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1.
J Vis Exp ; (168)2021 02 23.
Artigo em Inglês | MEDLINE | ID: mdl-33720124

RESUMO

Cancer stem cells (CSCs) are an important cause of tumor growth, metastasis, and recurrence. Isolation and identification of CSCs are of great significance for tumor research. Currently, several techniques are used for the identification and purification of CSCs from tumor tissues and tumor cell lines. Separation and analysis of side population (SP) cells are two of the commonly used methods. The methods rely on the ability of CSCs to rapidly expel fluorescent dyes, such as Hoechst 33342. The efflux of the dye is associated with the ATP-binding cassette (ABC) transporters and can be inhibited by ABC transporter inhibitors. Methods for staining cultured tumor cells with Hoechst 33342 and analyzing the proportion of their SP cells by flow cytometry are described. This assay is convenient, fast, and cost-effective. Data generated in this assay can contribute to a better understanding of the effect of genes or other extracellular and intracellular signals on the stemness properties of tumor cells.


Assuntos
Neoplasias/patologia , Células da Side Population/patologia , Benzimidazóis/metabolismo , Linhagem Celular Tumoral , Análise de Dados , Citometria de Fluxo , Corantes Fluorescentes/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição STAT3/metabolismo , Células da Side Population/metabolismo , Coloração e Rotulagem
2.
Nucleic Acids Res ; 49(5): 2488-2508, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33533919

RESUMO

The ubiquitous family of dimeric transcription factors AP-1 is made up of Fos and Jun family proteins. It has long been thought to operate principally at gene promoters and how it controls transcription is still ill-understood. The Fos family protein Fra-1 is overexpressed in triple negative breast cancers (TNBCs) where it contributes to tumor aggressiveness. To address its transcriptional actions in TNBCs, we combined transcriptomics, ChIP-seqs, machine learning and NG Capture-C. Additionally, we studied its Fos family kin Fra-2 also expressed in TNBCs, albeit much less. Consistently with their pleiotropic effects, Fra-1 and Fra-2 up- and downregulate individually, together or redundantly many genes associated with a wide range of biological processes. Target gene regulation is principally due to binding of Fra-1 and Fra-2 at regulatory elements located distantly from cognate promoters where Fra-1 modulates the recruitment of the transcriptional co-regulator p300/CBP and where differences in AP-1 variant motif recognition can underlie preferential Fra-1- or Fra-2 bindings. Our work also shows no major role for Fra-1 in chromatin architecture control at target gene loci, but suggests collaboration between Fra-1-bound and -unbound enhancers within chromatin hubs sometimes including promoters for other Fra-1-regulated genes. Our work impacts our view of AP-1.


Assuntos
Elementos Facilitadores Genéticos , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas c-fos/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Sítios de Ligação , Linhagem Celular Tumoral , Cromatina/química , Cromatina/metabolismo , Epigênese Genética , Antígeno 2 Relacionado a Fos/metabolismo , Humanos , Motivos de Nucleotídeos , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/fisiologia , Fator de Transcrição AP-1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Fatores de Transcrição de p300-CBP/metabolismo
3.
Cancer Sci ; 112(3): 1235-1250, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33393145

RESUMO

Acute lymphoblastic leukemia (ALL) is an aggressive hematological cancer that mainly affects children. Relapse and chemoresistance result in treatment failure, underlining the need for improved therapies. BTB and CNC homology 2 (BACH2) is a lymphoid-specific transcription repressor recognized as a tumor suppressor in lymphomas, but little is known about its function and regulatory network in pediatric ALL (p-ALL). Herein, we found aberrant BACH2 expression at new diagnosis not only facilitated risk stratification of p-ALL but also served as a sensitive predictor of early treatment response and clinical outcome. Silencing BACH2 in ALL cells increased cell proliferation and accelerated cell cycle progression. BACH2 blockade also promoted cell adhesion to bone marrow stromal cells and conferred cytarabine (Ara-C)-resistant properties to leukemia cells by altering stromal microenvironment. Strikingly, we identified FOS, a transcriptional activator competing with BACH2, as a novel downstream target repressed by BACH2. Blocking FOS by chemical compounds enhanced the effect of Ara-C treatment in both primary p-ALL cells and pre-B-ALL-driven leukemia xenografts and prolonged the survival of tumor-bearing mice. These data highlight an interconnected network of BACH2-FOS, disruption of which could render current chemotherapies more effective and offer a promising therapeutic strategy to overcome Ara-C resistance in p-ALL.


Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Fatores de Transcrição de Zíper de Leucina Básica/genética , Proliferação de Células/genética , Criança , Pré-Escolar , Citarabina/farmacologia , Citarabina/uso terapêutico , Feminino , Regulação Leucêmica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Lactente , Masculino , Células-Tronco Mesenquimais , Camundongos , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangue , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Cultura Primária de Células , Células Tumorais Cultivadas , Microambiente Tumoral/genética , Ensaios Antitumorais Modelo de Xenoenxerto
4.
Arch Oral Biol ; 122: 105029, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33387850

RESUMO

OBJECTIVE: The flower of chrysanthemum, used worldwide as a medicinal and edible product, has shown various bioactivities, such as anti-inflammatory, antioxidant, anti-tumorigenic, and hepatoprotective activities, as well as cardiovascular protection. However, the effect of Chrysanthemum morifolium Ramat. on the regulation of osteoclast differentiation has not yet been reported. In this study, we aimed to investigate the inhibitory effect of Chrysanthemum morifolium Ramat. water extract (CME) on RANKL-induced osteoclast differentiation in mouse bone marrow-derived macrophages (BMMs). STUDY DESIGN: Bone marrow-derived macrophages (BMMs) isolated from the C57BL/6 J mice. The viability of BMMs was detected with MTT assays. Inhibitory effects of CME on osteoclast differentiation and bone resorption was measured by TRAP staining and Pit assay. Osteoclast differentiation-associated gene expression were assessed by Real-time quantitative polymerase chain reaction. Intracellular signaling molecules was assessed by western blot. RESULTS: CME significantly inhibited osteoclast differentiation in BMMs without cytotoxicity, besides inhibiting MAPK/c-fos and PLCγ2/CREB activation. The inhibitory effects of CME on differentiation-related signaling molecules resulted in significant repression of NFATc1 expression, which is a key transcription factor in osteoclast differentiation, fusion, and activation. CONCLUSION: Our results confirmed the inhibition of RANKL-induced PLCγ2/CREB/c-fos/NFATc1 activation by CME during osteoclast differentiation. The findings collectively suggested CME as a traditional therapeutic agent for osteoporosis, RA, and periodontitis.


Assuntos
Reabsorção Óssea , Diferenciação Celular/efeitos dos fármacos , Chrysanthemum/química , Osteoclastos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Ligante RANK/metabolismo , Animais , Células da Medula Óssea , Camundongos , Camundongos Endogâmicos C57BL , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Proteínas Proto-Oncogênicas c-fos/metabolismo
5.
Phytomedicine ; 80: 153397, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33130475

RESUMO

BACKGROUND: The fruit of Zanthoxylum piperitum (ZP) is an herbal medicine as well as a spice agent in Asia to treat carminative, stomachic, anthelmintic and degenerative diseases. Z. piperitum was reported to have anti-oxidant, anti-inflammatory, anti-osteoarthritic and osteosarcoma proliferation-control effects. PURPOSE AND STUDY DESIGN: This study was conducted to determine the anti-osteoporotic effects and mechanisms of action of ZP. METHODS: Female ICR mice underwent ovariectomies (OVX) and were orally administered ZP at 1, 10 and 100 mg/kg for 6 weeks. The femoral and tibial bones were assessed by dual-energy X-ray absorptiometry and histology to analyze the bone mineral density (BMD) and the number of osteoclasts. Raw 264.7 cells were stimulated by 100 ng/ml receptor activator of nuclear factor-κB ligand (RANKL) for 7 days in the presence of ZP. RANKL-induced signaling molecules were analyzed in osteoclasts. RESULTS: The levels of femoral and tibial BMD were significantly increased by ZP administration. Serum biomarkers such as osteocalcin, calcium, alkaline phosphatase and bone-specific alkaline phosphatase concentrations were markedly recovered to normal levels in ZP-treated osteoporotic mice. In addition, the number of osteoclasts in the head, trochanter and body of the femur was obviously decreased in the ZP treatment groups. Moreover, ZP treated-cells showed a reduction in the number of TRAP-positive multinuclear cells in RANKL-stimulated Raw 264.7 cells. ZP decreased the RANKL-activated NFATc1 and c-fos, transcription factors of osteoclast formation. The nuclear translocation of NF-κB and phosphorylation of ERK42/44 were inhibited by the ZP treatment in RANKL-induced osteoclasts. CONCLUSION: Collectively, ZP exerts its inhibitory effect against bone resorption by regulating RANKL-mediated c-fos/NFATc1/NF-κB in osteoclast. ZP may prove to be a therapeutic agent for osteoporosis.


Assuntos
Osteoclastos/efeitos dos fármacos , Osteoporose/tratamento farmacológico , Extratos Vegetais/farmacologia , Zanthoxylum/química , Animais , Densidade Óssea/efeitos dos fármacos , Reabsorção Óssea/tratamento farmacológico , Reabsorção Óssea/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos ICR , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/metabolismo , Osteoporose/etiologia , Osteoporose/metabolismo , Ovariectomia/efeitos adversos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ligante RANK/metabolismo , Células RAW 264.7
6.
Nat Commun ; 11(1): 5074, 2020 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-33033265

RESUMO

Touch and itch sensations are crucial for evoking defensive and emotional responses, and light tactile touch may induce unpleasant itch sensations (mechanical itch or alloknesis). The neural substrate for touch-to-itch conversion in the spinal cord remains elusive. We report that spinal interneurons expressing Tachykinin 2-Cre (Tac2Cre) receive direct Aß low threshold mechanoreceptor (LTMR) input and form monosynaptic connections with GRPR neurons. Ablation or inhibition markedly reduces mechanical but not acute chemical itch nor noxious touch information. Chemogenetic inhibition of Tac2Cre neurons also displays pronounced deficit in chronic dry skin itch, a type of chemical itch in mice. Consistently, ablation of gastrin-releasing peptide receptor (GRPR) neurons, which are essential for transmitting chemical itch, also abolishes mechanical itch. Together, these results suggest that innocuous touch and chemical itch information converge on GRPR neurons and thus map an exquisite spinal circuitry hard-wired for converting innocuous touch to irritating itch.


Assuntos
Rede Nervosa/fisiopatologia , Prurido/fisiopatologia , Tato/fisiologia , Animais , Comportamento Animal , Injeções Espinhais , Luz , Potenciais da Membrana , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptores da Bombesina/metabolismo , Pele/patologia , Medula Espinal/fisiopatologia , Sinapses/metabolismo , Taquicininas/metabolismo
7.
Chem Biol Interact ; 331: 109235, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32971123

RESUMO

Ellagic acid (EA) is a naturally occurring polyphenolic compound that has been shown to exhibit diverse beneficial pharmacological activities including anti-osteoclastogenesis effect. However, the molecular mechanism by which EA inhibits osteoclastogenesis remains to be elucidated. The protein-protein interaction between receptor activator of nuclear factor (NF)-κB ligand (RANKL) and its receptor RANK contributes to osteoclast differentiation and activation in bone remodeling, and is regarded as an important therapeutic target for the treatment of osteoporosis. The current study is focused on investigating whether EA can directly bind to RANKL and/or RANK and block the interaction between RANKL and RANK, thereby inhibiting downstream signaling pathways. Interestingly, we found that EA had strong affinities to RANK and RANKL, with the estimated equilibrium dissociation constants (KD) of 2.485 × 10-11 and 1.688 × 10-9 M, respectively, and could disrupt the interaction between RANKL and RANK, thereby inhibiting RANKL-induced canonical RANK signaling pathways (p65, JNK, ERK, and p38) and expression of downstream master transcriptional factors (NFATc1 and c-Fos) and osteoclast-specific genes and proteins (TRAP, c-Src, and cathepsin K), which could ultimately suppress RANKL-induced osteoclast differentiation and F-actin ring formation. Taken together, our results revealed that EA could block RANKL-RANK interaction and suppress RANKL-induced osteoclastogenesis by inhibiting RANK signaling pathways in RAW 264.7 murine macrophages.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Ácido Elágico/farmacologia , Osteogênese/efeitos dos fármacos , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Actinas/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Cinética , Camundongos , Fatores de Transcrição NFATC/metabolismo , Osteoclastos/citologia , Osteoclastos/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ligante RANK/farmacologia , Células RAW 264.7 , Fosfatase Ácida Resistente a Tartarato/metabolismo
8.
Life Sci ; 259: 118374, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32891613

RESUMO

OBJECTIVE: Dipeptidyl peptidase IV (DPP4) has been indicated as a possible prognostic biomarker in papillary thyroid cancer (PTC). However, the mechanism of DPP4 during metastasis of PTC remains unclear. In this study, we investigated whether lysine acetyltransferase 5 (KAT5) and FBJ murine osteosarcoma viral oncogene homolog B (FosB) synergistically regulate high DPP4 expression in PTC. METHODS: PTC tissues and matched paracancerous tissues were harvested, followed by the establishment of IHH-4 and TPC-1 cells with downregulation of DPP4. The relevance of DPP4 on the metastasis of PTC cells was assessed. Subsequently, the effect of KAT5 on the transcription of DPP4 was verified. The binding relationship between FosB and DPP4 was predicted by a bioinformatics website. Functional rescue experiments were performed to evaluate cell activities after overexpression of KAT5 or FosB in cells with DPP4 knockdown. RESULTS: DPP4 was overexpressed in PTC tissues and cell lines, which was correlated with higher risks for metastases and poorer survival. DPP4 downregulation curtailed cell growth and metastasis. Moreover, KAT5 acetylated DPP4 promoter histone, which promoted transcription activation of DPP4. Subsequently, FosB recruited KAT5 at the DPP4 promoter, thereby enhancing DPP4 transcriptional activation. Further overexpression of KAT5 or FosB in cells with low expression of DPP4 promoted cell activity. Finally, DPP4 expedited p62 nuclear translocation to elevate Keap1/Nrf2 expression, thus facilitating the growth and metastasis of PTC cells. CONCLUSION: FosB enhanced the growth and metastasis of PTC cells by recruiting histone acetyltransferases KAT5 to increase DPP4 transcription and activate the p62/Keap1/Nrf2 signaling.


Assuntos
Dipeptidil Peptidase 4/metabolismo , Lisina Acetiltransferase 5/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Animais , Western Blotting , Linhagem Celular Tumoral , Feminino , Imunofluorescência , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Transplante de Neoplasias , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Câncer Papilífero da Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo
9.
J Headache Pain ; 21(1): 101, 2020 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-32799798

RESUMO

BACKGROUND: Although migraine is one of the most common primary headaches, its therapy is still limited in many cases. The use of animal models is crucial in the development of novel therapeutic strategies, but unfortunately, none of them show all aspects of the disease, therefore, there is a constant need for further improvement in this field. The application of inflammatory agents on the dura mater is a widely accepted method to mimic neurogenic inflammation in rodents, which plays a key role in the pathomechanism of migraine. Complete Freund's Adjuvant (CFA), and a mixture of inflammatory mediators, called inflammatory soup (IS) are often used for this purpose. METHODS: To examine the activation pattern that is caused by chemical stimulation of dura mater, we applied CFA or IS over the right parietal lobe. After 2 h and 4 h (CFA groups), or 2.5 h and 4 h (IS groups), animals were perfused, and c-Fos immunoreactive cells were counted in the caudal trigeminal nucleus. To explore every pitfall, we examined whether our surgical procedure (anesthetic drug, stereotaxic apparatus, local lidocaine) can alter the results under the same experimental settings. c-Fos labeled cells were counted in the second-order neuron area based on the somatotopic organization of the trigeminal nerve branches. RESULTS: We could not find any difference between the CFA and physiological saline group neither 2 h, nor 4 h after dural stimulation. IS caused significant difference after both time points between IS treated and control group, and between treated (right) and control (left) side. Stereotaxic frame usage had a substantial effect on the obtained results. CONCLUSIONS: Counting c-Fos immunoreactive cells based on somatotopic organization of the trigeminal nerve helped to examine the effect of chemical stimulation of dura in a more specific way. As a result, the use of IS over the parietal lobe caused activation in the area of the ophthalmic nerve. To see this effect, the use of lidocaine anesthesia is indispensable. In conclusion, application of IS on the dura mater induces short-term, more robust c-Fos activation than CFA, therefore it might offer a better approach to model acute migraine headache in rodents.


Assuntos
Dura-Máter/efeitos dos fármacos , Núcleo Inferior Caudal do Nervo Trigêmeo/efeitos dos fármacos , Animais , Adjuvante de Freund , Cefaleia , Inflamação , Lidocaína/farmacologia , Masculino , Transtornos de Enxaqueca/tratamento farmacológico , Neurônios , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Estimulação Química , Nervo Trigêmeo
10.
Acta Cir Bras ; 35(7): e202000705, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32785417

RESUMO

Purpose Studies have demonstrated that star fruit consumption by nephropathic patients triggers severe neurotoxic effects that can lead to convulsions or even death. Brain areas likely susceptible to star fruit poisoning have not been investigated. The objective of the present study was to map possible epileptogenic areas susceptible to star fruit intoxication in nephropathic rats. Methods The study analyzed 25 rats (5 groups). Rats in the experimental group underwent bilateral ureteral obstruction surgery and orogastric gavages with star fruit juice. An electroencephalogram was used to confirm convulsive seizures. Urea and creatinine levels were used to confirm the uremia model. Immunohistochemical analysis was used to map cells with c-Fos protein (c-Fos+ cells) to identify brain areas with increased neuronal activity. Control groups included non-nephropathic and nephropathic rats that did not receive star fruit. Results A statistically significant increase (p<0.01) in c-Fos+ cells was noted in nephropathic animals receiving star fruit juice compared to control groups, in brain areas commonly related to epileptogenic neural circuits including the hippocampus, amygdala, rhinal cortex, anterior cingulate area, piriform area, and medial dorsal thalamus. Conclusion These data corroborate the neurotoxic capacity of star fruit in nephropathic patients.


Assuntos
Frutas , Nefropatias , Proteínas Proto-Oncogênicas c-fos , Animais , Encéfalo , Córtex Cerebral , Frutas/envenenamento , Hipocampo , Humanos , Nefropatias/complicações , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos
11.
Science ; 369(6505): 842-846, 2020 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-32792398

RESUMO

How is neuropathic pain regulated in peripheral sensory neurons? Importins are key regulators of nucleocytoplasmic transport. In this study, we found that importin α3 (also known as karyopherin subunit alpha 4) can control pain responsiveness in peripheral sensory neurons in mice. Importin α3 knockout or sensory neuron-specific knockdown in mice reduced responsiveness to diverse noxious stimuli and increased tolerance to neuropathic pain. Importin α3-bound c-Fos and importin α3-deficient neurons were impaired in c-Fos nuclear import. Knockdown or dominant-negative inhibition of c-Fos or c-Jun in sensory neurons reduced neuropathic pain. In silico screens identified drugs that mimic importin α3 deficiency. These drugs attenuated neuropathic pain and reduced c-Fos nuclear localization. Thus, perturbing c-Fos nuclear import by importin α3 in peripheral neurons can promote analgesia.


Assuntos
Dor Crônica/fisiopatologia , Neuralgia/fisiopatologia , Células Receptoras Sensoriais/fisiologia , alfa Carioferinas/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Benzofenonas/farmacologia , Dor Crônica/genética , Perfilação da Expressão Gênica , Técnicas de Silenciamento de Genes , Isoxazóis/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Neuralgia/genética , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fator de Transcrição AP-1/metabolismo , alfa Carioferinas/genética
12.
Toxicol Lett ; 333: 140-149, 2020 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-32755622

RESUMO

Chrysotile is the only type of asbestos still widely exploited, and all kinds of asbestos including chrysotile was classified as a group I carcinogen by the IARC. There is a wealth of evidence that chrysotile can cause a range of cancers, including cancer of the lung, larynx, ovary, and mesothelioma. As the second largest chrysotile producer, China is at great risk of occupational exposure. Moreover, our previous experiment and some other studies have shown that the toxicity of mineral fibre from various mining areas may be different. To explore the oncogenic potential of chrysotile from different mining areas of China, Wistar rats were administered 0.5 mL chrysotile asbestos suspension of 2.0 mg/mL (from Akesai, Gansu; Mangnai, Qinghai; XinKang, Sichuan; and Shannan, Shaanxi) dissolved in saline by intratracheal instillation once-monthly and were sacrificed at 1 mo, 6 mo, and 12 mo. Our results found that chrysotile caused lung inflammation and lung tissue damage. Moreover, prolonged exposure of chrysotile can induce inactivation of the tumor suppressor gene P53 and P16 and activation of the protooncogene C-JUN and C-FOS both in the messenger RNA and protein level. In addition, chrysotile from Shannan and XinKang has a stronger effect which may link to cancer than that from Akesai and Mangnai.


Assuntos
Asbestos Serpentinas/toxicidade , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Poluentes Ambientais/toxicidade , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Pulmão/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Asbestos Serpentinas/química , Líquido da Lavagem Broncoalveolar/citologia , China , Inibidor p16 de Quinase Dependente de Ciclina/genética , Citocinas/metabolismo , Poluentes Ambientais/química , Expressão Gênica/efeitos dos fármacos , Exposição por Inalação/efeitos adversos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Contagem de Leucócitos , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Masculino , Fibras Minerais/toxicidade , Proteínas Proto-Oncogênicas c-fos/genética , Ratos Wistar , Proteína Supressora de Tumor p53/genética
13.
Int J Nanomedicine ; 15: 4705-4716, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32636626

RESUMO

Purpose: Ultra-small gold nanoclusters (AuNCs), as emerging fluorescent nanomaterials with excellent biocompatibility, have been widely investigated for in vivo biomedical applications. However, their effects in guiding osteogenic differentiation have not been investigated, which are important for osteoporosis therapy and bone regeneration. Herein, for the first time, lysozyme-protected AuNCs (Lys-AuNCs) are used to stimulate osteogenic differentiation, which have the potential for the treatment of bone disease. Methods: Proliferation of MC3T3E-1 is important for osteogenic differentiation. First, the proliferation rate of MC3T3E-1 was studied by Cell Counting Kit-8 (CCK8) assays. Signaling pathways of PI3K/Akt play central roles in controlling proliferation throughout the body. The expression of PI3K/Akt was investigated in the presence of lysozyme, and lysozyme-protected AuNCs (Lys-AuNCs) by Western blot (WB) and intracellular cell imaging to evacuate the osteogenic differentiation mechanisms. Moreover, the formation of osteoclasts (OC) plays a negative role in the differentiation of osteoblasts. Nuclear factor κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) signaling pathways are used to understand the negative influence of the osteogenic differentiation by the investigation of Raw 264.7 cell line. Raw 264.7 (murine macrophage-like) cells and NIH/3T3 (mouse fibroblast) cells were treated with tyloxapol, and the cell viability was assessed. Raw 264.7 cells have also been used for in vitro studies, on understanding the osteoclast formation and function. The induced osteoclasts were identified by TRAP confocal fluorescence imaging. These key factors in osteoclast formation, such as (NFATc-1, c-Fos, V-ATPase-2 and CTSK), were investigated by Western blot. Results: Based on the above investigation, Lys-AuNCs were found to promote osteogenic differentiation and decrease osteoclast activity. It is noteworthy that the lysozyme (protected template), AuNPs, or the mixture of Lysozyme and AuNPs have negligible effects on osteoblastic differentiation compared to Lys-AuNCs. Conclusion: This study opens up a novel avenue to develop a new gold nanomaterial for promoting osteogenic differentiation. The possibility of using AuNCs as nanomedicines for the treatment of osteoporosis can be expected.


Assuntos
Nanopartículas Metálicas/química , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Animais , Diferenciação Celular/efeitos dos fármacos , Ouro/farmacologia , Nanopartículas Metálicas/administração & dosagem , Camundongos , Muramidase/química , Muramidase/metabolismo , Fatores de Transcrição NFATC/metabolismo , Nanomedicina/métodos , Osteoblastos/citologia , Osteoclastos/citologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Células RAW 264.7
14.
Nature ; 583(7816): 441-446, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32641826

RESUMO

Connections between the gut and brain monitor the intestinal tissue and its microbial and dietary content1, regulating both physiological intestinal functions such as nutrient absorption and motility2,3, and brain-wired feeding behaviour2. It is therefore plausible that circuits exist to detect gut microorganisms and relay this information to areas of the central nervous system that, in turn, regulate gut physiology4. Here we characterize the influence of the microbiota on enteric-associated neurons by combining gnotobiotic mouse models with transcriptomics, circuit-tracing methods and functional manipulations. We find that the gut microbiome modulates gut-extrinsic sympathetic neurons: microbiota depletion leads to increased expression of the neuronal transcription factor cFos, and colonization of germ-free mice with bacteria that produce short-chain fatty acids suppresses cFos expression in the gut sympathetic ganglia. Chemogenetic manipulations, translational profiling and anterograde tracing identify a subset of distal intestine-projecting vagal neurons that are positioned to have an afferent role in microbiota-mediated modulation of gut sympathetic neurons. Retrograde polysynaptic neuronal tracing from the intestinal wall identifies brainstem sensory nuclei that are activated during microbial depletion, as well as efferent sympathetic premotor glutamatergic neurons that regulate gastrointestinal transit. These results reveal microbiota-dependent control of gut-extrinsic sympathetic activation through a gut-brain circuit.


Assuntos
Microbioma Gastrointestinal/fisiologia , Intestinos/inervação , Neurônios/fisiologia , Sistema Nervoso Simpático/citologia , Sistema Nervoso Simpático/fisiologia , Animais , Disbiose/fisiopatologia , Feminino , Gânglios Simpáticos/citologia , Gânglios Simpáticos/fisiologia , Motilidade Gastrointestinal , Vida Livre de Germes , Intestinos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Vias Neurais/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Transcriptoma
15.
Proc Natl Acad Sci U S A ; 117(32): 19556-19565, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32694207

RESUMO

Opioid addiction is a chronic, relapsing disorder associated with persistent changes in brain plasticity. Reconfiguration of neuronal connectivity may explain heightened abuse liability in individuals with a history of chronic drug exposure. To characterize network-level changes in neuronal activity induced by chronic opiate exposure, we compared FOS expression in mice that are morphine-naïve, morphine-dependent, or have undergone 4 wk of withdrawal from chronic morphine exposure, relative to saline-exposed controls. Pairwise interregional correlations in FOS expression data were used to construct network models that reveal a persistent reduction in connectivity strength following opiate dependence. Further, we demonstrate that basal gene expression patterns are predictive of changes in FOS correlation networks in the morphine-dependent state. Finally, we determine that regions of the hippocampus, striatum, and midbrain are most influential in driving transitions between opiate-naïve and opiate-dependent brain states using a control theoretic approach. This study provides a framework for predicting the influence of specific therapeutic interventions on the state of the opiate-dependent brain.


Assuntos
Encéfalo/fisiopatologia , Dependência de Morfina/fisiopatologia , Rede Nervosa/fisiopatologia , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/efeitos adversos , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Conectoma , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos Neurológicos , Morfina/administração & dosagem , Morfina/efeitos adversos , Dependência de Morfina/metabolismo , Rede Nervosa/efeitos dos fármacos , Rede Nervosa/metabolismo , Plasticidade Neuronal/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Síndrome de Abstinência a Substâncias/genética , Síndrome de Abstinência a Substâncias/metabolismo , Síndrome de Abstinência a Substâncias/fisiopatologia
16.
PLoS One ; 15(7): e0236039, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702030

RESUMO

BACKGROUND: The bidirectional selection of high and low anxiety-like behavior is a valuable tool for understanding the neurocircuits that are responsible for anxiety disorders. Our group developed two breeding lines of rats, known as Carioca High- and Low-conditioned Freezing (CHF and CLF), based on defensive freezing in the contextual fear conditioning paradigm. A random selected line was employed as a control (CTL) comparison group for both CHF and CLF lines of animals. The present study performed Fos immunochemistry to investigate changes in neural activity in different brain structures among CHF and CLF rats when they were exposed to contextual cues that were previously associated with footshock. RESULTS: The study indicated that CHF rats expressed high Fos expression in the locus coeruleus, periventricular nucleus of the hypothalamus (PVN), and lateral portion of the septal area and low Fos expression in the medial portion of the septal area, dentate gyrus, and prelimbic cortex (PL) compared to CTL animals. CLF rats exhibited a decrease in Fos expression in the PVN, PL, and basolateral nucleus of the amygdala and increase in the cingulate and perirhinal cortices compared to CTL animals. CONCLUSIONS: Both CHF and CLF rats displayed Fos expression changes key regions of the anxiety brain circuitry. The two bidirectional lines exhibit different pattern of neural activation and inhibition with opposing influences on the PVN, the main structure involved in regulating the hypothalamic-pituitary-adrenal neuroendocrine responses observed in anxiety disorders.


Assuntos
Encéfalo/metabolismo , Condicionamento Psicológico , Proteínas Proto-Oncogênicas c-fos/metabolismo , Animais , Ansiedade/metabolismo , Ansiedade/psicologia , Masculino , Ratos
17.
Oncogene ; 39(34): 5616-5632, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32661324

RESUMO

Increasing evidence indicates that long non-coding RNAs (lncRNAs) play vital roles in the tumorigenesis and progression of cancers. However, the functions and regulatory mechanisms of lncRNAs in nasopharyngeal carcinoma (NPC) are still largely unknown. Our previous lncRNA expression profiles identified that LINC01503 was overexpressed in NPC. Here, we verified that LINC01503 was highly expressed in NPC and correlated with poor prognosis. LINC01503 promoted NPC cell proliferation, migration, and invasion in vitro, and facilitated tumor growth and metastasis in vivo. Mechanistically, LINC01503 recruited splicing factor proline-and glutamine-rich (SFPQ) to activate Fos like 1 (FOSL1) transcription, and ectopic expression of FOSL1 reversed the suppressive effect of LINC01503 knockdown on NPC progression. Moreover, androgen receptor (AR)-mediated transcription activation was responsible for the overexpression of LINC01503, and AR ligand-dependent cell growth, migration, and invasion in NPC cells. Taken together, our findings reveal that AR-induced LINC01503 can promote NPC progression through the SFPQ-FOSL1 axis, which represents a novel prognostic biomarker and therapeutic target for NPC patients.


Assuntos
Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica , Carcinoma Nasofaríngeo/genética , Neoplasias Nasofaríngeas/genética , Fator de Processamento Associado a PTB/genética , Proteínas Proto-Oncogênicas c-fos/genética , RNA Longo não Codificante/genética , Receptores Androgênicos/genética , Animais , Linhagem Celular , Linhagem Celular Tumoral , Células HEK293 , Humanos , Estimativa de Kaplan-Meier , Metástase Linfática , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/terapia , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/terapia , Fator de Processamento Associado a PTB/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Interferência de RNA , Receptores Androgênicos/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos
18.
J Headache Pain ; 21(1): 72, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32522232

RESUMO

BACKGROUND: Vestibular migraine has recently been recognized as a novel subtype of migraine. However, the mechanism that relate vestibular symptoms to migraine had not been well elucidated. Thus, the present study investigated vestibular dysfunction in a rat model of chronic migraine (CM), and to dissect potential mechanisms between migraine and vertigo. METHODS: Rats subjected to recurrent intermittent administration of nitroglycerin (NTG) were used as the CM model. Migraine- and vestibular-related behaviors were analyzed. Immunofluorescent analyses and quantitative real-time polymerase chain reaction were employed to detect expressions of c-fos and calcitonin gene-related peptide (CGRP) in the trigeminal nucleus caudalis (TNC) and vestibular nucleus (VN). Morphological changes of vestibular afferent terminals was determined under transmission electron microscopy. FluoroGold (FG) and CTB-555 were selected as retrograde tracers and injected into the VN and TNC, respectively. Lentiviral vectors comprising CGRP short hairpin RNA (LV-CGRP) was injected into the trigeminal ganglion. RESULTS: CM led to persistent thermal hyperalgesia, spontaneous facial pain, and prominent vestibular dysfunction, accompanied by the upregulation of c-fos labeling neurons and CGRP immunoreactivity in the TNC (c-fos: vehicle vs. CM = 2.9 ± 0.6 vs. 45.5 ± 3.4; CGRP OD: vehicle vs. CM = 0.1 ± 0.0 vs. 0.2 ± 0.0) and VN (c-fos: vehicle vs. CM = 2.3 ± 0.8 vs. 54.0 ± 2.1; CGRP mRNA: vehicle vs. CM = 1.0 ± 0.1 vs. 2.4 ± 0.1). Furthermore, FG-positive neurons was accumulated in the superficial layer of the TNC, and the number of c-fos+/FG+ neurons were significantly increased in rats with CM compared to the vehicle group (vehicle vs. CM = 25.3 ± 2.2 vs. 83.9 ± 3.0). Meanwhile, CTB-555+ neurons dispersed throughout the VN. The structure of vestibular afferent terminals was less pronounced after CM compared with the peripheral vestibular dysfunction model. In vivo knockdown of CGRP in the trigeminal ganglion significantly reduced the number of c-fos labeling neurons (LV-CGRP vs. LV-NC = 9.9 ± 3.0 vs. 60.0 ± 4.5) and CGRP mRNA (LV-CGRP vs. LV-NC = 1.0 ± 0.1 vs. 2.1 ± 0.2) in the VN, further attenuating vestibular dysfunction after CM. CONCLUSIONS: These data demonstrates the possibility of sensitization of vestibular nucleus neurons to impair vestibular function after CM, and anti-CGRP treatment to restore vestibular dysfunction in patients with CM.


Assuntos
Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Transtornos de Enxaqueca/fisiopatologia , Núcleos Vestibulares/metabolismo , Animais , Hiperalgesia/metabolismo , Masculino , Nitroglicerina/farmacologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Núcleo Inferior Caudal do Nervo Trigêmeo/metabolismo , Gânglio Trigeminal/metabolismo
19.
Proc Natl Acad Sci U S A ; 117(27): 16000-16008, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571910

RESUMO

Carbonic anhydrases (CAs; EC 4.2.1.1) are metalloenzymes present in mammals with 16 isoforms that differ in terms of catalytic activity as well as cellular and tissue distribution. CAs catalyze the conversion of CO2 to bicarbonate and protons and are involved in various physiological processes, including learning and memory. Here we report that the integrity of CA activity in the brain is necessary for the consolidation of fear extinction memory. We found that systemic administration of acetazolamide, a CA inhibitor, immediately after the extinction session dose-dependently impaired the consolidation of fear extinction memory of rats trained in contextual fear conditioning. d-phenylalanine, a CA activator, displayed an opposite action, whereas C18, a membrane-impermeable CA inhibitor that is unable to reach the brain tissue, had no effect. Simultaneous administration of acetazolamide fully prevented the procognitive effects of d-phenylalanine. Whereas d-phenylalanine potentiated extinction, acetazolamide impaired extinction also when infused locally into the ventromedial prefrontal cortex, basolateral amygdala, or hippocampal CA1 region. No effects were observed when acetazolamide or d-phenylalanine was infused locally into the substantia nigra pars compacta. Moreover, systemic administration of acetazolamide immediately after the extinction training session modulated c-Fos expression on a retention test in the ventromedial prefrontal cortex of rats trained in contextual fear conditioning. These findings reveal that the engagement of CAs in some brain regions is essential for providing the brain with the resilience necessary to ensure the consolidation of extinction of emotionally salient events.


Assuntos
Anidrases Carbônicas/metabolismo , Medo/fisiologia , Memória/fisiologia , Animais , Complexo Nuclear Basolateral da Amígdala/fisiologia , Região CA1 Hipocampal/fisiologia , Emoções , Aprendizagem , Masculino , Camundongos , Córtex Pré-Frontal/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Wistar
20.
Nat Neurosci ; 23(8): 952-958, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32514139

RESUMO

In Alzheimer's disease (AD), hippocampus-dependent memories underlie an extensive decline. The neuronal ensemble encoding a memory, termed engram, is partially recapitulated during memory recall. Artificial activation of an engram can restore memory in a mouse model of early AD, but its fate and the factors that render the engram nonfunctional are yet to be revealed. Here, we used repeated two-photon in vivo imaging to analyze fosGFP transgenic mice (which express enhanced GFP under the Fos promoter) performing a hippocampus-dependent memory task. We found that partial reactivation of the CA1 engram during recall is preserved under AD-like conditions. However, we identified a novelty-like ensemble that interfered with the engram and thus compromised recall. Mimicking a novelty-like ensemble in healthy mice was sufficient to affect memory recall. In turn, reducing the novelty-like signal rescued the recall impairment under AD-like conditions. These findings suggest a novel mechanistic process that contributes to the deterioration of memories in AD.


Assuntos
Doença de Alzheimer/fisiopatologia , Hipocampo/fisiologia , Rememoração Mental/fisiologia , Proteínas Proto-Oncogênicas c-fos/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/fisiologia , Optogenética , Proteínas Proto-Oncogênicas c-fos/genética
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