK1) restores ischemic post-conditioning (IPostC) protection in hypertrophic myocardium following ischemia/reperfusion (I/R) injury. METHODS: C57Bl/6 mice received recombinant adeno-associated virus type 9 (rAAV9)-mediated activated MEK1 gene delivery systemically, then following the induction of cardiac hypertrophy via transverse aortic constriction for 4 weeks. In a Langendorff model, hypertrophic hearts were subjected to 40 min/60 min I/R or with IPostC intervention consisting of 6 cycles of 10 s reperfusion and 10 s no-flow before a 60-min reperfusion. Hemodynamics, infarct size (IS), myocyte apoptosis and changes in expression of reperfusion injury salvage kinase (RISK) pathway were examined. RESULTS: rAAV9-MEK1 gene delivery led to a 4.3-fold and 2.7-fold increase in MEK1 mRNA and protein expression in the heart versus their control values. I/R resulted in a larger IS in hypertrophic than in non-hypertrophic hearts (52.3 ± 4.7% vs. 40.0 ± 2.5%, P < 0.05). IPostC mediated IS reduction in non-hypertrophic hearts (27.6 ± 2.6%, P < 0.05), while it had no significant effect in hypertrophic hearts (46.5 ± 3.1%, P=NS) compared with the IS in non-hypertrophic or hypertrophic hearts subjected to I/R injury only, respectively. Hemodynamic decline induced by I/R was preserved by IPostC in non-hypertrophic hearts but not in hypertrophic hearts. rAAV9-MEK1 gene delivery restored IPostC protection in hypertrophic hearts evidenced by reduced IS (32.0 ± 2.8% vs. 46.5 ± 3.1%) and cardiac cell apoptosis and largely preserved hemodynamic parameters. These protective effects were associated with significantly increased phosphorylation of ERK1/2 and ribosomal protein S6 kinases (p70S6K), but it had no influence on Akt and glycogen synthase kinase-3ß. CONCLUSION: These results demonstrated that rAAV9-mediated activated MEK1 expression restores IPostC protection in the hypertrophic heart against I/R injury through the activation of ERK pathway.


Assuntos
Dependovirus/genética , Técnicas de Transferência de Genes , Vetores Genéticos , Hipertrofia Ventricular Esquerda/terapia , Pós-Condicionamento Isquêmico , MAP Quinase Quinase 1/biossíntese , Infarto do Miocárdio/prevenção & controle , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Miocárdio/enzimologia , Animais , Apoptose , Modelos Animais de Doenças , Indução Enzimática , Hipertrofia Ventricular Esquerda/enzimologia , Hipertrofia Ventricular Esquerda/genética , Hipertrofia Ventricular Esquerda/patologia , Preparação de Coração Isolado , MAP Quinase Quinase 1/genética , Masculino , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Infarto do Miocárdio/enzimologia , Infarto do Miocárdio/genética , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/genética , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Fosforilação , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo
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