Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations.
(2008)
Journal - Biological procedures online (Canada )
Abstract :
B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties and limiting cell numbers. We therefore employed mini-scale protocols to assess lipid signaling, particularly that of diacylglycerol and inositol trisphosphate, with as few as 0.5x10(6) purified early (T1) and late (T2) transitional B cells. Additionally, utilizing flow cytometric techniques, we determined levels of phosphatidylinositol bisphosphate and calcium mobilization in T1 and T2 cells, as well as mature follicular and marginal zone B cells using less than 1x10(6 )primary B cells. Thus, these biochemical and flow cytometric methodologies can be used to analyse signal-induced changes in phosphatidylinositol bisphosphate levels, diacylglycerol and inositol triphosphate production and calcium in each B cell population.
B-cell antigen receptor activates transcription factors NFAT (nuclear factor of activated T-cells) and NF-kappaB (nuclear factor kappaB) via a mechanism that involves diacylglycerol.
(2004)
Journal - Biochemical Society transactions (England )
Abstract :
Engagement of the B-cell antigen receptor (BCR) induces the activation of various transcription factors, including NFAT (nuclear factor of activated T-cells) and NF-kappaB (nuclear factor kappaB), which participate in long-term biological responses such as proliferation, survival and differentiation of B-lymphocytes. We addressed the biochemical basis of this process using the DT40 chicken B-cell lymphoma. We discovered that Bruton's tyrosine kinase (BTK) and phospholipase C-gamma2 (PLC-gamma2) are required to activate NFAT and NF-kappaB, and to produce the lipid second messenger diacylglycerol in response to BCR cross-linking. Therefore the functional integrity of the BTK/PLC-gamma2/diacylglycerol signalling axis is crucial for BCR-directed activation of both transcription factors NFAT and NF-kappaB.
| ISSN : | 0300-5127 |
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| Mesh Heading : | Animals Cell Line Chickens Chromatography, Thin Layer DNA-Binding Proteins Diglycerides NF-kappa B NFATC Transcription Factors Protein-Tyrosine Kinases Receptors, Antigen, B-Cell Transcription Factors metabolism |
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| Mesh Heading Relevant : | Nuclear Proteins metabolism metabolism metabolism metabolism metabolism |
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B cell receptor directs the activation of NFAT and NF-kappaB via distinct molecular mechanisms.
(2003)
Journal - Experimental cell research (United States )
Abstract :
BCR engagement initiates intracellular calcium ([Ca2+]i) mobilization which is critical for the activation of multiple transcription factors including NF-kappaB and NFAT. Previously, we showed that Bruton's tyrosine kinase (BTK)-deficient (btk-/-) B cells, which display a modestly reduced calcium response to BCR crosslinking, do not activate NF-kappaB. Here we show that BTK is also essential for the activation of NFAT following BCR engagement. Pharmacological mobilization of [Ca2+]i in BTK-deficient DT40 B cells (DT40.BTK) does not rescue BCR directed activation of NF-kappaB and only partially that of NFAT, suggesting existence of additional BTK-signaling pathways in this process. Therefore, we investigated a requirement for BTK in the production of diacylglycerol (DAG). We found that DT40.BTK B cells do not produce DAG in response to BCR engagement. Pharmacological inhibition of PKC isozymes and Ras revealed that the BCR-induced activation of NF-kappaB requires conventional PKCbeta, whereas that of NFAT may involve non-conventional PKCdelta and Ras pathways. Consistent with an essential role for BTK in the regulation of NFAT, B cells from btk-/- mice display defective expression of CD5, a gene under the control of NFAT. Together, these results suggest that BCR employs distinct BTK-dependent molecular mechanisms to regulate the activation of NF-kappaB versus NFAT.
| ISSN : | 0014-4827 |
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| Mesh Heading : | Animals Antigens, CD5 B-Lymphocytes Calcium Signaling Cell Line Chickens DNA-Binding Proteins Diglycerides Enzyme Inhibitors Isoenzymes Lymphocyte Activation Mice Mice, Inbred C57BL Mice, Knockout NF-kappa B NFATC Transcription Factors Phospholipase C gamma Protein Kinase C Protein-Tyrosine Kinases Receptors, Antigen, B-Cell Transcription Factors Type C Phospholipases ras Proteins biosynthesis genetics drug effects metabolism drug effects physiology drug effects biosynthesis pharmacology antagonists & inhibitors metabolism drug effects drug effects antagonists & inhibitors metabolism genetics drug effects drug effects antagonists & inhibitors metabolism antagonists & inhibitors metabolism |
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| Mesh Heading Relevant : | Nuclear Proteins metabolism physiology metabolism deficiency metabolism metabolism |
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Micro-scale flow cytometry-based and biochemical analysis of lipid signaling in primary B cell subpopulations
(2007)
Journal - Biological Procedures Online
Abstract :
B cell subpopulations in the spleen have been extensively characterized phenotypically; however, biochemical properties of these cell populations following B cell antigen receptor engagement have not been fully determined due to technical difficulties and limiting cell numbers. We therefore employed mini-scale protocols to assess lipid signaling, particularly that of diacylglycerol and inositol trisphosphate, with as few as 0.5x106 purified early (T1) and late (T2) transitional B cells. Additionally, utilizing flow cytometric techniques, we determined levels of phosphatidylinositol bisphosphate and calcium mobilization in T1 and T2 cells, as well as mature follicular and marginal zone B cells using less than 1x106 primary B cells. Thus, these biochemical and flow cytometric methodologies can be used to analyse signal-induced changes in phosphatidylinositol bisphosphate levels, diacylglycerol and inositol triphosphate production and calcium in each B cell population.
| Keywords : | receptors, antigen, b-cell,immunomagnetic separation |
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