scholarly journals Polarized Cytokine Release Triggered by P2X7 Receptor from Retinal Pigmented Epithelial Cells Dependent on Calcium Influx

Cells ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 2537
Author(s):  
Xiaolei Shao ◽  
Sonia Guha ◽  
Wennan Lu ◽  
Keith E. Campagno ◽  
Jonathan M. Beckel ◽  
...  
Keyword(s):  
P2x7 Receptor ◽  
Calcium Influx ◽  
Inflammatory Cells ◽  
Cytokine Signaling ◽  
Cytokine Release ◽  
Rpe Cells ◽  
Primary Mouse ◽  
Retinal Neurodegeneration ◽  
Apical Membranes

Cytokine release from non-inflammatory cells is a key step in innate immunity, and agonists triggering cytokine release are central in coordinating responses. P2X7 receptor (P2X7R) stimulation by extracellular ATP is best known to active the NLRP3 inflammasome and release IL-1β, but stimulation also leads to release of other cytokines. As cytokine signaling by retinal pigmented epithelial (RPE) cells is implicated in retinal neurodegeneration, the role of P2X7R in release of cytokine IL-6 from RPE cells was investigated. P2X7R stimulation triggered IL-6 release from primary mouse RPE, human iPS-RPE and human ARPE-19 cells. IL-6 release was polarized, with predominant rise across apical membranes. IL-6 release was inhibited by P2X7R antagonists A438079, A839977, and AZ10606120, but not the NRTI lamivudine (3TC), P2X1R antagonist NF279, or P2Y1R antagonist MRS2179. P2X7R-mediated IL-6 release required extracellular Ca2+ and was blocked by Ca2+ chelator BAPTA. IL-6 release and Ca2+ elevation occurred rapidly, consistent with vesicular IL-6 staining in unstimulated cells. P2X7R stimulation did not trigger IL-1β release in these unprimed cells. P2X7R-mediated IL-6 release was enhanced in RPE cells from the ABCA4−/− mouse model of retinal degeneration. In summary, P2X7R stimulation triggers rapid Ca2+-dependent IL-6 release across the apical membrane of RPE cells.

scholarly journals C-CBL Is a Critical Negative Regulator of Megakaryopoesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1581-1581
Author(s):  
Sebastian J. Saur ◽  
Melanie Märklin ◽  
Alexandra Poljak ◽  
Manuela Ganser ◽  
David E. James ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Cytokine Signaling ◽  
Wild Type ◽  
Platelet Factor ◽  
Physiological Level ◽  
Hematopoietic Stem ◽  
Primary Mouse

Abstract Megakaryopoiesis is controlled by a variety of hematopoietic growth factors in order to maintain a physiological level of circulating platelets. Thrombopoietin (TPO) is the main regulator of megakaryopoiesis modulating megakaryocyte differentiation, promoting endomitosis and proplatelet formation and as such supports the self-renewal and survival of hematopoietic stem cells. To allow proper proliferation and differentiation of different hematopoetic lineages, TPO signal transduction must be tightly regulated. Several mechanisms negatively modulating hematopoiesis and differentiation of the megakaryocytic lineage have previously been identified. Among those are suppressors cytokine signaling, protein phosphatases as well as a multitude of negative regulatory signaling pathways. However, one of the most effective mechanisms to permanently disable activated signaling proteins is by targeted degradation via lysosomes or proteasomes. In this study, we investigated the mechanisms that regulate TPO-mediated MPL degradation in primary mouse cells. Previous studies have identified CBL as an E3 ligase responsible for the ubiquitination of MPL in cell lines. In order to determine the potential role of c-CBL in murine thrombopoiesis, we used Cre/loxP technology to specifically delete c-CBL in the megakaryocytic lineage. Mice expressing two floxed c-CBL alleles were crossed to mice expressing Cre recombinase under the control of the platelet factor 4 (PF4) promoter. This yielded progeny with the desired genotype of c-CBLfl/fl PF4-Cre (CBL ko) after two generations of breeding. The desired cohort exhibited a quantitative absence of c-CBL in megakaryocytes and platelets as assessed by western blotting compared with wild type C57/BL6 mice. The expression of CBL in other hematopoietic cells such as B cells, T cells, neutrophils, monocytes and dendritic cells remained unaffected in this conditional ko strain. The experimental cohort showed significantly higher numbers of megakaryocytes in the bone marrow and of platelets in the peripheral blood as compared to wild type mice (1.2 mio vs. 1.8 mio cells/µl, p<0.0001). In addition, the platelets from the mutant mouse strain were of significantly smaller size (43 vs. 38 fL, p=0.0022). To evaluate the role of c-CBL in mature megakaryocytes, total bone marrow was collected from 12 wk old CBL ko mice and grown in TPO-containing culture medium for 72 h. Megakaryocytes derived from the bone marrow of wild type mice served as controls. Mature megakaryocytes were eventually isolated on a BSA-density gradient. Subsequent Western Blot analysis revealed a significant reduction of MPL ubiquitination in the CBL ko mice as compared to wild type mice, thereby identifying c-CBL as a critical negative regulator of megakaryopoesis. Taken together, we have successfully ablated c-CBL specifically from the megakaryocyte lineage and could demonstrate that this has profound effects on platelet counts and platelet size. In addition, we were able to show that c-CBL ablation leads to reduced ubiquitination of MPL and a consecutively longer half life of this protein culminating in substantially increased megakaryopoiesis in the c-CBL ko cohort. In summary, these data enhance our understanding of the regulation of TPO signaling and the physiological role of CBL in the megakaryocytic lineage. Disclosures No relevant conflicts of interest to declare.

The role of neuraminidase 1 (NEU1) in cytokine release by primary mouse mesangial cells and disease outcomes in murine lupus nephritis

Autoimmunity ◽  
2021 ◽  
pp. 1-13
Author(s):  
Jessalyn Rodgers ◽  
Kamala Sundararaj ◽  
Evelyn Bruner ◽  
Bethany Wolf ◽  
Tamara K. Nowling
Keyword(s):  
Lupus Nephritis ◽  
Mesangial Cells ◽  
Cytokine Release ◽  
Murine Lupus ◽  
Disease Outcomes ◽  
Primary Mouse

scholarly journals The role of P2X7 receptor in ATP-mediated human leukemia cell death: calcium influx-independent

2009 ◽  
Vol 41 (5) ◽  
pp. 362-369 ◽  
Author(s):  
Xiujun Zhang ◽  
Lijun Meng ◽  
Baoling He ◽  
Jing Chen ◽  
Peng Liu ◽  
...  
Keyword(s):  
Cell Death ◽  
P2x7 Receptor ◽  
Calcium Influx ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Human Leukemia Cell

Different Abilities of Thrombin Receptor Activating Peptide and Thrombin to Induce Platelet Calcium Rise and Full Release Reaction

1995 ◽  
Vol 74 (05) ◽  
pp. 1323-1328 ◽  
Author(s):  
Dominique Lasne ◽  
José Donato ◽  
Hervé Falet ◽  
Francine Rendu
Keyword(s):  
Essential Role ◽  
Calcium Influx ◽  
Dense Granule ◽  
Receptor Interaction ◽  
Thrombin Receptor ◽  
Release Reaction ◽  
External Calcium ◽  
Maximum Rise ◽  
Granule Release

SummarySynthetic peptides (TRAP or Thrombin Receptor Activating Peptide) corresponding to at least the first five aminoacids of the new N-terminal tail generated after thrombin proteolysis of its receptor are effective to mimic thrombin. We have studied two different TRAPs (SFLLR, and SFLLRN) in their effectiveness to induce the different platelet responses in comparison with thrombin. Using Indo-1/AM- labelled platelets, the maximum rise in cytoplasmic ionized calcium was lower with TRAPs than with thrombin. At threshold concentrations allowing maximal aggregation (50 μM SFLLR, 5 μM SFLLRN and 1 nM thrombin) the TRAPs-induced release reaction was about the same level as with thrombin, except when external calcium was removed by addition of 1 mM EDTA. In these conditions, the dense granule release induced by TRAPs was reduced by over 60%, that of lysosome release by 75%, compared to only 15% of reduction in the presence of thrombin. Thus calcium influx was more important for TRAPs-induced release than for thrombin-induced release. At strong concentrations giving maximal aggregation and release in the absence of secondary mediators (by pretreatment with ADP scavengers plus aspirin), SFLLRN mobilized less calcium, with a fast return towards the basal level and induced smaller lysosome release than did thrombin. The results further demonstrate the essential role of external calcium in triggering sustained and full platelet responses, and emphasize the major difference between TRAP and thrombin in mobilizing [Ca2+]j. Thus, apart from the proteolysis of the seven transmembrane receptor, another thrombin binding site or thrombin receptor interaction is required to obtain full and complete responses.

The Role of Cytokines in Interactions of Mesenchymal Stem Cells and Breast Cancer Cells

2020 ◽  
Vol 15 ◽  
Author(s):  
Hariharan Jayaraman ◽  
Nalinkanth V. Ghone ◽  
Ranjith Kumaran R ◽  
Himanshu Dashora
Keyword(s):  
Breast Cancer ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Tumor Microenvironment ◽  
Cancer Cells ◽  
Cell Communication ◽  
Extra Cellular Matrix ◽  
Cytokine Signaling ◽  
Cellular Matrix

: Mesenchymal stem cells because of its high proliferation, differentiation, regenerative capacity, and ease of availability have been a popular choice in cytotherapy. Mesenchymal Stem Cells (MSCs) have a natural tendency to home in a tumor microenvironment and acts against it, owing to the similarity of the latter to an injured tissue environment. Several studies have confirmed the recruitment of MSCs by tumor through various cytokine signaling that brings about phenotypic changes to cancer cells, thereby promoting migration, invasion, and adhesion of cancer cells. The contrasting results on MSCs as a tool for cancer cytotherapy may be due to the complex cell to cell interaction in the tumor microenvironment, which involves various cell types such as cancer cells, immune cells, endothelial cells, and cancer stem cells. Cell to cell communication can be simple or complex and it is transmitted through various cytokines among multiple cell phenotypes, mechano-elasticity of the extra-cellular matrix surrounding the cancer cells, and hypoxic environments. In this article, the role of the extra-cellular matrix proteins and soluble mediators that acts as communicators between mesenchymal stem cells and cancer cells has been reviewed specifically for breast cancer, as it is the leading member of cancer malignancies. The comprehensive information may be beneficial in finding a new combinatorial cytotherapeutic strategy using MSCs by exploiting the cross-talk between mesenchymal stem cells and cancer cells for treating breast cancer.

scholarly journals Cyclo-oxygenase 2, a putative mediator of vessel remodeling, is expressed in the brain AVM vessels and associates with inflammation

2021 ◽  
Author(s):  
Sara Keränen ◽  
Santeri Suutarinen ◽  
Rahul Mallick ◽  
Johanna P. Laakkonen ◽  
Diana Guo ◽  
...  
Keyword(s):  
Quantitative Polymerase Chain Reaction ◽  
Rank Correlation ◽  
Inflammatory Cells ◽  
Vessel Remodeling ◽  
Brain Avm ◽  
Inflammatory Drugs ◽  
Polymerase Chain ◽  
The Brain ◽  
Cox2 Expression

Abstract Background Brain arteriovenous malformations (bAVM) may rupture causing disability or death. BAVM vessels are characterized by abnormally high flow that in general triggers expansive vessel remodeling mediated by cyclo-oxygenase-2 (COX2), the target of non-steroidal anti-inflammatory drugs. We investigated whether COX2 is expressed in bAVMs and whether it associates with inflammation and haemorrhage in these lesions. Methods Tissue was obtained from surgery of 139 bAVMs and 21 normal Circle of Willis samples. The samples were studied with immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR). Clinical data was collected from patient records. Results COX2 expression was found in 78% (109/139) of the bAVMs and localized to the vessels’ lumen or medial layer in 70% (95/135) of the bAVMs. Receptors for prostaglandin E2, a COX2-derived mediator of vascular remodeling, were found in the endothelial and smooth muscle cells and perivascular inflammatory cells of bAVMs. COX2 was expressed by infiltrating inflammatory cells and correlated with the extent of inflammation (r = .231, p = .007, Spearman rank correlation). COX2 expression did not associate with haemorrhage. Conclusion COX2 is induced in bAVMs, and possibly participates in the regulation of vessel wall remodelling and ongoing inflammation. Role of COX2 signalling in the pathobiology and clinical course of bAVMs merits further studies.

scholarly journals Involvement of HECTD1 in LPS-induced astrocyte activation via σ-1R-JNK/p38-FOXJ2 axis

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ying Tang ◽  
Mengchun Zhou ◽  
Rongrong Huang ◽  
Ling Shen ◽  
Li Yang ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Astrocyte Activation ◽  
Hect Domain ◽  
P38 Inhibitor ◽  
Ubiquitin Protein Ligase ◽  
Primary Mouse ◽  
Lps Treatment

Abstract Background Astrocytes participate in innate inflammatory responses within the mammalian central nervous system (CNS). HECT domain E3 ubiquitin protein ligase 1 (HECTD1) functions during microglial activation, suggesting a connection with neuroinflammation. However, the potential role of HECTD1 in astrocytes remains largely unknown. Results Here, we demonstrated that HECTD1 was upregulated in primary mouse astrocytes after 100 ng/ml lipopolysaccharide (LPS) treatment. Genetic knockdown of HECTD1 in vitro or astrocyte-specific knockdown of HECTD1 in vivo suppressed LPS-induced astrocyte activation, whereas overexpression of HECTD1 in vitro facilitated LPS-induced astrocyte activation. Mechanistically, we established that LPS activated σ-1R-JNK/p38 pathway, and σ-1R antagonist BD1047, JNK inhibitor SP600125, or p38 inhibitor SB203580 reversed LPS-induced expression of HECTD1, thus restored LPS-induced astrocyte activation. In addition, FOXJ2 functioned as a transcription factor of HECTD1, and pretreatment of primary mouse astrocytes with BD1047, SB203580, and SP600125 significantly inhibited LPS-mediated translocation of FOXJ2 into the nucleus. Conclusions Overall, our present findings suggest that HECTD1 participates in LPS-induced astrocyte activation by activation of σ-1R-JNK/p38-FOXJ2 pathway and provide a potential therapeutic strategy for neuroinflammation induced by LPS or any other neuroinflammatory disorders.

scholarly journals Transient Receptor Potential C 1/4/5 Is a Determinant of MTI-101 Induced Calcium Influx and Cell Death in Multiple Myeloma

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1490
Author(s):  
Osama M. Elzamzamy ◽  
Brandon E. Johnson ◽  
Wei-Chih Chen ◽  
Gangqing Hu ◽  
Reinhold Penner ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Death ◽  
Transient Receptor Potential ◽  
Cyclic Peptide ◽  
Calcium Influx ◽  
Acquired Resistance ◽  
Treatment Strategies ◽  
Prognostic Indicators ◽  
Causative Role

Multiple myeloma (MM) is a currently incurable hematologic cancer. Patients that initially respond to therapeutic intervention eventually relapse with drug resistant disease. Thus, novel treatment strategies are critically needed to improve patient outcomes. Our group has developed a novel cyclic peptide referred to as MTI-101 for the treatment of MM. We previously reported that acquired resistance to HYD-1, the linear form of MTI-101, correlated with the repression of genes involved in store operated Ca2+ entry (SOCE): PLCβ, SERCA, ITPR3, and TRPC1 expression. In this study, we sought to determine the role of TRPC1 heteromers in mediating MTI-101 induced cationic flux. Our data indicate that, consistent with the activation of TRPC heteromers, MTI-101 treatment induced Ca2+ and Na+ influx. However, replacing extracellular Na+ with NMDG did not reduce MTI-101-induced cell death. In contrast, decreasing extracellular Ca2+ reduced both MTI-101-induced Ca2+ influx as well as cell death. The causative role of TRPC heteromers was established by suppressing STIM1, TRPC1, TRPC4, or TRPC5 function both pharmacologically and by siRNA, resulting in a reduction in MTI-101-induced Ca2+ influx. Mechanistically, MTI-101 treatment induces trafficking of TRPC1 to the membrane and co-immunoprecipitation studies indicate that MTI-101 treatment induces a TRPC1-STIM1 complex. Moreover, treatment with calpeptin inhibited MTI-101-induced Ca2+ influx and cell death, indicating a role of calpain in the mechanism of MTI-101-induced cytotoxicity. Finally, components of the SOCE pathway were found to be poor prognostic indicators among MM patients, suggesting that this pathway is attractive for the treatment of MM.

scholarly journals New Insights on the Role of TRP Channels in Calcium Signalling and Immunomodulation: Review of Pathways and Implications for Clinical Practice

2021 ◽  
Author(s):  
Saied Froghi ◽  
Charlotte R. Grant ◽  
Radhika Tandon ◽  
Alberto Quaglia ◽  
Brian Davidson ◽  
...  
Keyword(s):  
Immune System ◽  
Calcium Release ◽  
Transient Receptor Potential ◽  
Trp Channels ◽  
Calcium Influx ◽  
Calcium Signalling ◽  
Chronic Fatigue ◽  
Diverse Range ◽  
Immune System Activation

AbstractCalcium is the most abundant mineral in the human body and is central to many physiological processes, including immune system activation and maintenance. Studies continue to reveal the intricacies of calcium signalling within the immune system. Perhaps the most well-understood mechanism of calcium influx into cells is store-operated calcium entry (SOCE), which occurs via calcium release-activated channels (CRACs). SOCE is central to the activation of immune system cells; however, more recent studies have demonstrated the crucial role of other calcium channels, including transient receptor potential (TRP) channels. In this review, we describe the expression and function of TRP channels within the immune system and outline associations with murine models of disease and human conditions. Therefore, highlighting the importance of TRP channels in disease and reviewing potential. The TRP channel family is significant, and its members have a continually growing number of cellular processes. Within the immune system, TRP channels are involved in a diverse range of functions including T and B cell receptor signalling and activation, antigen presentation by dendritic cells, neutrophil and macrophage bactericidal activity, and mast cell degranulation. Not surprisingly, these channels have been linked to many pathological conditions such as inflammatory bowel disease, chronic fatigue syndrome and myalgic encephalomyelitis, atherosclerosis, hypertension and atopy.

scholarly journals Functional Specificity of the Members of the Sos Family of Ras-GEF Activators: Novel Role of Sos2 in Control of Epidermal Stem Cell Homeostasis

Cancers ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 2152
Author(s):  
Fernando C. Baltanás ◽  
Cynthia Mucientes-Valdivieso ◽  
L. Francisco Lorenzo-Martín ◽  
Natalia Fernández-Parejo ◽  
Rósula García-Navas ◽  
...  
Keyword(s):  
Stem Cell ◽  
Layer Structure ◽  
3D Culture ◽  
Hair Follicles ◽  
Specific Cell ◽  
Epidermal Stem Cell ◽  
Primary Keratinocytes ◽  
Cell Homeostasis ◽  
Primary Mouse

Prior reports showed the critical requirement of Sos1 for epithelial carcinogenesis, but the specific functionalities of the homologous Sos1 and Sos2 GEFs in skin homeostasis and tumorigenesis remain unclear. Here, we characterize specific mechanistic roles played by Sos1 or Sos2 in primary mouse keratinocytes (a prevalent skin cell lineage) under different experimental conditions. Functional analyses of actively growing primary keratinocytes of relevant genotypes—WT, Sos1-KO, Sos2-KO, and Sos1/2-DKO—revealed a prevalent role of Sos1 regarding transcriptional regulation and control of RAS activation and mechanistic overlapping of Sos1 and Sos2 regarding cell proliferation and survival, with dominant contribution of Sos1 to the RAS-ERK axis and Sos2 to the RAS-PI3K/AKT axis. Sos1/2-DKO keratinocytes could not grow under 3D culture conditions, but single Sos1-KO and Sos2-KO keratinocytes were able to form pseudoepidermis structures that showed disorganized layer structure, reduced proliferation, and increased apoptosis in comparison with WT 3D cultures. Remarkably, analysis of the skin of both newborn and adult Sos2-KO mice uncovered a significant reduction of the population of stem cells located in hair follicles. These data confirm that Sos1 and Sos2 play specific, cell-autonomous functions in primary keratinocytes and reveal a novel, essential role of Sos2 in control of epidermal stem cell homeostasis.

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