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Biochemistry and Cell Biology =... Dec 2023GPRC5A is the first member of a new class of orphan receptors coupled to G proteins, which also includes GPRC5B, GPRC5C, and GPRC5D. Since its cloning and identification... (Review)
Review
GPRC5A is the first member of a new class of orphan receptors coupled to G proteins, which also includes GPRC5B, GPRC5C, and GPRC5D. Since its cloning and identification in the 1990s, substantial progress has been made in understanding the possible functions of this receptor. has been implicated in a variety of cellular events, such as cytoskeleton reorganization, cell proliferation, cell cycle regulation, migration, and survival. It appears to be a central player in different pathological processes, including tumorigenesis, inflammation, immune response, and tissue damage. The levels of expression differ depending on the type of cancer, with increased expression in colon, pancreas, and prostate cancers; decreased expression in lung cancer; and varied results in breast cancer. In this review, we discuss the early discovery of as a phorbol ester-induced gene and later as a retinoic acid-induced gene, its regulation, and its participation in important canonical pathways related to numerous types of tumors and inflammatory processes. represents a potential new target for cancer, inflammation, and immunity therapies.
Topics: Male; Humans; Receptors, Retinoic Acid; Phorbol Esters; Receptors, G-Protein-Coupled; Lung Neoplasms; Inflammation; Tretinoin
PubMed: 37467514
DOI: 10.1139/bcb-2022-0352 -
Journal of the American Academy of... Nov 2023
PubMed: 37460064
DOI: 10.1016/j.jaad.2023.06.053 -
PloS One 2023THP-1 monocyte, which can be differentiated into macrophages by PMA, is widely used in researches on pathogen infection and host innate immunity, but reports on the...
THP-1 monocyte, which can be differentiated into macrophages by PMA, is widely used in researches on pathogen infection and host innate immunity, but reports on the induction methods of PMA are different and lack a unified standard, and the transcriptome characteristics of macrophage compared with THP-1 cells remains unclear. In this research, we examined the differentiation effect of three factors including induction time, cell seeding density and PMA concentration by detecting the positive rate of CD14 expression. The concentration of 80ng/ml of PMA, the induction time of 24h, and the cell seeding density of 5×105 cells/ml, could respectively facilitates a relatively higher CD14 positive rate in THP-1 cells. Under this optimized conditions, the CD14 positive rate of THP-1 cells can reach 66.52%. Transcriptome sequencing showed that after the above induction, the mRNA expression of 3113 genes which were closely related to cell communication, signal transduction, cell response to stimulus, signaling receptor binding and cytokine activity were up-regulated, and the top 10 genes were RGS1, SPP1, GDF15, IL-1B, HAVCR2, SGK1, EGR2, TRAC, IL-8 and EBI3. While the mRNA expression of 2772 genes which were associated with cell cycle process, DNA binding and replication and cell division, were down-regulated, and the top genes were SERPINB10, TRGC2, SERPINB2, TRGC1, MS4A3, MS4A4E, TRGJP1, MS4A6A, TRGJP2, MS4A4A. This research optimized the induction method on THP-1 cell differentiation from three aspects and delineated the transcriptomic profile of PMA-induced THP-1 cells, laying a foundation for the construction method of cell model and for the functional study of macrophage.
Topics: Humans; Transcriptome; THP-1 Cells; Tetradecanoylphorbol Acetate; Macrophages; Monocytes; Cell Differentiation; RNA, Messenger
PubMed: 37459313
DOI: 10.1371/journal.pone.0286056 -
Zhongguo Xue Xi Chong Bing Fang Zhi Za... Jul 2023To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps...
OBJECTIVE
To investigate the mechanisms underlying allergic conjunctivitis caused by conjunctival epithelial cell damage, neutrophil migration and neutrophil extracellular traps (NETs) formation induced by crude extracts of mite (CDM).
METHODS
Human conjunctival epithelial cells were stimulated with 500, 1 000, 2 000, 4 000 ng/mL, and the expression levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ) and IL-8 were detected using quantitative real-time PCR (qPCR) assay and enzyme-linked immunosorbent assay (ELISA). The culture supernatant of human conjunctival epithelial cells was collected and co-cultured with neutrophils. Neutrophil migration was measured using Transwell migration assay, and the expression of NETs markers myeloperoxidase (MPO) and citrullinated histone H3 (CitH3) was quantified using immunofluorescence staining. Neutrophils were stimulated with phorbol 12-myristate 13-acetate (PMA), and then NETs were collected for treatment of human conjunctival epithelial cells. Cell apoptosis was detected using flow cytometry, and the levels of IL-6, TNF-α, IFN-γ and IL-8 were measured in the cell culture supernatant using ELISA.
RESULTS
Treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL up-regulated IL-6, TNF-α, IFN-γ and IL-8 expression in human conjunctival epithelial cells. Following treatment with CDM at concentrations of 2 000 ng/mL and 4 000 ng/mL, the culture supernatant of human conjunctival epithelial cells promoted neutrophil migration and induced increases in the staining intensity of MPO and CitH3. In addition, increased NETs triggered the apoptosis of human conjunctival epithelial cells and IL-6, TNF-α, IFN-γ and IL-8 secretion in the culture supernatant of human conjunctival epithelial cells.
CONCLUSIONS
CDM induces human conjunctival epithelial cell damages, thereby promoting neutrophil migration and NETs formation, while the release of NETs further aggravates human conjunctival epithelial cell damages.
Topics: Animals; Humans; Extracellular Traps; Neutrophils; Interleukin-8; Dermatophagoides farinae; Interleukin-6; Tumor Necrosis Factor-alpha; Epithelial Cells; Interferon-gamma; Tetradecanoylphorbol Acetate
PubMed: 37455098
DOI: 10.16250/j.32.1374.2023032 -
Biochemical and Biophysical Research... Oct 2023Naturally occurring protein kinase C (PKC) activators such as phorbol esters, teleocidins, and aplysiatoxins, have the potential to become anti-cancer agents, since they...
Naturally occurring protein kinase C (PKC) activators such as phorbol esters, teleocidins, and aplysiatoxins, have the potential to become anti-cancer agents, since they are anti-proliferative against specific cancer cell lines in vitro. However, their potent tumor-promoting and proinflammatory activities have hampered their clinical uses. Recently, we developed 10-methyl-aplog-1 (1), a simplified analog of tumor-promoting debromoaplysiatoxin (DAT), which retained anti-proliferative activity comparable to DAT, but induced neither tumorigenesis nor inflammation on mouse skin. Our previous study suggested that PKCα and δ were involved in the cell line-selective anti-proliferative activity of 1, but the downstream signaling of PKC isozymes remained unknown. In this study, we confirmed that 1 inhibited the growth of three aplog-sensitive cancer cell lines (NCI-H460, HCC-2998, and HBC-4) without severe side effects in mice xenograft models. In addition, in vitro analysis using A549, one of the aplog-sensitive cell lines in vitro, revealed that PKCα induced PP2A-mediated attenuation of the Akt/S6 signaling axis. Since S6 inhibition in A549 was reported to result in G1 arrest, this pathway could be involved in the PKCα-dependent anti-proliferative activity of 1.
Topics: Humans; Mice; Animals; Protein Kinase C-alpha; Carcinoma, Hepatocellular; Structure-Activity Relationship; Cell Proliferation; Liver Neoplasms; Signal Transduction; Protein Kinase C; Cell Line, Tumor
PubMed: 37437496
DOI: 10.1016/j.bbrc.2023.07.008 -
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 -
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 -
Redox Biology Aug 2023Irisin is a newly discovered myokine which links exercise to inflammation and inflammation-related diseases through macrophage regulation. However, the effect of irisin...
INTRODUCTION
Irisin is a newly discovered myokine which links exercise to inflammation and inflammation-related diseases through macrophage regulation. However, the effect of irisin on the activity of inflammation related immune cells (such as neutrophils) has not been clearly described.
OBJECTIVES
The objective of our study was to explore the effect of irisin on the neutrophil extracellular traps (NETs) formation.
METHODS
Phorbol-12-myristate-13-acetate (PMA) was used to construct a classic neutrophil inflammation model that was used to observe the formation of NETs in vitro. We studied the effect of irisin on NETs formation and its regulation mechanism. Subsequently, acute pancreatitis (AP) was used to verify the protective effect of irisin in vivo, which was an acute aseptic inflammatory response disease model closely related to NETs.
RESULTS
Our study found that addition of irisin significantly reduced the formation of NETs via regulation of the P38/MAPK pathway through integrin αVβ5, which might be the one of key pathways in NETs formation, and which could theoretically offset the immunoregulatory effect of irisin. Systemic treatment with irisin reduced the severity of tissue damage common in the disease and inhibited the formation of NETs in pancreatic necrotic tissue of two classical AP mouse models.
CONCLUSION
The findings confirmed for the first time that irisin could inhibit NETs formation and protect mice from pancreatic injury, which further elucidated the protective effect of exercise on acute inflammatory injury.
Topics: Mice; Animals; Extracellular Traps; Pancreatitis; Fibronectins; Acute Disease; Neutrophils; Inflammation; Tetradecanoylphorbol Acetate
PubMed: 37392517
DOI: 10.1016/j.redox.2023.102787 -
Scientific Reports Jun 2023The defining biology that distinguishes neutrophil extracellular traps (NETs) from other forms of cell death is unresolved, and techniques which unambiguously identify...
The defining biology that distinguishes neutrophil extracellular traps (NETs) from other forms of cell death is unresolved, and techniques which unambiguously identify NETs remain elusive. Raman scattering measurement provides a holistic overview of cell molecular composition based on characteristic bond vibrations in components such as lipids and proteins. We collected Raman spectra from NETs and freeze/thaw necrotic cells using a custom built high-throughput platform which is able to rapidly measure spectra from single cells. Principal component analysis of Raman spectra from NETs clearly distinguished them from necrotic cells despite their similar morphology, demonstrating their fundamental molecular differences. In contrast, classical techniques used for NET analysis, immunofluorescence microscopy, extracellular DNA, and ELISA, could not differentiate these cells. Additionally, machine learning analysis of Raman spectra indicated subtle differences in lipopolysaccharide (LPS)-induced as opposed to phorbol myristate acetate (PMA)-induced NETs, demonstrating the molecular composition of NETs varies depending on the stimulant used. This study demonstrates the benefits of Raman microscopy in discriminating NETs from other types of cell death and by their pathway of induction.
Topics: Humans; Extracellular Traps; Neutrophils; Tetradecanoylphorbol Acetate; Microscopy, Fluorescence; Necrosis
PubMed: 37344494
DOI: 10.1038/s41598-023-36667-3 -
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi =... Jun 2023Objective To investigate the molecular mechanism of taurine regulating the polarization of M2 macrophages by mitophagy. Methods THP-1 cells were divided into four...
Objective To investigate the molecular mechanism of taurine regulating the polarization of M2 macrophages by mitophagy. Methods THP-1 cells were divided into four groups: M0 group (THP-1 cells were treated by 100 nmol/L phorbol myristate ester for 48 hours to polarize into M0), M2 group (THP-1 cells were induced to polarize into M2 macrophages by 20 ng/mL interferon-4 (IL-4) for 48 hours), M2 combined with taurine groups (added with 40 or 80 mmol/L taurine on the basis of M2 macrophages). The mRNA expression of mannose receptor C type 1(MRC-1), C-C motif chemokine ligand 22(CCL22) and dendritic cell-specific ICAM-3 grabbing non-integrin (CD209) in M2 macrophages were detected by quantitative real-time PCR. Mitochondrial and lysosome probes were used to detect the number of mitochondria and lysosomes by multifunction microplate reader and confocal laser scanning microscope. The level of mitochondrial membrane potential (MMP) was detected by JC-1 MMP assay kit. The expression of mitophagy-related proteins PTEN-induced putative kinase 1 (PINK1) and microtubule-associated protein 1 light chain 3 (LC3) were detected by Western blot analysis. Results Compared with M0 group, the expression of MRC-1, CCL22, CD209 and PINK1, the number of mitochondria and the level of MMP in M2 group were significantly increased, whereas the number of lysosomes and LC3II/LC3I ratio were decreased. Compared with M2 group, the expressions of MRC-1, CCL22 and CD209, the number of mitochondria and the level of MMP in M2 combined with taurine group dropped significantly while the number of lysosomes was found increased, and the protein expression of PINK1 and LC3II/LC3I ratio were also increased. Conclusions The polarization of M2 macrophages is regulated by taurine to prevent excessive polarization via reducing the level of MMP, improving the level of mitophagy, reducing the number of mitochondria, and inhibiting the mRNA expression of polarization markers in M2 macrophages.
Topics: Mitophagy; Taurine; Macrophages; Protein Kinases; RNA, Messenger
PubMed: 37340916
DOI: No ID Found