Reuben Tooze

Blimp1 Defines a Progenitor Population that Governs Cellular Input to the Sebaceous Gland

Epidermal lineage commitment occurs when multipotent stem cells are specified to three lineages: the epidermis, the hair follicle, and the sebaceous gland (SG). How and when a lineage becomes specified remains unknown. Here, we report the existence of a population of unipotent progenitor cells that reside in the SG and express the transcriptional repressor Blimp1. Using cell-culture studies and genetic lineage tracing, we demonstrate that Blimp1-expressing cells are upstream from other cells of the SG lineage. Blimp1 appears to govern cellular input into the gland since its loss leads to elevated c-myc expression, augmented cell proliferation, and SG hyperplasia. Finally, BrdU labeling experiments demonstrate that the SG defects associated with loss of Blimp1 lead to enhanced bulge stem cell activity, suggesting that when normal SG homeostasis is perturbed, multipotent stem cells in the bulge can be mobilized to correct this imbalance.

CD19 as a Membrane-Anchored Adaptor Protein of B Lymphocytes: Costimulation of Lipid and Protein Kinases by Recruitment of Vav

CD19 is a coreceptor that amplifies signaling by membrane immunoglobulin (mIg) to promote responses of the B lymphocyte to T-dependent antigens. Vav is a guanine nucleotide exchange factor for the Rho, Rac, Cdc42 family of small GTPases. We found that coligating mIg and CD19 causes a synergistic increase in the tyrosine phosphorylation of CD19. Phosphorylated tyrosine-391 of CD19 binds Vav to mediate a sustained increase in intracellular Ca2+ concentration. This response correlates with activation by the CD19-Vav complex of phosphatidylinositol 4-phosphate 5-kinase for the synthesis of phosphatidylinositol 4,5-bisphosphate. Interaction of CD19 with Vav also mediates the synergistic activation of the mitogen-activated protein kinase JNK. Therefore, CD19 is a membrane adaptor protein that recruits Vav for the activation of lipid and protein kinases.

Signal transduction through Vav-2 participates in humoral immune responses and B cell maturation.

B and T lymphocytes develop normally in mice lacking the guanine nucleotide exchange factor Vav-2. However, the immune responses to type II thymus-independent antigen as well as the primary response to thymus-dependent (TD) antigen are defective. Vav-2–deficient mice are also defective in their ability to switch immunoglobulin class, form germinal centers and generate secondary immune responses to TD antigens. Mice lacking both Vav-1 and Vav-2 contain reduced numbers of B lymphocytes and display a maturational block in the development of mature B cells. B cells from Vav-1−/−Vav-2−/− mice respond poorly to antigen receptor triggering, both in terms of proliferation and calcium release. These studies show the importance of Vav-2 in humoral immune responses and B cell maturation.

Reprogramming Primordial Germ Cells into Pluripotent Stem Cells

Background Specification of primordial germ cells (PGCs) results in the conversion of pluripotent epiblast cells into monopotent germ cell lineage. Blimp1/Prmt5 complex plays a critical role in the specification and maintenance of the early germ cell lineage. However, PGCs can be induced to dedifferentiate back to a pluripotent state as embryonic germ (EG) cells when exposed to exogenous signaling molecules, FGF-2, LIF and SCF. Methodology and Principal Findings Here we show that Trichostatin A (TSA), an inhibitor of histone deacetylases, is a highly potent agent that can replace FGF-2 to induce dedifferentiation of PGCs into EG cells. A key early event during dedifferentiation of PGCs in response to FGF-2 or TSA is the down-regulation of Blimp1, which reverses and apparently relieves the cell fate restriction imposed by it. Notably, the targets of Blimp1, which include c-Myc and Klf-4, which represent two of the key factors known to promote reprogramming of somatic cells to pluripotent state, are up-regulated. We also found early activation of the LIF/Stat-3 signaling pathway with the translocation of Stat-3 into the nucleus. By contrast, while Prmt5 is retained in EG cells, it translocates from the nucleus to the cytoplasm where it probably has an independent role in regulating pluripotency. Conclusions/Significance We propose that dedifferentiation of PGCs into EG cells may provide significant mechanistic insights on early events associated with reprogramming of committed cells to a pluripotent state.

Counterregulation by the Coreceptors CD19 and CD22 of MAP Kinase Activation by Membrane Immunoglobulin

The signaling pathways linked to membrane immunoglobulin (mIg) that are regulated by the coreceptors CD19 and CD22 are not known. The mitogen-activated protein (MAP) kinases ERK2, JNK, and p38 couple extracellular signals to transcriptional responses. The capacity of mIg to activate these MAP kinases is synergistically amplified by coligating CD19, and this effect requires that CD19 be juxtaposed to mIg. CD22 suppresses MAP kinase activation when cross-linked to mIg alone or to the coligated complex of mIg and CD19. Separate ligation and sequestration of CD22 from mIg enhances MAP kinase activation, probably reflecting release of mIg from constitutive down-regulation. Thus, CD19 and CD22 have counterregulatory effects on MAP kinase activation by mIg, which are dependent on their proximity to the antigen receptor.

CO-RECEPTORS OF B LYMPHOCYTES

The past year has seen advances in our understanding of accessory membrane proteins that modulate the B cell response to antigen-receptor stimulation. The generation of complement receptor deficient mice has reinforced our appreciation of the importance of complement receptors in the B cell response to antigen. The association of inositol polyphosphate 5-phosphatase with FcgammaRIIB suggests another mechanism, in addition to recruitment of the phosphotyrosine phosphatase SHP-1, by which secreted immunoglobulin can limit further response to antigen. The in vivo function of CD22 in regulating the threshold of antigen-receptor signalling has been shown using CD22-deficient mice. Lastly, B cell receptor signalling in the B-1 subset of B lymphocytes has been demonstrated to be negatively regulated by CD5.

Vav -promoter regulated oncogenic fusion protein NPM-ALK in transgenic mice causes B-cell lymphomas with hyperactive Jun kinase

Anaplastic large-cell lymphoma is associated with a chromosomal translocation generating an oncogenic fusion protein: the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). We have generated several independent lines of human NPM-ALK transgenic mice using the haematopoietic cell-specific Vav promoter. Lymphomas develop in two transgenic lines in which the Vav promoter regulates NPM-ALK expression. The transgenic line with higher copy number displays an early-onset phenotype in which all mice succumb to aggressive lymph node tumours with intestinal involvement, whereas the second line displays late-onset tumour development in the spleen and/or liver. Lymphomas from both lines are phenotypically distinct and display B-lineage characteristics with aberrant coexpression of myeloid markers. The NPM-ALK kinase is active in primary tumour tissue and forms a multimeric complex with tyrosine-phosphorylated proteins, that is, Shc. Jun and ERK kinase activities in tumours are elevated by up to 30-fold and fivefold, respectively, in comparison with sIgM-stimulated primary B cells. The new transgenic models provide a system for investigating the oncogenic events mediated by NPM-ALK in situ and a physiologically relevant context for developing tyrosine kinase inhibitor therapies of potential use in the clinic.

Development of T-leukaemias in CD45 tyrosine phosphatase-deficient mutant lck mice

The CD45 tyrosine phosphatase lowers T‐cell antigen receptor signalling thresholds by its positive actions on p56lck tyrosine kinase function. We now show that mice expressing active lckF505 at non‐oncogenic levels develop aggressive thymic lymphomas on a CD45−/− background. CD45 suppresses the tumorigenic potential of the kinase by dephosphorylation of the Tyr394 autophosphorylation site. In CD45−/− thymocytes the kinase is switched to a hyperactive oncogenic state, resulting in increased resistance to apoptosis. Transformation occurs in early CD4−CD8− thymocytes during the process of TCR‐β chain rearrangement by a recombinase‐independent mechanism. Our findings represent the first example in which a tyrosine phosphatase in situ prevents the oncogenic actions of a Src family tyrosine kinase.

BLIMP-1 is a target of cellular stress and downstream of the unfolded protein response.

B lymphocyte‐induced maturation protein‐1 (BLIMP‐1) acts during differentiation of B cells and monocytes, but was originally identified as a repressor of the IFN‐β promoter induced during viral infection. A central regulator of the intracellular response to viral infection is the interferon‐inducible double‐stranded RNA activated protein kinase (PKR). PKR belongs to a family of kinases that phosphorylate the eukaryotic translation initiation factor 2‐alpha (eIF2α) and activate common downstream signaling pathways. PERK, the endoplasmic reticulum resident PKR‐homologue, is activated during the unfolded protein response (UPR), a stress response involved in both macrophage activation and terminal B‐cell differentiation. This suggested that BLIMP‐1 might be a target of stress responses involving PERK. We demonstrate that BLIMP‐1 is rapidly up‐regulated during the UPR in human myeloid and B‐cell lines. This response is conserved in murine B‐cells and murine macrophages, in which mimics of physiological stress and classical activation stimuli also induce Blimp‐1. During the UPR, BLIMP‐1 mRNA is induced at the level of transcription. This response is dependent on an intact PERK signaling pathway, independent of new protein synthesis and blocked by an inhibitor of NF‐κB. Our data provide evidence for a novel pathway linking cellular stress to BLIMP‐1, a regulator of differentiation in macrophages and B cells.

Whole genome expression profiling based on paraffin embedded tissue can be used to classify diffuse large B‐cell lymphoma and predict clinical outcome

This study tested the validity of whole‐genome expression profiling (GEP) using RNA from formalin‐fixed, paraffin‐embedded (FFPE) tissue to sub‐classify Diffuse Large B‐cell Lymphoma (DLBCL), in a population based cohort of 172 patients. GEP was performed using Illumina Whole Genome cDNA‐mediated Annealing, Selection, extension & Ligation, and tumours were classified into germinal centre (GCB), activated B‐cell (ABC) and Type‐III subtypes. The method was highly reproducible and reliably classified cell lines of known phenotype. GCB and ABC subtypes were each characterized by unique gene expression signatures consistent with previously published data. A significant relationship between subtype and survival was observed, with ABC having the worst clinical outcome and in a multivariate survival model only age and GEP class remained significant. This effect was not seen when tumours were classified by immunohistochemistry. There was a significant association between age and subtype (mean ages ABC – 72·8 years, GC – 68·4 years, Type‐III – 64·5 years). Older patients with ABC subtype were also over‐represented in patients who died soon after diagnosis. The relationship between prognosis and subtype improved when only patients assigned to the three categories with the highest level of confidence were analysed. This study demonstrates that GEP‐based classification of DLBCL can be applied to RNA extracted from routine FFPE samples and has potential for use in stratified medicine trials and clinical practice.