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Journal of Visualized Experiments : JoVE May 2021Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form...
Membrane ruffling is the formation of motile plasma membrane protrusions containing a meshwork of newly polymerized actin filaments. Membrane ruffles may form spontaneously or in response to growth factors, inflammatory cytokines, and phorbol esters. Some of the membrane protrusions may reorganize into circular membrane ruffles that fuse at their distal margins and form cups that close and separate into the cytoplasm as large, heterogeneous vacuoles called macropinosomes. During the process, ruffles trap extracellular fluid and solutes that internalize within macropinosomes. High-resolution scanning electron microscopy (SEM) is a commonly used imaging technique to visualize and quantify membrane ruffle formation, circular protrusions, and closed macropinocytic cups on the cell surface. The following protocol describes the cell culture conditions, stimulation of the membrane ruffle formation in vitro, and how to fix, dehydrate, and prepare cells for imaging using SEM. Quantification of membrane ruffling, data normalization, and stimulators and inhibitors of membrane ruffle formation are also described. This method can help answer key questions about the role of macropinocytosis in physiological and pathological processes, investigate new targets that regulate membrane ruffle formation, and identify yet uncharacterized physiological stimulators as well as novel pharmacological inhibitors of macropinocytosis.
Topics: Actin Cytoskeleton; Cell Membrane; Cell Surface Extensions; Microscopy, Electron, Scanning; Pinocytosis
PubMed: 34125102
DOI: 10.3791/62658 -
Epigenetics 2020The transcriptional program that dictates haematopoietic cell fate and differentiation requires an epigenetic regulatory and memory function, provided by a network of...
The transcriptional program that dictates haematopoietic cell fate and differentiation requires an epigenetic regulatory and memory function, provided by a network of epigenetic factors that regulate DNA methylation, post-translational histone modifications and chromatin structure. Disturbed epigenetic regulation causes perturbations in the blood cell differentiation program that results in various types of haematopoietic disorders. Thus, accurate epigenetic regulation is essential for functional haematopoiesis. In this study, we used a CRISPR-Cas9 screening approach to identify new epigenetic regulators in myeloid differentiation. We designed a Chromatin-UMI CRISPR guide library targeting 1092 epigenetic regulators. Phorbol 12-myristate 13-acetate (PMA) treatment of the chronic myeloid leukaemia cell line K-562 was used as a megakaryocytic myeloid differentiation model. Both previously described developmental epigenetic regulators and novel factors were identified in our screen. In this study, we validated and characterized a role for the chromatin remodeller CHD2 in myeloid proliferation and megakaryocytic differentiation.
Topics: Cell Proliferation; Chromatin Assembly and Disassembly; DNA-Binding Proteins; Humans; K562 Cells; Megakaryocytes; Myelopoiesis; Tetradecanoylphorbol Acetate
PubMed: 31900031
DOI: 10.1080/15592294.2019.1710913 -
Cells Mar 2022Nociceptin and the nociceptin receptor (NOP) have been described as targets for treatment of pain and inflammation, whereas toll-like receptors (TLRs) play key roles in...
Nociceptin and the nociceptin receptor (NOP) have been described as targets for treatment of pain and inflammation, whereas toll-like receptors (TLRs) play key roles in inflammation and impact opioid receptors and endogenous opioids expression. In this study, interactions between the nociceptin and TLR systems were investigated. Human THP-1 cells were cultured with or without phorbol myristate acetate (PMA 5 ng/mL), agonists specific for TLR2 (lipoteichoic acid, LTA 10 µg/mL), TLR4 (lipopolysaccharide, LPS 100 ng/mL), TLR7 (imiquimod, IMQ 10 µg/mL), TLR9 (oligonucleotide (ODN) 2216 1 µM), PMA+TLR agonists, or nociceptin (0.01−100 nM). Prepronociceptin (ppNOC), NOP, and TLR mRNAs were quantified by RT-qPCR. Proteins were measured using flow cytometry. PMA upregulated ppNOC mRNA, intracellular nociceptin, and cell membrane NOP proteins (all p < 0.05). LTA and LPS prevented PMA’s upregulating effects on ppNOC mRNA and nociceptin protein (both p < 0.05). IMQ and ODN 2216 attenuated PMA’s effects on ppNOC mRNA. PMA, LPS, IMQ, and ODN 2216 increased NOP protein levels (all p < 0.05). PMA+TLR agonists had no effects on NOP compared to PMA controls. Nociceptin dose-dependently suppressed TLR2, TLR4, TLR7, and TLR9 proteins (all p < 0.01). Antagonistic effects observed between the nociceptin and TLR systems suggest that the nociceptin system plays an anti-inflammatory role in monocytes under inflammatory conditions.
Topics: Humans; Inflammation; Lipopolysaccharides; Opioid Peptides; RNA, Messenger; Tetradecanoylphorbol Acetate; Toll-Like Receptor 2; Toll-Like Receptor 4; Toll-Like Receptor 7; Toll-Like Receptor 9; Toll-Like Receptors; Nociceptin
PubMed: 35406649
DOI: 10.3390/cells11071085 -
Arteriosclerosis, Thrombosis, and... Sep 2023Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic...
BACKGROUND
Platelets and neutrophils are the first blood cells accumulating at sites of arterial thrombus formation, and both cell types contribute to the pathology of thrombotic events. We aimed to identify key interaction mechanisms between these cells using microfluidic approaches.
METHODS
Whole-blood perfusion was performed over a collagen surface at arterial shear rate. Platelet and leukocyte (in majority neutrophil) activation were microscopically visualized using fluorescent markers. The contributions of platelet-adhesive receptors (integrin, P-selectin, CD40L) and chemokines were studied by using inhibitors or antibodies and using blood from patients with GT (Glanzmann thrombasthenia) lacking platelet-expressed αIIbβ3.
RESULTS
We observed (1) an unknown role of activated platelet integrin αIIbß3 preventing leukocyte adhesion, which was overcome by short-term flow disturbance provoking massive adhesion; (2) that platelet-expressed CD40L controls the crawling pattern and thrombus fidelity of the cells on a thrombus; (3) that continued secretion of platelet substances promotes activation of identified neutrophils, as assessed by (fMLP [-formylmethionyl-leucyl-phenylalanine, a potent chemotactic agent and leukocyte activator] induced) [Ca] rises and antigen expression; (4) and that platelet-released chemokines activate the adhered cells in the order of CXCL7>CCL5>CXCL4. Furthermore, postsilencing of the platelets in a thrombus suppressed the leukocyte activation. However, the leukocytes on thrombi did no more than limitedly form neutrophil extracellular traps, unless stimulated with phorbol ester or lipopolysaccharide.
CONCLUSIONS
Together, these findings reveal a multifaceted regulation of adhesion and activation of neutrophils by platelets in a thrombus, with a balanced role of several platelet-adhesive receptors and a promoting role of platelet-released substances. This multivalent nature of neutrophil-thrombus interactions offers novel prospects for pharmacological intervention.
Topics: Blood Platelets; Platelet Glycoprotein GPIIb-IIIa Complex; Chemokines; Thrombosis; Neutrophil Activation; CD40 Ligand; Neutrophils; Cell Adhesion; Humans; Arteries
PubMed: 37409530
DOI: 10.1161/ATVBAHA.122.318767 -
Veterinary Sciences Sep 2021L. has gained importance as a source of seed oil for biodiesel production. The meal contained about 60% protein with a good balance of essential amino acids, containing... (Review)
Review
L. has gained importance as a source of seed oil for biodiesel production. The meal contained about 60% protein with a good balance of essential amino acids, containing various bioactive compounds, including saponins, phytic acids, trypsin inhibitors, lectins, phenolics, and flavonoids, which render it as a potential biofeed for animal production. The meal demonstrated various biological activities, including antioxidant, antibacterial, and anti-inflammatory effects which enhance its property as a bio-feed. The levels of these bioactive compounds in the seeds are dependent on the genotypes. The possessed different varieties which are either toxic or non-toxic according to the presence of phorbol esters. The presence of phorbol esters in the meal confirmed the toxic variety of resulting in the limited application of meal as a biofeed. The meal devoid of phorbol esters could be applied as a biofeed in the animal production industry, and for the toxic varieties, various techniques such as physicochemical and biological treatments have been introduced to the industry to remove the phorbol esters from meal. Several studies employing various cells and animals confirmed the toxicity of the phorbol esters. The molecular mechanism of action of phorbol esters is through up-regulation of PKC-β II gene, overexpression of down-stream proto-oncogenes resulted in inflammation and oxidative stress ending by apoptotic cell death. Despite the presence of valuable bioactive compounds in the meal, its nutritional application is not recommended unless the phorbol esters are completely removed.
PubMed: 34564573
DOI: 10.3390/vetsci8090179 -
Biomolecules & Therapeutics Mar 2022In this study, we investigated the influence of galangin on vascular contractibility and to determine the mechanism underlying the relaxation. Isometric contractions of...
In this study, we investigated the influence of galangin on vascular contractibility and to determine the mechanism underlying the relaxation. Isometric contractions of denuded aortic muscles were recorded and combined with western blot analysis which was performed to measure the phosphorylation of phosphorylation-dependent inhibitory protein of myosin phosphatase (CPI-17) and myosin phosphatase targeting subunit 1 (MYPT1) and to evaluate the effect of galangin on the RhoA/ROCK/CPI-17 pathway. Galangin significantly inhibited phorbol ester-, fluoride- and thromboxane mimetic-induced vasoconstrictions regardless of endothelial nitric oxide synthesis, suggesting its direct effect on vascular smooth muscle. Galangin significantly inhibited the fluoride-dependent increase in pMYPT1 and pCPI-17 levels and phorbol 12,13-dibutyrate-dependent increase in pERK1/2 level, suggesting repression of ROCK and MEK activity and subsequent phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that galangin-induced relaxation involves myosin phosphatase reactivation and calcium desensitization, which appears to be mediated by CPI-17 dephosphorylation via not PKC but ROCK inactivation.
PubMed: 34231489
DOI: 10.4062/biomolther.2021.087 -
Scientific Reports Sep 202012-O-Tetradecanoylphorbol-13-acetate (TPA) is the most widely used diacylglycerol (DAG) mimetic agent and inducer of protein kinase C (PKC)-mediated cellular response in...
12-O-Tetradecanoylphorbol-13-acetate (TPA) is the most widely used diacylglycerol (DAG) mimetic agent and inducer of protein kinase C (PKC)-mediated cellular response in biomedical studies. TPA has been proposed as a pluripotent cell differentiation factor, but results obtained have been inconsistent. In the present study we show that TPA can be applied as a cardiomyogenesis-promoting factor for the differentiation of mouse embryonic stem (mES) cells in vitro. The mechanism of TPA action is mediated by the induction of extracellular signal-regulated kinase (ERK) activity and the subsequent phosphorylation of GATA4 transcription factor. Interestingly, general mitogens (FGF, EGF, VEGF and serum) or canonical WNT signalling did not mimic the effect of TPA. Moreover, on the basis of our results, we postulate that a TPA-sensitive population of cardiac progenitor cells exists at a certain time point (after days 6-8 of the differentiation protocol) and that the proposed treatment can be used to increase the multiplication of ES cell-derived cardiomyocytes.
Topics: Animals; Cell Differentiation; Embryonic Stem Cells; Extracellular Signal-Regulated MAP Kinases; Mice; Myocytes, Cardiac; Phosphorylation; Protein Kinase C; Signal Transduction; Tetradecanoylphorbol Acetate
PubMed: 32985604
DOI: 10.1038/s41598-020-73074-4 -
The Biochemical Journal Jan 2021Protein kinase signalling, which transduces external messages to mediate cellular growth and metabolism, is frequently deregulated in human disease, and specifically in... (Review)
Review
Protein kinase signalling, which transduces external messages to mediate cellular growth and metabolism, is frequently deregulated in human disease, and specifically in cancer. As such, there are 77 kinase inhibitors currently approved for the treatment of human disease by the FDA. Due to their historical association as the receptors for the tumour-promoting phorbol esters, PKC isozymes were initially targeted as oncogenes in cancer. However, a meta-analysis of clinical trials with PKC inhibitors in combination with chemotherapy revealed that these treatments were not advantageous, and instead resulted in poorer outcomes and greater adverse effects. More recent studies suggest that instead of inhibiting PKC, therapies should aim to restore PKC function in cancer: cancer-associated PKC mutations are generally loss-of-function and high PKC protein is protective in many cancers, including most notably KRAS-driven cancers. These recent findings have reframed PKC as having a tumour suppressive function. This review focusses on a potential new mechanism of restoring PKC function in cancer - through targeting of its negative regulator, the Ser/Thr protein phosphatase PHLPP. This phosphatase regulates PKC steady-state levels by regulating the phosphorylation of a key site, the hydrophobic motif, whose phosphorylation is necessary for the stability of the enzyme. We also consider whether the phosphorylation of the potent oncogene KRAS provides a mechanism by which high PKC expression may be protective in KRAS-driven human cancers.
Topics: Antineoplastic Agents; Genes, Tumor Suppressor; Humans; Neoplasms; Nuclear Proteins; Phosphoprotein Phosphatases; Phosphorylation; Protein Kinase C; Proto-Oncogene Proteins p21(ras)
PubMed: 33502516
DOI: 10.1042/BCJ20190765 -
Theranostics 2020Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease characterized by autoantibody production by hyper-activated B cells. Although mesenchymal stem...
Systemic lupus erythematosus (SLE) is a multi-organ autoimmune disease characterized by autoantibody production by hyper-activated B cells. Although mesenchymal stem cells (MSCs) ameliorate lupus symptoms by inhibiting T cells, whether they inhibit B cells has been controversial. Here we address this issue and reveal how to prime MSCs to inhibit B cells and improve the efficacy of MSCs in SLE. We examined the effect of MSCs on purified B cells and the therapeutic efficacy of MSCs in lupus-prone MRL. mice. We screened chemicals for their ability to activate MSCs to inhibit B cells. Mouse bone marrow-derived MSCs inhibited mouse B cells in a CXCL12-dependent manner, whereas human bone marrow-derived MSCs (hMSCs) did not inhibit human B (hB) cells. We used a chemical approach to overcome this hurdle and found that phorbol myristate acetate (PMA), phorbol 12,13-dibutyrate, and ingenol-3-angelate rendered hMSCs capable of inhibiting IgM production by hB cells. As to the mechanism, PMA-primed hMSCs attracted hB cells in a CXCL10-dependent manner and induced hB cell apoptosis in a PD-L1-dependent manner. Finally, we showed that PMA-primed hMSCs were better than naïve hMSCs at ameliorating SLE progression in MRL. mice. Taken together, our data demonstrate that phorbol esters might be good tool compounds to activate MSCs to inhibit B cells and suggest that our chemical approach might allow for improvements in the therapeutic efficacy of hMSCs in SLE.
Topics: Animals; Apoptosis; B-Lymphocytes; Cells, Cultured; Female; Humans; Lupus Erythematosus, Systemic; Male; Mesenchymal Stem Cell Transplantation; Mesenchymal Stem Cells; Mice; Mice, Inbred C3H; Phorbol Esters; T-Lymphocytes
PubMed: 32929342
DOI: 10.7150/thno.46835 -
The Effect of Luteolin on the Modulation of Vascular Contractility via ROCK and CPI-17 Inactivation.Biomolecules & Therapeutics Mar 2023In this investigation, we made a study of the efficacy of luteolin (a flavonoid found in plants such as vegetables, herbs and fruits) on vascular contractibility and to...
In this investigation, we made a study of the efficacy of luteolin (a flavonoid found in plants such as vegetables, herbs and fruits) on vascular contractibility and to elucidate the mechanism underlying the relaxation. Isometric contractions of denuded muscles were stored and combined with western blot analysis which was conducted to assess the phosphorylation of myosin phosphatase targeting subunit 1 (MYPT1) and phosphorylation-dependent inhibitory protein for myosin phosphatase (CPI-17) and to examine the effect of luteolin on the RhoA/ROCK/CPI-17 pathway. Luteolin significantly alleviated phorbol ester-, fluoride- and thromboxane mimetic-elicited contractions regardless of endothelial nitric oxide synthesis, implying its direct effect on smooth muscle. It also significantly alleviated the fluoride-elicited elevation in pCPI-17 and pMYPT1 levels and phorbol 12,13-dibutyrate-elicited increase in pERK1/2 level, suggesting depression of ROCK and PKC/MEK activity and ensuing phosphorylation of MYPT1, CPI-17 and ERK1/2. Taken together, these results suggest that luteolin-elicited relaxation includes myosin phosphatase reactivation and calcium desensitization, which seems to be arbitrated by CPI-17 dephosphorylation via ROCK/PKC inhibition.
PubMed: 36065763
DOI: 10.4062/biomolther.2022.087