Critical roles for Rac1 and Rac2 GTPases in B cell development and signaling

The Rac1 guanosine triphosphatase (GTPase) has been implicated in multiple cellular functions, including actin dynamics, proliferation, apoptosis, adhesion, and migration resulting from signaling by multiple receptors, including the B cell antigen receptor (BCR). We used conditional gene targeting to generate mice with specific Rac1 deficiency in the B cell lineage. In the absence of both Rac1 and the highly related Rac2, B cell development was almost completely blocked. Both GTPases were required to transduce BCR signals leading to proliferation, survival and up-regulation of BAFF-R, a receptor for BAFF, a key survival molecule required for B cell development and maintenance.     

Requirement for B cell linker protein (BLNK) in B cell development

Linker proteins function as molecular scaffolds to localize enzymes with substrates. In B cells, B cell linker protein (BLNK) links the B cell receptor (BCR)-activated Syk kinase to the phosphoinositide and mitogen-activated kinase pathways. To examine the in vivo role of BLNK, mice deficient in BLNK were generated. B cell development in BLNK-/- mice was blocked at the transition from B220+CD43+ progenitor B to B220+CD43- precursor B cells. Only a small percentage of immunoglobulin M++ (IgM++), but not mature IgMloIgDhi, B cells were detected in the periphery. Hence, BLNK is an essential component of BCR signaling pathways and is required to promote B cell development.     

B cell antigen receptor signaling: roles in cell development and disease

Signals propagated through the B cell antigen receptor (BCR) are vital for the development and survival of B lymphocytes in both the bone marrow and the periphery. These signals not only guide maturation and activation but also affect the removal of potentially self-reactive B lymphocytes. Interestingly, these signals are known to be either ligand-independent ("tonic" signals) or induced by ligand (antigen) binding to the BCR. We focus on the problems that occur in B cell development due to defects in signals emanating from the BCR. In addition, we present the B Cell Antigen Receptor Pathway, an STKE Connections Map that illustrates the events involved in B cell signaling.     

Variability and robustness in T cell activation from regulated heterogeneity in protein levels

In T cells, the stochasticity of protein expression could contribute to the useful diversification of biological functions within a clonal population or interfere with accurate antigen discrimination. Combining computer modeling and single-cell measurements, we examined how endogenous variation in the expression levels of signaling proteins might affect antigen responsiveness during T cell activation. We found that the CD8 co-receptor fine-tunes activation thresholds, whereas the soluble hematopoietic phosphatase 1 (SHP-1) digitally regulates cell responsiveness. Stochastic variation in the expression of these proteins generates substantial diversity of activation within a clonal population of T cells, but co-regulation of CD8 and SHP-1 levels ultimately limits this very diversity. These findings reveal how eukaryotic cells can draw on regulated variation in gene expression to achieve phenotypic variability in a controlled manner.     

Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors

Phospholipase C gamma 1 (PLC gamma 1) and p21ras guanosine triphosphatase (GTPase) activating protein (GAP) bind to and are phosphorylated by activated growth factor receptors. Both PLC gamma 1 and GAP contain two adjacent copies of the noncatalytic Src homology 2 (SH2) domain. The SH2 domains of PLC gamma 1 synthesized individually in bacteria formed high affinity complexes with the epidermal growth factor (EGF)- or platelet derived growth factor (PDGF)-receptors in cell lysates, and bound synergistically to activated receptors when expressed together as one bacterial protein. In vitro complex formation was dependent on prior growth factor stimulation and was competed by intracellular PLC gamma 1. Similar results were obtained for binding of GAP SH2 domains to the PDGF-receptor. The isolated SH2 domains of other signaling proteins, such as p60src and Crk, also bound activated PDGF-receptors in vitro. SH2 domains, therefore, provide a common mechanism by which enzymatically diverse regulatory proteins can physically associate with the same activated receptors and thereby couple growth factor stimulation to intracellular signal transduction pathways.     

Imaging of germinal center selection events during affinity maturation

The germinal center (GC) is an important site for the generation and selection of B cells bearing high-affinity antibodies, yet GC cell migration and interaction dynamics have not been directly observed. Using two-photon microscopy of mouse lymph nodes, we revealed that GC B cells are highly motile and extend long cell processes. They transited between GC dark and light zones and divided in both regions, although these B cells resided for only several hours in the light zone where antigen is displayed. GC B cells formed few stable contacts with GC T cells despite frequent encounters, and T cells were seen to carry dead B cell blebs. On the basis of these observations, we propose a model in which competition for T cell help plays a more dominant role in the selection of GC B cells than previously appreciated.     

TCR-induced transmembrane signaling by peptide/MHC class II via associated Ig-alpha/beta dimers

Previous findings suggest that during cognate T cell-B cell interactions, major histocompatability complex (MHC) class II molecules transduce signals, leading to Src-family kinase activation, Ca2+ mobilization, and proliferation. Here, we show that antigen stimulation of resting B cells induces MHC class II molecules to associate with Immunoglobulin (Ig)-alpha/Ig-beta (CD79a/CD79b) heterodimers, which function as signal transducers upon MHC class II aggregation by the T cell receptor (TCR). The B cell receptor (BCR) and MHC class II/Ig-alpha/Ig-beta are distinct complexes, yet class II-associated Ig-alpha/beta appears to be derived from BCR. Hence, Ig-alpha/beta are used in a sequential fashion for transduction of antigen and cognate T cell help signals.     

Live-cell imaging of enzyme-substrate interaction reveals spatial regulation of PTP1B

Endoplasmic reticulum-localized protein-tyrosine phosphatase PTP1B terminates growth factor signal transduction by dephosphorylation of receptor tyrosine kinases (RTKs). But how PTP1B allows for RTK signaling in the cytoplasm is unclear. In order to test whether PTP1B activity is spatially regulated, we developed a method based on F?rster resonant energy transfer for imaging enzyme-substrate (ES) intermediates in live cells. We observed the establishment of a steady-state ES gradient across the cell. This gradient exhibited robustness to cell-to-cell variability, growth factor activation, and RTK localization, which demonstrated spatial regulation of PTP1B activity. Such regulation may be important for generating distinct cellular environments that permit RTK signal transduction and that mediate its eventual termination.     

Mcl-1 is essential for germinal center formation and B cell memory

Lymphocyte survival during immune responses is controlled by the relative expression of pro- and anti-apoptotic molecules, regulating the magnitude, quality, and duration of the response. We investigated the consequences of deleting genes encoding the anti-apoptotic molecules Mcl1 and Bcl2l1 (Bcl-x(L)) from B cells using an inducible system synchronized with expression of activation-induced cytidine deaminase (Aicda) after immunization. This revealed Mcl1 and not Bcl2l1 to be indispensable for the formation and persistence of germinal centers (GCs). Limiting Mcl1 expression reduced the magnitude of the GC response with an equivalent, but not greater, effect on memory B cell formation and no effect on persistence. Our results identify Mcl1 as the main anti-apoptotic regulator of activated B cell survival and suggest distinct mechanisms controlling survival of GC and memory B cells.     

Role of adaptor TRIF in the MyD88-independent toll-like receptor signaling pathway

Stimulation of Toll-like receptors (TLRs) triggers activation of a common MyD88-dependent signaling pathway as well as a MyD88-independent pathway that is unique to TLR3 and TLR4 signaling pathways leading to interferon (IFN)-beta production. Here we disrupted the gene encoding a Toll/IL-1 receptor (TIR) domain-containing adaptor, TRIF. TRIF-deficient mice were defective in both TLR3- and TLR4-mediated expression of IFN-beta and activation of IRF-3. Furthermore, inflammatory cytokine production in response to the TLR4 ligand, but not to other TLR ligands, was severely impaired in TRIF-deficient macrophages. Mice deficient in both MyD88 and TRIF showed complete loss of nuclear factor kappa B activation in response to TLR4 stimulation. These findings demonstrate that TRIF is essential for TLR3- and TLR4-mediated signaling pathways facilitating mammalian antiviral host defense.     

Ca2+ entry through plasma membrane IP3 receptors

Inositol 1,4,5-trisphosphate receptors (IP3Rs) release calcium ions, Ca2+, from intracellular stores, but their roles in mediating Ca2+ entry are unclear. IP3 stimulated opening of very few (1.9 +/- 0.2 per cell) Ca2+-permeable channels in whole-cell patch-clamp recording of DT40 chicken or mouse B cells. Activation of the B cell receptor (BCR) in perforated-patch recordings evoked the same response. IP3 failed to stimulate intracellular or plasma membrane (PM) channels in cells lacking IP3R. Expression of IP3R restored both responses. Mutations within the pore affected the conductances of IP3-activated PM and intracellular channels similarly. An impermeant pore mutant abolished BCR-evoked Ca2+ signals, and PM IP3Rs were undetectable. After introduction of an alpha-bungarotoxin binding site near the pore, PM IP3Rs were modulated by extracellular alpha-bungarotoxin. IP(3)Rs are unusual among endoplasmic reticulum proteins in being also functionally expressed at the PM, where very few IP3Rs contribute substantially to the Ca2+ entry evoked by the BCR.     

Expression of BMP Receptors in Porcine Granulosa Cells （GCs） and Their Regulation by Luteinizing Hormone （LH）

Bone morphogenetic proteins (BMPs) play critical roles in follicle growth and development. BMPs initiate signaling by assembling BMP receptors and activating Smads, which in turn alter expression of target enes. The mechanism underlying the regulation of the expression of BMP receptors and Smads during follicle development in pigs is still unknown. By quantitative real-time PCR, the mRNA expression of BMP receptors and Smads in granulosa cells (GC) was investigated.Cells were obtained from small porcine follicles (SF; <3 mm diameter) and dominant follicles (DF; >6 mm diameter); ActRIA and BMPR2 mRNA levels in DF were significantly higher (P<0.05) than that in SF, whereas BMPRIB, Smad4 and Smad7 expression tended to decrease (P>0.05). The levels of BMPRIA, ActR2, Smadl, Smad5, and Smad8 mRNA did not differ between DFs and SFs. To investigate the effect of LH on BMP receptors in GC, cells obtained from porcine DFs were cultured in medium supplemented with different doses of luteinizing hormone (LH). High doses of LH (4 IU mL-1)significantly decreased the concentration of estradiol (E2) and progesterone (P4) in medium and the expression of Cyp19a1 (P450 aromatase, P450arom) and Cypl lal (cholesterol side-chain cleavage enzyme P450, P450scc), while significantly increased viable cell numbers and up-regulated expression of cyclin dependent kinase-4 (CDK4) and cyclin D2. However,LH had no effect on the expression of BMP receptor genes. Thus, the present study indicates that the expression of members of the BMP signaling pathway in porcine GC is regulated during follicle development and the expression of BMP receptors are not regulated by LH in porcine GCs.     

A network of control mediated by regulator of calcium/calmodulin-dependent signaling

Calmodulin (CaM) is a major effector for the intracellular actions of Ca2+ in nearly all cell types. We identified a CaM-binding protein, designated regulator of calmodulin signaling (RCS). G protein-coupled receptor (GPCR)-dependent activation of protein kinase A (PKA) led to phosphorylation of RCS at Ser55 and increased its binding to CaM. Phospho-RCS acted as a competitive inhibitor of CaM-dependent enzymes, including protein phosphatase 2B (PP2B, also called calcineurin). Increasing RCS phosphorylation blocked GPCR- and PP2B-mediated suppression of L-type Ca2+ currents in striatal neurons. Conversely, genetic deletion of RCS significantly increased this modulation. Through a molecular mechanism that amplifies GPCR- and PKA-mediated signaling and attenuates GPCR- and PP2B-mediated signaling, RCS synergistically increases the phosphorylation of key proteins whose phosphorylation is regulated by PKA and PP2B.     

A distinct signaling pathway used by the IgG-containing B cell antigen receptor

The immunoglobulin G (IgG)-containing B lymphocyte antigen receptor (IgG-BCR) transmits a signal distinct from that of IgM-BCR or IgD-BCR, although all three use the same signal-transducing component, Igalpha/Igbeta. Here we demonstrate that the inhibitory coreceptor CD22 down-modulates signaling through IgM-BCR and IgD-BCR, but not that through IgG-BCR, because of the IgG cytoplasmic tail, which prevents CD22 phosphorylation. These results suggest that the cytoplasmic tail of IgG specifically enhances IgG-BCR signaling by preventing CD22-mediated signal inhibition. Enhanced signaling through IgG-BCR may be involved in efficient IgG production, which is crucial for immunity to pathogens.     

胃癌SOCS-1基因异常甲基化及其意义

目的：探讨SOCS-1基因在胃癌和癌旁组织中的表达及其启动子甲基化状态与胃癌发生、发展和转移等的关系。方法：采集45例胃癌病人的肿瘤标本、18例癌旁胃粘膜组织以及10例正常胃粘膜组织,运用甲基化特异性PCR反应研究胃癌组织中SOCS-1基因CpG岛甲基化状态,同时运用实时定量PCR分析SOCS-1基因的表达。结果：45例胃癌标本中有21例（46．7％）SOCS-1基因呈CpG岛甲基化,癌旁组织中为2例（11．1％）,而10例正常胃粘膜组织中则未发现SOCS-1基因CpG岛甲基化;SOCS-1基因CpG岛甲基化组的SOCS-1基因表达量与无SOCS-1基因CpG岛甲基化组相比,其基因相对表达量明显减少（P〈0．05）,表明SOCS-1基因CpG岛甲基化可抑制SOCS-1基因表达。与病人临床病理特征相结合比较,发现SOCS-1基因CpG岛甲基化与年龄、性别无关,与肿瘤分化程度及TNM分期等因素有关。结论：在胃癌中存在SOCS-1基因CpG岛甲基化,且由于CpG岛甲基化而促使基因表达抑制。SOCS-1基因CpG岛甲基化在胃癌的发生、发展中可能具有一定的意义。     

OSBP is a cholesterol-regulated scaffolding protein in control of ERK 1/2 activation

Oxysterol-binding protein (OSBP) is the founding member of a family of sterol-binding proteins implicated in vesicle transport, lipid metabolism, and signal transduction. Here, OSBP was found to function as a cholesterol-binding scaffolding protein coordinating the activity of two phosphatases to control the extracellular signal-regulated kinase (ERK) signaling pathway. Cytosolic OSBP formed a approximately 440-kilodalton oligomer with a member of the PTPPBS family of tyrosine phosphatases, the serine/threonine phosphatase PP2A, and cholesterol. This oligomer had dual specific phosphatase activity for phosphorylated ERK (pERK). When cell cholesterol was lowered, the oligomer disassembled and the level of pERK rose. The oligomer also disassembled when exposed to oxysterols. Increasing the amount of OSBP oligomer rendered cells resistant to the effects of cholesterol depletion and decreased the basal level of pERK. Thus, cholesterol functions through its interaction with OSBP outside of membranes to regulate the assembly of an oligomeric phosphatase that controls a key signaling pathway in the cell.     

Mitogenic influence of human R-spondin1 on the intestinal epithelium

Several described growth factors influence the proliferation and regeneration of the intestinal epithelium. Using a transgenic mouse model, we identified a human gene, R-spondin1, with potent and specific proliferative effects on intestinal crypt cells. Human R-spondin1 (hRSpo1) is a thrombospondin domain-containing protein expressed in enteroendocrine cells as well as in epithelial cells in various tissues. Upon injection into mice, the protein induced rapid onset of crypt cell proliferation involving beta-catenin stabilization, possibly by a process that is distinct from the canonical Wnt-mediated signaling pathway. The protein also displayed efficacy in a model of chemotherapy-induced intestinal mucositis and may have therapeutic application in gastrointestinal diseases.     

斑马鱼中Wnt信号通过调控侧线基板形成影响侧线发育机制

鱼类侧线系统源自胚胎期的侧线基板,侧线基板的特化受到多种信号的综合作用。利用转基因斑马鱼hsp:wnt8a-egfp和hsp:dkk1-egfp,增强或抑制Wnt信号,通过YO-PRO-1染色和毛细胞计数,研究斑马鱼中Wnt信号对侧线系统发育的影响。热激诱导Dkk1过表达后,斑马鱼侧线系统的神经丘不能形成。碱性磷酸酶染色和整体原位杂交结果表明:Wnt信号是神经丘毛细胞前体细胞以及侧线基板标记基因Eya1和Six2b表达所必需的。综上结果:Wnt信号可能通过调控侧线基板形成影响斑马鱼的侧线发育。