Gene profiling techniques allow the assay of transcripts from organs, tissues, and cells with an unprecedented level of coverage. However, most of these approaches are still limited by the fact that organs and tissues are composed of multiple cell types that are each unique in their patterns of gene expression. To identify the transcriptome from a single cell type in a complex tissue, investigators have relied upon physical methods to separate cell types or in situ hybridization and immunohistochemistry. Here, we describe a strategy to rapidly and efficiently isolate ribosome-associated mRNA transcripts from any cell type in vivo. We have created a mouse line, called RiboTag, which carries an Rpl22 allele with a floxed wild-type C-terminal exon followed by an identical Cterminal exon that has three copies of the hemagglutinin (HA) epitope inserted before the stop codon. When the RiboTag mouse is crossed to a cell-type-specific Cre recombinase-expressing mouse, Cre recombinase activates the expression of epitopetagged ribosomal protein RPL22 HA , which is incorporated into actively translating polyribosomes. Immunoprecipitation of polysomes with a monoclonal antibody against HA yields ribosomeassociated mRNA transcripts from specific cell types. We demonstrate the application of this technique in brain using neuronspecific Cre recombinase-expressing mice and in testis using a Sertoli cell Cre recombinase-expressing mouse.gene profiling ͉ immunopreciptation ͉ mouse genetics
NF-kappa B signaling is required for the maintenance of normal B lymphocytes, whereas dysregulated NF-kappa B activation contributes to B cell lymphomas. The events that regulate NF-kappa B signaling in B lymphocytes are poorly defined. Here, we demonstrate that PKC-beta is specifically required for B cell receptor (BCR)-mediated NF-kappa B activation. B cells from protein kinase C-beta (PKC-beta)-deficient mice failed to recruit the I kappa B kinase (IKK) complex into lipid rafts, activate IKK, degrade I kappa B or up-regulate NF-kappa B-dependent survival signals. Inhibition of PKC-beta promoted cell death in B lymphomas characterized by exaggerated NF-kappa B activity. Together, these data define an essential role for PKC-beta in BCR survival signaling and highlight PKC-beta as a key therapeutic target for B-lineage malignancies.
Signaling through the Ag receptor is required for peripheral B lymphocyte maturation and maintenance. Defects in components of the B cell receptor (BCR) signalosome result in developmental blocks at the transition from immature (heat-stable Ag (HSA)high) to mature (HSAlow) B cells. Recent studies have subdivided the immature, or transitional, splenic B cells into two subsets, transitional 1 (T1) and transitional 2 (T2) cells. T1 and T2 cells express distinct surface markers and are located in distinct anatomic locations. In this report, we evaluated the BCR signaling capacity of T1 and T2 B cell subsets. In response to BCR engagement, T2 cells rapidly entered cell cycle and resisted cell death. In contrast, T1 cells did not proliferate and instead died after BCR stimulation. Correlating with these results, T2 cells robustly induced expression of the cell cycle regulator cyclin D2 and the antiapoptotic factors A1/Bfl-1 and Bcl-xL and exhibited activation of Akt. In contrast, T1 cells failed to up-regulate these markers. BCR stimulation of T2 cells also led to down-regulation of CD21 and CD24 (HSA) expression, resulting in a mature B cell phenotype. In addition, T2 cells from Bruton’s tyrosine kinase-deficient Xid mice failed to generate these proliferative and survival responses, suggesting a requirement for the BCR signalosome specifically at the T2 stage. Taken together, these data clearly demonstrate that T2 immature B cells comprise a discrete developmental subset that mediates BCR-dependent proliferative, prosurvival, and differentiation signals. Their distinct BCR-dependent responses suggest unique roles for T1 vs T2 cells in peripheral B cell selection.
A targeted disruption of the RI␣ isoform of protein kinase A (PKA) was created by using homologous recombination in embryonic stem cells. Unlike the other regulatory and catalytic subunits of PKA, RI␣ is the only isoform that is essential for early embryonic development. RI␣ homozygous mutant embryos fail to develop a functional heart tube at E8.5 and are resorbed at approximately E10.5. Mutant embryos show significant growth retardation and developmental delay compared with wild type littermates from E7.5 to E10.5. The anterior-posterior axis of RI␣ mutants is well developed, with a prominent head structure but a reduced trunk. PKA activity measurements reveal an increased basal PKA activity in these embryos. Brachyury mRNA expression in the primitive streak of RI␣ mutants is significantly reduced, consistent with later deficits in axial, paraxial, and lateral plate mesodermal derivatives. This defect in the production and migration of mesoderm can be completely rescued by crossing RI␣ mutants to mice carrying a targeted disruption in the C␣ catalytic subunit, demonstrating that unregulated PKA activity rather than a specific loss of RI␣ is responsible for the phenotype. Primary embryonic fibroblasts from RI␣ mutant embryos display an abnormal cytoskeleton and an altered ability to migrate in cell culture. Our results demonstrate that unregulated PKA activity negatively affects growth factor-mediated mesoderm formation during early mouse development.
AKAP5 (also referred to as AKAP150 in rodents and AKAP79 in humans) is a scaffolding protein that is highly expressed in neurons and targets a variety of signaling molecules to dendritic membranes. AKAP5 interacts with PKA holoenzymes containing RIIα or RIIβ as well as calcineurin (PP2B), PKC, calmodulin, adenylyl cyclase type V/VI, L-type calcium channels, and β-adrenergic receptors. AKAP5 has also been shown to interact with members of the MAGUK family of PSD-scaffolding proteins including PSD95 and SAP97 and target signaling molecules to receptors and ion channels in the postsynaptic density (PSD). We created two lines of AKAP5 mutant mice: a knockout of AKAP5 (KO) and a mutant that lacks the PKA binding domain of AKAP5 (D36). We find that PKA is delocalized in both the hippocampus and striatum of KO and D36 mice indicating that other neural AKAPs cannot compensate for the loss of PKA binding to AKAP5. In AKAP5 mutant mice, a significant fraction of PKA becomes localized to dendritic shafts and this correlates with increased binding to microtubule associated protein-2 (MAP2). Electrophysiological and behavioral analysis demonstrated more severe deficits in both synaptic plasticity and operant learning in the D36 mice compared with the complete KO animals. Our results indicate that the targeting of calcineurin or other binding partners of AKAP5 in the absence of the balancing kinase, PKA, leads to a disruption of synaptic plasticity and results in learning and memory defects.
The TCL1 protooncogene is overexpressed in many mature B cell lymphomas, especially from AIDS patients. To determine whether aberrant expression promotes B cell transformation, we generated a murine model in which a TCL1 transgene was overexpressed at similar levels in both B and T cells. Strikingly, transgenic mice developed Burkitt-like lymphoma (BLL) and diffuse large B cell lymphoma (DLBCL) with attendant Bcl-6 expression and mutated J H gene segments at a very high penetrance beginning at 4 months of age. In contrast, only one mouse developed a T cell malignancy at 15 months, consistent with a longer latency for transformation of T cells by TCL1. Activation of premalignant splenic B cells by means of B cell antigen receptor (BCR) engagement resulted in significantly increased proliferation and augmented AKT-dependent signaling, including increased S6 ribosomal protein phosphorylation. Transgenic spleen cells also survived longer than wild-type spleen cells in long-term culture. Together these data demonstrate that TCL1 is a powerful oncogene that, when overexpressed in both B and T cells, predominantly yields mature B cell lymphomas.T he TCL1 (T cell leukemia 1) protooncogene is expressed in CD3 Ϫ CD4 Ϫ CD8 Ϫ precursor T cells and is extinguished at the CD4 ϩ CD8 ϩ stage of thymocyte development (1). In B cells, TCL1 is first expressed in pro-B cells and remains high in naive mantle zone B cells of peripheral lymphoid tissues (1-4). Downregulation of TCL1 expression in follicle center centroblasts and centrocytes is followed by gene extinction in post-germinal center (GC) memory B cells and plasma cells (4, 5).Continued high-level TCL1 expression, because of chromosomal rearrangements, was implicated in mature peripheral T cell malignancies (6, 7). Polyclonal and oligoclonal T cell expansions preceded clonal outgrowth by many years, suggesting that additional lesions were required for transformation (8,9). Supporting this tumorigenic mechanism, transgenic mice expressing TCL1-familymember proteins exclusively in T cells developed polyclonal T cell expansions before the evolution of clonal malignancies at 15 to 20 months (10, 11). Overexpression of TCL1, or MTCP1 (mature T cell proliferation 1), in mouse T cells did not affect B cell development or produce B cell lymphomas. These findings indicate that aberrant expression of TCL1 or MTCP1 in T cells perturbs T cell homeostasis through cell autonomous pathways without inducing premalignant or malignant changes in bystander B cells.About 15% of AIDS patients develop aggressive B cell nonHodgkin lymphoma (AIDS-NHL) (12, 13). Most AIDS-NHL originate from GC or post-GC B cells, but the early events leading to AIDS-NHL remain poorly defined (13,14). Diffuse large B cell lymphoma (DLBCL) is the most prevalent type of AIDS-NHL, and these tumors generally lack consistent genetic and͞or viral tumorpromoting alterations. Recently, abundant TCL1 expression was shown in a high percentage of AIDS-NHL of post-GC origin (3,4). This discovery led us to postulate that TCL1 dy...
Splenic peripheral B-cell development and the events regulating this functionally significant but relatively poorly defined developmental process have become a major focus in recent studies in B-cell immunology. Following the exit from the bone marrow, peripheral B cells develop through transitional type 1 (T1) and transitional type 2 (T2) B-cell stages. Emerging data suggest that the T2 subset is the immediate precursor of the mature B-cell populations present in the spleen. In this review, we first elaborate on the evidence describing the unique properties of CD21hiCD24hiCD23hiIgMhiIgDhi T2 B cells. T2 cells uniquely activate a proliferative, pro-survival, and differentiation program in response to B-cell antigen receptor (BCR) engagement. The potential mechanisms leading to the differential BCR responsiveness of T1 versus T2 B cells are discussed. We also review evidence that distinguishes key BCR-dependent signaling pathways operative in T2 and mature B cells. These signaling cascades include a protein kinase Cbeta (PKCbeta)-dependent cell-survival pathway and a second PKCbeta-independent pathway essential for BCR-driven differentiation. Finally, we discuss recent intriguing results suggesting that the type of signal(s) encountered by T2 cells leads to their differential maturation toward the follicular mature versus marginal zone mature B-cell populations. These combined observations suggest important implications with regard to B-cell selection and tolerance, potential novel therapeutic targets for B-cell lymphomas, and how the intricate balance of commensal organisms and other microenvironmental signals interact to promote the generation of 'innate-like' versus adaptive effector B-cell populations.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.