RESUMO
Pleiotrophin (PTN) is a secreted growth factor recently proposed to act as a neuromodulatory peptide in the Central Nervous System. PTN appears to be involved in neurodegenerative diseases and neural disorders, and it has also been implicated in learning and memory. Specifically, PTN-deficient mice exhibit a lower threshold for LTP induction in the hippocampus, which is attenuated in mice overexpressing PTN. However, there is little information about the signaling systems recruited by PTN to modulate neural activity. To address this issue, the gene expression profile in hippocampus of mice lacking PTN was analyzed using microarrays of 22,000 genes. In addition, we corroborated the effect of the absence of PTN on the expression of these genes by silencing this growth factor in primary neuronal cultures in vitro. The microarray analysis identified 102 genes that are differentially expressed (z-score>3.0) in PTN null mice, and the expression of eight of those modified in the hippocampus of KO mice was also modified in vitro after silencing PTN in cultured neurons with siRNAs. The data obtained indicate that the absence of PTN affects AKT pathway response and modulates the expression of genes related with neuroprotection (Mgst3 and Estrogen receptor 1, Ers 1) and cell differentiation (Caspase 6, Nestin, and Odz4), both in vivo and in vitro.
Assuntos
Proteínas de Transporte/metabolismo , Cerebelo/metabolismo , Citocinas/metabolismo , Hipocampo/metabolismo , Neurônios/metabolismo , Transcriptoma , Animais , Proteínas de Transporte/genética , Caspase 6/genética , Caspase 6/metabolismo , Células Cultivadas , Cerebelo/citologia , Citocinas/deficiência , Citocinas/genética , Hipocampo/citologia , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Ribossômico 18S/genética , RNA Ribossômico 18S/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Sinaptofisina/genética , Sinaptofisina/metabolismoRESUMO
SCOPE: This study evaluated the capacity of dietary catechin (C), quercetin (Q), and the combination of both (CQ), to attenuate adipose inflammation triggered by high fructose (HFr) consumption in rats and by tumor necrosis factor alpha (TNF-α) in 3T3-L1 adipocytes. METHODS AND RESULTS: In rats, HFr consumption for 6 wk caused dyslipidemia, insulin resistance, reduced plasma adiponectin, adiposity, and adipose tissue inflammation. Dietary supplementation with 20 mg/kg/day of C, Q, and CQ improved all these parameters. In 3T3-L1 adipocytes, C and Q attenuated TNF-α-induced elevated protein carbonyls, increased proinflammatory cytokine expression (MCP-1, resistin), and decreased adiponectin. The protective effects of C and Q on adipose inflammation are in part associated with their capacity to (i) decrease the activation of the mitogen-activated kinases (MAPKs) JNK and p38; and (ii) prevent the downregulation of PPAR-γ. In summary, C and Q, and to a larger extent the combination of both, attenuated adipose proinflammatory signaling cascades and regulated the balance of molecules that improve (adiponectin) or impair (TNF-α, MCP-1, resistin) insulin sensitivity. CONCLUSION: Together, these findings suggest that dietary Q and C may have potential benefits in mitigating MetS-associated adipose inflammation, oxidative stress, and insulin resistance.
Assuntos
Adipócitos/efeitos dos fármacos , Catequina/farmacologia , Frutose/efeitos adversos , Inflamação/tratamento farmacológico , Quercetina/farmacologia , Células 3T3-L1 , Adiponectina/genética , Adiponectina/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Regulação para Baixo , Inflamação/induzido quimicamente , Resistência à Insulina , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Ratos , Resistina/genética , Resistina/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Inhibition of basal Jun kinase (JNK) activity by small interfering RNAs (siRNAs) enhances cisplatin sensitivity and decreases DNA repair in T98G glioblastoma cells. Although the JNK pathway has been extensively studied in recent years, little is known concerning the signaling pathways that control their expression in glioma cells. The aim of the present study was to assess the role of c-Jun-NH2-terminal kinases (JNKs) in the regulation of T98G glioblastoma cells treated with cisplatin in the presence or absence of siRNAs against JNK1 and JNK2. Addition of either small interfering JNK1-siRNA or JNK2-siRNA induced decreased DNA repair and sensitized the T98G glioblastoma cells to the DNA damaging drug cisplatin (cis-diamminedichloroplatinum). This effect was associated with reduced cell survival and loss of anchorageindependent colony formation. The results indicate that effective inhibition of the JNK pathway significantly sensitizes glioblastoma cells to cisplatin, a compound of proven clinical value whose spectrum of application is limited by resistance phenomena.
Assuntos
Neoplasias Encefálicas/tratamento farmacológico , Cisplatino/farmacologia , Reparo do DNA/efeitos dos fármacos , Glioblastoma/tratamento farmacológico , Proteínas Quinases JNK Ativadas por Mitógeno/genética , RNA Interferente Pequeno/farmacologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Reparo do DNA/genética , Técnicas de Silenciamento de Genes , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , MAP Quinase Quinase 7/genética , MAP Quinase Quinase 7/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , RNA Interferente Pequeno/genética , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The plant Cecropia pachystachya Trécul has been used in Brazilian folk medicine to treat hypertension, bladder and kidney inflammation and renal diseases. The aim of this study was to evaluate the potential of the aqueous fraction from the ethanolic extract of Cecropia pachystachya (FCP) in the management of hypertension, inflammation and progressive renal disease in rats submitted to 5/6 nephrectomy. MATERIALS AND METHODS: Thirty male Wistar rats submitted to 5/6 nephrectomy (5/6 NE) were untreated (NE) or treated (NE+FCP) with the FCP (0.5g/kg/day). The treatment started 15 days after surgery, and the rats were followed for a period of 60 days. Systolic blood pressure (SBP) and albuminuria were evaluated from 15-60 days after the surgical procedure. Function and estructural renal changes, TGF-ß (transforming growth factor ß), MCP-1 (monocyte chemoattractant protein-1) and nitric oxide (NO) urinary excretion were analyzed. Expression and activity of the renal enzymes arginase (ARG), angiotensin converting enzyme (ACE), and MAP kinase p-JNK expression also were analyzed. RESULTS: The nephrectomized rats developed progressive albuminuria and increased SBP that was less intense in the treated group. There was a reduction in the glomerular filtration rate (GFR) in the nephrectomized rats, which was attenuated by treatment with FCP extract. The treatment with FCP also attenuated the histological changes, reduced the expression and activity of renal arginase, the number of macrophages (ED-1 positive cells) and the p-JNK expression in the renal cortex of the rats submitted to 5/6 NE. The urinary excretion of TGF-ß was less intense in the treated group and was associated with the reduction of the expression and activity of the renal arginase. CONCLUSIONS: These results suggest that the reduction of renal arginase activity, p-JNK and TGF-ß expression can explain the mechanism by which the treatment with C. pachystachya reduced the inflammation and improved renal function. This study presents the potential use of Cecropia pachystachya in the treatment of chronic renal diseases.
Assuntos
Cecropia/química , Inflamação/tratamento farmacológico , Nefropatias/tratamento farmacológico , Extratos Vegetais/farmacologia , Albuminúria/tratamento farmacológico , Animais , Arginase/metabolismo , Brasil , Progressão da Doença , Taxa de Filtração Glomerular/efeitos dos fármacos , Hipertensão/tratamento farmacológico , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Rim/efeitos dos fármacos , Rim/enzimologia , Rim/fisiopatologia , Nefropatias/enzimologia , Masculino , Medicina Tradicional , Nefrectomia , Ratos , Ratos Wistar , Fator de Crescimento Transformador beta/genéticaRESUMO
Hepatocyte growth factor (HGF) is a protective factor in myocardial injury, but its mechanisms of action have not yet been fully elucidated. Nexilin, which locates specifically to the Z-disc, is a novel Z-disc protein that enables the Z-discs to persistently withstand the extreme mechanical forces generated during muscle contraction. Therefore, we investigated the role of HGF in modulating nexilin expression in hypoxia-reoxygenation (H/R)-treated cardiomyocytes. We cultured neonatal cardiomyocytes and treated them with HGF. The mRNA and protein levels of nexilin were determined by RT-PCR and Western blotting. H/R treatment decreased nexilin mRNA expression and nexilin protein levels in cardiomyocytes. Furthermore, treatment with HGF upregulated nexilin expression and the JNK inhibitor SP600125 partly inhibited HGF-induced nexilin upregulation. In conclusion, our results suggest that ischemia-reperfusion injury may downregulate nexilin expression in cardiomyocytes, and HGF may exert its protective role during myocardial ischemic injury through upregulation of nexilin expression in cardiomyocytes.
Assuntos
Fator de Crescimento de Hepatócito/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas dos Microfilamentos/genética , Miócitos Cardíacos/efeitos dos fármacos , Oxigênio/farmacologia , RNA Mensageiro/genética , Animais , Animais Recém-Nascidos , Antracenos/farmacologia , Butadienos/farmacologia , Hipóxia Celular , Flavonoides/farmacologia , Regulação da Expressão Gênica , Ventrículos do Coração/citologia , Ventrículos do Coração/efeitos dos fármacos , Ventrículos do Coração/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas dos Microfilamentos/agonistas , Proteínas dos Microfilamentos/antagonistas & inibidores , Proteínas dos Microfilamentos/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Nitrilas/farmacologia , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Ratos , Transdução de SinaisRESUMO
Cisplatin has been the most accepted drug for the treatment of ovarian cancer for almost 40 years. Although the majority of patients with ovarian cancer respond to front-line platinum combination chemotherapy, many patients will develop cisplatin-resistance disease, which is extremely rapid and fatal. Although various mechanisms of cisplatin resistance have been postulated, the key molecules involved in such resistance have not been identified. MiRNAs are endogenously expressed small non-coding RNAs, which are evolutionarily conserved and function as post-transcriptional regulators of gene expression. Dysregulation of miRNAs have been associated with cancer initiation, progression and drug resistance. The oncogenic miRNA-21, one of the best-studied miRNAs, is upregulated in almost all human cancers. However, the regulation of miR-21 in cisplatin resistant ovarian cancer cells has not been assessed. In this study, we measured the miR-21 expression by real-time PCR and found upregulation of miR-21 in cisplatin resistant compared with cisplatin sensitive ovarian cancer cells. Chromatin immunoprecipitation studies demonstrated the association of the c-Jun transcription factor to the pri-mir-21 DNA promoter regions. Blocking the JNK-1, the major activator of c-Jun phosphorylation, reduced the expression of pre-mir-21 and increased the expression of its well-known target gene, PDCD4. Overexpression of miR-21 in cisplatin sensitive cells decreased PDCD4 levels and increased cell proliferation. Finally, targeting miR-21 reduced cell growth, proliferation and invasion of cisplatin resistant ovarian cancer cells. These results suggest that the JNK-1/c-Jun/miR-21 pathway contributes to the cisplatin resistance of ovarian cancer cells and demonstrated that miR-21 is a plausible target to overcome cisplatin resistance.
Assuntos
Antineoplásicos/farmacologia , Proteínas Reguladoras de Apoptose/metabolismo , Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Neoplasias Ovarianas/genética , Proteínas de Ligação a RNA/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/genética , Western Blotting , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Imunoprecipitação da Cromatina , Feminino , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteína Quinase 8 Ativada por Mitógeno/genética , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/patologia , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
The p21Waf1/Cip1 protein (hereafter, p21) and the cJun N-terminal kinase (JNK) are two well-characterized cell modulators that play a crucial role in cell differentiation, senescence and apoptosis. Here, we report that transcription of the p21Waf1/Cip1 and JNK-1 genes is affected by inhibition of the early growth response-1 (Egr-1) in response to a small interfering RNA [siRNA)-Egr-1] in LNCaP and PC-3 prostate carcinoma cell lines. The expression levels of protein were determined by western blotting, and apoptosis was measured by propidium iodide staining and flow cytometric analysis. Inhibition of Egr-1, p21 and JNK-1 was carried out by siRNAs. LNCaP and PC-3 cells exhibited readily detectable Egr-1, JNK and p21, even in low serum medium without the addition of other exogenous agents. The expression of Egr-1, p21 and JNK was strongly increased after treatment of the cells with TPA, tumor necrosis factor-α (TNF-α) or arsenite. Suppression of Egr-1 expression by siRNA abrogated the ability of TPA to induce Egr-1 and JNK-1 activities, moderately increasing the p21 activity and abrogating the anti-apoptotic effect of Egr-1 observed in the prostate cancer cell lines. Moreover, blockade of p21 and JNK was unable to decrease the activity of Egr-1, while siRNA against p21 abrogated the proapoptotic effect of p21. The results demonstrated that Egr-1 acts as a key player in prostate tumor cell growth and survival, while p21 plays a key proapoptotic role in LNCaP and PC-3 prostate carcinoma cell lines.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/genética , Proteína 1 de Resposta de Crescimento Precoce/antagonistas & inibidores , Proteína 1 de Resposta de Crescimento Precoce/genética , Expressão Gênica/genética , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Neoplasias da Próstata/genética , RNA Interferente Pequeno/genética , Apoptose/genética , Linhagem Celular Tumoral , Humanos , Masculino , Transcrição Gênica/genéticaRESUMO
OBJECTIVE: The double-stranded RNA-dependent protein kinase (PKR) was recently implicated in regulating molecular integration of nutrient- and pathogen-sensing pathways in obese mice. However, its modulation in human tissues in situations of insulin resistance has not been investigated. The present study was performed to first determine the tissue expression and phosphorylation levels of PKR in the liver, muscle, and adipose tissue in obese humans, and also the modulation of this protein in the adipose tissue of obese patients after bariatric surgery. DESIGN AND METHODS: Eleven obese subjects who were scheduled to undergo Roux-en-Y Gastric Bypass Procedure participated in this study. Nine apparently healthy lean subjects as a control group were also included. RESULTS: Our data show that PKR is activated in liver, muscle, and adipose tissue of obese humans and, after bariatric surgery, there is a clear reduction in PKR activation accompanied by a decrease in protein kinase-like endoplasmic reticulum kinase, c-Jun N-terminal kinase, inhibitor of kappa ß kinase, and insulin receptor substrate-1 serine 312 phosphorylation in subcutaneous adipose tissue from these patients. CONCLUSION: Thus, it is proposed that PKR is an important mediator of obesity-induced insulin resistance and a potential target for the therapy.
Assuntos
Resistência à Insulina , Obesidade/enzimologia , eIF-2 Quinase/metabolismo , Adulto , Antropometria , Glicemia/metabolismo , Índice de Massa Corporal , Estudos de Casos e Controles , Feminino , Derivação Gástrica , Humanos , Insulina/sangue , Proteínas Substratos do Receptor de Insulina/genética , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Fígado/enzimologia , Masculino , Músculo Esquelético/enzimologia , Obesidade/cirurgia , Fosforilação , Gordura Subcutânea/enzimologia , eIF-2 Quinase/genéticaRESUMO
6-Hydroxydopamine (6-OHDA) is a neurotoxin that generates an experimental model of Parkinson's disease in rodents and is commonly employed to induce a lesion in dopaminergic pathways. The characterization of those molecular mechanisms linked to 6-OHDA-induced early toxicity is needed to better understand the cellular events further leading to neurodegeneration. The present work explored how 6-OHDA triggers early downstream signaling pathways that activate neurotoxicity in the rat striatum. Mitochondrial function, caspases-dependent apoptosis, kinases signaling (Akt, ERK 1/2, SAP/JNK and p38) and crosstalk between nuclear factor kappa B (NF-κB) and nuclear factor-erythroid-2-related factor 2 (Nrf2) were evaluated at early times post-lesion. We found that 6-OHDA initiates cell damage via mitochondrial complex I inhibition, cytochrome c and apoptosis-inducing factor (AIF) release, as well as activation of caspases 9 and 3 to induce apoptosis, kinase signaling modulation and NF-κB-mediated inflammatory responses, accompanied by inhibition of antioxidant systems regulated by the Nrf2 pathway. Our results suggest that kinases SAP/JNK and p38 up-regulation may play a role in the early stages of 6-OHDA toxicity to trigger intrinsic pathways for apoptosis and enhanced NF-κB activation. In turn, these cellular events inhibit the activation of cytoprotective mechanisms, thereby leading to a condition of general damage.
Assuntos
Apoptose/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , NF-kappa B/metabolismo , Oxidopamina/toxicidade , Animais , Fator de Indução de Apoptose/metabolismo , Caspase 3/metabolismo , Caspase 9/metabolismo , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/patologia , Citocromos c/metabolismo , Inflamação/induzido quimicamente , Inflamação/patologia , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Masculino , Mitocôndrias/patologia , Síndromes Neurotóxicas/etiologia , Síndromes Neurotóxicas/patologia , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
We investigated whether quercetin protects from steatosis and limits the expression of proinflammatory and fibrogenic genes in C57BL/6J mice with nonalcoholic steatohepatitis (NASH) induced by feeding a methionine-choline-deficient (MCD) diet. Quercetin (50 mg/kg) was given by oral route daily. Mice were randomly divided into 4 groups that received for 2 or 4 wk: the control diet plus vehicle, control diet plus quercetin, MCD diet plus vehicle, and MCD diet plus quercetin. At both 2 and 4 wk, feeding the MCD diet resulted in liver steatosis, inflammatory cell accumulation, oxidative stress evaluated by the concentration of TBARS, and fibrosis evidenced by the staining of α-smooth muscle actin-positive cells in the liver. At both 2 and 4 wk, the MCD diet induced an increase in the mRNA levels of Il6, Tnf, Ptgs2, and Hmgb1 and increased the protein concentrations of Toll-like receptor-4, c-Jun terminal kinase, and p65 NFκB subunit compared with control rats. Feeding the mice the MCD diet also triggered an increase of Col1a1, Col3a1, Plod3, Tgfb1, Smad3, Smad7, Pdgfb, Ctgf, Areg, Mmp9, and Timp1 mRNA levels. These effects were totally or partially prevented by treatment with quercetin. The data obtained suggest that attenuation of multiple profibrotic and proinflammatory gene pathways contributes to the beneficial effects of quercetin in mice with MCD diet-induced steatohepatitis.