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1.
Nat Commun ; 12(1): 5068, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417460

RESUMO

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.


Assuntos
Acetilglucosamina/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Restrição Calórica , Linhagem Celular , Colforsina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epinefrina/metabolismo , Glucagon/metabolismo , Glucocorticoides/metabolismo , Gluconeogênese/efeitos dos fármacos , Glicosilação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/metabolismo , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
2.
Phytomedicine ; 91: 153701, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34438230

RESUMO

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is characterized by limited airflow due to pulmonary and alveolar abnormalities from exposure to cigarette smoke (CS). Current therapeutic drugs are limited and the development of novel treatments to prevent disease progression is challenging. Isoforskolin (ISOF) from the plant Coleus forskohlii is an effective activator of adenylyl cyclase (AC) isoforms. Previously we found ISOF could attenuate acute lung injury in animal models, while the effect of ISOF on COPD has not been elucidated. PURPOSE: In this study, we aimed to evaluate the efficacy of ISOF on COPD and reveal its potential mechanisms. METHODS: A rat model of COPD was established by long-term exposure to CS, then the rats were orally administered with ISOF (0.5, 1 and 2 mg/kg). The pulmonary function, lung morphology, inflammatory cells and cytokines in serum or bronchoalveolar lavage fluid (BALF) were evaluated. Transcriptomics, proteomics and network pharmacology analysis were utilized to identify potential mechanisms of ISOF. Droplet digital PCR was used to detect the mRNA expression of AC1-10 in donor lung tissues. AC activation was determined in recombinant human embryonic kidney 293 (HEK293) cells stably expressing human AC isoforms. In addition, ISOF caused trachea relaxation ex vivo were assessed in isolated trachea rings from guinea pigs. RESULTS: ISOF significantly ameliorated pathological damage of lung tissue and improved pulmonary function in COPD rats. ISOF treatment decreased the number of inflammatory cells in peripheral blood, and also the levels of pro-inflammatory cytokines in serum and BALF. Consistent with omics-based analyses, ISOF markedly downregulated the mTOR level in lung tissue. Flow cytometry analysis revealed that ISOF treatment reduced the ratio of Th17/Treg cells in peripheral blood. Furthermore, the expression levels of AC1 and AC2 are relatively higher than other AC isoforms in normal lung tissues, and ISOF could potently activate AC1 and AC2 in vitro and significantly relax isolated guinea pig trachea. CONCLUSION: Collectively, our studies suggest that ISOF exerts its anti-COPD effect by improving lung function, anti-inflammation and trachea relaxation, which may be related to AC activation, mTOR signaling and Th17/Treg balance.


Assuntos
Adenilil Ciclases , Colforsina/farmacologia , Doença Pulmonar Obstrutiva Crônica , Fumaça , Animais , Coleus/química , Cobaias , Células HEK293 , Humanos , Compostos Fitoquímicos/farmacologia , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Ratos , Fumaça/efeitos adversos , Fumar
3.
Int J Mol Sci ; 22(12)2021 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-34199197

RESUMO

In the cochlea, non-sensory supporting cells are directly connected to adjacent supporting cells via gap junctions that allow the exchange of small molecules. We have previously shown that the pharmacological regulation of gap junctions alleviates cisplatin (CDDP)-induced ototoxicity in animal models. In this study, we aimed to identify specific small molecules that pass through gap junctions in the process of CDDP-induced auditory cell death and suggest new mechanisms to prevent hearing loss. We found that the cyclic adenosine monophosphate (cAMP) inducer forskolin (FSK) significantly attenuated CDDP-induced auditory cell death in vitro and ex vivo. The activation of cAMP/PKA/CREB signaling was observed in organ of Corti primary cells treated with FSK, especially in supporting cells. Co-treatment with gap junction enhancers such as all-trans retinoic acid (ATRA) and quinoline showed potentiating effects with FSK on cell survival via activation of cAMP/PKA/CREB. In vivo, the combination of FSK and ATRA was more effective for preventing ototoxicity compared to either single treatment. Our study provides the new insight that gap junction-mediated intercellular communication of cAMP may prevent CDDP-induced ototoxicity.


Assuntos
Comunicação Celular , Cisplatino/efeitos adversos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Junções Comunicantes/metabolismo , Ototoxicidade/metabolismo , Transdução de Sinais , Células A549 , Animais , Comunicação Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Colforsina/farmacologia , Colforsina/uso terapêutico , Conexina 26/metabolismo , Junções Comunicantes/efeitos dos fármacos , Células Ciliadas Auditivas/metabolismo , Células HeLa , Perda Auditiva/induzido quimicamente , Perda Auditiva/tratamento farmacológico , Perda Auditiva/prevenção & controle , Humanos , Camundongos , Substâncias Protetoras/farmacologia , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , Gânglio Espiral da Cóclea/efeitos dos fármacos , Gânglio Espiral da Cóclea/patologia , Tretinoína/farmacologia , Tretinoína/uso terapêutico
4.
Cell Death Dis ; 12(7): 635, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34155192

RESUMO

FURIN is a pro-protein convertase previously shown to be important for placental syncytialisation (Zhou et al. [1]), a process of cell fusion whereby placental cytotrophoblast cells fuse to form a multinucleated syncytium. This finding has been broadly accepted however, we have evidence suggesting the contrary. Spontaneously syncytialising term primary human trophoblast cells and BeWo choriocarcinoma cells were treated with either FURIN siRNA or negative control siRNA or the protease inhibitor, DEC-RVKR-CMK, or vehicle. Cells were then left to either spontaneously syncytialise (primary trophoblasts) or were induced to syncytialise with forskolin (BeWo). Effects on syncytialisation were measured by determining human chorionic gonadotrophin secretion and E-cadherin protein levels. We showed that FURIN is not important for syncytialisation in either cell type. However, in primary trophoblasts another protease also inhibited by DEC-RVKR-CMK, may be involved. Our results directly contrast with those published by Zhou et al. Zhou et al. however, used first trimester villous explants to study syncytialisation, and we used term primary trophoblasts. Therefore, we suggest that FURIN may be involved in syncytialisation of first trimester trophoblasts, but not term trophoblasts. What is more concerning is that our results using BeWo cells do not agree with their results, even though for the most part, we used the same experimental design. It is unclear why these experiments yielded different results, however we wanted to draw attention to simple differences in measuring syncytialisation or flaws in method reporting (including omission of cell line source and passage numbers, siRNA concentration and protein molecular weights) and choice of immunoblot loading controls, that could impact on experimental outcomes. Our study shows that careful reporting of methods by authors and thorough scrutiny by referees are vital. Furthermore, a universal benchmark for measuring syncytialisation is required so that various studies of syncytialisation can be validated.


Assuntos
Fusão Celular , Furina/metabolismo , Placentação , Trofoblastos/enzimologia , Clorometilcetonas de Aminoácidos/farmacologia , Antígenos CD/metabolismo , Caderinas/metabolismo , Linhagem Celular Tumoral , Gonadotropina Coriônica/metabolismo , Colforsina/farmacologia , Feminino , Furina/antagonistas & inibidores , Furina/genética , Humanos , Placentação/efeitos dos fármacos , Gravidez , Primeiro Trimestre da Gravidez , Inibidores de Serino Proteinase/farmacologia , Nascimento a Termo , Trofoblastos/efeitos dos fármacos
5.
Biomed Res Int ; 2021: 6687551, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34104650

RESUMO

In the present study, we examined the synergetic effect of forskolin and mevastatin administration on lipid profile and lipid metabolism in omental adipose tissue in dyslipidemic rats. The study was conducted on forty male albino rats. The rats were randomly classified into four main groups of ten animals in each group as follows: group A, served as control nontreated; group B, rats that received Triton WR 1339 (500 mg/kg); group C, rats that received Triton WR 1339 with forskolin (100% FSK extract 0.5 mg/kg/day) for four weeks; and group D, dyslipidemic rats received both mevastatin and forskolin. At the end of the experimental period, blood and omental adipose tissue samples were collected, preserved, and used for biochemical determination of lipid profile and mRNA expression profile of adenylate cyclase (AC), hormone-sensitive lipase, respectively (HSL), and adenosine monophosphate-activated protein kinase (AMPK). The results showed a significant decline in the serum concentration of total cholesterol, LDL-cholesterol, and triglycerides, although there was a significant increase in serum levels of HDL-cholesterol and glycerol in rats received forskolin alone or with mevastatin when compared with control and dyslipidemic groups. The mRNA expression levels of AC, HSL, and AMPK were significantly increased in omental adipose tissue of rats received forskolin when compared with other groups. In conclusion, forskolin acts synergistically with mevastatin to lower lipid profile and improve lipid metabolism in dyslipidemic rats through upregulation of AMPK expression.


Assuntos
Monofosfato de Adenosina/metabolismo , Colforsina/farmacologia , Dislipidemias/tratamento farmacológico , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipídeos/fisiologia , Lovastatina/análogos & derivados , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , HDL-Colesterol/metabolismo , Dieta Hiperlipídica/efeitos adversos , Dislipidemias/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Lovastatina/farmacologia , Masculino , Ratos , Triglicerídeos/metabolismo , Regulação para Cima/efeitos dos fármacos
6.
PLoS Biol ; 19(6): e3001149, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34153028

RESUMO

Synaptic plasticity is a cellular model for learning and memory. However, the expression mechanisms underlying presynaptic forms of plasticity are not well understood. Here, we investigate functional and structural correlates of presynaptic potentiation at large hippocampal mossy fiber boutons induced by the adenylyl cyclase activator forskolin. We performed 2-photon imaging of the genetically encoded glutamate sensor iGluu that revealed an increase in the surface area used for glutamate release at potentiated terminals. Time-gated stimulated emission depletion microscopy revealed no change in the coupling distance between P/Q-type calcium channels and release sites mapped by Munc13-1 cluster position. Finally, by high-pressure freezing and transmission electron microscopy analysis, we found a fast remodeling of synaptic ultrastructure at potentiated boutons: Synaptic vesicles dispersed in the terminal and accumulated at the active zones, while active zone density and synaptic complexity increased. We suggest that these rapid and early structural rearrangements might enable long-term increase in synaptic strength.


Assuntos
Fibras Musgosas Hipocampais/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Colforsina/farmacologia , Ácido Glutâmico/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência por Excitação Multifotônica , Fibras Musgosas Hipocampais/efeitos dos fármacos , Fibras Musgosas Hipocampais/ultraestrutura , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/efeitos dos fármacos , Vesículas Sinápticas/metabolismo
7.
Am J Physiol Cell Physiol ; 321(1): C158-C175, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34038243

RESUMO

In whole cell patch clamp recordings, it was discovered that normal human adrenal zona glomerulosa (AZG) cells express members of the three major families of K+ channels. Among these are a two-pore (K2P) leak-type and a G protein-coupled, inwardly rectifying (GIRK) channel, both inhibited by peptide hormones that stimulate aldosterone secretion. The K2P current displayed properties identifying it as TREK-1 (KCNK2). This outwardly rectifying current was activated by arachidonic acid and inhibited by angiotensin II (ANG II), adrenocorticotrophic hormone (ACTH), and forskolin. The activation and inhibition of TREK-1 was coupled to AZG cell hyperpolarization and depolarization, respectively. A second K2P channel, TASK-1 (KCNK3), was expressed at a lower density in AZG cells. Human AZG cells also express inwardly rectifying K+ current(s) (KIR) that include quasi-instantaneous and time-dependent components. This is the first report demonstrating the presence of KIR in whole cell recordings from AZG cells of any species. The time-dependent current was selectively inhibited by ANG II, and ACTH, identifying it as a G protein-coupled (GIRK) channel, most likely KIR3.4 (KCNJ5). The quasi-instantaneous KIR current was not inhibited by ANG II or ACTH and may be a separate non-GIRK current. Finally, AZG cells express a voltage-gated, rapidly inactivating K+ current whose properties identified as KV1.4 (KCNA4), a conclusion confirmed by Northern blot. These findings demonstrate that human AZG cells express K2P and GIRK channels whose inhibition by ANG II and ACTH is likely coupled to depolarization-dependent secretion. They further demonstrate that human AZG K+ channels differ fundamentally from the widely adopted rodent models for human aldosterone secretion.


Assuntos
Hormônio Adrenocorticotrópico/farmacologia , Angiotensina II/farmacologia , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/genética , Canal de Potássio Kv1.4/genética , Proteínas do Tecido Nervoso/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Zona Glomerulosa/metabolismo , Adolescente , Adulto , Aldosterona/biossíntese , Ácido Araquidônico/farmacologia , Autopsia , Criança , Colforsina/farmacologia , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G/metabolismo , Expressão Gênica , Humanos , Canal de Potássio Kv1.4/antagonistas & inibidores , Canal de Potássio Kv1.4/metabolismo , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/metabolismo , Técnicas de Patch-Clamp , Canais de Potássio de Domínios Poros em Tandem/antagonistas & inibidores , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Cultura Primária de Células , Zona Glomerulosa/citologia , Zona Glomerulosa/efeitos dos fármacos
8.
Nat Biomed Eng ; 5(8): 864-879, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33737730

RESUMO

Muscle loss and impairment resulting from traumatic injury can be alleviated by therapies using muscle stem cells. However, collecting sufficient numbers of autologous myogenic stem cells and expanding them efficiently has been challenging. Here we show that myogenic stem cells (predominantly Pax7+ cells)-which were selectively expanded from readily obtainable dermal fibroblasts or skeletal muscle stem cells using a specific cocktail of small molecules and transplanted into muscle injuries in adult, aged or dystrophic mice-led to functional muscle regeneration in the three animal models. We also show that sustained release of the small-molecule cocktail in situ through polymer nanoparticles led to muscle repair by inducing robust activation and expansion of resident satellite cells. Chemically induced stem cell expansion in vitro and in situ may prove to be advantageous for stem cell therapies that aim to regenerate skeletal muscle and other tissues.


Assuntos
Músculo Esquelético/fisiologia , Regeneração , Células Satélites de Músculo Esquelético/citologia , Animais , Reprogramação Celular/efeitos dos fármacos , Colforsina/farmacologia , Meios de Cultura/química , Meios de Cultura/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Camundongos , Doenças Musculares/terapia , Nanopartículas/química , Fator de Transcrição PAX7/metabolismo , Polímeros/química , Células Satélites de Músculo Esquelético/metabolismo , Células Satélites de Músculo Esquelético/transplante , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Ácido Valproico/farmacologia
9.
Biomed Res Int ; 2021: 8881843, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33564685

RESUMO

Background: Diabetic cardiomyopathy is one of the cardiac complications in diabetes patients, eventually resulting in heart failure and increasing morbidity and mortality. Oxidative stress is a critical pathological feature in diabetic hearts, contributing to the development of DCM. Forskolin (FSK) was shown to reduce oxidative stress. This study was aimed at investigating the effects of FSK on diabetic hearts and the relevant molecular mechanisms. Methods: Streptozotocin- (STZ-) induced diabetes in mice was treated with FSK through intraperitoneal injection. Cardiac functions were evaluated by echocardiography. Hematoxylin-eosin and Masson trichrome staining was employed to determine heart morphological changes and cardiac fibrosis, respectively. Cardiac fibrosis-related markers were detected by western blot. Superoxide dismutase activity, reduced/oxidized glutathione ratio, and malondialdehyde concentration in left ventricles were determined using respective commercial kits. Results: Abnormal cardiac diastolic dysfunction and cardiac fibrosis were observed in diabetic hearts. FSK treatment significantly improved the cardiac diastolic function and attenuated the abnormal morphological change in diabetic hearts. Moreover, FSK treatment in diabetic mice decreased the expression of fibronectin, collagen I, TGF-ß, and α-SMA and reduced myocardial fibrosis. Furthermore, we observed that FSK significantly blocked oxidative stress in diabetic hearts. Conclusions: Our study demonstrates that FSK protects against the development of DCM in STZ-induced diabetes in mice. Our study suggests that FSK might be a potential target for drug development in treating DCM.


Assuntos
Colforsina/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Cardiomiopatias Diabéticas/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Actinas/genética , Animais , Apoptose/efeitos dos fármacos , Diabetes Mellitus Experimental/fisiopatologia , Cardiomiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Fibrose/tratamento farmacológico , Fibrose/fisiopatologia , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiopatologia , Humanos , Camundongos , Miocárdio/patologia , Fator de Crescimento Transformador beta/genética
10.
Int J Mol Sci ; 22(3)2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33499110

RESUMO

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes.


Assuntos
Mutação , Peptídeo Natriurético Tipo C/metabolismo , Neoplasias Hipofisárias/metabolismo , Receptores do Fator Natriurético Atrial/metabolismo , Acromegalia/metabolismo , Animais , Gatos , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Estrogênios/metabolismo , Feminino , Masculino , Fenótipo , Hipófise/metabolismo , Ratos , Ratos Wistar , Hormônio Liberador de Tireotropina/farmacologia
11.
Blood ; 137(4): 500-512, 2021 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-33507291

RESUMO

Glucocorticoid (GC) resistance remains a clinical challenge in pediatric acute lymphoblastic leukemia where response to GC is a reliable prognostic indicator. To identify GC resistance pathways, we conducted a genome-wide, survival-based, short hairpin RNA screen in murine T-cell acute lymphoblastic leukemia (T-ALL) cells. Genes identified in the screen interfere with cyclic adenosine monophosphate (cAMP) signaling and are underexpressed in GC-resistant or relapsed ALL patients. Silencing of the cAMP-activating Gnas gene interfered with GC-induced gene expression, resulting in dexamethasone resistance in vitro and in vivo. We demonstrate that cAMP signaling synergizes with dexamethasone to enhance cell death in GC-resistant human T-ALL cells. We find the E prostanoid receptor 4 expressed in T-ALL samples and demonstrate that prostaglandin E2 (PGE2) increases intracellular cAMP, potentiates GC-induced gene expression, and sensitizes human T-ALL samples to dexamethasone in vitro and in vivo. These findings identify PGE2 as a target for GC resensitization in relapsed pediatric T-ALL.


Assuntos
AMP Cíclico/fisiologia , Dexametasona/farmacologia , Dinoprostona/farmacologia , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Sistemas do Segundo Mensageiro/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linhagem Celular Tumoral , Criança , Cromograninas/antagonistas & inibidores , Colforsina/farmacologia , AMP Cíclico/farmacologia , Dexametasona/administração & dosagem , Dinoprostona/administração & dosagem , Dinoprostona/antagonistas & inibidores , Dinoprostona/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Subunidades alfa Gs de Proteínas de Ligação ao GTP/antagonistas & inibidores , Subunidades alfa Gs de Proteínas de Ligação ao GTP/deficiência , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Modelos Animais , Terapia de Alvo Molecular , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Quimera por Radiação , Receptores de Glucocorticoides/biossíntese , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/fisiologia , Receptores de Prostaglandina E Subtipo EP4/biossíntese , Receptores de Prostaglandina E Subtipo EP4/genética , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Phytochemistry ; 184: 112654, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33461046

RESUMO

To understand the compatibility of three native endophytic fungi Phialemoniopsis cornearis (SF1), Macrophomina pseudophaseolina (SF2) and Fusarium redolens (RF1) with Trichoderma viride (TV1) on Coleus forskohlii in enhancing plant growth and forskolin content, field experiments were conducted. Co-inoculation of RF1+TV1 showed significant improvement in plant growth (52%), root biomass (67%), and in-planta forskolin content (94%), followed by treatment with SF2+TV1 and SF1+TV1. qRT-PCR was carried out to quantify expression of five key forskolin biosynthetic pathway genes (CfTPS2, CfTPS3, CfTPS4, CfCYP76AH15, and CfACT1-8) in RF1+TV1 treated C. forskohlii plants. Elevated expression of CfTPS2, CfTPS4, CfCYP76AH15 and CfACT1-8 genes was observed with RF1+TV1 combination as compared to uninoculated C. forskohlii plants. Besides, RF1+TV1 treatment considerably reduced the severity of nematode infection of C. forskohlii plants under field conditions. Thus, congruent properties of F. redolens (RF1) were witnessed with co-inoculation of T. viride (TV1) under field conditions which resulted in enhanced forskolin content, root biomass, and reduced nematode infections in C. forskohlii. Overall, this approach could be an economical and sustainable step towards cultivation of commercially important medicinal plants.


Assuntos
Plectranthus , Trichoderma , Ascomicetos , Colforsina/farmacologia , Endófitos , Fusarium , Hypocreales , Raízes de Plantas
13.
Nat Commun ; 12(1): 305, 2021 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-33436646

RESUMO

Apelin and arginine-vasopressin (AVP) are conversely regulated by osmotic stimuli. We therefore hypothesized that activating the apelin receptor (apelin-R) with LIT01-196, a metabolically stable apelin-17 analog, may be beneficial for treating the Syndrome of Inappropriate Antidiuresis, in which AVP hypersecretion leads to hyponatremia. We show that LIT01-196, which behaves as a potent full agonist for the apelin-R, has an in vivo half-life of 156 minutes in the bloodstream after subcutaneous administration in control rats. In collecting ducts, LIT01-196 decreases dDAVP-induced cAMP production and apical cell surface expression of phosphorylated aquaporin 2 via AVP type 2 receptors, leading to an increase in aqueous diuresis. In a rat experimental model of AVP-induced hyponatremia, LIT01-196 subcutaneously administered blocks the antidiuretic effect of AVP and the AVP-induced increase in urinary osmolality and induces a progressive improvement of hyponatremia. Our data suggest that apelin-R activation constitutes an original approach for hyponatremia treatment.


Assuntos
Apelina/análogos & derivados , Apelina/metabolismo , Arginina Vasopressina/efeitos adversos , Diurese , Hiponatremia/patologia , Hiponatremia/fisiopatologia , Sequência de Aminoácidos , Animais , Apelina/administração & dosagem , Apelina/sangue , Receptores de Apelina/metabolismo , Arginina Vasopressina/sangue , Glicemia/metabolismo , Pressão Sanguínea/efeitos dos fármacos , Linhagem Celular , Colforsina/farmacologia , AMP Cíclico/biossíntese , Desamino Arginina Vasopressina/farmacologia , Modelos Animais de Doenças , Diurese/efeitos dos fármacos , Eletrólitos/sangue , Meia-Vida , Hiponatremia/sangue , Hiponatremia/urina , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Túbulos Renais Coletores/fisiopatologia , Masculino , Camundongos , Modelos Biológicos , Contração Miocárdica/efeitos dos fármacos , Peptídeos/química , Peptídeos/farmacologia , Fosforilação/efeitos dos fármacos , Ratos Sprague-Dawley , Tolvaptan/farmacologia
14.
Cancer Res ; 81(3): 619-633, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33218969

RESUMO

Metastases largely rely on hematogenous dissemination of tumor cells via the vascular system and significantly limit prognosis of patients with solid tumors. To colonize distant sites, circulating tumor cells must destabilize the endothelial barrier and transmigrate across the vessel wall. Here we performed a high-content screen to identify drugs that block tumor cell extravasation by testing 3,520 compounds on a transendothelial invasion coculture assay. Hits were further characterized and validated using a series of in vitro assays, a zebrafish model enabling three-dimensional (3D) visualization of tumor cell extravasation, and mouse models of lung metastasis. The initial screen advanced 38 compounds as potential hits, of which, four compounds enhanced endothelial barrier stability while concurrently suppressing tumor cell motility. Two compounds niclosamide and forskolin significantly reduced tumor cell extravasation in zebrafish, and niclosamide drastically impaired metastasis in mice. Because niclosamide had not previously been linked with effects on barrier function, single-cell RNA sequencing uncovered mechanistic effects of the drug on both tumor and endothelial cells. Importantly, niclosamide affected homotypic and heterotypic signaling critical to intercellular junctions, cell-matrix interactions, and cytoskeletal regulation. Proteomic analysis indicated that niclosamide-treated mice also showed reduced levels of kininogen, the precursor to the permeability mediator bradykinin. Our findings designate niclosamide as an effective drug that restricts tumor cell extravasation through modulation of signaling pathways, chemokines, and tumor-endothelial cell interactions. SIGNIFICANCE: A high-content screen identified niclosamide as an effective drug that restricts tumor cell extravasation by enhancing endothelial barrier stability through modulation of molecular signaling, chemokines, and tumor-endothelial cell interactions. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/3/619/F1.large.jpg.


Assuntos
Colforsina/farmacologia , Endotélio Vascular , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes , Niclosamida/farmacologia , Migração Transendotelial e Transepitelial/efeitos dos fármacos , Animais , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Células Endoteliais/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Cininogênios/análise , Masculino , Metabolômica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Proteômica , Peixe-Zebra
15.
Nat Chem Biol ; 17(2): 205-212, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33106662

RESUMO

Momilactones from rice have allelopathic activity, the ability to inhibit growth of competing plants. Transferring momilactone production to other crops is a potential approach to combat weeds, yet a complete momilactone biosynthetic pathway remains elusive. Here, we address this challenge through rapid gene screening in Nicotiana benthamiana, a heterologous plant host. This required us to solve a central problem: diminishing intermediate and product yields remain a bottleneck for multistep diterpene pathways. We increased intermediate and product titers by rerouting diterpene biosynthesis from the chloroplast to the cytosolic, high-flux mevalonate pathway. This enabled the discovery and reconstitution of a complete route to momilactones (>10-fold yield improvement in production versus rice). Pure momilactone B isolated from N. benthamiana inhibited germination and root growth in Arabidopsis thaliana, validating allelopathic activity. We demonstrated the broad utility of this approach by applying it to forskolin, a Hedgehog inhibitor, and taxadiene, an intermediate in taxol biosynthesis (~10-fold improvement in production versus chloroplast expression).


Assuntos
Diterpenos/metabolismo , Lactonas/metabolismo , Plantas/metabolismo , Transdução de Sinais/genética , Alcenos/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Cloroplastos/genética , Colforsina/farmacologia , Citosol/metabolismo , Diterpenos/farmacologia , Ácido Mevalônico/metabolismo , Oryza/genética , Paclitaxel/biossíntese , Folhas de Planta/química , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Tabaco/metabolismo
16.
Methods Mol Biol ; 2201: 117-125, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975794

RESUMO

Quantitative measurement of receptor signaling by different ligands is important for understanding the mechanism of drug action and screening of drugs. Here, we describe a simple and cost-effective method of measuring adenylyl cyclase inhibition, one of the hallmarks of opioid receptor activation. The assay is based on bioluminescence resonance energy transfer (BRET) that involves transfection of a biosensor in human embryonic kidney (HEK)-293 cells stably transfected with µ-opioid receptor (µ receptor), enabling real-time measurement of cAMP levels.


Assuntos
Inibidores de Adenilil Ciclases/análise , Técnicas de Transferência de Energia por Ressonância de Bioluminescência/métodos , Imagem Molecular/métodos , Toxina Adenilato Ciclase , Inibidores de Adenilil Ciclases/metabolismo , Adenilil Ciclases/metabolismo , Analgésicos Opioides , Animais , Colforsina/farmacologia , AMP Cíclico , Transferência de Energia , Células HEK293 , Humanos , Receptores Opioides/química , Receptores Opioides/metabolismo , Receptores Opioides mu
17.
PLoS One ; 15(12): e0239937, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33259490

RESUMO

Our previous work has shown myometrial PKA activity declines in term and twin-preterm labour in association with an increase in the expression of the oxytocin receptor (OTR). Here we investigate the action of cAMP/PKA in basal conditions, with the addition of progesterone (P4) and/or IL-1ß to understand how cAMP/PKA acts to maintain pregnancy and whether the combination of cAMP and P4 would be a viable therapeutic combination for the prevention of preterm labour (PTL). Further, given that we have previously found that cAMP enhances P4 action we wanted to test the hypothesis that changes in the cAMP effector system are responsible for the functional withdrawal of myometrial P4 action. Myometrial cells were grown from biopsies obtained from women at the time of elective Caesarean section before the onset of labour. The addition of forskolin, an adenylyl cyclase activator, repressed basal OTR mRNA levels at all doses and P4 only enhanced this effect at its highest dose. Forskolin repressed the IL-1ß-induced increase in OTR mRNA and protein levels in a PKA-dependent fashion and repressed IL-1ß-activation and nuclear transfer of NFκB and AP-1. P4 had similar effects and the combination P4 and forskolin had greater effects on OTR and NFκB than forskolin alone. While PKA knockdown had no effect on the ability of P4 to repress IL-1ß-induced OTR expression it reversed the repressive effect of the combination of P4 and forskolin and resulted in a greater increase than observed with IL-1ß alone. These studies suggest that cAMP acts via PKA to repress inflammation-driven OTR expression, but that when PKA activity is reduced, the combination of cAMP and P4 actually enhances the OTR response to inflammation, promoting the onset of labour and suggesting that changes in the cAMP effector system can induce a functional P4 withdrawal.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Miométrio/metabolismo , Receptores de Ocitocina/metabolismo , Adulto , Colforsina/farmacologia , AMP Cíclico/metabolismo , Feminino , Humanos , Interleucina-1beta/farmacologia , Miométrio/efeitos dos fármacos , Progesterona/farmacologia , Receptores de Ocitocina/genética
18.
Mol Pain ; 16: 1744806920970368, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33307981

RESUMO

The embryonic rat dorsal root ganglion (DRG) neuron-derived 50B11 cell line is a promising sensory neuron model expressing markers characteristic of NGF and GDNF-dependent C-fibre nociceptors. Whether these cells have the capacity to develop into distinct nociceptive subtypes based on NGF- or GDNF-dependence has not been investigated. Here we show that by augmenting forskolin (FSK) and growth factor supplementation with NGF or GDNF, 50B11 cultures can be driven to acquire differential functional responses to common nociceptive agonists capsaicin and ATP respectively. In addition, to previous studies, we also demonstrate that a differentiated neuronal phenotype can be maintained for up to 7 days. Western blot analysis of nociceptive marker proteins further demonstrates that the 50B11 cells partially recapitulate the functional phenotypes of classical NGF-dependent (peptidergic) and GDNF-dependent (non-peptidergic) neuronal subtypes described in DRGs. Further, 50B11 cells differentiated with NGF/FSK, but not GDNF/FSK, show sensitization to acute prostaglandin E2 treatment. Finally, RNA-Seq analysis confirms that differentiation with NGF/FSK or GDNF/FSK produces two 50B11 cell subtypes with distinct transcriptome expression profiles. Gene ontology comparison of the two subtypes of differentiated 50B11 cells to rodent DRG neurons studies shows significant overlap in matching or partially matching categories. This transcriptomic analysis will aid future suitability assessment of the 50B11 cells as a high-throughput nociceptor model for a broad range of experimental applications. In conclusion, this study shows that the 50B11 cell line is capable of partially recapitulating features of two distinct types of embryonic NGF and GDNF-dependent nociceptor-like cells.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Gânglios Espinais/citologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/farmacologia , Fator de Crescimento Neural/farmacologia , Nociceptores/citologia , Potenciais de Ação/efeitos dos fármacos , Trifosfato de Adenosina/farmacologia , Animais , Biomarcadores/metabolismo , Capsaicina/farmacologia , Diferenciação Celular/genética , Linhagem Celular , Forma Celular/efeitos dos fármacos , Colforsina/farmacologia , Dinoprostona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Variação Genética , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nociceptores/efeitos dos fármacos , Fenótipo , Análise de Componente Principal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Canais de Sódio/metabolismo
19.
Biomolecules ; 10(12)2020 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-33322066

RESUMO

Human placenta-derived multipotent stem cells (PDMCs) resembling embryonic stem cells can differentiate into three germ layer cells, including ectodermal lineage cells, such as neurons, astrocytes, and oligodendrocytes. The favorable characteristics of noninvasive cell harvesting include fewer ethical, religious, and legal considerations as well as accessible and limitless supply. Thus, PDMCs are attractive for cell-based therapy. The Schwann cell (SC) is the most common cell type used for tissue engineering such as nerve regeneration. However, the differentiation potential of human PDMCs into SCs has not been demonstrated until now. In this study, we evaluated the potential of PDMCs to differentiate into SC-like cells in a differentiation medium. After induction, PDMCs not only exhibited typical SC spindle-shaped morphology but also expressed SC markers, including S100, GFAP, p75, MBP, and Sox 10, as revealed by immunocytochemistry. Moreover, a reverse transcription-quantitative polymerase chain reaction analysis revealed the elevated gene expression of S100, GFAP, p75, MBP, Sox-10, and Krox-20 after SC induction. A neuroblastoma cell line, SH-SY5Y, was cultured in the conditioned medium (CM) collected from PDMC-differentiated SCs. The growth rate of the SH-SY5Y increased in the CM, indicating the function of PDMC-induced SCs. In conclusion, human PDMCs can be differentiated into SC-like cells and thus are an attractive alternative to SCs for cell-based therapy in the future.


Assuntos
Colforsina/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Células-Tronco Multipotentes/efeitos dos fármacos , Neuregulina-1/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Células de Schwann/efeitos dos fármacos , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Feminino , Expressão Gênica , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Proteína Básica da Mielina/genética , Proteína Básica da Mielina/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Placenta/citologia , Placenta/metabolismo , Gravidez , Cultura Primária de Células , Proteínas S100/genética , Proteínas S100/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Células de Schwann/citologia , Células de Schwann/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
PLoS One ; 15(12): e0244253, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33347508

RESUMO

In order to elucidate involvement of cyclic AMP and intracellular Ca2+,[Ca2+]i, in the modulation of aqueous humour formation (AHF), we studied the effects of terbutaline, forskolin and 8-Br-cAMP in the isolated bovine eye. We also studied the interaction of cAMP on calcium signaling in cultured ciliary epithelial (CE) cells. Drug effects on AHF were measured by fluorescein dilution. Drug effects on [Ca2+]i were studied by the fura-2 fluorescence ratio technique. Terbutaline (100 nmol-100 M), forskolin (30 nM-100 M) or 8-Br-cAMP (100 nM- 10 µM), administered in the arterial perfusate produced significant reductions in AHF. The AH reducing effect of terbutaline was blocked by a selective inhibitor of protein kinase A (KT-5720). ATP (100 M) caused a rapid, transient (peak) increase in [Ca2+]i followed by a sustained plateau phase lasting more than 5 minutes. Preincubation of the cells (6 min) with terbutaline, forskolin or 8-Br-cAMP significantly reduced the peak calcium response to ATP. The sustained plateau phase of the response, on the other hand, was augmented by each of the agents. KT-5720 partially reversed the inhibitory effect of terbutaline on the peak and totally inhibited its effect on the plateau phase. These data indicate: (a) that AHF in the bovine eye can be manipulated through cyclic AMP, operating via protein kinase A, (b) that protein kinase A can affect [Ca2+]i homeostasis, (c) that calcium release from the intracellular store, not the entry, affects AHF, and (d) that interaction of [Ca2+]i with cAMP plays a role in modulating AH secretion.


Assuntos
Humor Aquoso/metabolismo , Secreções Corporais/efeitos dos fármacos , Cálcio/metabolismo , Colforsina/farmacologia , AMP Cíclico/farmacologia , Terbutalina/farmacologia , Animais , Humor Aquoso/efeitos dos fármacos , Broncodilatadores/farmacologia , Bovinos , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo
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