Annexin-1 modulates T-cell activation and differentiation

Annexin-1 is an anti-inflammatory protein that plays an important homeostatic role in innate immunity; however, its potential actions in the modulation of adaptive immunity have never been explored. Although inactive by itself, addition of annexin-1 to stimulated T cells augmented anti-CD3/CD28-mediated CD25 and CD69 expression and cell proliferation. This effect was paralleled by increased nuclear factor-kappaB (NF-kappaB), nuclear factor of activated T cells (NFATs), and activator protein-1 (AP-1) activation and preceded by a rapid T-cell receptor (TCR)-induced externalization of the annexin-1 receptor. Interestingly, differentiation of naive T cells in the presence of annexin-1 increased skewing in Th1 cells; in the collagen-induced arthritis model, treatment of mice with annexin-1 during the immunization phase exacerbated signs and symptoms at disease onset. Consistent with these findings, blood CD4+ cells from patients with rheumatoid arthritis showed a marked up-regulation of annexin-1 expression. Together these results demonstrate that annexin-1 is a molecular "tuner" of TCR signaling and suggest this protein might represent a new target for the development of drugs directed to pathologies where an unbalanced Th1/Th2 response or an aberrant activation of T cells is the major etiologic factor.     

IQGAP1 and IQGAP2 are Reciprocally Altered in Hepatocellular Carcinoma

Background  

IQGAP1 and IQGAP2 are homologous members of the IQGAP family of scaffold proteins. Accumulating evidence implicates IQGAPs in tumorigenesis. We recently reported that IQGAP2 deficiency leads to the development of hepatocellular carcinoma (HCC) in mice. In the current study we extend these findings, and investigate IQGAP1 and IQGAP2 expression in human HCC.     

Essential role of B-Raf in ERK activation during extraembryonic development

The kinases of the Raf family have been intensively studied as activators of the mitogen-activated protein kinase kinase/extra-cellular signal-regulated kinase (ERK) module in regulated and deregulated proliferation. Genetic evidence that Raf is required for ERK activation in vivo has been obtained in lower organisms, which express only one Raf kinase, but was hitherto lacking in mammals, which express more than one Raf kinase. Ablation of the two best studied Raf kinases, B-Raf and Raf-1, is lethal at midgestation in mice, hampering the detailed study of the essential functions of these proteins. Here, we have combined conventional and conditional gene ablation to show that B-Raf is essential for ERK activation and for vascular development in the placenta. B-Raf-deficient placentae show complete absence of phosphorylated ERK and strongly reduced HIF-1alpha and VEGF levels, whereas all these parameters are normal in Raf-1-deficient placentae. In addition, neither ERK phosphorylation nor development are affected in B-raf-deficient embryos that are born alive obtained by epiblast-restricted gene inactivation. The data demonstrate that B-Raf plays a nonredundant role in ERK activation during extraembyronic mammalian development in vivo.     

IQGAP1蛋白在呼吸系统疾病中的研究进展

IQGAP1蛋白为小的真核细胞蛋白家族中的一员,在细胞增殖、分化、转移等过程中起着重要作用.最近研究发现其参与了气道炎性损伤、肺癌侵袭转移等呼吸系统疾病变化.本文就IQGAP1蛋白的结构和功能、在呼吸系统疾病中的调控作用作一综述.     

TAAR1 activation modulates monoaminergic neurotransmission, preventing hyperdopaminergic and hypoglutamatergic activity

The trace amine-associated receptor 1 (TAAR1), activated by endogenous metabolites of amino acids like the trace amines p-tyramine and β-phenylethylamine, has proven to be an important modulator of the dopaminergic system and is considered a promising target for the treatment of neuropsychiatric disorders. To decipher the brain functions of TAAR1, a selective TAAR1 agonist, RO5166017, was engineered. RO5166017 showed high affinity and potent functional activity at mouse, rat, cynomolgus monkey, and human TAAR1 stably expressed in HEK293 cells as well as high selectivity vs. other targets. In mouse brain slices, RO5166017 inhibited the firing frequency of dopaminergic and serotonergic neurons in regions where Taar1 is expressed (i.e., the ventral tegmental area and dorsal raphe nucleus, respectively). In contrast, RO5166017 did not change the firing frequency of noradrenergic neurons in the locus coeruleus, an area devoid of Taar1 expression. Furthermore, modulation of TAAR1 activity altered the desensitization rate and agonist potency at 5-HT(1A) receptors in the dorsal raphe, suggesting that TAAR1 modulates not only dopaminergic but also serotonergic neurotransmission. In WT but not Taar1(-/-) mice, RO5166017 prevented stress-induced hyperthermia and blocked dopamine-dependent hyperlocomotion in cocaine-treated and dopamine transporter knockout mice as well as hyperactivity induced by an NMDA antagonist. These results tie TAAR1 to the control of monoamine-driven behaviors and suggest anxiolytic- and antipsychotic-like properties for agonists such as RO5166017, opening treatment opportunities for psychiatric disorders.     

RhoC和IQGAP1蛋白在原发性肝细胞癌中的表达及意义

目的:探讨RhoC和IQGAP1蛋白在原发性肝细胞癌(hepatocellular carcinoma,HCC)组织中的表达及其临床病理意义.方法:采用免疫组织化学法分别检测56例原发性肝细胞癌和15例正常肝组织中RhoC及IQGAP1蛋白的表达,并分析两者的相关性及与临床病理因素的关系.结果:RhoC和IQGAP1在...     

Constitutive activation of B-Raf in the mouse germ line provides a model for human cardio-facio-cutaneous syndrome

RASopathies are a class of developmental syndromes that result from congenital mutations in key elements of the RAS/RAF/MEK signaling pathway. A well-recognized RASopathy is the cardio-facio-cutaneous (CFC) syndrome characterized by a distinctive facial appearance, heart defects, and mental retardation. Clinically diagnosed CFC patients carry germ-line mutations in four different genes, B-RAF, MEK1, MEK2, and K-RAS. B-RAF is by far the most commonly mutated locus, displaying mutations that most often result in constitutive activation of the B-RAF kinase. Here, we describe a mouse model for CFC generated by germ-line expression of a B-RafLSLV600E allele. This targeted allele allows low levels of expression of B-RafV600E, a constitutively active B-Raf kinase first identified in human melanoma. B-Raf+/LSLV600E mice are viable and display several of the characteristic features observed in CFC patients, including reduced life span, small size, facial dysmorphism, cardiomegaly, and epileptic seizures. These mice also show up-regulation of specific catecholamines and cataracts, two features detected in a low percentage of CFC patients. In addition, B-Raf+/LSLV600E mice develop neuroendocrine tumors, a pathology not observed in CFC patients. These mice may provide a means of better understanding the pathophysiology of at least some of the clinical features present in CFC patients. Moreover, they may serve as a tool to evaluate the potential therapeutic efficacy of B-RAF inhibitors and establish the precise window at which they could be effective against this congenital syndrome.     

Mixed-lineage kinase 3 regulates B-Raf through maintenance of the B-Raf/Raf-1 complex and inhibition by the NF2 tumor suppressor protein

The Ras --> Raf --> MEK1/2 --> extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase (MAPK) pathway couples mitogenic signals to cell proliferation. B-Raf and Raf-1 function within an oligomer wherein they are regulated in part by mutual transactivation. The MAPK kinase kinase (MAP3K) mixed-lineage kinase 3 (MLK3) is required for mitogen activation of B-Raf and cell proliferation. Here we show that the kinase activity of MLK3 is not required for support of B-Raf activation. Instead, MLK3 is a component of the B-Raf/Raf-1 complex and is required for maintenance of the integrity of this complex. We show that the activation of ERK and the proliferation of human schwannoma cells bearing a loss-of-function mutation in the neurofibromatosis 2 (NF2) gene require MLK3. We find that merlin, the product of NF2, blunts the activation of both ERK and c-Jun N-terminal kinase (JNK). Finally, we demonstrate that merlin and MLK3 can interact in situ and that merlin can disrupt the interactions between B-Raf and Raf-1 or those between MLK3 and either B-Raf or Raf-1. Thus, MLK3 is part of a multiprotein complex and is required for ERK activation. The levels of this complex may be negatively regulated by merlin.     

PKC activation by melatonin modulates vimentin intermediate filament organization in N1E-115 cells

Melatonin enters cells and causes cytoskeletal rearrangements in unicellular organisms, plants and vertebrates. This pineal secretory product causes microtubule enlargement and neurite outgrowth by a calmodulin antagonism in N1E-115 cells. Recently, direct in vitro activation of protein kinase C by melatonin was described. Vimentin intermediate filaments are attached to microtubules and their organization depends on both microtubule distribution and phosphorylation of specific proteins. Protein kinase C is a serine threonine kinase which phosphorylates vimentin and through this mechanism causes intermediate filament disassembly. In this work the effects of melatonin on protein kinase C activation, content, and subcellular distribution were studied in N1E-115 cells. Also, melatonin effects on vimentin phosphorylation and subcellular distribution were explored. The results show that melatonin both activates and increases protein kinase C content in the membrane cytoskeletal fraction. Melatonin protein kinase C activation was followed by an increase in both vimentin phosphorylation and by vimentin subcellular redistribution. Moreover, staurosporine, a serine threonine kinase inhibitor, prevented increased vimentin phosphorylation elicited by melatonin. Similar effects to those caused by melatonin were obtained with the protein kinase C activator phorbol 12-myristate 13-acetate. Data support the idea that melatonin modulates vimentin organization through protein kinase C activation.     

Endocannabinoid signaling negatively modulates stress-induced activation of the hypothalamic-pituitary-adrenal axis

Activation of the hypothalamic-pituitary-adrenal (HPA) axis is critical for the adaptation and survival of animals upon exposure to stressful stimuli, and data suggest that endocannabinoid (eCB) signaling modulates neuroendocrine function. We have explored the role of eCB signaling in the modulation of stress-induced HPA axis activation. Administration of the CB1 receptor antagonist/inverse agonist SR141716 (0.01, 0.1, 1, and 5 mg/kg, i.p.) to male mice produced a small, dose-dependent increase in the serum corticosterone (CORT) concentration. Despite this effect, the highest dose of SR141716 did not significantly increase neuronal activity within the paraventricular nucleus of the hypothalamus, as measured by the induction of Fos protein. Similarly, exposure of mice to 30 min of restraint increased serum CORT concentrations, but did not produce a consistent, statistically significant increase in Fos expression within the PVN. However, pretreatment of mice with SR141716 before restraint stress robustly potentiated restraint-induced CORT release and Fos expression within the PVN. Pretreatment of mice with either the CB1 receptor agonist CP55940, the eCB transport inhibitor AM404, or the fatty acid amide hydrolase inhibitor URB597 significantly decreased or eliminated restraint-induced CORT release. Upon exposure to acute restraint, hypothalamic 2-arachidonylglycerol content was reduced compared with the control value; however, after 5 d of restraint exposure (which resulted in an attenuated CORT response), the hypothalamic 2-arachidonylglycerol content was increased compared with the control value. These data indicate that eCB signaling negatively modulates HPA axis function in a context-dependent manner and suggest that pharmacological augmentation of eCB signaling could serve as a novel approach to the treatment of anxiety-related disorders.     

Adenosine A3 receptor activation modulates the capillary endothelial glycocalyx

The endothelial glycocalyx is a dynamic extracellular matrix composed of cell surface proteoglycans, glycoproteins, and adsorbed serum proteins that has been implicated in the regulation and modulation of capillary tube hematocrit, permeability, and hemostasis. High tissue adenosine levels have been shown to adversely affect microvascular function and tissue survival after an ischemic episode, and previous work in this laboratory has shown that adenosine causes arteriolar constriction and degranulation of mast cells via the A3 receptor (A3AR). We hypothesized that adenosine exerts at least part of its effect through modification of the glycocalyx via the A3AR. We used an in vivo cremaster model (hamster and mouse) in which circulating plasma was labeled with a 70-kDa FITC-dextran, and the capillaries were examined before and after superfusion with varying concentrations of adenosine (or other vasoactive molecules). Measurements of the dextran exclusion from an endothelial cell surface layer and red cell separation from the endothelial cell surface were made for up to 30 minutes. Our data indicate that adenosine causes a rapid and profound decrease in the ability of the glycocalyx to exclude dextran but only affects red blood cell exclusion at pharmacological levels. Knockout mice deficient in the A3AR were completely protected from glycocalyx changes attributable to adenosine. These data show a potential link between a known vasoactive tissue metabolite, adenosine, and regulation of the glycocalyx, which may be important during (patho)physiological changes in microvascular function during inflammatory insults.     

Proteasome inhibitor bortezomib modulates TLR4-induced dendritic cell activation

Evidence from the animal model suggests that proteasome inhibitors may have immunosuppressive properties; however, their effects on the human immune system remain poorly investigated. Here, we show that bortezomib, a proteasome inhibitor with anticancer activity, impairs several immune properties of human monocyte-derived dendritic cells (DCs). Namely, exposure of DCs to bortezomib reduces their phagocytic capacity, as shown by FITC-labeled dextran internalization and mannose-receptor CD206 down-regulation. DCs treated with bortezomib show skewed phenotypic maturation in response to stimuli of bacterial (lipopolysaccharide [LPS]) and endogenous sources (including TNF-alpha and CD40L), as well as reduced cytokine production and immunostimulatory capacity. LPS-induced CCL-2/MCP-1 and CCL5/RANTES secretions by DCs were prevented by DC treatment with bortezomib. Finally, CCR7 up-regulation in DCs exposed to LPS as well as migration toward CCL19/MIP-3beta were strongly impaired. As a suitable mechanism for these effects, bortezomib was found to down-regulate MyD88, an essential adaptor for TLR signaling, and to relieve LPS-induced activation of NF-kappaB, IRF-3, and IRF-8 and of the MAP kinase pathway. In summary, inhibition of DC function may represent a novel mechanism by which proteasome inhibitors exert immunomodulatory effects. These compounds could prove useful for tuning TLR signaling and for the treatment of inflammatory and immune-mediated disorders.     

Tumor-derived lactic acid modulates dendritic cell activation and antigen expression

The tumor milieu can influence dendritic cell (DC) differentiation. We analyzed DC differentiation in a 3-dimensional tumor model and propose a new mechanism of DC modulation by the tumor environment. Monocytes were cultured in the presence of IL-4 and GM-CSF within multicellular tumor spheroids (MCTSs) generated from different tumor cell lines. Monocytes invaded the MCTSs and differentiated into tumor-associated dendritic cells (TADCs). The antigen expression was altered on TADCs independent of the culture conditions (immature/mature DCs, Langerhans cells) and IL-12 secretion was reduced. Supernatants of MCTSs could partially transfer the suppressive effect. Conditioned media from urothelial carcinoma cell lines contained high levels of M-CSF and IL-6, both cytokines known to modulate DC differentiation. In contrast, melanoma and prostate carcinoma MCTS cocultures produced little M-CSF and IL-6, but high levels of lactic acid. Indeed, addition of lactic acid during DC differentiation in vitro induced a phenotype comparable with TADCs generated within melanoma and prostate carcinoma MCTSs. Blocking of lactic acid production in melanoma MCTS cocultures reverted the TADC phenotype to normal. We therefore conclude that tumor-derived lactic acid is an important factor modulating the DC phenotype in the tumor environment, which may critically contribute to tumor escape mechanisms.     

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters.It is well-established that different populations of T lymphocytes can recognize not only peptides in the context of major histocompatibility complex (MHC) class I (MHCI) and MHCII molecules but also, foreign and self-lipids in association with CD1 proteins (1), antigen-presenting molecules that share structural similarities with MHCI molecules. Of five CD1 isoforms, CD1d restricts the activity of a family of cells known as invariant natural killer T (iNKT) cells because of their semiinvariant T-cell receptor (TCR) use (1). To date, the exogenous glycolipid α-GalactosylCeramide (α-GalCer) represents the best characterized CD1d-restricted agonist for iNKT cells (2). Unlike conventional peptide-specific T cells, iNKT cells react against CD1d⁺ antigen-presenting cells (APCs) in the absence of exogenous antigens, a feature defined as autoreactivity (3). iNKT cell autoreactivity underpins the constitutive memory phenotype of iNKT cells and their ability to be activated during a variety of immune responses from infections to cancer and autoimmunity (1). Some of the endogenous antigens known to elicit iNKT cell autoreactivity belong to glycosphingolipid families, with a mix of α- and β-anomeric configurations (4–7). How iNKT cell autoreactivity is fine-tuned to prevent autoimmunity is subject of much investigation. Previous results have shown that exposure of APCs to Toll-like receptor (TLR) agonists enhances iNKT cell autoreactivity (8, 9), consistent with the proposed mechanism by which ligand availability is regulated by lysosomal glycosidases (4, 6).The recent application of advanced optical techniques (10–13) in combination with substrate patterning and functionalization (14, 15) is providing detailed information on how the lateral organization of a variety of molecules located on both sides of the immunological synapse contributes to controlling T-cell activation. Specifically, single-molecule dynamic approaches and superresolution optical nanoscopy experiments have provided indisputable proof that many receptors on the cell membrane organize in small nanoclusters before ligand activation (16). Membrane nanodomains enriched in cholesterol and sphingolipids (17), protein–protein interactions (18), and interactions between transmembrane proteins and the cytoskeleton (19, 20) have been all implicated in regulating receptor dynamics and nanoclustering. An emerging concept attributes the actin cytoskeleton the ability of imposing barriers or fences on the cell membrane, restricting the lateral mobility of transmembrane proteins (19–21). This transient restriction would, in turn, increase the local concentration of transmembrane proteins, leading to protein nanoclusters. For instance, it has been shown that the actin cytoskeleton promotes the dimerization rate of EGF receptors and facilitates ligand binding and signaling activation (18, 22). Confinement of CD36 has also been observed as a result of its diffusion along linear channels dependent on the integrity of the cortical cytoskeleton (23). This constrained diffusion promotes CD36 clustering, influencing CD36-mediated signaling and internalization. A similar mechanism has been proposed for the maintenance of MHCI clusters on the cell membrane by the actin cytoskeleton, with loss of MHCI clustering resulting in a decreased CD8 T-cell activation (24, 25).Recent confocal microscopy studies have revealed that the association between agonist-loaded CD1d molecules and lipid rafts might contribute to the regulation of iNKT cell activation (26). This elegant study for the first time, to our knowledge, linked the spatial organization of CD1d molecules on the cell membrane of APCs with the activation profile of iNKT cells. However, it remains unclear whether the results of these experiments obtained using mouse cells can be extended to human cells and whether additional insights can be obtained by using higher-resolution microscopy. Indeed, it is not yet known whether surface-expressed CD1d molecules exist as monomers or nanoclusters and whether the actin cytoskeleton might regulate CD1d lateral organization and iNKT cell activation. Interestingly, it has been recently reported that the actin cytoskeleton impairs antigen presentation by CD1d and that disruption of F actin or inhibition of the ρ-associated protein kinase enhances CD1d-mediated antigen presentation (27). These results suggest that the actin cytoskeleton might regulate, in a not yet known manner, antigen presentation by CD1d molecules.Here, we combined dual-color single-molecule dynamic approaches with superresolution optical nanoscopy to characterize for the first time, to our knowledge, the spatiotemporal behavior of CD1d on living human myeloid cells. We find that α-GalCer–loaded human CD1d (hCD1d) molecules are organized in nanoclusters on the cell membrane of APCs. We report that the actin cytoskeleton prevents enhanced hCD1d nanoclustering by hindering physical encountering between hCD1d diffusing nanoclusters, thus reducing basal iNKT cell activation. Furthermore, we observed an increase in nanocluster density on activation of APCs with inflammatory stimuli, such as TLR stimulation, mirroring the increased iNKT cell stimulation. Notably, even during inflammation, the actin cytoskeleton retains an important role to limit hCD1d cluster size and iNKT cell activation. Overall, our results suggest that regulation of CD1d nanoclustering through the actin cytoskeleton represents a previously unidentified mechanism to fine-tune peripheral iNKT cell autoreactivity.     

Dimerization of complement factor H-related proteins modulates complement activation in vivo

The complement system is a key component regulation influences susceptibility to age-related macular degeneration, meningitis, and kidney disease. Variation includes genomic rearrangements within the complement factor H-related (CFHR) locus. Elucidating the mechanism underlying these associations has been hindered by the lack of understanding of the biological role of CFHR proteins. Here we present unique structural data demonstrating that three of the CFHR proteins contain a shared dimerization motif and that this hitherto unrecognized structural property enables formation of both homodimers and heterodimers. Dimerization confers avidity for tissue-bound complement fragments and enables these proteins to efficiently compete with the physiological complement inhibitor, complement factor H (CFH), for ligand binding. Our data demonstrate that these CFHR proteins function as competitive antagonists of CFH to modulate complement activation in vivo and explain why variation in the CFHRs predisposes to disease.