Glycosyltransferase regulation mediated by pre-TCR signaling in early thymocyte development

CT1 is a carbohydrate moiety of CD45 that is expressed on fetal thymocytes in vivo. Examination of CT1 expression on thymocyte subsets revealed that primarily pro-T cells (CD44+ CD25+) and pre-T cells (CD44- CD25+) expressed CT1. Interestingly, non-T-lineage committed lymphoid progenitors (CD44+ CD25-) lacked CT1 indicating temporal regulation of expression of this determinant in early T-lineage committed development. In addition, CT1 was expressed by the majority of thymocytes in RAG-2(-/-) mice where thymocyte development is blocked at the CD44- CD25+ stage. Since late pre-T cells (CD44- CD25-) lacked the CT1 epitope we tested whether pre-TCR triggering regulated CT1 expression. Injection of CD3epsilon-specific mAb into RAG-2(-/-) mice induces differentiation of immature thymocytes to the double-positive stage of thymocyte development. Using this system, we demonstrated that expression of CT1 by RAG-2(-/-) thymocytes was rapidly lost from pre-T cells following anti-CD3 mAb treatment. Furthermore, the decline in CT1 expression induced by CD3 signaling paralleled a loss of mRNA for the glycosyltransferase responsible for the addition of CT1 to CD45. Flow cytometric analysis also revealed that the loss of the CT1 epitope was inversely correlated with an increase in peanut agglutinin ligand expression, demonstrating a complex regulation of cell surface glycosylation at a critical juncture in thymocyte development.      

Notch regulation of early thymocyte development

Notch signaling plays multiple roles in T cell development. Following thymic entry, Notch signals are required to specify the T cell fate from a multipotent hematopoietic progenitor. At subsequent steps in early T cell development, Notch provides important differentiation, survival, proliferation and metabolic signals. This review focuses on the multiple functions of Notch in early T cell development, from T cell specification in the thymus through beta selection.     

p38 MAPK信号通路参与受体介导的细胞内吞的调控

目的：观察p38 MAPK信号转导通路在受体介导的细胞内吞中的作用。方法：利用Alexa 594标记的转铁蛋白作为受体介导的内吞作用的观察指标，来研究p38特异性抑制剂SB203580或ERK通路特异性抑制剂PD98059预处理以及p38基因敲除对该过程的影响。 结果：在受体介导的细胞内吞中，p38被磷酸化激活。SB203580的预处理或p38基因的敲除都能阻断受体介导的细胞内吞，而PD98059的预处理对该过程没有影响。 结论：p38 MAPK信号转导通路参与了受体介导的细胞内吞的调控。     

Temporal and spatial regulation of bone morphogenetic protein signaling in late lung development.

Bone morphogenetic proteins (BMPs) play important roles in early lung development. No study to date has addressed a role for BMP signaling in late lung development. We describe changes in the expression and localization of BMP receptors (Bmpr1a, Bmpr1b, and Bmpr2) and Smad (Smad1, Smad4, Smad5, and Smad8) intracellular signaling proteins during the saccular and alveolarization stages of late lung development. BMP signaling, assessed by Smad1/5 phosphorylation, nuclear translocation, and induction of id1, id2, and id3 gene expression, was evident throughout late lung development. Our data indicate that BMP signaling is active during late lung development, and points to roles for the BMP system in septal and vascular development, and in the homeostasis of the epithelial layer of large conducting airways in the mature lung.     

Nuclear Notch1 signaling and the regulation of dendritic development

To understand the function of Notch in the mammalian brain, we examined Notch1 signaling and its cellular consequences in developing cortical neurons. We found that the cytoplasmic domain of endogenous Notch1 translocated to the nucleus during neuronal differentiation. Notch1 cytoplasmic-domain constructs transfected into cortical neurons were present in multiple phosphorylated forms, localized to the nucleus and could induce CBF1-mediated transactivation. Molecular perturbation experiments suggested that Notch1 signaling in cortical neurons promoted dendritic branching and inhibited dendritic growth. These observations show that Notch1 signaling to the nucleus exerts an important regulatory influence on the specification of dendritic morphology in neurons.     

Rac1信号通路在小鼠可卡因情景线索记忆过程中的作用

目的:探讨Rac1信号通路在小鼠可卡因情景线索记忆过程中的作用。方法:将成年雄性C57BL/6J小鼠随机分为对照组(control)、可卡因配对的条件性位置偏爱组(CPP)和Rac1抑制剂NSC23766处理组(NSC),利用CPP实验检测可卡因是否可诱导小鼠产生CPP,利用立体定位技术注射AAV-GFP病毒于小鼠海马CA1区并做免疫荧光染色观察海马CA1区神经元的形态;利用Western Blot及GST-pull down方式检测蛋白表达及活性变化;采用Rac1抑制剂NSC23766阻断信号通路探讨其对小鼠可卡因诱导的CPP的影响。结果:可卡因可以诱导小鼠形成CPP,并导致Rac1 GTPase活性增强及其下游信号分子cofilin磷酸化活性升高,并最终导致海马CA1区锥体神经元发生神经可塑性变化。结论:Rac1信号通路通过影响小鼠海马CA1区锥体神经元发生结构可塑性,从而参与调控可卡因情景线索记忆。     

RasGRP is essential for mouse thymocyte differentiation and TCR signaling

The Ras signaling pathway plays a critical role in thymopoiesis and T cell activation, but the mechanism of Ras regulation is controversial. At least one mode of Ras regulation in T cells involves the messenger diacylglycerol (DAG). RasGRP, a Ras activator with a DAG-binding C1 domain, is expressed in T cells and thymocytes. Here we show that thymi of RasGRP-null mutant mice have approximately normal numbers of immature thymocytes but a marked deficiency of mature, single-positive (CD4+CD8- and CD4-CD8+) thymocytes. In Ras signaling and proliferation assays, mutant thymocytes showed a complete lack of response to DAG analogs or T cell receptor (TCR) stimulation by antibodies. Thus, TCR and DAG are linked through RasGRP to Ras signaling.     

Transcription factor NF-Y is involved in regulation of the JNK pathway during Drosophila thorax development

The CCAAT motif-binding factor, nuclear factor Y (NF-Y) consists of three different subunits, NF-YA, NF-YB and NF-YC. Knockdown of Drosophila NF-YA (dNF-YA) in the notum compartment of wing discs by a pannir -GAL4 and UAS- dNF-YAIR mainly resulted in a thorax disclosed phenotype. Reduction of the Drosophila c-Jun N-terminal kinase (JNK) basket ( bsk ) gene dose enhanced the knockdown of dNF-YA-induced phenotype. Monitoring of JNK activity in the wing disc by LacZ expression in a puckered ( puc ) -LacZ enhancer trap line revealed reduction in the level of the JNK reporter, puc-LacZ signals, in dNF-YA RNAi clones. In addition, expression of wild-type Bsk effectively suppressed the phenotype induced by knockdown of dNF-YA. The bsk gene promoter contains a CCAAT motif and this motif plays a positive role in the promoter activity. We performed chromatin immunoprecipitation (ChIP) assays in S2 cells with anti-dNF-YA IgG and quantitative real-time PCR. The bsk gene promoter region containing the CCAAT boxes was effectively amplified in the immunoprecipitates by PCR. However, this region was not amplified in the immunoprecipitates from dNF-YA knockdown cells. Furthermore, the level of endogenous bsk mRNA is reduced in the dNF-YA knockdown larvae. These results suggest that dNF-Y is necessary for proper bsk expression and activity of JNK pathway during thorax development.     

Silkworm ferritin 1 heavy chain homolog is involved in defense against bacterial infection through regulation of haemolymph iron homeostasis

Iron functions as a nutrient and a potential toxin in all organisms. It plays a key role in the interaction between microbes and their hosts as well. Microbial infection disrupts iron homeostasis in the host; meanwhile the host endeavors to keep the homeostasis through iron transport and storage. Transferrins and ferritins are the major iron-binding proteins that affect iron distribution in insects. In this study, we investigated a possible involvement of Bombyx mori ferritin 1 (BmFer1) heavy chain homolog in the defense against bacterial infection in the silkworm larvae. The BmFer1 mRNA abundance was up-regulated in hemocytes, but not in fat body, after Pseudomonas aeruginosa or Staphylococcus aureus infection. The infection resulted in elevated iron levels in the hemolymph. Injection of recombinant BmFer1 protein into hemocoel reduced the plasma iron level after infection, limited the bacterial growth in the hemolymph, and resulted in a lower mortality caused by infection. Our study indicated that B. mori ferritin-1 may restrict iron access of the invading bacteria to block their growth as a defense strategy.     

Somatostatin is expressed in the murine thymus and enhances thymocyte development

A functional interaction between the immune and the nervous system has been suggested, with neuropeptides acting as immunomodulators. Somatostatin (SOM) is a neuropeptide, mainly produced in the brain, that binds to five different receptors (SSTR). It is believed that SOM along with one of its receptors, SSTR2, is expressed in the murine thymus, although their exact localization is unresolved. We found that SOM is highly expressed in both cortical and medullary epithelial cells whereas its receptor SSTR2 is expressed on thymocytes. In order to elucidate its role in thymopoiesis, SOM was added in fetal thymic organ culture (FTOC) and found to increase thymocyte numbers and enhance maturation. SOM increased the cellular proliferation of total splenocytes but inhibited proliferation of thymocytes and purified splenic T cells. Furthermore, SOM was able to induce the migration of thymocytes. We also investigated the effect of four other neuropeptides in FTOC and found that, vasoactive intestinal peptide had a marginal effect, whereas substance P increased thymic cellularity, at intermediate but not at low or high concentrations. In contrast, both neuropeptide Y and calcitonin gene-related peptide reduced thymocyte numbers. This study supports the hypothesis for a role of neuropeptides, particularly somatostatin, in immune regulation and development.     

Fgfr1 regulates development through the combinatorial use of signaling proteins

Fibroblast growth factor (Fgf) signaling governs multiple processes important in development and disease. Many lines of evidence have implicated Erk1/2 signaling induced through Frs2 as the predominant effector pathway downstream from Fgf receptors (Fgfrs), but these receptors can also signal through other mechanisms. To explore the functional significance of the full range of signaling downstream from Fgfrs in mice, we engineered an allelic series of knock-in point mutations designed to disrupt Fgfr1 signaling functions individually and in combination. Analysis of each mutant indicates that Frs2 binding to Fgfr1 has the most pleiotropic functions in development but also that the receptor uses multiple proteins additively in vivo. In addition to Frs2, Crk proteins and Plcγ also contribute to Erk1/2 activation, affecting axis elongation and craniofacial and limb development and providing a biochemical mechanism for additive signaling requirements. Disruption of all known signaling functions diminished Erk1/2 and Plcγ activation but did not recapitulate the peri-implantation Fgfr1-null phenotype. This suggests that Erk1/2-independent signaling pathways are functionally important for Fgf signaling in vivo.     

Inhibition of Notch signaling biases rat thymocyte development towards the NK cell lineage

Notch receptors are involved in directing the choice between alternative cell fates in developmental scenarios such as thymopoiesis. By pharmacological interference in rat fetal thymus organ culture we show that inhibition of Notch signaling arrests T cell development at an early double-negative stage and is accompanied by a dramatic increase in the number of NK cells. These cells show an activated phenotype, lack recombination of the TCR beta gene locus and express perforin. Similarly, in thymic lobes reconstituted with fetal liver cells, progenitors predominantly develop into NK cells both after pharmacological interference of Notch and after treatment with a recombinant rat Notch1/Fc chimera. Collectively, this identifies the lineage decision of NK/T precursor cells as an important site of Notch action in rat thymocytes.     

EphrinB1-EphB signaling regulates thymocyte-epithelium interactions involved in functional T cell development

The Eph and ephrin families are involved in numerous developmental processes. Recently, an increasing body of evidence has related these families with some aspects of T cell development. In the present study, we show that the addition of either EphB2-Fc or ephrinB1-Fc fusion proteins to fetal thymus organ cultures established from 17-day-old fetal mice decreases the numbers of both double-positive (CD4(+)CD8(+)) and single-positive (both CD4(+)CD8(-) and CD4(-)CD8(+)) thymocytes, in correlation with increased apoptosis. By using reaggregate thymus organ cultures formed by fetal thymic epithelial cells (TEC) and CD4(+)CD8(+) thymocytes, we have also demonstrated that ephrinB1-Fc proteins are able to disorganize the three-dimensional epithelial network that in vivo supports the T cell maturation, and to alter the thymocyte interactions. In addition, in an in vitro model, Eph/ephrinB-Fc treatment also decreases the formation of cell conjugates by CD4(+)CD8(+) thymocytes and TEC as well as the TCR-dependent signaling between both cell types. Finally, immobilized EphB2-Fc and ephrinB1-Fc modulate the anti-CD3 antibody-induced apoptosis of CD4(+)CD8(+) thymocytes in a process dependent on concentration. These results therefore support a role for Eph/ephrinB in the processes of development and selection of thymocytes as well as in the establishment of the three-dimensional organization of TEC.     

Somatic activation of beta-catenin bypasses pre-TCR signaling and TCR selection in thymocyte development

Mutation or ablation of T cell factor 1 and lymphocyte enhancer factor 1 indicated involvement of the Wnt pathway in thymocyte development. The central effector of the Wnt pathway is beta-catenin, which undergoes stabilization upon binding of Wnt ligands to frizzled receptors. We report here that conditional stabilization of beta-catenin in immature thymocytes resulted in the generation of single positive T cells that lacked the alpha beta TCR and developed in the absence of pre-TCR signaling and TCR selection. Although active beta-catenin induced differentiation in the absence of TCRs, its action was associated with reduced proliferation and survival when compared to developmental changes induced by the pre-TCR or the alpha beta TCR.     

Ras signaling is involved in the expression of Fas-L in glioma

Fas-L expresses on a variety of tumors and is suspected to modify the dialog between tumor and the immune system. However, the cellular abnormality in tumor cells leading to an aberrant expression of Fas-L is unclear. In this study, we demonstrate the involvement of Ras signaling in the Fas-L expression in several ways. First, the activated Ha-rasval12 gene enhanced the Fas-L expression of primary human glial cells. Second, blocking the Ras signal pathway in glioma cells by lovastatin or the Ha-rasAsn17 dominant-negative mutant gene resulted in reduced Fas-L expression. Transfection of the Ha-rasAsn17 into glioma cells also inhibited the activation of NFKB, which is a downstream component of Ras signaling. Accordingly, the membrane-permeable NFKB competitor suppressed the Fas-L expression. Furthermore, the Fas-L expression coincided with the Ras activity in the murine 212 cells, in which the Ras activity could be induced by isopropyl 3-D-thiogalactoside. In summary, these results suggest that the enhanced Ras signaling with consequential NFKB activation, which is a frequent defect found in tumors, could mediate the Fas-L expression of tumors.     

Calcineurin B1 is essential for positive but not negative selection during thymocyte development

During development, discrete cell fates often result from variation in the intensity of a particular signal. The mechanisms underlying these seemingly analog-to-digital switches are not understood. In developing T lymphocytes, low-intensity signals through the antigen receptor result in positive selection while more intense signals give rise to negative selection. By deleting the genetic locus encoding the regulatory B1 subunit of calcineurin specifically in thymocytes, we found an absolute requirement for calcineurin in positive selection. In contrast, calcineurin activity was dispensable in several models of negative selection. Unexpectedly, we found that removal of calcineurin activity from thymocytes results in inefficient ERK activation at the double-positive stage of thymocyte development, when selection occurs. These studies clarify the mechanism by which graded signals are converted to discrete outcomes in T cell development and further indicate that the developmental roles of calcineurin likely contribute to immunosuppression by calcineurin inhibitors.