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1.
Genes Dev ; 34(1-2): 72-86, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31831627

RESUMO

Yes-associated protein (YAP) and its homolog transcriptional coactivator with PDZ-binding motif (TAZ) are key effectors of the Hippo pathway to control cell growth and organ size, of which dysregulation yields to tumorigenesis or hypertrophy. Upon activation, YAP/TAZ translocate into the nucleus and bind to TEAD transcription factors to promote transcriptional programs for proliferation or cell specification. Immediate early genes, represented by AP-1 complex, are rapidly induced and control later-phase transcriptional program to play key roles in tumorigenesis and organ maintenance. Here, we report that YAP/TAZ directly promote FOS transcription that in turn contributes to the biological function of YAP/TAZ. YAP/TAZ bind to the promoter region of FOS to stimulate its transcription. Deletion of YAP/TAZ blocks the induction of immediate early genes in response to mitogenic stimuli. FOS induction contributes to expression of YAP/TAZ downstream target genes. Genetic deletion or chemical inhibition of AP-1 suppresses growth of YAP-driven cancer cells, such as Lats1/2-deficient cancer cells as well as Gαq/11 mutated uveal melanoma. Furthermore, AP-1 inhibition almost completely abrogates the hepatomegaly induced by YAP overexpression. Our findings reveal a feed-forward interplay between immediate early transcription of AP-1 and Hippo pathway function.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Neoplásica da Expressão Gênica , Transativadores/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Deleção de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes fos/genética , Células HEK293 , Humanos , Fígado/metabolismo , Melanoma/fisiopatologia , Camundongos , Mitógenos/farmacologia , Tamanho do Órgão/genética , Regiões Promotoras Genéticas/genética , Neoplasias Uveais/fisiopatologia
2.
J Basic Microbiol ; 59(12): 1238-1247, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31613018

RESUMO

Penicillium griseoroseum lectin was 80-fold purified by successive DEAE Sepharose anion exchange and Sephadex G-100 gel permeation chromatography. P. griseoroseum lectin exhibited haemagglutination activity towards protease-treated rabbit erythrocytes. It showed specificity towards various carbohydrates such as d-mannose, N-acetyl-d-glucosamine, mucins, and so forth. P. griseoroseum lectin was found as a glycoprotein with glycan content of 4.33%. Purified P. griseoroseum lectin is homodimeric having a molecular mass of 57 kDa with subunit molecular mass of 28.6 kDa. Haemagglutination activity of purified P. griseoroseum lectin was completely stable from 25°C to 35°C at a pH range of 6-7.5. Lectin activity was not influenced by divalent metal ions and denaturants. P. griseoroseum lectin manifested mitogenicity towards mice splenocytes and activity reached a peak at 75 µg/ml of lectin concentration. P. griseoroseum lectin in microgram concentrations stimulated proliferation of mice splenocytes. Thus, P. griseoroseum lectin exhibits potential mitogenicity, which can be exploited for further biomedical applications.


Assuntos
Lectinas/química , Lectinas/isolamento & purificação , Mitógenos/química , Mitógenos/isolamento & purificação , Penicillium/química , Animais , Carboidratos/química , Cátions/metabolismo , Proliferação de Células/efeitos dos fármacos , Quelantes , Glicoproteínas/química , Hemaglutinação , Concentração de Íons de Hidrogênio , Lectinas/farmacologia , Masculino , Camundongos , Mitógenos/farmacologia , Peso Molecular , Multimerização Proteica , Estabilidade Proteica , Temperatura
3.
Protein Pept Lett ; 26(12): 887-892, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31544688

RESUMO

BACKGROUND: Lectins have been studied in recent years due to their immunomodulatory activities. OBJECTIVE: We purified a lectin named OniL from tilapia fish (Oreochromis niloticus) and here we analyzed the cell proliferation and cytokine production in Balb/c mice splenocytes. METHODS: Cells were stimulated in vitro in 24, 48, 72 hours and 6 days with different concentrations of OniL and Con A. Evaluation of cell proliferation was performed through [3H]-thymidine incorporation, cytokines were investigated using ELISA assay and cell viability assay was performed by investigation of damage through signals of apoptosis and necrosis. RESULTS: OniL did not promote significant cell death, induced high mitogenic activity in relation to control and Con A and stimulated the cells to release high IL-2 and IL-6 cytokines. CONCLUSION: These findings suggest that, like Con A, OniL lectin can be used as a mitogenic agent in immunostimulatory assays.


Assuntos
Proliferação de Células/efeitos dos fármacos , Lectinas de Ligação a Manose/farmacologia , Mitógenos/farmacologia , Baço/citologia , Animais , Morte Celular , Linhagem Celular , Sobrevivência Celular , Concanavalina A/farmacologia , Citocinas/biossíntese , Masculino , Camundongos Endogâmicos BALB C , Tilápia
4.
Mater Sci Eng C Mater Biol Appl ; 104: 109924, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31499991

RESUMO

We report the first experimental evidence for the mitogenic action of cerium(IV) oxide and cerium(III) fluoride nanoparticles (CONs and CFNs) on the regeneration of a whole organism - freshwater flatworms Schmidtea mediterranea (planarian). Both types of cerium-containing nanoparticles are shown to be a highly potent mitogen for planaria. Both CONs and CFNs, in micro- and nanomolar concentrations, markedly accelerate planarian blastema growth, due to the enhancement of cellular proliferation, causing an increase in the mitotic index and in the quantity of blastema cells in regenerating planaria. CONs provided maximum activity at concentrations which were two orders of magnitude lower than those for CeF3. The valence state of cerium in cerium-containing nanoparticles plays a significant role in the planarian regeneration mechanism: CeO2 nanoparticles containing predominantly Ce4+ species presumably scavenge wound induced reactive oxygen species and moderately activate gene expression processes, while the regenerative action of CeF3 nanoparticles containing only Ce3+ species is manifested in the pronounced expression of the genes involved in cell division, differentiation and migration. This is the first report on the effect of cerium-containing nanoparticles on tissue regeneration in vivo, further revealing the mechanisms of their biological action, which enhances the possibility of their use in cellular technologies.


Assuntos
Cério/farmacologia , Fluoretos/farmacologia , Compostos Inorgânicos/farmacologia , Mitógenos/farmacologia , Nanopartículas/química , Planárias/citologia , Planárias/fisiologia , Regeneração/efeitos dos fármacos , Animais , Morte Celular/efeitos dos fármacos , DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Instabilidade Genômica , Cabeça , Mitose/efeitos dos fármacos , Mutagênicos/toxicidade , Planárias/efeitos dos fármacos , Planárias/genética , Testes de Toxicidade
5.
Mol Cell ; 76(4): 562-573.e4, 2019 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-31543423

RESUMO

Cells escape the need for mitogens at a restriction point several hours before entering S phase. The restriction point has been proposed to result from CDK4/6 initiating partial Rb phosphorylation to trigger a bistable switch whereby cyclin E-CDK2 and Rb mutually reinforce each other to induce Rb hyperphosphorylation. Here, using single-cell analysis, we unexpectedly found that cyclin E/A-CDK activity can only maintain Rb hyperphosphorylation starting at the onset of S phase and that CDK4/6 activity, but not cyclin E/A-CDK activity, is required to hyperphosphorylate Rb throughout G1 phase. Mitogen removal in G1 results in a gradual loss of CDK4/6 activity with a high likelihood of cells sustaining Rb hyperphosphorylation until S phase, at which point cyclin E/A-CDK activity takes over. Thus, it is short-term memory, or transient hysteresis, in CDK4/6 activity following mitogen removal that sustains Rb hyperphosphorylation, demonstrating a probabilistic rather than an irreversible molecular mechanism underlying the restriction point.


Assuntos
Proliferação de Células , Quinase 4 Dependente de Ciclina/metabolismo , Quinase 6 Dependente de Ciclina/metabolismo , Células Epiteliais/efeitos dos fármacos , Pontos de Checagem da Fase G1 do Ciclo Celular , Mitógenos/farmacologia , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Células Epiteliais/enzimologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/enzimologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Camundongos , Modelos Biológicos , Fosforilação , Proteínas de Ligação a Retinoblastoma/metabolismo , Transdução de Sinais , Fatores de Tempo , Ubiquitina-Proteína Ligases/metabolismo
6.
Appl Microbiol Biotechnol ; 103(17): 7017-7027, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31289905

RESUMO

Fibroblast growth factor (FGF) 13, a member of the FGF11 subfamily, is a kind of intracrine protein similar to other family members including FGF11, FGF12, and FGF14. Unlike classical FGF, FGF13 exerts its bioactivities independent of fibroblast growth factor receptors (FGFRs). However, the effect of exogenous administration of FGF13 still remains further investigated. In the present study, we established an Escherichia coli expression system for the large-scale production of FGF13 and then obtained two isoform proteins including recombinant human FGF13A (rhFGF13A) and rhFGF13B with a purity greater than 90% by column chromatography, respectively. Otherwise, soluble analysis indicated that both rhFGF13A and rhFGF13B expressed in E. coli BL21 (DE3) pLysS were soluble. Furthermore, cellular-based experiments demonstrated that rhFGF13A, rather than rhFGF13B, could promote the proliferation of NIH3T3 cells in the presence of heparin. Mechanistically, the mitogenic effect of FGF13 was mediated by activation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK), but not p38. Moreover, blockage of FGFRs also significantly attenuated the mitogenic effects of rhFGF13A, implying that FGFRs are still related to FGF13. Thus, our research shows that exogenous FGF13 can act as secreted FGF to participate in cell signal transmission and heparin is still required as an ancillary cofactor for the mitogenic effects of FGF13, which may help people to discover more potential functions of FGF13 in cell life activities.


Assuntos
Escherichia coli/metabolismo , Fatores de Crescimento de Fibroblastos/isolamento & purificação , Fatores de Crescimento de Fibroblastos/farmacologia , Mitógenos/isolamento & purificação , Mitógenos/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/isolamento & purificação , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Heparina/farmacologia , Humanos , Camundongos , Mitógenos/genética , Mitógenos/metabolismo , Células NIH 3T3 , Isoformas de Proteínas , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais
7.
Nat Commun ; 10(1): 2016, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31043605

RESUMO

Appropriate therapeutic modulation of endothelial proliferation and sprouting is essential for the effective inhibition of angiogenesis in cancer or its induction in cardiovascular disease. The current view is that an increase in growth factor concentration, and the resulting mitogenic activity, increases both endothelial proliferation and sprouting. Here, we modulate mitogenic stimuli in different vascular contexts by interfering with the function of the VEGF and Notch signalling pathways at high spatiotemporal resolution in vivo. Contrary to the prevailing view, our results indicate that high mitogenic stimulation induced by VEGF, or Notch inhibition, arrests the proliferation of angiogenic vessels. This is due to the existence of a bell-shaped dose-response to VEGF and MAPK activity that is counteracted by Notch and p21, determining whether endothelial cells sprout, proliferate, or become quiescent. The identified mechanism should be considered to achieve optimal therapeutic modulation of angiogenesis.


Assuntos
Endotélio Vascular/efeitos dos fármacos , Mitógenos/farmacologia , Neovascularização Patológica/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Endotélio Vascular/patologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Knockout , Neovascularização Patológica/patologia , Receptores Notch/antagonistas & inibidores , Receptores Notch/metabolismo , Retina , Vasos Retinianos , Transdução de Sinais/genética , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo
8.
Int J Mol Sci ; 20(5)2019 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-30818819

RESUMO

Allergic conjunctivitis (AC) is one of the most common ophthalmological disorders seen in clinical practice. Growing evidence from recent years suggests that a subset of IL-10-expressing B cells is involved in inflammatory allergic diseases. In this study, we aimed to evaluate the potential involvement of blood Bregs cells in perennial allergic conjunctivitis (PAC), and interleukins (IL)-1ß, IL-6, IL-8, IL-10, and IL-12, and tumor necrosis factor (TNF)-α, were measured in tear samples and compared with healthy controls (HC) using flow cytometry. Non-significant differences in CD19⁺IL-10⁺ cell frequency between PAC patients and healthy controls (HC) were observed. Nevertheless, when we analyzed the mean fluorescence intensity (MFI) of IL-10 on CD19⁺CD38Lo/Med/Hi-gated cells, we observed a significant decrease in MFI in all Bregs subsets in PAC patients. Additionally, tear cytokines showed 2.8 times lower levels of IL-10 than TNF-α in PAC patients when compared to HC. Our findings demonstrate an immunological dysregulation in patients with allergic conjunctivitis, characterized by the low expression of IL-10 in circulating CD19⁺CD38⁺ Bregs subsets and an inverted tear IL-10/TNF-α ratio, promoting a local pro-inflammatory microenvironment. These findings highlight the novel pathologic changes involved in ocular allergic diseases. Understanding systemic and local mechanisms will aid the design of immunomodulating therapeutics at different levels.


Assuntos
Linfócitos B Reguladores/metabolismo , Conjuntivite Alérgica/imunologia , Conjuntivite Alérgica/metabolismo , Interleucina-10/metabolismo , Lágrimas/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Adolescente , Estudos de Casos e Controles , Criança , Feminino , Humanos , Subpopulações de Linfócitos/metabolismo , Masculino , Mitógenos/farmacologia
9.
Dev Cell ; 48(6): 853-863.e5, 2019 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-30713073

RESUMO

Attaining proper organ size during development and regeneration hinges on the activity of mitogenic factors. Here, we performed a large-scale chemical screen in embryonic zebrafish to identify cardiomyocyte mitogens. Although commonly considered anti-proliferative, vitamin D analogs like alfacalcidol had rapid, potent mitogenic effects on embryonic and adult cardiomyocytes in vivo. Moreover, pharmacologic or genetic manipulation of vitamin D signaling controlled proliferation in multiple adult cell types and dictated growth rates in embryonic and juvenile zebrafish. Tissue-specific modulation of vitamin D receptor (VDR) signaling had organ-restricted effects, with cardiac VDR activation causing cardiomegaly. Alfacalcidol enhanced the regenerative response of injured zebrafish hearts, whereas VDR blockade inhibited regeneration. Alfacalcidol activated cardiac expression of genes associated with ErbB2 signaling, while ErbB2 inhibition blunted its effects on cell proliferation. Our findings identify vitamin D as mitogenic for cardiomyocytes and other cell types in zebrafish and indicate a mechanism to regulate organ size and regeneration.


Assuntos
Coração/anatomia & histologia , Coração/fisiologia , Miócitos Cardíacos/citologia , Regeneração/efeitos dos fármacos , Vitamina D/farmacologia , Peixe-Zebra/anatomia & histologia , Peixe-Zebra/fisiologia , Animais , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Coração/efeitos dos fármacos , Mitógenos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Tamanho do Órgão/efeitos dos fármacos , Especificidade de Órgãos , Transdução de Sinais/efeitos dos fármacos , Peixe-Zebra/embriologia , Proteínas de Peixe-Zebra/metabolismo
10.
J Vet Intern Med ; 33(2): 889-896, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30693587

RESUMO

BACKGROUND: Equine protozoal myeloencephalitis (EPM) is a common and devastating neurologic disease of horses in the United States. Because some EPM-affected horses have decreased immune responses, immunomodulators such as levamisole have been proposed as supplemental treatments. However, little is known about levamisole's effects or its mechanism of action in horses. OBJECTIVE: Levamisole in combination with another mitogen will stimulate a macrophage 1 (M1), dendritic cell 1 (DC1), T-helper 1 (CD4 Th1), and T-cytotoxic (CD8) immune response in equine peripheral blood mononuclear cells (PBMCs) in vitro as compared to mitogen alone. ANIMALS: Ten neurologically normal adult horses serologically negative for Sarcocystis neurona. METHODS: Prospective study. Optimal conditions for levamisole were determined based on cellular proliferation. Peripheral blood mononuclear cells were then cultured using optimal conditions of mitogen and levamisole to identify the immune phenotype, based on subset-specific activation markers, intracellular cytokine production, and cytokine concentrations in cell supernatants. Subset-specific proliferation was determined using a vital stain. RESULTS: Concanavalin A (conA) with levamisole, but not levamisole alone, resulted in a significant decrease (P < .05) in PBMC proliferation compared to conA alone. Levamisole alone did not elicit a specific immune phenotype different than that induced by conA. CONCLUSION AND CLINICAL IMPORTANCE: Levamisole co-cultured with conA significantly attenuated the PBMC proliferative response as compared with conA. If the mechanisms by which levamisole modulates the immune phenotype can be further defined, levamisole may have potential use in the treatment of inflammatory diseases.


Assuntos
Adjuvantes Imunológicos/farmacologia , Cavalos/imunologia , Leucócitos Mononucleares/efeitos dos fármacos , Levamisol/farmacologia , Animais , Células Cultivadas , Concanavalina A/farmacologia , Feminino , Leucócitos Mononucleares/imunologia , Masculino , Mitógenos/farmacologia , Estudos Prospectivos , Regulação para Cima
11.
Methods Mol Biol ; 1881: 27-34, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30350195

RESUMO

Chromosome analysis of chronic lymphocytic leukemia (CLL) is an important clinical tool for evaluating prognosis and disease progression. Visualizing chromosomes microscopically using traditional cytogenetic techniques requires dividing cells to be arrested during metaphase. The major challenge for performing this analysis on CLL samples is stimulating the cells to divide in culture. Stimulation of CLL cells with CpG oligodeoxynucleotides has improved our ability to perform chromosome analysis for this leukemia. This protocol should help the reader successfully culture CLL samples for clinical chromosome analysis.


Assuntos
Leucemia Linfocítica Crônica de Células B/genética , Oligodesoxirribonucleotídeos/farmacologia , Cultura Primária de Células/métodos , Linfócitos B/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Humanos , Cariotipagem/instrumentação , Cariotipagem/métodos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/patologia , Metáfase/efeitos dos fármacos , Mitógenos/farmacologia , Cultura Primária de Células/instrumentação , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Células Tumorais Cultivadas
12.
Cell Stem Cell ; 24(1): 79-92.e6, 2019 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-30581080

RESUMO

In many tissues, homeostasis is maintained by physical contact between stem cells and an anatomically defined niche. However, how stem cell homeostasis is achieved in environments where cells are motile and dispersed among their progeny remains unknown. Using murine spermatogenesis as a model, we find that spermatogenic stem cell density is tightly regulated by the supply of fibroblast growth factors (FGFs) from lymphatic endothelial cells. We propose that stem cell homeostasis is achieved through competition for a limited supply of FGFs. We show that the quantitative dependence of stem cell density on FGF dosage, the biased localization of stem cells toward FGF sources, and stem cell dynamics during regeneration following injury can all be predicted and explained within the framework of a minimal theoretical model based on "mitogen competition." We propose that this model provides a generic and robust mechanism to support stem cell homeostasis in open, or facultative, niche environments.


Assuntos
Fator 5 de Crescimento de Fibroblastos/fisiologia , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Homeostase , Mitógenos/farmacologia , Espermatogênese , Espermatozoides/citologia , Células-Tronco/citologia , Animais , Diferenciação Celular , Autorrenovação Celular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Espermatozoides/fisiologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologia
13.
J Endocrinol Invest ; 42(3): 271-282, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29934772

RESUMO

PURPOSE: Thyroid disorders are clinically associated with impaired fertility in women, and these abnormalities can be improved by restoring the euthyroid state. The exact mechanisms of thyroid effect on female fertility are not well known; however, it is conceivable that thyroid hormones (THs) might act on ovarian physiology via receptors in granulosa cells. This work is aimed at evaluating the effects of THs on non-tumoral granulosa cells and follicles. METHODS: Freshly isolated rat ovarian follicles and granulosa cells were exposed to T3 or T4 (THs). Cell growth and viability were evaluated by cell counting and the MTT assay, respectively, follicle growth was evaluated by volume measurements. Apoptosis was evaluated by the TUNEL assay and active Caspase 3 staining. rGROV cells were exposed to T3, and apoptosis was induced by serum deprivation. Bcl2, Bcl-2-associated X protein (BAX), Akt and pAkt expression were evaluated by western blot. RESULTS: T3 induced a 40% increase in follicle volume (after 7 days). This increase was presumably due to the observed decrease (33%) in the apoptotic rate of the granulosa cell population. Both T3 and T4 caused a dose-dependent increase in rat granulosa cell number and viability. In addition, THs decreased the cell apoptotic rate in a dose-dependent manner. In both conditions, T3 appeared to be more efficient. In rGROV cells, 100 nM T3 induced cell growth and, in the absence of growth factors, reduced cell apoptosis by 40%, downregulating Caspase 3 and BAX. This effect was associated with an increase in pAkt levels. The involvement of the PI3 K pathway was confirmed by the ability of the PI3 K specific inhibitor (LY-294,002) to abolish T3 pro-survival action. CONCLUSIONS: THs influence cell survival of ovarian granulosa cells. This effect likely contributes to the TH-induced follicle volume increase.


Assuntos
Proliferação de Células/efeitos dos fármacos , Células da Granulosa/citologia , Mitógenos/farmacologia , Folículo Ovariano/citologia , Tiroxina/farmacologia , Tri-Iodotironina/farmacologia , Animais , Apoptose , Células Cultivadas , Feminino , Células da Granulosa/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Ratos , Ratos Wistar
14.
Genes Dev ; 32(21-22): 1398-1419, 2018 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-30366908

RESUMO

The transcription factor MYC (also c-Myc) induces histone modification, chromatin remodeling, and the release of paused RNA polymerase to broadly regulate transcription. MYC is subject to a series of post-translational modifications that affect its stability and oncogenic activity, but how these control MYC's function on the genome is largely unknown. Recent work demonstrates an intimate connection between nuclear compartmentalization and gene regulation. Here, we report that Ser62 phosphorylation and PIN1-mediated isomerization of MYC dynamically regulate the spatial distribution of MYC in the nucleus, promoting its association with the inner basket of the nuclear pore in response to proliferative signals, where it recruits the histone acetyltransferase GCN5 to bind and regulate local gene acetylation and expression. We demonstrate that PIN1-mediated localization of MYC to the nuclear pore regulates MYC target genes responsive to mitogen stimulation that are involved in proliferation and migration pathways. These changes are also present at the chromatin level, with an increase in open regulatory elements in response to stimulation that is PIN1-dependent and associated with MYC chromatin binding. Taken together, our study indicates that post-translational modification of MYC controls its spatial activity to optimally regulate gene expression in response to extrinsic signals in normal and diseased states.


Assuntos
Poro Nuclear/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-myc/metabolismo , Ativação Transcricional , Animais , Linhagem Celular , Células Cultivadas , Cromatina/metabolismo , Humanos , Camundongos , Camundongos Knockout , Mitógenos/farmacologia , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-myc/química , Serina/metabolismo , Cicatrização , Fatores de Transcrição de p300-CBP/metabolismo
15.
Dig Dis Sci ; 63(12): 3382-3397, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30196390

RESUMO

BACKGROUND AND AIMS: Concanavalin A is known to activate T cells and to cause liver injury and hepatitis, mediated in part by secretion of TNFα from macrophages. Poly(ADP-ribose) polymerase-1 (PARP-1) inhibitors have been shown to prevent tissue damage in various animal models of inflammation. The objectives of this study were to evaluate the efficacy and mechanism of the PARP-1 inhibitor 3-aminobenzamide (3-AB) in preventing concanavalin A-induced liver damage. METHODS: We tested the in vivo effects of 3-AB on concanavalin A-treated mice, its effects on lipopolysaccharide (LPS)-stimulated macrophages in culture, and its ability to act as a scavenger in in vitro assays. RESULTS: 3-AB markedly reduced inflammation, oxidative stress, and liver tissue damage in concanavalin A-treated mice. In LPS-stimulated RAW264.7 macrophages, 3-AB inhibited NFκB transcriptional activity and subsequent expression of TNFα and iNOS and blocked NO production. In vitro, 3-AB acted as a hydrogen peroxide scavenger. The ROS scavenger N-acetylcysteine (NAC) and the ROS formation inhibitor diphenyleneiodonium (DPI) also inhibited TNFα expression in stimulated macrophages, but unlike 3-AB, NAC and DPI were unable to abolish NFκB activity. PARP-1 knockout failed to affect NFκB and TNFα suppression by 3-AB in stimulated macrophages. CONCLUSIONS: Our results suggest that 3-AB has a therapeutic effect on concanavalin A-induced liver injury by inhibiting expression of the key pro-inflammatory cytokine TNFα, via PARP-1-independent NFκB suppression and via an NFκB-independent anti-oxidative mechanism.


Assuntos
Benzamidas/farmacologia , Hepatite , Macrófagos , Doença Aguda , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Células Cultivadas , Concanavalina A/farmacologia , Modelos Animais de Doenças , Hepatite/metabolismo , Hepatite/prevenção & controle , Macrófagos/efeitos dos fármacos , Macrófagos/fisiologia , Camundongos , Mitógenos/farmacologia , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/metabolismo
16.
Cell Biol Int ; 42(11): 1511-1522, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30080297

RESUMO

Proliferation of the adult hepatocyte population represents a central feature of tissue regeneration after liver injury and resection. This process could be driven by a diverse range of mitogens, such as hepatocyte growth factor (HGF) and fibroblast growth factor (FGF). Among FGF family, FGF2 is closely related to wound repair and cell proliferation. FGF2 does function in the process of angiogenesis in regenerating liver, while fewer reports are concerned with the impact and underlying mechanism of FGF2 on liver cell proliferation. To this end, an immortalized human normal hepatocyte L02 and mouse primary hepatocytes were exposed to FGF2 in this study. We demonstrate that FGF2 significantly enhances liver cell proliferation. Treatment with FGF2 obviously increases the phosphorylation level of extracellular signal-regulated kinases 1 and 2 (ERK1/2) and c-Jun N-terminal kinase (JNK). Activity inhibition or expression down-regulation prove that both ERK1/2 and JNK signaling are required for FGF2-mediated effect on liver cell proliferation. Interestingly, interfering of ERK1/2 signaling results in marked decrease of JNK activation under FGF2 treatment, and JNK signaling is also involved in regulation of FGF2-induced ERK1/2 activation, suggesting that cross-talk between ERK1/2 and JNK signaling is important for FGF2 mitogenic activity. Both ERK1/2 and JNK signal via CREB to function in proliferation impact of FGF2 on liver cells. Taken together, this study reveals that ERK and JNK pathways synergistically regulate FGF2-induced liver cell proliferation via phosphorylating CREB, which will contribute to the understanding of FGF2 impact on liver cell proliferation and liver regeneration.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator 2 de Crescimento de Fibroblastos/farmacologia , Fígado/citologia , Sistema de Sinalização das MAP Quinases , Mitógenos/farmacologia , Animais , Proliferação de Células/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Modelos Biológicos
17.
Clin Sci (Lond) ; 132(14): 1615-1627, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30006481

RESUMO

Increased airway smooth muscle (ASM) mass is observed in chronic obstructive pulmonary disease (COPD), which is correlated with disease severity and negatively affects lung function in these patients. Thus, there is clear unmet clinical need for finding new therapies which can target airway remodeling and disease progression in COPD. Apoptosis signal-regulating kinase 1 (ASK1) is a ubiquitously expressed mitogen-activated protein kinase (MAPK) kinase kinase (MAP3K) activated by various stress stimuli, including reactive oxygen species (ROS), tumor necrosis factor (TNF)-α, and lipopolysaccharide (LPS) and is known to regulate cell proliferation. ASM cells from COPD patients are hyperproliferative to mitogens in vitro However, the role of ASK1 in ASM growth is not established. Here, we aim to determine the effects of ASK1 inhibition on ASM growth and pro-mitogenic signaling using ASM cells from COPD patients. We found greater expression of ASK1 in ASM bundles of COPD lung when compared with non-COPD. Pre-treatment of ASM cells with highly selective ASK1 inhibitor, TC ASK 10 resulted in a dose-dependent reduction in mitogen (FBS, PDGF, and EGF; 72 h)-induced ASM growth as measured by CyQUANT assay. Further, molecular targetting of ASK1 using siRNA in ASM cells prevented mitogen-induced cell growth. In addition, to anti-mitogenic potential, ASK1 inhibitor also prevented TGFß1-induced migration of ASM cells in vitro Immunoblotting revealed that anti-mitogenic effects are mediated by C-Jun N-terminal kinase (JNK) and p38MAP kinase-signaling pathways as evident by reduced phosphorylation of downstream effectors JNK1/2 and p38MAP kinases, respectively, with no effect on extracellular signal-regulated kinase (ERK) 1/2 (ERK1/2). Collectively, these findings establish the anti-mitogenic effect of ASK1 inhibition and identify a novel pathway that can be targetted to reduce or prevent excessive ASM mass in COPD.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Pulmão/metabolismo , MAP Quinase Quinase Quinase 5/genética , Miócitos de Músculo Liso/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Humanos , Pulmão/citologia , MAP Quinase Quinase Quinase 5/antagonistas & inibidores , MAP Quinase Quinase Quinase 5/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Mitógenos/farmacologia , Músculo Liso/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , Interferência de RNA , Fator de Crescimento Transformador beta1/farmacologia
18.
J Neuroimmunol ; 321: 12-23, 2018 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-29957382

RESUMO

Regulation of µ-, δ- and κ-opioid receptor protein level in spleen lymphocytes when stimulated by mitogen is not known. To answer the question whether these cells do express opioid receptor (OR) proteins, primary, fresh rat spleen lymphocytes were prepared and stimulated for 48 h with mitogenic dose of Con A. The unstimulated lymphocytes did not express µ- and δ-OR proteins in detectable amounts, however, stimulation with Con A resulted in appearance of clearly detectable immunoblot signals of both µ-OR and δ-OR. κ-OR were detected already in primary cells and increased 2.4-fold in Con A-stimulated cells. These results were supported by data obtained by flow cytometry analysis indicating a dramatic increase in number of µ-, δ- and κ-OR expressing cells after mitogen stimulation. The newly synthesized µ-, δ- and κ-OR in Con A-stimulated spleen lymphocytes were present in the cells interior and not functionally mature, at least in terms of their ability to enhance activity of trimeric G proteins determined by three different protocols of agonist-stimulated, high-affinity [35S]GTPγS binding assay. The up-regulation of µ-, δ- and κ-OR was associated with specific decrease of their cognate trimeric G proteins, Gi1α/Gi2α; the other Gα and Gß subunits were unchanged. The level of ß-arrestin-1/2 was also decreased in Con A-stimulated splenocytes. We conclude that up-regulation of OR expression level in spleen lymphocytes by Con A proceeds in conjunction with down-regulation of their intracellular signaling partners, Gi1α/Gi2α proteins and ß-arrestin-1/2. These regulatory proteins are expressed in high amounts already in unstimulated cells and decreased by mitogen stimulation.


Assuntos
Linfócitos/metabolismo , Receptores Opioides delta/biossíntese , Receptores Opioides kappa/biossíntese , Receptores Opioides mu/biossíntese , Baço/metabolismo , Animais , Concanavalina A/farmacologia , Linfócitos/efeitos dos fármacos , Masculino , Mitógenos/farmacologia , Ratos , Ratos Wistar , Receptores Opioides delta/efeitos dos fármacos , Receptores Opioides kappa/efeitos dos fármacos , Receptores Opioides mu/efeitos dos fármacos , Baço/citologia , Baço/efeitos dos fármacos , Regulação para Cima
19.
Sci Rep ; 8(1): 7881, 2018 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-29777119

RESUMO

The aim of the study was to clarify the effect of long-term anti-TNF therapy on T cell function in patients with rheumatologic immune-mediated inflammatory diseases (IMID). The production of IFNγ by T cells was evaluated at baseline and after 1, 2, 4, and 8 years of anti-TNF agents by means of a QuantiFERON-TB Gold In-Tube assay. The T cell proliferation and surface co-expression of CD25/CD134 in response to phytohaemagglutinin together with the in vitro impact of anti-TNF therapy on the functional capacity of T cells were evaluated after 8 years from the onset of the biological treatment. Age-matched healthy donors were enrolled as controls. The quantitative mitogen-induced IFNγ responses significantly increased with respect to baseline at each time point, apart from the determination after 4 years. We found an increased expression of CD25/CD134 in CD4+ compared to CD8+ T cells both in patients and controls. The in vitro addition of anti-TNF agents induced a significant decrease of both the IFNγ response and of CD25/CD134, whereas no effect on the intensity of the proliferative response was observed. Our data provide a biological basis for the reassuring issues on the safety of long-term anti-TNF treatment in patients with IMID.


Assuntos
Adalimumab/uso terapêutico , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Etanercepte/uso terapêutico , Doenças Reumáticas/tratamento farmacológico , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Adulto , Idoso , Artrite Psoriásica/tratamento farmacológico , Artrite Psoriásica/patologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Doença Crônica , Feminino , Humanos , Interferon gama/metabolismo , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Mitógenos/farmacologia , Receptores OX40/metabolismo , Doenças Reumáticas/patologia
20.
Mol Cell Endocrinol ; 476: 37-47, 2018 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-29680290

RESUMO

Airway smooth muscle (ASM) cell hyperplasia driven by persistent inflammation is a hallmark feature of remodeling in asthma. Sex steroid signaling in the lungs is of considerable interest, given epidemiological data showing more asthma in pre-menopausal women and aging men. Our previous studies demonstrated that estrogen receptor (ER) expression increases in asthmatic human ASM; however, very limited data are available regarding differential roles of ERα vs. ERß isoforms in human ASM cell proliferation. In this study, we evaluated the effect of selective ERα and ERß modulators on platelet-derived growth factor (PDGF)-stimulated ASM proliferation and the mechanisms involved. Asthmatic and non-asthmatic primary human ASM cells were treated with PDGF, 17ß-estradiol, ERα-agonist and/or ERß-agonist and/or G-protein-coupled estrogen receptor 30 (GPR30/GPER) agonist and proliferation was measured using MTT and CyQuant assays followed by cell cycle analysis. Transfection of small interfering RNA (siRNA) ERα and ERß significantly altered the human ASM proliferation. The specificity of siRNA transfection was confirmed by Western blot analysis. Gene and protein expression of cell cycle-related antigens (PCNA and Ki67) and C/EBP were measured by RT-PCR and Western analysis, along with cell signaling proteins. PDGF significantly increased ASM proliferation in non-asthmatic and asthmatic cells. Treatment with PPT showed no significant effect on PDGF-induced proliferation, whereas WAY interestingly suppressed proliferation via inhibition of ERK1/2, Akt, and p38 signaling. PDGF-induced gene expression of PCNA, Ki67 and C/EBP in human ASM was significantly lower in cells pre-treated with WAY. Furthermore, WAY also inhibited PDGF-activated PCNA, C/EBP, cyclin-D1, and cyclin-E. Overall, we demonstrate ER isoform-specific signaling in the context of ASM proliferation. Activation of ERß can diminish remodeling in human ASM by inhibiting pro-proliferative signaling pathways, and may point to a novel perception for blunting airway remodeling.


Assuntos
Receptor beta de Estrogênio/metabolismo , Pulmão/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Transdução de Sinais , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/metabolismo , Receptor beta de Estrogênio/agonistas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucina-13/farmacologia , Antígeno Ki-67/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitógenos/farmacologia , Miócitos de Músculo Liso/efeitos dos fármacos , Antígeno Nuclear de Célula em Proliferação/metabolismo , RNA Interferente Pequeno/metabolismo , Receptores Estrogênicos/metabolismo , Receptores Acoplados a Proteínas-G/agonistas , Receptores Acoplados a Proteínas-G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
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