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Experimental and Molecular Pathology Apr 2022Neutrophils stand sentinel over infection and possess diverse antimicrobial weapons, including neutrophil extracellular traps (NETs). NETs are composed of web-like...
Neutrophils stand sentinel over infection and possess diverse antimicrobial weapons, including neutrophil extracellular traps (NETs). NETs are composed of web-like extracellular DNA decorated with antimicrobial substances and can trap and eliminate invading microorganisms. Although phorbol 12-myristate 13-acetate (PMA) is a potent NET inducer, previous studies have demonstrated that not all neutrophils exhibit NET formation even if stimulated by PMA at high concentrations. This study first showed that some neutrophils stimulated by PMA displayed a swollen nucleus but not NET formation and that hypoxic environments suppressed the NET release. Next, characterization of PMA-stimulated neutrophils with a swollen nucleus was accomplished by differentiating between suicidal-type NETosis and apoptosis. Furthermore, the significance of the phenomenon was examined using formalin-fixed, paraffin-embedded human lung disease tissues with and without pneumonia. As a result, histone H3 citrullination, DNA outflow, propidium iodide labeling, resistance to DNase I, and suspended actin rearrangement were characteristics of PMA-stimulated neutrophils with a swollen nucleus distinct from neutrophils that underwent either suicidal-type NETosis or apoptosis. Neutrophils stimulated by PMA under hypoxic conditions secreted matrix metalloproteinase-9 cytotoxic to human lung-derived fibroblasts. Further, deposition of neutrophil-derived citrullinated histone H3 chromatin substances in pulmonary lesions was greater in patients with pneumonia than in patients without pneumonia and positively correlated with hypoxia-inducible factor-1α expression. The collective findings suggested that neutrophils activated under hypoxic conditions could be putative modulators of hypoxia-related disease manifestations.
Topics: Acetates; DNA; Extracellular Traps; Histones; Humans; Hypoxia; Lung Diseases; Myristic Acid; Neutrophils; Phorbols; Tetradecanoylphorbol Acetate
PubMed: 35259405
DOI: 10.1016/j.yexmp.2022.104754 -
Virology Mar 1999Smubp-2 is a novel transcription factor that was first identified through its interaction with the immunoglobulin Smu region (Mizuta et al., 1993) and has been cloned by...
Smubp-2 is a novel transcription factor that was first identified through its interaction with the immunoglobulin Smu region (Mizuta et al., 1993) and has been cloned by virtue of its binding to two 12-O-tetradecanoylphorbol-13-acetate-responsive elements in the Epstein-Barr virus immediate-early BZLF1 promoter (Gulley et al., 1997). In this report, we examined the effect of Smubp-2 overexpression on BZLF1 prom oter activity. Overexpression of Smubp-2 in the B lymphocyte cell line BJAB caused repression of the BZLF1 gene promoter. A 14-bp region that partially overlaps with a 12-O-tetradecanoylphorbol-13-acetate-responsive element was required for maximal repression by Smubp-2, but some repression was also seen with a minimal promoter containing only the BZLF1 promoter TATA box and an initiation site. A 30-bp fragment containing the 14-bp region could transfer Smubp-2-mediated repression to heterologous promoters. Smubp-2 was found to associate with the basal transcription factor TATA binding protein (TBP) and to disrupt the formation of a stable TBP-TFIIA-DNA complex on the BZLF1 promoter TATA box and the adenovirus E1B promoter TATA box. Repression of the BZLF1 promoter by overexpressed Smubp-2 was rescued by overexpression of the basal factor TFIIA. These results suggest that complete repression of the BZLF1 promoter by Smubp-2 involves disruption of a functional TBP-TFIIA-TATA box complex and requires the -93 bp-to--79 bp region of the promoter.
Topics: Cell Line; DNA-Binding Proteins; Electrophoresis, Polyacrylamide Gel; Humans; Promoter Regions, Genetic; TATA Box; TATA-Box Binding Protein; Tetradecanoylphorbol Acetate; Trans-Activators; Transcription Factor TFIIA; Transcription Factors; Viral Proteins
PubMed: 10049831
DOI: 10.1006/viro.1998.9588 -
Molecules and Cells Mar 2016The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative...
The mechanism by which 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using human diploid fibroblast (HDF) cell replicative senescence as a model. Upon TPA treatment, protein kinase C (PKC) α and PKCβ1 exerted differential effects on the nuclear translocation of cytoplasmic pErk1/2, a protein which maintains senescence. PKCα accompanied pErk1/2 to the nucleus after freeing it from PEA-15pS(104) via PKCβ1 and then was rapidly ubiquitinated and degraded within the nucleus. Mitogen-activated protein kinase docking motif and kinase activity of PKCα were both required for pErk1/2 transport to the nucleus. Repetitive exposure of mouse skin to TPA downregulated PKCα expression and increased epidermal and hair follicle cell proliferation. Thus, PKCα downregulation is accompanied by in vivo cell proliferation, as evidenced in 7, 12-dimethylbenz(a)anthracene (DMBA)-TPA-mediated carcinogenesis. The ability of TPA to reverse senescence was further demonstrated in old HDF cells using RNA-sequencing analyses in which TPA-induced nuclear PKCα degradation freed nuclear pErk1/2 to induce cell proliferation and facilitated the recovery of mitochondrial energy metabolism. Our data indicate that TPA-induced senescence reversal and carcinogenesis promotion share the same molecular pathway. Loss of PKCα expression following TPA treatment reduces pErk1/2-activated SP1 biding to the p21(WAF1) gene promoter, thus preventing senescence onset and overcoming G1/S cell cycle arrest in senescent cells.
Topics: Animals; Cell Proliferation; Cellular Senescence; Fibroblasts; Gene Expression Regulation; Humans; MAP Kinase Signaling System; Mice; Phosphorylation; Protein Kinase C beta; Protein Kinase C-alpha; Protein Transport; Tetradecanoylphorbol Acetate
PubMed: 26912086
DOI: 10.14348/molcells.2016.2362 -
Journal of Virology Mar 1996Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS. The simian immunodeficiency virus (SIV) causes a similar syndrome in macaques. The product of...
Human immunodeficiency virus type 1 (HIV-1) is the causative agent of AIDS. The simian immunodeficiency virus (SIV) causes a similar syndrome in macaques. The product of the nef gene of SIV has been shown to be important for virus replication and disease progression in vivo. In vitro, both SIV and HIV Nef downregulate surface expression of CD4 and accelerate total CD4 turnover. The mechanism by which Nef downregulates CD4 has not been established. A current model suggests that Nef enhances cell surface CD4 endocytosis and degradation in lysosomes. However, this was recently challenged when CD4 was found to accumulate in early endosomes of cells expressing Nef. Because inhibition of Nef function might halt virus replication and disease progression, we tested two macrolide antibiotics for their ability to inhibit Nef function. Concanamycin B (ConB) and bafilomycin A1 (BFLA1) are specific inhibitors of acidification of cell endosomes and lysosomes and, unlike other inhibitors, do not affect transport. Although ConB (25 nM) and BFLA1 (100 nM) blocked phorbol myristate acetate- and Nef-induced CD4 degradation in human monocyte U937 cells, CD4 surface expression was not recovered. Instead, CD4 accumulated in lysosomes. To determine if Nef is directly responsible for CD4 degradation or if they bind to each other in a manner similar to Vpu, transcripts of human CD4 and HIV-1 nef were cotranslated in vitro. Our results indicate that under our experimental conditions, Nef does not affect CD4 stability and does not associate with CD4 in this in vitro system. Our data suggest that (i) CD4 downregulation by Nef results in degradation of CD4 in lysosomes, (ii) inhibition of CD4 degradation by macrolide antibiotics does not restore surface expression, and (iii) the inhibition of CD4 expression by Nef appears to be indirect and is likely to involve cellular factors.
Topics: Anti-Bacterial Agents; CD4 Antigens; Cell Line; Cell-Free System; Down-Regulation; Gene Expression; Gene Products, nef; HIV-1; Humans; Lysosomes; Macrolides; Tetradecanoylphorbol Acetate; nef Gene Products, Human Immunodeficiency Virus
PubMed: 8627671
DOI: 10.1128/JVI.70.3.1527-1534.1996 -
Infection and Immunity Feb 1998The mechanisms involved in coiling phagocytosis are not yet known, and it is not even clear whether this phenomenon is either an incidental event or a specific response....
The mechanisms involved in coiling phagocytosis are not yet known, and it is not even clear whether this phenomenon is either an incidental event or a specific response. Therefore, the phagocytic uptake of Borrelia burgdorferi and other spirochetes by human monocytes in vitro was used to investigate the involvement of both sides--microbes and phagocytes--in coiling phagocytosis. As seen with electron microscopy, morphologically similar Borrelia, Leptospira and Treponema strains induced markedly different frequencies of coiling phagocytosis. The monocytes used coiling phagocytosis for both live (motile) and killed (nonmotile) B. burgdorferi, but pseudopod coils were observed neither with fragmented B. burgdorferi nor with cell-free supernatant from B. burgdorferi cultures. Investigation of the relationship of coiling phagocytosis with other pseudopod-based cellular mechanisms revealed that the use of bioreagents that inhibit conventional phagocytosis also inhibited coiling phagocytis but did not affect membrane ruffling. Bioreagents that increase membrane ruffling did not affect phagocytosis of B. burgdorferi, except for granulocyte-macrophage colony-stimulating factor and phorbol myristate acetate, which increased coiling phagocytosis selectively. These results demonstrate that coiling phagocytosis is not induced by microbial motility, viability, or a certain morphology and that it is not a random event. Rather, it is a selective uptake mechanism actively driven by the phagocytes. However, whether coiling phagocytosis represents an independent alternative to conventional phagocytosis or, alternatively, a fault in conventional phagocytosis remains to be determined.
Topics: Borrelia burgdorferi Group; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Phagocytosis; Tetradecanoylphorbol Acetate
PubMed: 9453619
DOI: 10.1128/IAI.66.2.627-635.1998 -
Biochimica Et Biophysica Acta.... Jan 2023Neutrophil extracellular trap formation (NETosis) has been irrefutably referred to as a distinct and unique form of active cell death with the purpose to counteract...
Neutrophil extracellular trap formation (NETosis) has been irrefutably referred to as a distinct and unique form of active cell death with the purpose to counteract invading pathogens or augmenting the inflammatory cascade. Since the discovery, consistent efforts have been made to understand the various aspects of the initiation and sustenance of NETosis. In this study, using a global metabolomics approach during the phorbol 12-myristate 13-acetate (PMA) induced NETosis in human neutrophils, various metabolic pathways were found to be altered which includes intermediates related to, carbohydrate metabolism, and redox related metabolites, nucleic acid metabolism, and amino acids metabolism. Enrichment analysis of the metabolite sets highlighted the importance of the pentose phosphate pathway (PPP) and glutathione metabolism PMA-induced NETotic neutrophils. Further, analysis of the glutathyniolation status of neutrophil proteins by Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) indicated six different glutathionylated proteins: among them, two metabolically important proteins were α-enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) with MALDI score 166 and 70 respectively. Other proteins were lactoferrin, β-actin, c-myc promoter-binding protein, and uracil DNA glycosylase with MALDI scores of 96, 167, 104, and 68 respectively. Besides, activation of signalling proteins involved in metabolic regulation is also correlated with NETosis. Altogether, a balance between reactive oxygen species-glutathione metabolism seems to regulate the activity of glycolytic enzymes such as GAPDH and α-enolase during PMA-induced NETosis in a time-dependent manner.
Topics: Humans; Extracellular Traps; Neutrophils; Tetradecanoylphorbol Acetate; Glyceraldehyde-3-Phosphate Dehydrogenases; Glutathione; Metabolome; Phosphopyruvate Hydratase
PubMed: 36265832
DOI: 10.1016/j.bbadis.2022.166581 -
European Journal of Pharmacology Sep 2012The dopamine transporter removes the neurotransmitter from the synapse, regulating dopamine availability. The transporter can be internalized and its function is blocked...
The dopamine transporter removes the neurotransmitter from the synapse, regulating dopamine availability. The transporter can be internalized and its function is blocked by cocaine and other ligands. Melittin inhibits dopamine transporter function and causes internalization of the recombinant transporter in stably transfected HEK-293 cells, but the specific pathways for internalization and disposition of the transporter are unknown. Here we report that melittin treatment increased both transporter internalization and colocalization with clathrin, effects that were blocked by pretreatment with cocaine. Density gradient centrifugation revealed that melittin treatment caused the dopamine transporter to associate with a density fraction containing the early endosome marker Rab 5A. Confocal microscopy revealed that melittin treatment also increased transporter colocalization with Rab 5A and decreased colocalization with the late endosome marker Rab 7 and the recycling endosome marker Rab 11. Following 60 min of melittin treatment, the transporter was trafficked back to the membrane. By comparison, phorbol ester treatment increased transporter colocalization with early endosome antigen 1 and Rab 7 in a time-dependent manner. Cocaine treatment alone does not affect transporter trafficking in these cells. Results indicate multiple dopamine transporter internalization and recycling pathways that depend on transporter-ligand interactions and post-translational modifications.
Topics: Biotinylation; Dopamine Plasma Membrane Transport Proteins; HEK293 Cells; Humans; Intracellular Space; Melitten; Protein Transport; Tetradecanoylphorbol Acetate; Transfection
PubMed: 22683840
DOI: 10.1016/j.ejphar.2012.05.020 -
Memorias Do Instituto Oswaldo Cruz Aug 2007We investigated the effect of two modulators of protein kinase C, sphingosine and phorbol-12-myristate-13-acetate (PMA), on the growth and dimethylsulfoxide...
Effect of sphingosine and phorbol-12-myristate-13-acetate on the growth and dimethylsulfoxide-induced differentiation in the insect trypanosomatid Herpetomonas samuelpessoai.
We investigated the effect of two modulators of protein kinase C, sphingosine and phorbol-12-myristate-13-acetate (PMA), on the growth and dimethylsulfoxide (DMSO)-induced differentiation in Herpetomonas samuelpessoai. Sphingosine did not stimulate the transformation of undifferentiated-promastigotes in differentiated-paramastigotes. PMA alone or in association with DMSO increased the number of paramastigotes in comparison to control cells. DMSO inhibited the parasite growth (35%) and several unusual morphological features resembling aberrant cell division were observed. Sphingosine did not significantly reduce the growth in contrast to PMA. Collectively, our results demonstrated that the reduction of the proliferation translates in an increase of the differentiation rate in the insect trypanosomatid H. samuelpessoai.
Topics: Animals; Cell Differentiation; Dimethyl Sulfoxide; Enzyme Activation; Protein Kinase C; Sphingosine; Tetradecanoylphorbol Acetate; Trypanosomatina
PubMed: 17710305
DOI: 10.1590/s0074-02762007005000059 -
Molecules and Cells Aug 2001The effects of forskolin (FSK) and phobol 12-myristate-13-acetate (PMA) on c-fos and c-jun mRNA expressions in rat C6 glioma cells were studied. Both FSK and PMA...
The effects of forskolin (FSK) and phobol 12-myristate-13-acetate (PMA) on c-fos and c-jun mRNA expressions in rat C6 glioma cells were studied. Both FSK and PMA increased the c-fos mRNA level. The C-jun mRNA level was decreased by FSK, whereas it was increased by PMA. The elevated c-fos mRNA level, induced by FSK or PMA, was significantly inhibited by dexamethasone (DEX). In contrast, DEX did not affect the FSK- and PMA-induced response of the c-jun mRNA level. Cycloheximide (CHX) caused a superinduction of the FSK- or PMA-induced c-fos mRNA level. Furthermore, CHX also potentiated the PMA-induced c-jun mRNA level. However, CHX did not affect the FSK-induced down-regulation of the c-jun mRNA level. When C6 glioma cells were incubated with PMA and FSK, the PMA-induced c-jun mRNA level was inhibited by FSK, whereas FSK did not affect the PMA-induced c-fos mRNA level. Our results suggest that the activations of PKA and PKC pathways have different roles in the regulation of the c-jun mRNA expression in rat C6 glioma cells. PKA activation can inhibit induction of the c-jun mRNA expression by PMA. In addition, DEX appears to have a selective inhibitory action against c-fos, but not c-jun, -mRNA expression that is regulated by PKA and PKC. On-going protein synthesis inhibition is required for the superinduction of the c-fos expression that is induced by PMA, or FSK and the PMA-induced c-jun mRNA level.
Topics: Animals; Blotting, Northern; Colforsin; Cycloheximide; Dexamethasone; Gene Expression Regulation; Genes, fos; Genes, jun; Glucocorticoids; Protein Synthesis Inhibitors; Proto-Oncogene Proteins c-fos; Proto-Oncogene Proteins c-jun; RNA, Messenger; Rats; Signal Transduction; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured
PubMed: 11561718
DOI: No ID Found -
Biomedical Research (Tokyo, Japan) 2012Glial cells missing Drosophila homolog a (GCMa) is a member of the GCM transcription factor family and plays critical roles in trophoblast differentiation and placental...
Glial cells missing Drosophila homolog a (GCMa) is a member of the GCM transcription factor family and plays critical roles in trophoblast differentiation and placental functions. It is well established that the cyclic AMP (cAMP)-dependent pathway induces the expression and transcriptional activity of GCMa by regulating post-translational modifications of GCMa, which results in enhancement of trophoblast differentiation. We previously observed that phorbol 12-myristate 13-acetate (PMA) stimulates phosphorylation of GCMa on serines 328, 378 and 383 through the protein kinase C (PKC)- and mitogen-activated protein kinase kinase (MEK)/extracellular signalregulated kinase (ERK)-dependent pathway, which decreases the protein stability of GCMa. Here we report that PMA increases the ubiquitination level of GCMa, dependent on the phosphorylation of GCMa on serines 328, 378 and 383. We found that this phosphorylation also stimulates the transcriptional activity of GCMa. Our data indicate that the PMA-induced PKC- and MEK/ERKdependent pathway enhances the degradation as well as the transcriptional activity of GCMa. We also examined the impact of this signaling pathway on trophoblasts and the results suggest that the PKC- and MEK/ERK-dependent pathway is involved in the regulation of trophoblast differentiation.
Topics: Cell Differentiation; DNA-Binding Proteins; Enzyme-Linked Immunosorbent Assay; HEK293 Cells; Humans; MAP Kinase Signaling System; Nuclear Proteins; Phosphorylation; Protein Kinase C; Protein Stability; Proteolysis; Serine; Tetradecanoylphorbol Acetate; Transcription Factors; Transcriptional Activation; Trophoblasts; Ubiquitination
PubMed: 22975632
DOI: 10.2220/biomedres.33.217