-
Journal of Leukocyte Biology Jul 2023CR3 (CD11b/CD18; αmβ2 integrin) is a conserved phagocytic receptor. The active conformation of CR3 binds the iC3b fragment of complement C3 as well as many host and...
CR3 (CD11b/CD18; αmβ2 integrin) is a conserved phagocytic receptor. The active conformation of CR3 binds the iC3b fragment of complement C3 as well as many host and microbial ligands, leading to actin-dependent phagocytosis. There are conflicting reports about how CR3 engagement affects the fate of phagocytosed substrates. Using imaging flow cytometry, we confirmed that binding and internalization of iC3b-opsonized polystyrene beads by primary human neutrophils was CR3-dependent. iC3b-opsonized beads did not stimulate neutrophil reactive oxygen species, and most beads were found in primary granule-negative phagosomes. Similarly, Neisseria gonorrhoeae that does not express phase-variable Opa proteins suppresses neutrophil reactive oxygen species and delays phagolysosome formation. Here, binding and internalization of Opa-deleted (Δopa) N. gonorrhoeae by adherent human neutrophils was inhibited using blocking antibodies against CR3 and by adding neutrophil inhibitory factor, which targets the CD11b I-domain. No detectable C3 was deposited on N. gonorrhoeae in the presence of neutrophils alone. Conversely, overexpressing CD11b in HL-60 promyelocytes enhanced Δopa N. gonorrhoeae phagocytosis, which required the CD11b I-domain. Phagocytosis of N. gonorrhoeae was also inhibited in mouse neutrophils that were CD11b-deficient or treated with anti-CD11b. Phorbol ester treatment upregulated surface CR3 on neutrophils in suspension, enabling CR3-dependent phagocytosis of Δopa N. gonorrhoeae. Neutrophils exposed to Δopa N. gonorrhoeae had limited phosphorylation of Erk1/2, p38, and JNK. Neutrophil phagocytosis of unopsonized Mycobacterium smegmatis, which also resides in immature phagosomes, was CR3-dependent and did not elicit reactive oxygen species. We suggest that CR3-mediated phagocytosis is a silent mode of entry into neutrophils, which is appropriated by diverse pathogens to subvert phagocytic killing.
Topics: Mice; Animals; Humans; Neutrophils; Reactive Oxygen Species; Phagocytosis; Macrophage-1 Antigen; Complement C3b; Receptors, Complement
PubMed: 36882066
DOI: 10.1093/jleuko/qiad028 -
Cells Sep 2022This review will briefly outline the major signaling pathways in PMA-activated neutrophils. PMA is widely used to understand neutrophil pathways and formation of NETs.... (Review)
Review
This review will briefly outline the major signaling pathways in PMA-activated neutrophils. PMA is widely used to understand neutrophil pathways and formation of NETs. PMA activates PKC; however, we highlight some isoforms that contribute to specific functions. PKC α, β and δ contribute to ROS production while PKC βII and PKC ζ are involved in cytoskeleton remodeling. Actin polymerization is important for the chemotaxis of neutrophils and its remodeling is connected to ROS balance. We suggest that, although ROS and production of NETs are usually observed together in PMA-activated neutrophils, there might be a regulatory mechanism balancing both. Interestingly, we suggest that serine proteases might determine the PAD4 action. PAD4 could be responsible for the activation of the NF-κB pathway that leads to IL-1β release, triggering the cleavage of gasdermin D by serine proteases such as elastase, leading to pore formation contributing to release of NETs. On the other hand, when serine proteases are inhibited, NETs are formed by citrullination through the PAD4 pathway. This review puts together results from the last 31 years of research on the effects of PMA on the neutrophil and proposes new insights on their interpretation.
Topics: Actins; Extracellular Traps; NF-kappa B; Neutrophils; Pancreatic Elastase; Reactive Oxygen Species; Serine Proteases; Tetradecanoylphorbol Acetate
PubMed: 36139464
DOI: 10.3390/cells11182889 -
Journal of Natural Products Aug 2022The kernels of the Australian blushwood tree () are the source of the veterinary anticancer drug tigilanol tiglate (, Stelfonta) and contain a concentration of phorboids... (Review)
Review
The kernels of the Australian blushwood tree () are the source of the veterinary anticancer drug tigilanol tiglate (, Stelfonta) and contain a concentration of phorboids significantly higher than croton oil, the only abundant source of these compounds previously known. The oily matrix of the blushwood kernels is composed of free fatty acids and not by glycerides as found in croton oil. By active partitioning, it was therefore possible to recover and characterize for the first time a cryptic tigliane fraction, that is, the diterpenoid fraction that, because of its lipophilicity, could not be obtained by solvent partition of crude extracts. The cryptic tigliane fraction accounted for ca. 30% of the tigliane kernel titer and was quantified by H NMR spectroscopy and profiled by HPLC-MS. Long-chain (linoleates and/or oleates) 20-acyl derivatives of the epoxytigliane diesters tigilanol tiglate (EBC-46, ), EBC-47 (), EBC-59 (), EBC-83 (), and EBC-177 () were identified. By chemoselective acylation of EBC-46 () and EBC-177 () the natural triesters and and a selection of analogues were prepared to assist identification of the natural compounds. The presence of a free C-20 hydroxy group is a critical requirement for PKC activation by phorbol esters. The unexpected activity of 20-linoleoyl triester in a cytotoxicity assay based on PKC activation was found to be related mainly to its hydrolysis to tigilanol tiglate () under the prolonged conditions of the assay, while other esters were inactive. Significant differences between the esterification profile of the epoxytigliane di- and triesters exist in , suggesting a precise, yet elusive, blueprint of acyl decoration for the tigliane polyol 5-hydroxyepoxyphorbol.
Topics: Australia; Croton Oil; Euphorbiaceae; Phorbols; Trees
PubMed: 35973043
DOI: 10.1021/acs.jnatprod.2c00226 -
Scientific Reports Jul 2023To further elucidate the expression, regulation and function of Signaling Lymphocytic Activation Molecule Family (SLAMF) protein members in human monocytes and...
To further elucidate the expression, regulation and function of Signaling Lymphocytic Activation Molecule Family (SLAMF) protein members in human monocytes and macrophages. Un-differentiated monocytic THP-1 cell (u-THP-1) and differentiated THP-1 macrophage (d-THP-1) were used as culture models in the study. Responses of cells to the differentiation agents phorbol ester (25 ng/ml) and TLR (Toll-like receptor) ligands were assessed. RT-PCR and Western blot analysis were used to determine mRNA and protein level. Pro-inflammatory cytokine mRNA expression levels and phagocytosis were used as functional markers. Data analyzed using t-test, one-way or two-way ANOVA followed by post hoc test. SLAMFs were differentially expressed in THP-1 cells. Differentiation of u-THP-1 to d-THP-1 led to significantly higher SLAMF7 mRNA and protein levels than other SLAMF. In addition, TLR stimuli increased SLAMF7 mRNA expression but not protein expression. Importantly, SLAMF7 agonist antibody and TLR ligands synergistically increased the mRNA expression levels of IL-1β, IL-6 and TNF-α, but had no effect on phagocytosis. SLAMF7 knocked-down in d-THP-1 significantly lowered TLR-induced mRNA expressions of pro-inflammatory markers. SLAM family proteins are differentially regulated by differentiation and TLRs. SLAMF7 enhanced TLR-mediated induction of pro-inflammatory cytokines in monocytes and macrophages but not phagocytosis.
Topics: Humans; Cytokines; Family; Ligands; Lipopolysaccharides; Macrophages; Monocytes; RNA, Messenger; Signaling Lymphocytic Activation Molecule Family; Toll-Like Receptors
PubMed: 37420084
DOI: 10.1038/s41598-023-37040-0 -
International Journal of Molecular... May 2023CD248 (endosialin) belongs to a glycoprotein family that also includes thrombomodulin (CD141), CLEC14A, and CD93 (AA4) stem cell markers. We analyzed the regulated...
CD248 (endosialin) belongs to a glycoprotein family that also includes thrombomodulin (CD141), CLEC14A, and CD93 (AA4) stem cell markers. We analyzed the regulated expression of CD248 in vitro using skin (HFFF) and synovial (FLS) mesenchymal stem cell lines, and in fluid and tissue samples of rheumatoid arthritis (RA) and osteoarthritis (OA) patients. Cells were incubated with either rhVEGF, bFGF, TGF-β1, IL1-β, TNF-α, TGFβ1, IFN-γ, or PMA (Phorbol ester). There was no statistically significant change in membrane expression. A soluble (s) form of cleaved CD248 (sCD248) was detected after cell treatment with IL1-β and PMA. Matrix metalloprotease (MMP) MMP-1 and MMP-3 mRNAs were significantly up-regulated by IL1-β and PMA. A broad MMP inhibitor blocked the release of soluble CD248. In RA synovial tissue, we identified CD90 perivascular MSCs double-stained for CD248 and VEGF. High sCD248 levels were detected in synovial fluid from RA. In culture, subpopulations of CD90 CD14 RA MSCs were either identified as CD248 or CD141 cells but CD93. CD248 is abundantly expressed by inflammatory MSCs and shed in an MMP-dependent manner in response to cytokines and pro-angiogenic growth factors. Both membrane-bound and soluble CD248 (acting as a decoy receptor) may contribute to RA pathogenesis.
Topics: Humans; Lectins, C-Type; Arthritis, Rheumatoid; Synovial Membrane; Cytokines; Mesenchymal Stem Cells; Cells, Cultured; Fibroblasts; Antigens, Neoplasm; Antigens, CD; Cell Adhesion Molecules
PubMed: 37298499
DOI: 10.3390/ijms24119546 -
Biochemistry Apr 2021Munc13-1 is a presynaptic active zone protein that acts as a master regulator of synaptic vesicle priming and neurotransmitter release in the brain. It has been...
Munc13-1 is a presynaptic active zone protein that acts as a master regulator of synaptic vesicle priming and neurotransmitter release in the brain. It has been implicated in the pathophysiology of several neurodegenerative diseases. Diacylglycerol and phorbol ester activate Munc13-1 by binding to its C1 domain. The objective of this study is to identify the structural determinants of ligand binding activity of the Munc13-1 C1 domain. Molecular docking suggested that residues Trp-588, Ile-590, and Arg-592 of Munc13-1 are involved in ligand interactions. To elucidate the role of these three residues in ligand binding, we generated W588A, I590A, and R592A mutants in full-length Munc13-1, expressed them as GFP-tagged proteins in HT22 cells, and measured their ligand-induced membrane translocation by confocal microscopy and immunoblotting. The extent of 1,2-dioctanoyl--glycerol (DOG)- and phorbol ester-induced membrane translocation decreased in the following order: wild type > I590A > W588A > R592A and wild type > W588A > I590A > R592A, respectively. To understand the effect of the mutations on ligand binding, we also measured the DOG binding affinity of the isolated wild-type C1 domain and its mutants in membrane-mimicking micelles using nuclear magnetic resonance methods. The DOG binding affinity decreased in the following order: wild type > I590A > R592A. No binding was detected for W588A with DOG in micelles. This study shows that Trp-588, Ile-590, and Arg-592 are essential determinants for the activity of Munc13-1 and the effects of the three residues on the activity are ligand-dependent. This study bears significance for the development of selective modulators of Munc13-1.
Topics: Binding Sites; Cell Line; Diglycerides; Humans; Models, Molecular; Nerve Tissue Proteins; Protein Binding; Protein Conformation
PubMed: 33818064
DOI: 10.1021/acs.biochem.1c00165 -
Advances in Biological Regulation Dec 2020Classical and novel protein kinase C (PKC) isozymes (c/nPKCs), members of the PKC family that become activated by the lipid second messenger diacylglycerol (DAG) and... (Review)
Review
Classical and novel protein kinase C (PKC) isozymes (c/nPKCs), members of the PKC family that become activated by the lipid second messenger diacylglycerol (DAG) and phorbol esters, exert a myriad of cellular effects that impact proliferative and motile cellular responses. While c/nPKCs have been indisputably associated with tumor promotion, their roles exceed by far their sole involvement as promoter kinases. Indeed, this original dogma has been subsequently redefined by the introduction of several new concepts: the identification of tumor suppressing roles for c/nPKCs, and their participation in early and late stages of carcinogenesis. This review dives deep into the intricate roles of c/nPKCs in cancer initiation as well as in the different stages of the metastatic cascade, with great emphasis in their involvement in cancer cell motility via regulation of small Rho GTPases, the production of extracellular matrix (ECM)-degrading proteases, and the epithelial-to-mesenchymal transition (EMT) program required for the acquisition of highly invasive traits. Here, we highlight functional interplays between either PKCα or PKCε and mesenchymal features that may ultimately contribute to anticancer drug resistance in cellular and animal models. We also introduce the novel hypothesis that c/nPKCs may be implicated in the control of immune evasion through the regulation of immune checkpoint protein expression. In summary, dissecting the colossal complexity of c/nPKC signaling in the wide spectrum of cancer progression may bring new opportunities for the development of meaningful tools aiding for cancer prognosis and therapy.
Topics: Animals; Carcinogenesis; Diglycerides; Epithelial-Mesenchymal Transition; Humans; Mutation; Neoplasm Metastasis; Neoplasms; Protein Conformation; Protein Isoforms; Protein Kinase C
PubMed: 33017725
DOI: 10.1016/j.jbior.2020.100755 -
Polish Journal of Veterinary Sciences Dec 2021The aim of this study was to analyze the chemiluminescence (CL) of peripheral blood in clinically healthy horses of different sexes and ages. The tests were carried out...
The aim of this study was to analyze the chemiluminescence (CL) of peripheral blood in clinically healthy horses of different sexes and ages. The tests were carried out on 119 half- -breed horses, representing various forms of use (66 recreational horses and 53 sport horses). The test material was peripheral blood, which was collected under resting conditions, i.e. before physical activity related to the use of these animals. In the blood samples, spontaneous and stimulated CL with zymosan and phorbol myristate acetate were determined. It has been found that regular training effort increases the blood's pro-oxidative potential, which was demonstrated by significantly higher (p⟨0.05) CL values in sport horses compared to recreational animals. Analysis of the results did not show any statistically significant correlation between sex or age of the horses with chemiluminescence values in peripheral blood. The result of the research suggests the need to optimize the results of blood CL measurements, taking into account the number of neutrophils and the concentration of hemoglobin in the blood of tested animals. Analysis of non-optimized blood CL results may lead to premature conclusions.
Topics: Animals; Horses; Luminescence; Luminescent Measurements; Neutrophils; Tetradecanoylphorbol Acetate; Zymosan
PubMed: 35179840
DOI: 10.24425/pjvs.2021.139978 -
BioRxiv : the Preprint Server For... Aug 2023Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt...
Activation of the Wnt pathway lies at the core of many human cancers. Wnt and macropinocytosis are often active in the same processes, and understanding how Wnt signaling and membrane trafficking cooperate should improve our understanding of embryonic development and cancer. Here we show that a macropinocytosis activator, the tumor promoter Phorbol 12-myristate 13-acetate (PMA), enhances Wnt signaling. Experiments using the embryo as an in vivo model showed marked cooperation between the PMA phorbol ester and Wnt signaling, which was blocked by inhibitors of macropinocytosis, Rac1 activity, and lysosome acidification. Human colorectal cancer tissue arrays and xenografts in mice showed a correlation of cancer progression with increased macropinocytosis/multivesicular body/lysosome markers and decreased GSK3 levels. The crosstalk between canonical Wnt, focal adhesions, lysosomes, and macropinocytosis suggests possible therapeutic targets for cancer progression in Wnt-driven cancers.
PubMed: 37333286
DOI: 10.1101/2023.06.02.543509 -
British Journal of Pharmacology Oct 2021Neurosteroids influence neuronal function and have multiple promising clinical applications. Direct modulation of postsynaptic neurotransmitter receptors by...
BACKGROUND AND PURPOSE
Neurosteroids influence neuronal function and have multiple promising clinical applications. Direct modulation of postsynaptic neurotransmitter receptors by neurosteroids is well characterized, but presynaptic effects remain poorly understood. Here, we report presynaptic glutamate release potentiation by neurosteroids pregnanolone and pregnanolone sulfate and compare their mechanisms of action to phorbol 12,13-dibutyrate (PDBu), a mimic of the second messenger DAG.
EXPERIMENTAL APPROACH
We use whole-cell patch-clamp electrophysiology and pharmacology in rat hippocampal microisland cultures and total internal reflection fluorescence (TIRF) microscopy in HEK293 cells expressing GFP-tagged vesicle priming protein Munc13-1, to explore the mechanisms of neurosteroid presynaptic modulation.
KEY RESULTS
Pregnanolone sulfate and pregnanolone potentiate glutamate release downstream of presynaptic Ca influx, resembling the action of a phorbol ester PDBu. PDBu partially occludes the effect of pregnanolone, but not of pregnanolone sulfate. Calphostin C, an inhibitor that disrupts DAG binding to its targets, reduces the effect PDBu and pregnanolone, but not of pregnanolone sulfate, suggesting that pregnanolone might interact with a well-known DAG/phorbol ester target Munc13-1. However, TIRF microscopy experiments found no evidence of pregnanolone-induced membrane translocation of GFP-tagged Munc13-1, suggesting that pregnanolone may regulate Munc13-1 indirectly or interact with other DAG targets.
CONCLUSION AND IMPLICATIONS
We describe a novel presynaptic effect of neurosteroids pregnanolone and pregnanolone sulfate to potentiate glutamate release downstream of presynaptic Ca influx. The mechanism of action of pregnanolone, but not of pregnanolone sulfate, partly overlaps with that of PDBu. Presynaptic effects of neurosteroids may contribute to their therapeutic potential in the treatment of disorders of the glutamate system.
Topics: Animals; Glutamic Acid; HEK293 Cells; Humans; Neurosteroids; Pregnanolone; Rats; Sulfates
PubMed: 33988248
DOI: 10.1111/bph.15529