Signal Transduction In B Lymphocytes: Identification Of Key Signaling Molecules

为了更好地了解GCK和GCKR在体内的作用，我们分离了小鼠GCK和GCKR基因。Gck-/-和Gckr -/-小鼠已被创建并回交到C57Bl/6背景上，用于产生双敲除（KO）小鼠。突变不影响小鼠的发育，因为Gck、Gckr和双KO小鼠出生时具有正常的孟德尔频率。对所有三种小鼠品系的免疫组织的评估显示了异常免疫反应性的证据。它们拥有大量生发中心和适度的脾肿大。Gck缺失对免疫反应性的影响大于Gckr缺失。Gck-/-和Gckr-/-小鼠对Toll受体样体（TLR）配体有增强的增殖反应。LPS刺激双KO脾细胞导致CD11b高、B220-和GR1阳性亚群（可能是中性粒细胞）中p38活化增加。在一项合作研究中，我们发现Gck-/-小鼠强烈破坏tlr刺激的巨噬细胞细胞因子和趋化因子的释放，并且小鼠对内毒素介导的致死性具有抗性。骨髓移植研究表明，造血细胞Gck信号对于全身性炎症至关重要。破坏Gck可显著降低tlr介导的巨噬细胞jun - n-末端激酶（JNK）和p38丝裂原活化蛋白激酶（MAPKs）的激活。细胞外信号调节激酶（ERK）和核因子κ b （nf - κ b）的激活在很大程度上不受影响。因此，GCK是连接JNK和p38的重要TLR效应物，而不是ERK或NF-kappaB。

To better understand the role of GCK and GCKR in vivo, the murine Gck and Gckr genes have been isolated. Both Gck-/- and Gckr -/- mice have been created and backcrossed on to a C57Bl/6 background used to generate double knock-out (KO) mice. The mutations did not affect mouse development as the Gck, Gckr, and double KO mice are born with normal Mendelian frequencies. Evaluation of the immune tissues from all three mouse strains revealed evidence of abnormal immune reactivity. They possess expanded numbers of germinal centers and modest splenomegaly. The loss of Gck affected immune reactivity more than did the loss of Gckr. Both the Gck-/- and the Gckr-/- mice have an augmented proliferative response to Toll receptor like (TLR) ligands. LPS stimulation of the double KO spleen cells led to increased p38 activation in a CD11b high, B220-, and GR1 positive subset (likely neutrophils). In a collaborative study we found that Gck-/- mice strongly have impaired TLR-stimulated macrophage cytokine and chemokine release and the mice are resistant to endotoxin-mediated lethality. Bone marrow transplantation studies showed that hematopoietic cell Gck signaling is essential for systemic inflammation. Disruption of Gck substantially reduced TLR-mediated activation of macrophage Jun-N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs). Extracellular signal-regulated kinase (ERK) and nuclear factor-kappaB (NF-kappaB) activation were largely unaffected. Thus, GCK is an essential TLR effector coupling JNK and p38, but not ERK or NF-kappaB to systemic inflammation. To examine the effects of Toll-like receptor signaling B cell chemotaxis and trafficking. We stimulated mouse B cells with lipopolysaccharide (LPS), which engages Toll like receptor 4. We found that LPS stimulation increased the expression of a variety of homing and chemokine receptors; increased the ratio between Gnai2 and Rgs1 expression; and augmented B cell chemotaxis. When transferred into recipient mice the lipopolysaccharide activated B cells homed to lymph nodes better than did non-stimulated cells. Two photon intravital imaging showed highly polarized cells in the centers of lymph node follicles. In vivo tracking studies revealed extensive B cell-B cell and B cell-stromal cell interactions. When germinal center were present the LPS-activated B cells accumulated in the dark zone. Over time the transferred LPS activated B cells accumulated within the splenic marginal zone and possessed a memory B cell phenotype. Exposure of cell to TLR ligands predominantly induces interferon (IFN) regulatory factors (IRFs), and NF-kB and AP1 activation. TLR signaling activates Traf6, which serves as an E3 ligase to ubiquitinate key proteins in the NF-kB signaling pathway. It also induces A20 expression, which is de-ubiquitinating enzyme involved in restricting TLRs signaling. Autophagy delivers cytoplasmic constituents to autolysosomes and has been linked to innate and adaptive immunity. TLR4 signaling is known to induce autophagy and it recruits a key protein in the autophagy pathway, Beclin 1, to the receptor complex. We have shown that Traf6-mediated lysine 63-linked ubiquitination of Beclin 1 is critical for TLR4 triggered autophagy in macrophages. Two Traf6 binding motifs in Beclin 1 facilitated Traf6 binding and its ubiquitination. An in vitro ubiquitination assay revealed that Traf6 directly ubiquitinates Beclin 1. Beclin 1 lysine 117, strategically located in the Beclin 1 BH3 domain is a major site for K63-linked ubiquitination and likely regulates the interaction between Beclin 1 and Bcl2, a known inhibitor of autophagy. A20 reduced Beclin 1 K63-linked ubiquitination, and limited the induction of autophagy following TLR signaling. These results indicate that Traf6 and A20 by controlling K63-ubiquitination of Beclin 1 play key roles in regulating autophagy during inflammatory responses. Our studies of chemokine receptor signaling have focused on the proximal elements in the signaling pathway. These receptors predominantly use the heterotrimeric G protein Gi to link to downstream signaling pathways. We have shown Gi alpha proteins regulate and co-localize with F-actin at actin-rich structures, including microspikes or filopodia, lamellipodia, adhesion sites, phagocytic cups, actin comet tails, sub-cortical ruffles and stress fibers. Consistently, reduction of Gi alpha protein expression altered cell morphology, modulated actin filaments and microtubules, and reduced cell migration. That Gi alpha facilitates the interplay of actin and microtubule was further supported by observations that Gi alpha depletion and PTX treatment both altered the dynamics and distribution of myosin X, Rab5, Rac, and the actin bundling protein fascin, which directs myosin X to tips of filopodia. Conversely, CXCL12 treatment induced the translocation of Gi alpha, myosin X, Rab5 and fascin to the plasma membrane, and increased their co-localization at filopodia, periphery ruffle and circular ruffle. Filopodia appear to function as sensors that explore and interact with a cell's surroundings. Since filopodia grow by polymerization of actin at their tips, these observations suggest that Gi alpha may play an important role in the molecular machinery that regulates actin polymerization at the filopodial tip. In addition to signaling through chemokine receptors, we have been interested in the signaling pathways initiated by another group of receptors, which have emerged as important regulators of lymphocyte trafficking. These receptors all bind the phospholipid sphingosine 1-phosphate (S1P). The S1P receptors function at the level of vascular endothelial cells to regulate lymph node egress by controlling access to the medullary sinus and directly on lymphocytes to promote lymph node exit. In addition, S1P receptors function in the positioning of B cells in the marginal zone of the spleen. Using a series of S1P analogues and receptor inhibitors we have shown that three different S1P receptors termed S1P1, S1P3, and S1P4 function to regulate lymphocyte responses to S1P. S1P1 functions predominantly to slow lymphocyte migration, while S1P3 and S1P4 act as chemoattractant receptors. B cells prepared from S1P3-/- mice exhibit normal chemotaxis to chemokines, but lack responsiveness to S1P, however, homing and egress from lymph nodes of S1P3-/- B cells is not impaired. We also showed the presence of lymphatic sinusoidal structures at the T-B boundary that support exit of B cells. We developed methodology for intravital imaging of the medullary sinsus and the cortical lymphatic structures. Intravital microscopy revealed B cells crossing into the lymphatics and following treatement with FTY720 the velocity of B lymphocytes declined and those cells in neighborhood of the lymphatic sinusoids failed to penetrate into lumen. After immunization or natural infection there are functional and morphological changes in local lymph nodes. We have noted the following changes in lymphatics in local lymph nodes 2-4 day after immunization: increase in level of expression of S1PR1 in immunized compared to control lymph nodes lymphatics, HEVs and lymphatics remodeling where HEVs expressed lymphatic marker (LYVE1), HEVs and lymphatics within activated B cell follicles, increased level of expression of CXCL13 in immunized lymph nodes lymphatics, and lymphatics that expressed higher level of adhesion molecules CD144 and associated intracellular -catenin.