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1.
Int J Mol Sci ; 22(18)2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34576127

RESUMO

Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO●) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.


Assuntos
Diferenciação Celular , Radicais Livres/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Proteínas/metabolismo , Acetofenonas/farmacologia , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Espectroscopia de Ressonância de Spin Eletrônica , Células HL-60 , Humanos , Radical Hidroxila , Monócitos/efeitos dos fármacos , NADP/metabolismo , Coloração e Rotulagem , Acetato de Tetradecanoilforbol/farmacologia , Células U937
2.
Gene ; 800: 145842, 2021 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-34274479

RESUMO

Treatment of serum-starved quiescent human cells with fetal bovine serum (FBS), epidermal growth factor (EGF), or the phorbol ester (12-O-tetradecanoylphorbol-13-acetate, TPA) activates the RAS-MAPK pathway which initiates a transcriptional program which drives cells toward proliferation. Stimulation of the RAS-MAPK pathway activates mitogen- and stress-activated kinases (MSK) 1 and 2, which phosphorylate histone H3 at S10 (H3S10ph) or S28 (H3S28ph) (nucleosomal response) located at the regulatory regions of immediate-early genes, setting in motion a series of chromatin remodeling events that result in transcription initiation. To investigate immediate-early genes regulated by the MSK, we have completed transcriptome analyses (RNA sequencing) of human normal fibroblast cells (CCD-1070Sk) stimulated with EGF or TPA ± H89, a potent MSK/PKA inhibitor. The induction of many immediate-early genes was independent of MSK activity. However, the induction of immediate-early genes attenuated with H89 also had reduced induction with the PKA inhibitor, Rp-cAMPS. Several EGF-induced genes, coding for transcriptional repressors, were further upregulated with H89 but not with Rp-cAMPS, suggesting a role for MSK in modulating the induction level of these genes.


Assuntos
Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Mitógenos/farmacologia , Linhagem Celular , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Fibroblastos/fisiologia , Perfilação da Expressão Gênica , Genes Precoces/efeitos dos fármacos , Humanos , Isoquinolinas/farmacologia , Reprodutibilidade dos Testes , Proteínas Quinases S6 Ribossômicas 90-kDa/antagonistas & inibidores , Sulfonamidas/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , Tionucleotídeos/farmacologia
3.
Cell Death Dis ; 12(7): 659, 2021 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-34193827

RESUMO

Cellular stress can lead to several human disease pathologies due to aberrant cell death. The p53 family (tp53, tp63, and tp73) and downstream transcriptional apoptotic target genes (PUMA/BBC3 and NOXA/PMAIP1) have been implicated as mediators of stress signals. To evaluate the importance of key stress response components in vivo, we have generated zebrafish null alleles in puma, noxa, p53, p63, and p73. Utilizing these genetic mutants, we have deciphered that the apoptotic response to genotoxic stress requires p53 and puma, but not p63, p73, or noxa. We also identified a delayed secondary wave of genotoxic stress-induced apoptosis that is p53/puma independent. Contrary to genotoxic stress, ER stress-induced apoptosis requires p63 and puma, but not p53, p73, or noxa. Lastly, the oxidative stress-induced apoptotic response requires p63, and both noxa and puma. Our data also indicate that while the neural tube is poised for apoptosis due to genotoxic stress, the epidermis is poised for apoptosis due to ER and oxidative stress. These data indicate there are convergent as well as unique molecular pathways involved in the different stress responses. The commonality of puma in these stress pathways, and the lack of gross or tumorigenic phenotypes with puma loss suggest that a inhibitor of Puma may have therapeutic application. In addition, we have also generated a knockout of the negative regulator of p53, mdm2 to further evaluate the p53-induced apoptosis. Our data indicate that the p53 null allele completely rescues the mdm2 null lethality, while the puma null completely rescues the mdm2 null apoptosis but only partially rescues the phenotype. Indicating Puma is the key mediator of p53-dependent apoptosis. Interestingly the p53 homozygous null zebrafish develop tumors faster than the previously described p53 homozygous missense mutant zebrafish, suggesting the missense allele may be hypomorphic allele.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose , Dano ao DNA , Estresse do Retículo Endoplasmático , Estresse Oxidativo , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Animais , Animais Geneticamente Modificados , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Proteínas Reguladoras de Apoptose/genética , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação da Expressão Gênica , Macrolídeos/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Fosfoproteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia , Tapsigargina/farmacologia , Fatores de Tempo , Transativadores/genética , Transcrição Genética , Proteína Supressora de Tumor p53/genética , Raios X , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
4.
Arch Biochem Biophys ; 710: 108988, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34274337

RESUMO

Monocytes are differentiated into macrophages. In this study, mitochondrial DNA copy number (mtDNAcn) levels and downstream events such as the expression of respiratory chain mRNAs were investigated during the phorbol 12-myristate 13-acetate (PMA)-induced differentiation of monocytes. Although PMA treatment increased mtDNAcn, the expression levels of mRNAs encoded in mtDNA were decreased. The levels of mitochondrial transcription factor A mRNA and protein were also decreased. The levels of coenzyme Q10 remained unchanged. These results imply that, although mtDNAcn is considered as a health marker, the levels of mtDNAcn may not always be consistent with the parameters of mitochondrial functions.


Assuntos
DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Macrófagos/citologia , Macrófagos/metabolismo , Monócitos/citologia , Monócitos/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Variações do Número de Cópias de DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Macrófagos/efeitos dos fármacos , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas Mitocondriais/metabolismo , Monócitos/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Células THP-1 , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/metabolismo , Ubiquinona/análogos & derivados , Ubiquinona/metabolismo
5.
Int J Mol Sci ; 22(10)2021 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-34063504

RESUMO

Protein kinase C (PKC) activation induces cellular reprogramming and differentiation in various cell models. Although many effectors of PKC physiological actions have been elucidated, the molecular mechanisms regulating oligodendrocyte differentiation after PKC activation are still unclear. Here, we applied a liquid chromatography-mass spectrometry (LC-MS/MS) approach to provide a comprehensive analysis of the proteome expression changes in the MO3.13 oligodendroglial cell line after PKC activation. Our findings suggest that multiple networks that communicate and coordinate with each other may finally determine the fate of MO3.13 cells, thus identifying a modular and functional biological structure. In this work, we provide a detailed description of these networks and their participating components and interactions. Such assembly allows perturbing each module, thus describing its physiological significance in the differentiation program. We applied this approach by targeting the Rho-associated protein kinase (ROCK) in PKC-activated cells. Overall, our findings provide a resource for elucidating the PKC-mediated network modules that contribute to a more robust knowledge of the molecular dynamics leading to this cell fate transition.


Assuntos
Diferenciação Celular/fisiologia , Oligodendroglia/citologia , Oligodendroglia/metabolismo , Proteína Quinase C/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Espectrometria de Massas/métodos , Oligodendroglia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Quinases Associadas a rho/metabolismo
6.
Front Immunol ; 12: 649600, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34135890

RESUMO

Using the optogenetic photo-manipulation of photoactivatable (PA)-Rac1, remarkable cell surface ruffling and the formation of a macropinocytic cup (premacropinosome) could be induced in the region of RAW264 macrophages irradiated with blue light due to the activation of PA-Rac1. However, the completion of macropinosome formation did not occur until Rac1 was deactivated by the removal of the light stimulus. Following PA-Rac1 deactivation, some premacropinosomes closed into intracellular macropinosomes, whereas many others transformed into long Rab10-positive tubules without forming typical macropinosomes. These Rab10-positive tubules moved centripetally towards the perinuclear Golgi region along microtubules. Surprisingly, these Rab10-positive tubules did not contain any endosome/lysosome compartment markers, such as Rab5, Rab7, or LAMP1, suggesting that the Rab10-positive tubules were not part of the degradation pathway for lysosomes. These Rab10-positive tubules were distinct from recycling endosomal compartments, which are labeled with Rab4, Rab11, or SNX1. These findings suggested that these Rab10-positive tubules may be a part of non-degradative endocytic pathway that has never been known. The formation of Rab10-positive tubules from premacropinosomes was also observed in control and phorbol myristate acetate (PMA)-stimulated macrophages, although their frequencies were low. Interestingly, the formation of Rab10-positive premacropinosomes and tubules was not inhibited by phosphoinositide 3-kinase (PI3K) inhibitors, while the classical macropinosome formation requires PI3K activity. Thus, this study provides evidence to support the existence of Rab10-positive tubules as a novel endocytic pathway that diverges from canonical macropinocytosis.


Assuntos
Endocitose/imunologia , Macrófagos/imunologia , Microtúbulos/metabolismo , Pinocitose/imunologia , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Endocitose/efeitos dos fármacos , Endocitose/efeitos da radiação , Complexo de Golgi/metabolismo , Microscopia Intravital , Luz , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Microtúbulos/imunologia , Microtúbulos/efeitos da radiação , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Optogenética , Pinocitose/efeitos dos fármacos , Pinocitose/efeitos da radiação , Células RAW 264.7 , Acetato de Tetradecanoilforbol/farmacologia , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
7.
Methods Mol Biol ; 2285: 111-119, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33928547

RESUMO

An important hallmark for the characterisation of Th cells is their capacity for cytokine expression. In this chapter, we describe how Th cells can be restimulated polyclonally to reveal their cytokine-producing potential that can then be analysed by intracellular staining and flow cytometry.


Assuntos
Citocinas/metabolismo , Citometria de Fluxo , Análise de Célula Única , Linfócitos T Auxiliares-Indutores/metabolismo , Animais , Células Cultivadas , Humanos , Ionomicina/farmacologia , Projetos de Pesquisa , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Fluxo de Trabalho
8.
Methods Mol Biol ; 2285: 191-200, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33928554

RESUMO

Flow cytometric evaluation of phosphorylation status of signal transduction molecules is a useful method to study T-cell signaling pathways. As mutations occurring in TCR complex molecules, common gamma chain family's cytokines, their receptors or molecules involved in these pathways can lead to severe immune system defects, the study of T-cell signal transduction can be applied to both basic and clinical/translational research areas. In the present chapter, we show two different protocols for the study of T- cell response to an antigen-like stimulus and to IL-2.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Citometria de Fluxo , Fosfoproteínas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Animais , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Células Cultivadas , Humanos , Interleucina-2/farmacologia , Fosforilação , Receptores de Antígenos de Linfócitos T/agonistas , Projetos de Pesquisa , Transdução de Sinais/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fluxo de Trabalho
9.
Cells ; 10(3)2021 03 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799840

RESUMO

Reactive oxygen species (ROS)-induced oxidative stress in adipose tissue is associated with inflammation and the development of obesity-related metabolic disorders. The aim of this study is to investigate the effects of hydrogen nano-bubble water (HW) on ROS generation, adipogenesis, and interleukin-6 (IL-6) secretion in hydrogen peroxide (H2O2) or phorbol 12-myristate 13-acetate (PMA)-stimulated OP9 adipocytes, and three-dimensional (3D) subcutaneous adipose equivalents. Nanoparticle tracking analysis showed that fresh HW contains 1.17 × 108/mL of nano-sized hydrogen bubbles. Even after 8 to 13 months of storage, approximately half of the bubbles still remained in the water. CellROX® staining showed that HW could diminish H2O2- or PMA-induced intracellular ROS generation in human keratinocytes HaCaT and OP9 cells. We discovered that PMA could markedly increase lipid accumulation to 180% and IL-6 secretion 2.7-fold in OP9 adipocytes. Similarly, H2O2 (5 µM) also significantly stimulated lipid accumulation in OP9 cells and the 3D adipose equivalents. HW treatment significantly repressed H2O2- or PMA-induced lipid accumulation and IL-6 secretion in OP9 adipocytes and the 3D adipose equivalents. In conclusion, HW showed a possibility of repressing oxidative stress, inflammatory response, and adipogenesis at cellular/tissue levels. It can be used for preventing the development of metabolic disorders amongst obese people.


Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Peróxido de Hidrogênio/farmacologia , Hidrogênio/farmacologia , Interleucina-6/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Gordura Subcutânea/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Água/farmacologia , Adipócitos/metabolismo , Animais , Técnicas de Cocultura , Humanos , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Nanopartículas , Estresse Oxidativo/efeitos dos fármacos , Transdução de Sinais , Gordura Subcutânea/citologia , Gordura Subcutânea/metabolismo
10.
Nutrients ; 13(3)2021 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-33802038

RESUMO

Many studies have highlighted the relationship between food and health status, with the aim of improving both disease prevention and life expectancy. Among the different food groups, fermented foods a have huge microbial biodiversity, making them an interesting source of metabolites that could exhibit health benefits. Our previous study highlighted the capacity of raw goat milk cheese, and some of the extracts recovered by the means of chemical fractionation, to increase the longevity of the nematode Caenorhabditis elegans. In this article, we pursued the investigation with a view toward understanding the biological mechanisms involved in this phenomenon. Using mutant nematode strains, we evaluated the implication of the insulin-like DAF-2/DAF-16 and the p38 MAPK pathways in the phenomenon of increased longevity and oxidative-stress resistance mechanisms. Our results demonstrated that freeze-dried raw goat milk cheese, and its extracts, induced the activation of the DAF-2/DAF-16 pathway, increasing longevity. Concerning oxidative-stress resistance, all the extracts increased the survival of the worms, but no evidence of the implication of both of the pathways was highlighted, except for the cheese-lipid extract that did seem to require both pathways to improve the survival rate. Simultaneously, the cheese-lipid extract and the dried extract W70, obtained with water, were able to reduce the reactive oxygen species (ROS) production in human leukocytes. This result is in good correlation with the results obtained with the nematode.


Assuntos
Caenorhabditis elegans/fisiologia , Queijo , Leucócitos/fisiologia , Estresse Oxidativo , Animais , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular , Alimentos em Conserva , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Liofilização , Regulação da Expressão Gênica , Longevidade , MAP Quinase Quinase 4/genética , MAP Quinase Quinase 4/metabolismo , Sistema de Sinalização das MAP Quinases , Leite , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol/farmacologia
11.
Int J Mol Sci ; 22(6)2021 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-33802689

RESUMO

Palmitoylethanolamide (PEA) is an endogenous lipid produced on demand by neurons and glial cells that displays neuroprotective properties. It is well known that inflammation and neuronal damage are strictly related processes and that microglia play a pivotal role in their regulation. The aim of the present work was to assess whether PEA could exert its neuroprotective and anti-inflammatory effects through the modulation of microglia reactive phenotypes. In N9 microglial cells, the pre-incubation with PEA blunted the increase of M1 pro-inflammatory markers induced by lipopolysaccharide (LPS), concomitantly increasing those M2 anti-inflammatory markers. Images of microglial cells were processed to obtain a set of morphological parameters that highlighted the ability of PEA to inhibit the LPS-induced M1 polarization and suggested that PEA might induce the anti-inflammatory M2a phenotype. Functionally, PEA prevented Ca2+ transients in both N9 cells and primary microglia and antagonized the neuronal hyperexcitability induced by LPS, as revealed by multi-electrode array (MEA) measurements on primary cortical cultures of neurons, microglia, and astrocyte. Finally, the investigation of the molecular pathway indicated that PEA effects are not mediated by toll-like receptor 4 (TLR4); on the contrary, a partial involvement of cannabinoid type 2 receptor (CB2R) was shown by using a selective receptor inverse agonist.


Assuntos
Amidas/farmacologia , Etanolaminas/farmacologia , Microglia/metabolismo , Fármacos Neuroprotetores/farmacologia , Ácidos Palmíticos/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Polaridade Celular/efeitos dos fármacos , Células Cultivadas , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Microglia/efeitos dos fármacos , NF-kappa B/metabolismo , Ratos , Receptor CB2 de Canabinoide/metabolismo , Células THP-1 , Acetato de Tetradecanoilforbol/farmacologia , Receptor 4 Toll-Like/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
12.
Nitric Oxide ; 109-110: 33-41, 2021 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-33667621

RESUMO

INTRODUCTION: THP-1 cells, a human leukemia monocytic cell line, differentiated by phorbol myristate acetate (PMA) are widely used as surrogate of human macrophages. Differentiated THP-1 cells acquire macrophage-like characteristics including more adherence and altered cell function. Nitric oxide (NO), an intracellular messenger, is critical in regulating cell differentiation. Here we elucidated whether NO relates to PMA-induced monocyte-to-macrophage differentiation of THP-1 cells. The mutual regulation of calcium and NO was also investigated. MATERIAL & METHODS: THP-1 cells were incubated with PMA for 24 h, followed by assay of adherence, morphological change, migration or IL-1ß release. L-NG-Nitroarginine methyl ester (l-NAME, a nitric oxide synthase inhibitor) or BAPTA-AM (a calcium chelator) was added before PMA stimulation, and levels of calcium and NO were measured. Furthermore, a selective inhibitor of inducible nitric oxide synthase (iNOS) activity was employed to study the role of iNOS. RESULTS AND DISCUSSION: Effects of PMA on upregulation of adherence, lipopolysaccharide-triggered IL-1ß, and migration ability of THP-1 cells were consistent with NO concentrations. Both l-NAME and BAPTA-AM mitigated effects of PMA on THP-1 cells differentiation. BAPTA-AM decreased levels of NO, while l-NAME had no effect on calcium levels. Of note, inhibition of iNOS activity decreased PMA-triggered upregulation of NO. CONCLUSION: PMA induced differentiation of THP-1 cells partially in a NO-dependent manner. The calcium signaling may mediate PMA-triggered upregulation of NO.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Óxido Nítrico/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Humanos , Macrófagos/metabolismo , Monócitos/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/metabolismo , Células THP-1 , Regulação para Cima/efeitos dos fármacos
13.
J Physiol Biochem ; 77(2): 321-329, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33704695

RESUMO

Lysophosphatidic acid (LPA) acts through the activation of G protein-coupled receptors, in a Ca2+-dependent manner. We show the effects of LPA on the plasma membrane Ca2+-ATPase (PMCA) from kidney proximal tubule cells. The Ca2+-ATPase activity was inhibited by nanomolar concentrations of LPA, with maximal inhibition (~50%) obtained with 20 nM LPA. This inhibitory action on PMCA activity was blocked by Ki16425, an antagonist for LPA receptors, indicating that this lipid acts via LPA1 and/or LPA3 receptor. This effect is PKC-dependent, since it is abolished by calphostin C and U73122, PKC, and PLC inhibitors, respectively. Furthermore, the addition of 10-8 M PMA, a well-known PKC activator, mimicked PMCA modulation by LPA. We also demonstrated that the PKC activation leads to an increase in PMCA phosphorylation. These results indicate that LPA triggers LPA1 and/or LPA3 receptors at the BLM, inducing PKC-dependent phosphorylation with further inhibition of PMCA. Thus, LPA is part of the regulatory lipid network present at the BLM and plays an important role in the regulation of intracellular Ca2+ concentration that may result in significant physiological alterations in other Ca2+-dependent events ascribed to the renal tissue.


Assuntos
Cálcio/metabolismo , Membrana Celular/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , Receptores de Ácidos Lisofosfatídicos/genética , Animais , Fracionamento Celular , Membrana Celular/metabolismo , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Estrenos/farmacologia , Regulação da Expressão Gênica , Transporte de Íons/efeitos dos fármacos , Isoxazóis/farmacologia , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/metabolismo , Naftalenos/farmacologia , Fosforilação/efeitos dos fármacos , ATPases Transportadoras de Cálcio da Membrana Plasmática/antagonistas & inibidores , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo , Cultura Primária de Células , Propionatos/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Pirrolidinonas/farmacologia , Receptores de Ácidos Lisofosfatídicos/antagonistas & inibidores , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Suínos , Acetato de Tetradecanoilforbol/farmacologia , Fosfolipases Tipo C/antagonistas & inibidores , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo
14.
Biochimie ; 184: 8-17, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33556471

RESUMO

Acquired drug-resistance, often involving downregulation or mutations in the target protein, is a major caveat in precision medicine. Understanding mechanisms of resistance to therapeutic drugs may unravel strategies to overcome or prevent them. We previously identified phorbol ester (PE) compounds such as TPA that induce Protein Kinase δ (PKCδ), thereby suppressing leukemogenesis. Here we identified erythroleukemia cell lines that resist PEs and showed that reduced PKCδ protein expression underlies drug resistance. Reduced level of PKCδ in resistant cell lines was due to its phosphorylation followed by protein degradation. Indeed, proteasome inhibition prevented PE-induced loss of PKCδ. Accordingly, a combination of TPA and the proteasome inhibitor ALLN significantly suppressed leukemia in a mouse model of leukemia. PKCδ downregulation by TPA was independent of the downstream MAPK/ERK/P38/JNK pathway. Instead, expression of ubiquitin-associated and SH3 domain-containing protein b (Ubash3b) was induced by TPA, which leads to PKCδ protein dephosphorylation and degradation. This specific degradation was blocked by RNAi-mediated depletion of Ubash3b. In drug-sensitive leukemic cells, TPA did not induce Ubash3b, and consequently, PKCδ levels remained high. A PE-resistant cell line derived from PE-treated sensitive cells exhibited very low PKCδ expression. In these drug resistance cells, a Ubash3b independent mechanism led to PKCδ degradation. Thus, PE compounds in combination with proteasome or specific inhibitors for Ubash3b, or other factors can overcome resistance to TPA, leading to durable suppression of leukemic growth. These results identify Ubash3b as a potential target for drug development.


Assuntos
Carcinogênese/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Leucemia/enzimologia , Proteínas de Neoplasias/metabolismo , Proteína Quinase C-delta/biossíntese , Proteínas Tirosina Fosfatases/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Carcinogênese/genética , Carcinogênese/metabolismo , Linhagem Celular Tumoral , Humanos , Leucemia/genética , Leucemia/patologia , Proteínas de Neoplasias/genética , Proteína Quinase C-delta/genética , Proteínas Tirosina Fosfatases/genética
15.
Methods Mol Biol ; 2270: 61-76, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33479893

RESUMO

IL-10 is the best known and most studied anti-inflammatory cytokine and, in the last 20 years, it has acquired even greater fame as it has been associated with the regulatory phenotype of B cells. Indeed, although great efforts have been made to find a unique marker, to date IL-10 remains the main way to follow both murine and human regulatory B cells, hence the need of precise and reproducible methods to identify and purify IL-10-producing B cells for both functional and molecular downstream assays. In this chapter, we present our protocols to isolate these cells from the murine spleen and peritoneum and from human peripheral blood. Since the production of IL-10 by B cells is not only a weapon to counteract the adverse effect of pro-inflammatory cytokines but also a response to cellular activation, we focused on those B cells that are prone to IL-10 production and detectable following a short-term stimulation with phorbol-12-myristate-13-acetate, ionomycin, and lipopolysaccharide (murine system) or CpG (human system).


Assuntos
Subpopulações de Linfócitos B/citologia , Linfócitos B Reguladores/citologia , Separação Celular/métodos , Animais , Subpopulações de Linfócitos B/imunologia , Citocinas/imunologia , Expressão Gênica/genética , Expressão Gênica/imunologia , Humanos , Interleucina-10/metabolismo , Ionomicina/farmacologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/imunologia , Camundongos , Ésteres de Forbol/farmacologia , Baço/citologia , Acetato de Tetradecanoilforbol/farmacologia
16.
Biochem J ; 478(3): 553-578, 2021 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-33459343

RESUMO

Autosomal dominant mutations in LRRK2 that enhance kinase activity cause Parkinson's disease. LRRK2 phosphorylates a subset of Rab GTPases including Rab8A and Rab10 within its effector binding motif. Here, we explore whether LRRK1, a less studied homolog of LRRK2 that regulates growth factor receptor trafficking and osteoclast biology might also phosphorylate Rab proteins. Using mass spectrometry, we found that in LRRK1 knock-out cells, phosphorylation of Rab7A at Ser72 was most impacted. This residue lies at the equivalent site targeted by LRRK2 on Rab8A and Rab10. Accordingly, recombinant LRRK1 efficiently phosphorylated Rab7A at Ser72, but not Rab8A or Rab10. Employing a novel phospho-specific antibody, we found that phorbol ester stimulation of mouse embryonic fibroblasts markedly enhanced phosphorylation of Rab7A at Ser72 via LRRK1. We identify two LRRK1 mutations (K746G and I1412T), equivalent to the LRRK2 R1441G and I2020T Parkinson's mutations, that enhance LRRK1 mediated phosphorylation of Rab7A. We demonstrate that two regulators of LRRK2 namely Rab29 and VPS35[D620N], do not influence LRRK1. Widely used LRRK2 inhibitors do not inhibit LRRK1, but we identify a promiscuous inhibitor termed GZD-824 that inhibits both LRRK1 and LRRK2. The PPM1H Rab phosphatase when overexpressed dephosphorylates Rab7A. Finally, the interaction of Rab7A with its effector RILP is not affected by LRRK1 phosphorylation and we observe that maximal stimulation of the TBK1 or PINK1 pathway does not elevate Rab7A phosphorylation. Altogether, these findings reinforce the idea that the LRRK enzymes have evolved as major regulators of Rab biology with distinct substrate specificity.


Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Fibroblastos , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/imunologia , Camundongos , Camundongos Knockout , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Fosfosserina/metabolismo , Proteínas Quinases/deficiência , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/farmacologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Organismos Livres de Patógenos Específicos , Acetato de Tetradecanoilforbol/farmacologia
17.
Int J Mol Sci ; 22(2)2021 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-33467555

RESUMO

Acute leukemias, the most common cancers in children, are characterized by excessive proliferation of malignant progenitor cells. As a consequence of impaired blood cell production, leukemia patients are susceptible to infectious complications-a major cause of non-relapse mortality. Neutrophil extracellular traps (NETs) are involved in various pathologies, from autoimmunity to cancer. Although aberrant NETs formation may be partially responsible for immune defects observed in acute leukemia, still little is known on the NET release in the course of leukemia. Here, we present the first comprehensive evaluation of NETs formation by neutrophils isolated from children with acute leukemia in different stages of the disease and treatment stimulated in vitro with phorbol 12-myristate 13-acetate (PMA), N-formyl-methionyl-leucyl-phenylalanine (fMLP), and calcium ionophore (CI). NETs release was measured using quantitative fluorescent method and visualized microscopically. In this setting, NETs release was significantly impaired in leukemic children both at the diagnosis and during the treatment, and full restoration of neutrophil function was achieved only after successful completion of the leukemia treatment. We suggest that neutrophil function impairment may result from both disease- and treatment-related factors. In this context, deficient innate immune response observed in acute leukemia patients may be present regardless of neutrophil count and contribute to secondary immunodeficiency observed in this population.


Assuntos
Armadilhas Extracelulares/imunologia , Imunidade Inata/imunologia , Leucemia/imunologia , Neutrófilos/imunologia , Doença Aguda , Adolescente , Ionóforos de Cálcio/farmacologia , Células Cultivadas , Criança , Pré-Escolar , Humanos , Imunidade Inata/efeitos dos fármacos , Lactente , Leucemia/sangue , Leucemia/tratamento farmacológico , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Acetato de Tetradecanoilforbol/farmacologia
18.
Curr Pharm Biotechnol ; 22(1): 159-167, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32216736

RESUMO

BACKGROUND: Tripterine (TRI), an active monomer in Tripterygium wilfordii, has significant pharmacological activities, such as anti-inflammatory, immunosuppressive and anti-tumor activities. TRI may be used to treat allergic diseases because of its characteristics of immunosuppression. OBJECTIVE: This study aims to explore the anti-allergic effect of TRI. METHODS: It was tested in vivo and in vitro in this study. RESULTS: The results showed that TRI could significantly inhibit histamine release from rat peritoneal mast cells; the inhibitory effect of TRI on histamine release was stronger than that of other known histamine inhibitors such as disodium cromoglyceride. TRI also significantly inhibited systemic anaphylactic shock induced by compound 48/80 and skin allergy induced by IgE, and inhibited the expression of inflammatory factors secreted by Human Mast Cells (HMC-1) induced by Phorbol 12-Myristate 13- Acetate (PMA) and calcium carrier A23187. In the animal model of allergic rhinitis induced by Ovalbumin (OA), the scores of friction, histamine, IgE, inflammatory factors and inflammatory cells decreased after TRI was administered orally or nasally. CONCLUSION: TRI, as an active immunoregulatory factor, has great potential in the treatment of mast cell-mediated allergic diseases.


Assuntos
Anafilaxia/tratamento farmacológico , Antialérgicos/farmacologia , Liberação de Histamina/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Rinite Alérgica/tratamento farmacológico , Triterpenos/farmacologia , Animais , Antialérgicos/uso terapêutico , Calcimicina/farmacologia , Citocinas/metabolismo , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/uso terapêutico , Humanos , Masculino , Mastócitos/metabolismo , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Ratos , Rinite Alérgica/imunologia , Acetato de Tetradecanoilforbol/farmacologia , Triterpenos/uso terapêutico , p-Metoxi-N-metilfenetilamina/farmacologia
19.
Mol Pharmacol ; 99(2): 104-113, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33239332

RESUMO

Cardiac fibrosis is characterized by accumulation and activation of fibroblasts and excessive production of extracellular matrix, which results in myocardial stiffening and eventually leads to heart failure. Although previous work suggests that protein kinase C (PKC) isoforms play a role in cardiac fibrosis and remodeling, the results are conflicting. Moreover, the potential of targeting PKC with pharmacological tools to inhibit pathologic fibrosis has not been fully evaluated. Here we investigated the effects of selected PKC agonists and inhibitors on cardiac fibroblast (CF) phenotype, proliferation, and gene expression using primary adult mouse CFs, which spontaneously transdifferentiate into myofibroblasts in culture. A 48-hour exposure to the potent PKC activator phorbol 12-myristate 13-acetate (PMA) at 10 nM concentration reduced the intensity of α-smooth muscle actin staining by 56% and periostin mRNA levels by 60% compared with control. The decreases were inhibited with the pan-PKC inhibitor Gö6983 and the inhibitor of classical PKC isoforms Gö6976, suggesting that classical PKCs regulate CF transdifferentiation. PMA also induced a 33% decrease in 5-bromo-2'-deoxyuridine-positive CFs, which was inhibited with Gö6983 but not with Gö6976, indicating that novel PKC isoforms (nPKCs) regulate CF proliferation. Moreover, PMA downregulated the expression of collagen-encoding genes Col1a1 and Col3a1 nPKC-dependently, showing that PKC activation attenuates matrix synthesis in CFs. The partial PKC agonist isophthalate derivative bis(1-ethylpentyl) 5-(hydroxymethyl)isophthalate induced parallel changes in phenotype, cell cycle activity, and gene expression. In conclusion, our results reveal distinct PKC-dependent regulation of CF transdifferentiation and proliferation and suggest that PKC agonists exhibit potential as an antifibrotic treatment. SIGNIFICANCE STATEMENT: Cardiac fibrosis is a pathological process that contributes to the development of heart failure. The molecular mechanisms regulating fibrosis in the heart are, however, not fully understood, which hinders the development of new therapies. Here, we demonstrate that classical and novel protein kinase C (PKC) isoforms distinctly regulate cardiac fibroblast transdifferentiation and proliferation, the two central processes in fibrosis. Our results indicate that pharmacological PKC activation may be a promising strategy to inhibit myocardial fibrosis.


Assuntos
Carbazóis/farmacologia , Indóis/farmacologia , Maleimidas/farmacologia , Miocárdio/citologia , Miofibroblastos/citologia , Proteína Quinase C/metabolismo , Acetato de Tetradecanoilforbol/farmacologia , Actinas/metabolismo , Animais , Moléculas de Adesão Celular/genética , Proliferação de Células/efeitos dos fármacos , Transdiferenciação Celular/efeitos dos fármacos , Células Cultivadas , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Fibrose , Humanos , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Miofibroblastos/efeitos dos fármacos , Miofibroblastos/metabolismo , Cultura Primária de Células , Proteína Quinase C/antagonistas & inibidores
20.
Biochimie ; 181: 169-175, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-33333171

RESUMO

We investigated whether docosahexaenoic acid (DHA), a dietary n-3 fatty acid, modulates calcium (Ca2+) signaling and cell cycle progression in human Jurkat T-cells. Our study demonstrates that DHA inhibited Jurkat T-cell cycle progression by blocking their passage from S phase to G2/M phase. In addition, DHA decreased the plasma membrane expression of TRPC3 and TRPC6 calcium channels during T-cell proliferation. Interestingly, this fatty acid increased plasma membrane expression of TRPC6 after 24 h of mitogenic stimulation by phorbol-13-myristate-12-acetate (PMA) and ionomycin. These variations in the membrane expression of TRPC3 and TRPC6 channels were not directly correlated with the mRNA expression, indicating that it was a post-translational phenomenon. DHA increased free intracellular calcium concentrations, [Ca2+]i, via opening TRPC3 and TRPC6 channels. We conclude that the anti-proliferative effect of DHA might involve the modulation of TRPC3 and TRPC6 channels in human T-cells.


Assuntos
Membrana Celular/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Linfócitos T/metabolismo , Canais de Cátion TRPC/biossíntese , Canal de Cátion TRPC6/biossíntese , Humanos , Ionomicina/farmacologia , Células Jurkat , Acetato de Tetradecanoilforbol/farmacologia
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