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1.
Molecules ; 26(20)2021 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-34684708

RESUMO

Elk-1 is a transcription factor that binds together with a dimer of the serum response factor (SRF) to the serum-response element (SRE), a genetic element that connects cellular stimulation with gene transcription. Elk-1 plays an important role in the regulation of cellular proliferation and apoptosis, thymocyte development, glucose homeostasis and brain function. The biological function of Elk-1 relies essentially on the interaction with other proteins. Elk-1 binds to SRF and generates a functional ternary complex that is required to activate SRE-mediated gene transcription. Elk-1 is kept in an inactive state under basal conditions via binding of a SUMO-histone deacetylase complex. Phosphorylation by extracellular signal-regulated protein kinase, c-Jun N-terminal protein kinase or p38 upregulates the transcriptional activity of Elk-1, mediated by binding to the mediator of RNA polymerase II transcription (Mediator) and the transcriptional coactivator p300. Strong and extended phosphorylation of Elk-1 attenuates Mediator and p300 recruitment and allows the binding of the mSin3A-histone deacetylase corepressor complex. The subsequent dephosphorylation of Elk-1, catalyzed by the protein phosphatase calcineurin, facilitates the re-SUMOylation of Elk-1, transforming Elk-1 back to a transcriptionally inactive state. Thus, numerous protein-protein interactions control the activation cycle of Elk-1 and are essential for its biological function.


Assuntos
Proteínas Elk-1 do Domínio ets/metabolismo , Proteínas Elk-1 do Domínio ets/fisiologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica/genética , Camundongos , Proteínas Nucleares/metabolismo , Fosforilação , Domínios e Motivos de Interação entre Proteínas/fisiologia , Mapeamento de Interação de Proteínas/métodos , Mapas de Interação de Proteínas/fisiologia , Proteínas Proto-Oncogênicas/metabolismo , Fator de Resposta Sérica/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/genética , Ativação Transcricional/genética , Proteínas Elk-1 do Domínio ets/genética
2.
Int J Mol Sci ; 22(19)2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34638857

RESUMO

IL-8/MCP-1 act as neutrophil/monocyte chemoattractants, respectively. Oxidative stress emerges as a key player in the pathophysiology of obesity. However, it remains unclear whether the TNF-α/oxidative stress interplay can trigger IL-8/MCP-1 expression and, if so, by which mechanism(s). IL-8/MCP-1 adipose expression was detected in lean, overweight, and obese individuals, 15 each, using immunohistochemistry. To detect the role of reactive oxygen species (ROS)/TNF-α synergy as a chemokine driver, THP-1 cells were stimulated with TNF-α, with/without H2O2 or hypoxia. Target gene expression was measured by qRT-PCR, proteins by flow cytometry/confocal microscopy, ROS by DCFH-DA assay, and signaling pathways by immunoblotting. IL-8/MCP-1 adipose expression was significantly higher in obese/overweight. Furthermore, IL-8/MCP-1 mRNA/protein was amplified in monocytic cells following stimulation with TNF-α in the presence of H2O2 or hypoxia (p ˂ 0.0001). Synergistic chemokine upregulation was related to the ROS levels, while pre-treatments with NAC suppressed this chemokine elevation (p ≤ 0.01). The ROS/TNF-α crosstalk involved upregulation of CHOP, ERN1, HIF1A, and NF-κB/ERK-1,2 mediated signaling. In conclusion, IL-8/MCP-1 adipose expression is elevated in obesity. Mechanistically, ROS/TNF-α crosstalk may drive expression of these chemokines in monocytic cells by inducing ER stress, HIF1A stabilization, and signaling via NF-κB/ERK-1,2. NAC had inhibitory effect on oxidative stress-driven IL-8/MCP-1 expression, which may have therapeutic significance regarding meta-inflammation.


Assuntos
Quimiocina CCL2/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Peróxido de Hidrogênio/farmacologia , Interleucina-8/genética , Monócitos/efeitos dos fármacos , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Tecido Adiposo/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimiocina CCL2/metabolismo , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-8/metabolismo , Masculino , Pessoa de Meia-Idade , Monócitos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células THP-1
3.
Int J Mol Sci ; 22(19)2021 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-34639236

RESUMO

We analyze the 7,8-dihydroxyflavone (DHF)/TrkB signaling activation of two main intracellular pathways, mitogen-activated protein kinase (MAPK)/ERK and phosphatidylinositol 3 kinase (PI3K)/AKT, in the neuroprotection of axotomized retinal ganglion cells (RGCs). METHODS: Adult albino Sprague-Dawley rats received left intraorbital optic nerve transection (IONT) and were divided in two groups. One group received daily intraperitoneal DHF (5 mg/kg) and another vehicle (1%DMSO in 0.9%NaCl) from one day before IONT until processing. Additional intact rats were employed as control (n = 4). At 1, 3 or 7 days (d) after IONT, phosphorylated (p)AKT, p-MAPK, and non-phosphorylated AKT and MAPK expression levels were analyzed in the retina by Western blotting (n = 4/group). Radial sections were also immunodetected for the above-mentioned proteins, and for Brn3a and vimentin to identify RGCs and Müller cells (MCs), respectively (n = 3/group). RESULTS: IONT induced increased levels of p-MAPK and MAPK at 3d in DHF- or vehicle-treated retinas and at 7d in DHF-treated retinas. IONT induced a fast decrease in AKT in retinas treated with DHF or vehicle, with higher levels of phosphorylation in DHF-treated retinas at 7d. In intact retinas and vehicle-treated groups, no p-MAPK or MAPK expression in RGCs was observed. In DHF- treated retinas p-MAPK and MAPK were expressed in the ganglion cell layer and in the RGC nuclei 3 and 7d after IONT. AKT was observed in intact and axotomized RGCs, but the signal intensity of p-AKT was stronger in DHF-treated retinas. Finally, MCs expressed higher quantities of both MAPK and AKT at 3d in both DHF- and vehicle-treated retinas, and at 7d the phosphorylation of p-MAPK was higher in DHF-treated groups. CONCLUSIONS: Phosphorylation and increased levels of AKT and MAPK through MCs and RGCs in retinas after DHF-treatment may be responsible for the increased and long-lasting RGC protection afforded by DHF after IONT.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Flavonas/farmacologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fármacos Neuroprotetores/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células Ganglionares da Retina/efeitos dos fármacos , Animais , Axotomia , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Regulação da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Proteínas Proto-Oncogênicas c-akt/genética , Ratos , Ratos Sprague-Dawley , Células Ganglionares da Retina/metabolismo
4.
Int J Mol Sci ; 22(19)2021 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-34639043

RESUMO

Studies have shown that bone marrow-derived mesenchymal stem cells (BMSCs) can differentiate into dermal fibroblasts to participate in skin-repairing. However, at present, little is known about how microgravity affects dermal fibroblastic differentiation of BMSCs in space. The aim of this study was to investigate the effect of simulated microgravity (SMG) on the differentiation of BMSCs into dermal fibroblasts and the related molecular mechanism. Here, using a 2D-clinostat device to simulate microgravity, we found that SMG inhibited the differentiation and suppressed the Wnt/ß-catenin signaling and phosphorylation of extracellular regulated protein kinases 1/2 (ERK1/2). After upregulating the Wnt/ß-catenin signaling with lithium chloride (LiCl) treatment, we found that the effect of the differentiation was restored. Moreover, the Wnt/ß-catenin signaling was upregulated when phosphorylation of ERK1/2 was activated with tert-Butylhydroquinone (tBHQ) treatment. Taken together, our findings suggest that SMG inhibits dermal fibroblastic differentiation of BMSCs by suppressing ERK/ß-catenin signaling pathway, inferring that ERK/ß-catenin signaling pathway may act as a potential intervention target for repairing skin injury under microgravity conditions.


Assuntos
Diferenciação Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Ausência de Peso , beta Catenina/metabolismo , Animais , Derme/citologia , Modelos Biológicos , Roedores , Transdução de Sinais
5.
Molecules ; 26(19)2021 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-34641584

RESUMO

Despite its classification as a non-life-threatening disease, increased skin pigmentation adversely affects quality of life and leads to loss of self-confidence. Until now, there are no recommended remedies with high efficacy and human safety for hyperpigmentation. This study aimed to investigate anti-melanogenic activity and underlying mechanism of cajanin, an isoflavonoid extracted from Dalbergia parviflora Roxb. (Leguminosae) in human melanin-producing cells. Culture with 50 µM cajanin for 48-72 h significantly suppressed proliferation in human melanoma MNT1 cells assessed via MTT viability assay. Interestingly, cajanin also efficiently diminished melanin content in MNT1 cells with the half maximum inhibitory concentration (IC50) at 77.47 ± 9.28 µM. Instead of direct inactivating enzymatic function of human tyrosinase, down-regulated mRNA and protein expression levels of MITF and downstream melanogenic enzymes, including tyrosinase, TRP-1 and Dct (TRP-2) were observed in MNT1 cells treated with 50 µM cajanin for 24-72 h. Correspondingly, treatment with cajanin modulated the signaling pathway of CREB and ERK which both regulate MITF expression level. Targeted suppression on MITF-related proteins in human melanin-producing cells strengthens the potential development of cajanin as an effective treatment for human hyperpigmented disorders.


Assuntos
Isoflavonas/farmacologia , Melanoma/tratamento farmacológico , Melanoma/metabolismo , Fator de Transcrição Associado à Microftalmia/efeitos dos fármacos , Fator de Transcrição Associado à Microftalmia/metabolismo , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Dalbergia/química , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Hiperpigmentação/tratamento farmacológico , Interferon Tipo I/metabolismo , Oxirredutases Intramoleculares/metabolismo , Isoflavonas/química , Melaninas/biossíntese , Melanócitos/efeitos dos fármacos , Melanócitos/enzimologia , Melanócitos/metabolismo , Melanoma/enzimologia , Monofenol Mono-Oxigenase/metabolismo , Extratos Vegetais/farmacologia , Proteínas da Gravidez/metabolismo , Qualidade de Vida
6.
J Immunol ; 207(11): 2688-2698, 2021 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-34697226

RESUMO

Regulation of BCR signaling has important consequences for generating effective Ab responses to pathogens and preventing production of autoreactive B cells during development. Currently defined functions of Fc receptor-like (FCRL) 1 include positive regulation of BCR-induced calcium flux, proliferation, and Ab production; however, the mechanistic basis of FCRL1 signaling and its contributions to B cell development remain undefined. Molecular characterization of FCRL1 signaling shows phosphotyrosine-dependent associations with GRB2, GRAP, SHIP-1, and SOS1, all of which can profoundly influence MAPK signaling. In contrast with previous characterizations of FCRL1 as a strictly activating receptor, we discover a role for FCRL1 in suppressing ERK activation under homeostatic and BCR-stimulated conditions in a GRB2-dependent manner. Our analysis of B cells in Fcrl1 -/- mice shows that ERK suppression by FCRL1 is associated with a restriction in the number of cells surviving splenic maturation in vivo. The capacity of FCRL1 to modulate ERK activation presents a potential for FCRL1 to be a regulator of peripheral B cell tolerance, homeostasis, and activation.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/imunologia , Proteína Adaptadora GRB2/imunologia , Proteínas de Membrana/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout
7.
PLoS One ; 16(9): e0256730, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34495991

RESUMO

INTRODUCTION: Recently, electric cigarettes with liquid (e-liquid) were introduced as an alternative to tobacco smoking. They were promoted as possible cessation aids and were considered to be potentially less harmful than traditional tobacco-based cigarettes. However, there is little information on the toxicants present in e-liquids and their possible carcinogenic effects. METHODS: Western blot analysis was performed to identify the protein levels of cancer progression related signal transducers. Patient-derived brain tumor cells (CSC2) were injected into mouse brains and tumor growth was then observed by performing magnetic resonance imaging (MRI) and hematoxylin and eosin (H&E) staining of the whole brain. Immunohistochemistry (IHC) staining and Immunofluorescence staining were performed to study the expression of pEGFR and pERK. RESULTS: Western blotting revealed that e-liquids increased pEGFR and pERK expression in a dose dependent manner. Animal experiments revealed that the e-liquid treated group had accelerated tumor growth and poor prognosis compared to the vehicle group. Histological staining showed activation of pEGFR and pERK in the e-liquid treated group. CONCLUSION: Our study revealed that e-liquid activates pEGFR and pERK, leading to accelerated brain tumor growth and poor prognosis.


Assuntos
Neoplasias Encefálicas/metabolismo , Carcinogênese/efeitos dos fármacos , Sistemas Eletrônicos de Liberação de Nicotina , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glioblastoma/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Nicotina/administração & dosagem , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Neoplasias Encefálicas/patologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Fumar Cigarros/metabolismo , Modelos Animais de Doenças , Receptores ErbB/metabolismo , Feminino , Glioblastoma/patologia , Xenoenxertos/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias/métodos , Fosforilação/efeitos dos fármacos , Prognóstico , Propilenoglicol/administração & dosagem , Soluções , Solventes/administração & dosagem , Carga Tumoral/efeitos dos fármacos
8.
Exp Cell Res ; 407(2): 112826, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34508742

RESUMO

INTS6 (integrator complex subunit 6) has been reported as a tumor suppressor in many cancers. However, the expression and biological function of INTS6 in colorectal cancer (CRC) has not been investigated yet. In this study, we found that INTS6 expression was significantly increased in CRC tissues when compared with normal tissues and was associated with poor prognosis. Downregulation of INTS6 induced G1/S-phase cell cycle arrest, and markedly suppressed the growth of CRC cells and the derived tumors, while overexpression of INTS6 showed opposite effect. Mechanism study revealed that INTS6 increased the levels of phosphorylated AKT (p-AKT) and ERK (p-ERK), and the growth-promoting effect of INTS6 was inhibited by AKT and ERK inhibitors. Besides, INTS6 also affected the expression of two targets of PI3K/AKT and MAPK signaling, c-Myc and CDK2, which contributed to cell cycle alteration. Altogether, the present study has revealed the oncogenic role of INTS6 in CRC, providing a novel therapeutic target for this malignant cancer.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , Pontos de Checagem da Fase G1 do Ciclo Celular , Humanos , Masculino , Camundongos , Camundongos Nus , Fosfatidilinositol 3-Quinases/genética , Prognóstico , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas de Ligação a RNA/genética , Taxa de Sobrevida , Células Tumorais Cultivadas , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto
9.
Int J Mol Sci ; 22(17)2021 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-34502316

RESUMO

Farrerol (FA) is a flavanone isolated from the Chinese herbal medicine "Man-shan-hong" (Rhododendron dauricum L.). In the present study, FA decreased the viability of SKOV3 cells in a dose- and time-dependent manner, and it induced G2/M cell cycle arrest and cell apoptosis. Cell cycle distribution analysis via flow cytometry showed that FA decreased G1 populations and increased G2/M populations in SKOV3 cells. Additionally, Western blotting confirmed an increase in the expression level of proteins involved in the cell cycle, e.g., CDK and cyclins. FA-induced apoptosis in SKOV3 cells was also investigated using a TUNEL assay, and increased expression levels of proapoptotic factors, including Caspase-3 and poly ADP ribose polymerase (PARP), through the Extracellular signal-regulated kinase (ERK)/MAPK pathway were investigated. Proinflammatory cytokines (e.g., IL-6, TNF-α, and IL-1) have been identified as a driver of the pathological mechanisms underlying involuntary weight loss and impaired physical function, i.e., cachexia, during cancer; in the present study, we showed that farrerol attenuates TNF-α-induced lipolysis and increases adipogenic differentiation in 3T3-L1 cells. Thus, farrerol could potentially be used as an anticancer agent or anticachetic drug.


Assuntos
Cromonas/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Lipólise/efeitos dos fármacos , Neoplasias Ovarianas/tratamento farmacológico , Fator de Necrose Tumoral alfa/farmacologia , Apoptose , Ciclo Celular , Proliferação de Células , Feminino , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Células Tumorais Cultivadas
10.
Life Sci ; 284: 119931, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34480934

RESUMO

AIMS: Retinal neovascularization is one of the visual disorders during the postmenopausal period or types two diabetes. Physical activities and also phytoestrogens with powerful antioxidant features have been widely considered to improve nervous system diseases. Therefore, this study investigated the effects of genistein, swimming exercise, and their co-treatment on retina angiogenesis, oxidative stress, and inflammation in diabetic-ovariectomized rats. MAIN METHODS: Wistar rats were randomly divided into six groups (n = 8 per group): sham, ovariectomized group (OVX), OVX + diabetes (OVX.D), OVX.D+ genistein (1 mg/kg, eight weeks; daily SC), OVX.D + exercise (eight weeks), and OVX.D+ genistein+exercise (eight weeks). At the end of 8 weeks, the retina was removed under anesthesia. The assessed effects of treatment were by measuring MiR-146a and miR-132 expression via RT-PCR, the protein levels of ERK, MMP-2, VEGF, and NF-κB via western blotting, inflammation, and oxidative stress markers levels via the Eliza. KEY FINDINGS: The results showed miR-132, miR-146b, and MMP-2, NF-κB, ERK, VEGF, TNF-α, IL-1ß proteins, and MDA factor in the OVX.D group were increased, but glutathione (GSH) was decreased in comparison with the sham and OVX groups. Both exercise and genistein treatment has reversed the disorder caused by diabetes. However, the combination of exercise and genistein was more effective than each treatment alone. SIGNIFICANCE: It can be concluded that the interaction of exercise and genistein on microRNAs and their target protein was affected in the inflammation, stress oxidative, and extracellular matrix metalloproteinase pathways, can leading to a decrease in impairment of retinal neovascularization of the ovariectomized diabetic rats.


Assuntos
Diabetes Mellitus Experimental/patologia , Genisteína/farmacologia , Inflamação/patologia , Ovariectomia , Estresse Oxidativo , Retina/patologia , Natação/fisiologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Biomarcadores/metabolismo , Diabetes Mellitus Experimental/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Genisteína/administração & dosagem , Glucose/metabolismo , Glutationa/metabolismo , Homeostase/efeitos dos fármacos , Mediadores da Inflamação/metabolismo , Malondialdeído/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , NF-kappa B/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos Wistar , Retina/efeitos dos fármacos
11.
Nutrients ; 13(9)2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34579029

RESUMO

Ferulic acid (FA) is a polyphenol pertaining to the class of hydroxycinnamic acids present in numerous foods of a plant origin. Its dietary consumption leads to the formation of several phase I and II metabolites in vivo, which represent the largest amount of ferulates in the circulation and in the intestine in comparison with FA itself. In this work, we evaluated their efficacy against the proinflammatory effects induced by lipopolysaccharide (LPS) in intestinal Caco-2 cell monolayers, as well as the mechanisms underlying their protective action. LPS-induced overexpression of proinflammatory enzymes such as inducible nitric oxide synthase (iNOS) and the consequent hyperproduction of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) were limited by physiological relevant concentrations (1 µM) of FA, its derivatives isoferulic acid (IFA) and dihydroferulic acid (DHFA), and their glucuronidated and sulfated metabolites, which acted upstream by limiting the activation of MAPK p38 and ERK and of Akt kinase, thus decreasing the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) translocation into the nucleus. Furthermore, the compounds were found to promote the expression of Nrf2, which may have contributed to the downregulation of NF-ĸB activity. The overall data show that phase I/II metabolites retain the efficacy of their dietary free form in contrasting inflammatory response.


Assuntos
Ácidos Cumáricos/farmacologia , Enterócitos/efeitos dos fármacos , Inflamação/induzido quimicamente , Lipopolissacarídeos/toxicidade , Células CACO-2 , Sobrevivência Celular/efeitos dos fármacos , Ácidos Cumáricos/metabolismo , GMP Cíclico/genética , GMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas I-kappa B/genética , Proteínas I-kappa B/metabolismo , Inflamação/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Óxido Nítrico/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
12.
Int J Mol Sci ; 22(18)2021 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-34576014

RESUMO

Follicle-stimulating hormone receptor (FSHR) plays a key role in reproduction through the activation of multiple signaling pathways. Low molecular weight (LMW) ligands composed of biased agonist properties are highly valuable tools to decipher complex signaling mechanisms as they allow selective activation of discrete signaling cascades. However, available LMW FSHR ligands have not been fully characterized yet. In this context, we explored the pharmacological diversity of three benzamide and two thiazolidinone derivatives compared to FSH. Concentration/activity curves were generated for Gαs, Gαq, Gαi, ß-arrestin 2 recruitment, and cAMP production, using BRET assays in living cells. ERK phosphorylation was analyzed by Western blotting, and CRE-dependent transcription was assessed using a luciferase reporter assay. All assays were done in either wild-type, Gαs or ß-arrestin 1/2 CRISPR knockout HEK293 cells. Bias factors were calculated for each pair of read-outs by using the operational model. Our results show that each ligand presented a discrete pharmacological efficacy compared to FSH, ranging from super-agonist for ß-arrestin 2 recruitment to pure Gαs bias. Interestingly, LMW ligands generated kinetic profiles distinct from FSH (i.e., faster, slower or transient, depending on the ligand) and correlated with CRE-dependent transcription. In addition, clear system biases were observed in cells depleted of either Gαs or ß-arrestin genes. Such LMW properties are useful pharmacological tools to better dissect the multiple signaling pathways activated by FSHR and assess their relative contributions at the cellular and physio-pathological levels.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/farmacologia , Receptores do FSH/agonistas , beta-Arrestina 2/farmacologia , AMP Cíclico/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HEK293 , Humanos , Cinética
13.
Chem Biol Interact ; 349: 109651, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34520753

RESUMO

Selenium is an essential trace element for human, and has anti-tumor effects. In this study, we investigated the anti-tumor activity of sodium selenite (Na2SeO3) and explored its possible mechanisms involved in a breast cancer cell line. We found that Na2SeO3 could inhibit the cell viability of MCF7 cells, yet with minimal damage to human umbilical vein endothelial cells (HUVECs). The results of Hoechst staining and Western Blot showed that Na2SeO3 induced apoptosis of MCF7 cells. Na2SeO3 activated endoplasmic reticulum stress (ERS), as evidenced by the up-regulation of ERS-related proteins, including ATF6, p-eIF2α, ATF4, and CHOP, and the down-regulation of PERK. ATF6, p-eIF2α and apoptosis were decreased by pre-treatment with an ERS inhibitor (4-PBA). Na2SeO3 activated oxidative stress (OS) through increasing ROS generation and decreasing mitochondrial membrane potential (MMP) which induced apoptosis. Pre-treatment with an antioxidant (NAC) attenuated Na2SeO3-induced OS and cell apoptosis. Furthermore, ERS and OS had mutual effects. Pre-treatment with 4-PBA could act against the up-regulation of ROS and the down-regulation of MMP. Pre-treatment with NAC attenuated the expression of ATF6. At the same time, we found that treatment with Na2SeO3 promoted the phosphorylation of p38 and JNK, while inhibiting the phosphorylation of ERK. However, the up-regulation was inhibited after pre-treatment of NAC, and pre-treatment with 4-PBA inhibited the increase only of p38. Based on these results, our study provides a mechanistic understanding of how Na2SeO3 has antitumor effects against MCF7 cells through the OS and ERS pathway. OS and ERS interact with each other, and p38 is regulated by them.


Assuntos
Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Selenito de Sódio/farmacologia , Sobrevivência Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Células MCF-7 , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Fenilbutiratos/farmacologia , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo
14.
Development ; 148(17)2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34473253

RESUMO

The STAT3 transcription factor, acting both in the nucleus and mitochondria, maintains embryonic stem cell pluripotency and promotes their proliferation. In this work, using zebrafish, we determined in vivo that mitochondrial STAT3 regulates mtDNA transcription in embryonic and larval stem cell niches and that this activity affects their proliferation rates. As a result, we demonstrated that import of STAT3 inside mitochondria requires Y705 phosphorylation by Jak, whereas its mitochondrial transcriptional activity, as well as its effect on proliferation, depends on the MAPK target S727. These data were confirmed using mouse embryonic stem cells: although the Y705-mutated STAT3 cannot enter mitochondria, the S727 mutation does not affect import into the organelle and is responsible for STAT3-dependent mitochondrial transcription. Surprisingly, STAT3-dependent increase of mitochondrial transcription appears to be independent from STAT3 binding to STAT3-responsive elements. Finally, loss-of-function experiments, with chemical inhibition of the JAK/STAT3 pathway or genetic ablation of stat3 gene, demonstrated that STAT3 is also required for cell proliferation in the intestine of zebrafish.


Assuntos
Proliferação de Células , Células-Tronco Embrionárias/citologia , Mitocôndrias/metabolismo , Fator de Transcrição STAT3/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Sistema Nervoso Central/embriologia , DNA Mitocondrial/metabolismo , Embrião não Mamífero , Células-Tronco Embrionárias/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Intestinos/embriologia , Janus Quinases/metabolismo , Mutação , Fosforilação , Fator de Transcrição STAT3/genética , Transdução de Sinais , Transcrição Genética , Ativação Transcricional , Peixe-Zebra , Proteínas de Peixe-Zebra/genética
15.
Molecules ; 26(18)2021 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-34577156

RESUMO

DPY19L3 has been identified as a C-mannosyltransferase for thrombospondin type-1 repeat domain-containing proteins. In this study, we focused on the role of DPY19L3 in the myogenic differentiation of C2C12 mouse myoblast cells. We carried out DPY19L3 gene depletion using the CRISPR/Cas9 system. The result showed that these DPY19L3-knockout cells could not be induced for differentiation. Moreover, the phosphorylation levels of MEK/ERK and p70S6K were suppressed in the DPY19L3-knockout cells compared with that of parent cells, suggesting that the protein(s) that is(are) DPY19L3-mediated C-mannosylated and regulate(s) MEK/ERK or p70S6K signaling is(are) required for the differentiation.


Assuntos
Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Manosiltransferases/genética , Manosiltransferases/fisiologia , Mioblastos/fisiologia , Animais , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Técnicas de Silenciamento de Genes , Glicosilação , Manosiltransferases/metabolismo , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mioblastos/citologia , Fosforilação/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo , Transdução de Sinais/genética , Regulação para Cima/genética
16.
Elife ; 102021 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-34569932

RESUMO

Accumulating evidence has shown transcranial low-intensity ultrasound can be potentially a non-invasive neural modulation tool to treat brain diseases. However, the underlying mechanism remains elusive and the majority of studies on animal models applying rather high-intensity ultrasound that cannot be safely used in humans. Here, we showed low-intensity ultrasound was able to activate neurons in the mouse brain and repeated ultrasound stimulation resulted in adult neurogenesis in specific brain regions. In vitro calcium imaging studies showed that a specific ultrasound stimulation mode, which combined with both ultrasound-induced pressure and acoustic streaming mechanotransduction, is required to activate cultured cortical neurons. ASIC1a and cytoskeletal proteins were involved in the low-intensity ultrasound-mediated mechanotransduction and cultured neuron activation, which was inhibited by ASIC1a blockade and cytoskeleton-modified agents. In contrast, the inhibition of mechanical-sensitive channels involved in bilayer-model mechanotransduction like Piezo or TRP proteins did not repress the ultrasound-mediated neuronal activation as efficiently. The ASIC1a-mediated ultrasound effects in mouse brain such as immediate response of ERK phosphorylation and DCX marked neurogenesis were statistically significantly compromised by ASIC1a gene deletion. Collated data suggest that ASIC1a is the molecular determinant involved in the mechano-signaling of low-intensity ultrasound that modulates neural activation in mouse brain.


Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Encéfalo/metabolismo , Mecanotransdução Celular , Neurogênese , Neurônios/metabolismo , Ondas Ultrassônicas , Canais Iônicos Sensíveis a Ácido/genética , Animais , Encéfalo/citologia , Células CHO , Sinalização do Cálcio , Cricetulus , Citoesqueleto/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Neuropeptídeos/metabolismo , Fosforilação , Pressão , Fatores de Tempo
17.
Molecules ; 26(17)2021 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-34500840

RESUMO

Maintaining skin homeostasis is one of the most important factors for skin health. UVB-induced skin photoaging is a difficult problem that has negative impacts on skin homeostasis. So far, a number of compounds have been discovered that improve human skin barrier function and hydration, and are thought to be effective ways to protect skin homeostasis. Potentilla glabra var. mandshurica (Maxim.) Hand.-Mazz. Ethanol Extract (Pg-EE) is a compound that has noteworthy anti-inflammatory properties. However, its skin-protective effects are poorly understood. Therefore, we evaluated the capacity of Pg-EE to strengthen the skin barrier and improve skin hydration. Pg-EE can enhance the expression of filaggrin (FLG), transglutaminase (TGM)-1, hyaluronic acid synthase (HAS)-1, and HAS-2 in human keratinocytes. Moreover, Pg-EE down-regulated the expression of pro-inflammatory cytokines and up-regulated the production of FLG, HAS-1, and HAS-2 suppressed by UVB through inhibition of p38 mitogen-activated protein kinase (MAPK) and extracellular signal-regulated kinase (ERK) pathways. Given the above, since Pg-EE can improve skin barrier, hydration and reduce the UVB-induced inflammation on skin, it could therefore be a valuable natural ingredient for cosmetics or pharmaceuticals to treat skin disorders.


Assuntos
Anti-Inflamatórios/química , Extratos Vegetais/química , Potentilla/química , Protetores contra Radiação/química , Envelhecimento da Pele/efeitos da radiação , Anti-Inflamatórios/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Descoberta de Drogas , Etanol/química , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HaCaT , Humanos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Extratos Vegetais/farmacologia , Protetores contra Radiação/farmacologia , Transdução de Sinais , Superóxido Dismutase-1/metabolismo , Espectrometria de Massas em Tandem , Raios Ultravioleta
18.
Nutrients ; 13(7)2021 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-34371902

RESUMO

There is ongoing debate as to whether or not α-hydroxyisocaproic acid (HICA) positively regulates skeletal muscle protein synthesis resulting in the gain or maintenance of skeletal muscle. We investigated the effects of HICA on mouse C2C12 myotubes under normal conditions and during cachexia induced by co-exposure to TNFα and IFNγ. The phosphorylation of AMPK or ERK1/2 was significantly altered 30 min after HICA treatment under normal conditions. The basal protein synthesis rates measured by a deuterium-labeling method were significantly lowered by the HICA treatment under normal and cachexic conditions. Conversely, myotube atrophy induced by TNFα/IFNγ co-exposure was significantly improved by the HICA pretreatment, and this improvement was accompanied by the inhibition of iNOS expression and IL-6 production. Moreover, HICA also suppressed the TNFα/IFNγ co-exposure-induced secretion of 3-methylhistidine. These results demonstrated that HICA decreases basal protein synthesis under normal or cachexic conditions; however, HICA might attenuate skeletal muscle atrophy via maintaining a low level of protein degradation under cachexic conditions.


Assuntos
Caquexia/tratamento farmacológico , Caproatos/farmacologia , Interferon gama/toxicidade , Interleucina-6/metabolismo , Fibras Musculares Esqueléticas/efeitos dos fármacos , Atrofia Muscular/tratamento farmacológico , Óxido Nítrico Sintase Tipo II/metabolismo , Fator de Necrose Tumoral alfa/toxicidade , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Caquexia/induzido quimicamente , Caquexia/metabolismo , Caquexia/patologia , Linhagem Celular , Regulação para Baixo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Metilistidinas/metabolismo , Camundongos , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/patologia , Atrofia Muscular/induzido quimicamente , Atrofia Muscular/metabolismo , Atrofia Muscular/patologia , Fosforilação , Biossíntese de Proteínas , Proteólise
19.
BMC Musculoskelet Disord ; 22(1): 680, 2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34380439

RESUMO

BACKGROUND: Skeletal muscle atrophy and fibrosis are pathological conditions that contribute to morbidity in numerous conditions including aging, cachexia, and denervation. Muscle atrophy is characterized as reduction of muscle fiber size and loss of muscle mass while muscle fibrosis is due to fibroblasts activation and excessive production of extracellular matrix. Purinergic receptor P2Y2 has been implicated in fibrosis. This study aims to elucidate the roles of P2Y2 in sleketal muscle atrophy and fibrosis. METHODS: Primary muscle fibroblasts were isolated from wild type and P2Y2 knockout (KO) mice and their proliferating and migrating abilities were assessed by CCK-8 and Transwell migration assays respectively. Fibroblasts were activated with TGF-ß1 and assessed by western blot of myofibroblast markers including α-SMA, CTGF, and collagen I. Muscle atrophy and fibrosis were induced by transection of distal sciatic nerve and assessed using Masson staining. RESULTS: P2Y2 KO fibroblasts proliferated and migrated significantly slower than WT fibroblasts with or without TGF-ß1.The proliferation and ECM production were enhanced by P2Y2 agonist PSB-1114 and inhibited by antagonist AR-C118925. TGF-ß1 induced fibrotic activation was abolished by P2Y2 ablation and inhibited by AKT, ERK, and PKC inhibitors. Ablation of P2Y2 reduced denervation induced muscle atrophy and fibrosis. CONCLUSIONS: P2Y2 is a promoter of skeletal muscle atrophy and activation of fibroblasts after muscle injury, which signaling through AKT, ERK and PKC. P2Y2 could be a potential intervention target after muscle injury.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Músculo Esquelético/patologia , Proteína Quinase C/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores Purinérgicos P2Y2/metabolismo , Animais , Células Cultivadas , Fibroblastos/patologia , Fibrose , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fator de Crescimento Transformador beta1/farmacologia
20.
Toxicol Lett ; 351: 65-77, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34454012

RESUMO

Phthalates are plasticizers widely found in the environment. They are potential endocrine disruptors. Bis(2-butoxyethyl) phthalate (BBOP) is a unique phthalate that contains oxygen atoms in the carbon backbone. Little is known about its reproductive and developmental toxicity. The objective of this study was to determine the effect of BBOP on fetal Leydig cell development after in utero exposure to rats. Sprague Dawley pregnant dams were randomly allocated into 6 groups, and were gavaged with BBOP (0, 10, 100, 250, 500, and 1000 mg/kg body weight/day) from gestational day (GD) 14-21. Seven of the 8 dams in the 1000 mg/kg BBOP group died before giving birth. Twelve of the 20 dams in the 500 mg/kg BBOP group had whole litter loss. BBOP significantly reduced the body weight of dams and male offspring and serum testosterone level and anogenital distance of male fetus on GD 21 at 500 mg/kg. BBOP markedly increased fetal Leydig cell proliferation and number at 500 mg/kg while inducing their abnormal aggregation at 250 and 500 mg/kg. BBOP down-regulated the expression of Lhcgr, Scarb1, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, Insl3, and Nr5a1 at various doses while up-regulating the expression of Sertoli cell gene Fshr and Sox9. The phosphorylation of AKT1, AKT2, and ERK1/2 was also markedly reduced by BBOP. In conclusion, BBOP in utero exposure can disrupt fetal Leydig cell development, possibly via the mechanism that may include inhibiting the phosphorylation of AKT1, AKT2, and ERK1/2.


Assuntos
Desenvolvimento Fetal/efeitos dos fármacos , Células Intersticiais do Testículo/efeitos dos fármacos , Ácidos Ftálicos/toxicidade , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Feto , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Masculino , Estrutura Molecular , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos
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