Protease-activated receptor 2 signalling promotes dendritic cell antigen transport and T-cell activation in vivo.

Deficiency of protease-activated receptor-2 (PAR2) modulates inflammation in several models of inflammatory and autoimmune disease, although the underlying mechanism(s) are not understood. PAR2 is expressed on endothelial and immune cells, and is implicated in dendritic cell (DC) differentiation. We investigated in vivo the impact of PAR2 activation on DCs and T cells in PAR2 wild-type (WT) and knockout (KO) mice using a specific PAR2 agonist peptide (AP2). PAR2 activation significantly increased the frequency of mature CD11c(high) DCs in draining lymph nodes 24 hr after AP2 administration. Furthermore, these DCs exhibited increased expression of major histocompatibility complex (MHC) class II and CD86. A significant increase in activated (CD44(+) CD62(-)) CD4(+) and CD8(+) T-cell frequencies was also observed in draining lymph nodes 48 hr after AP2 injection. No detectable change in DC or T-cell activation profiles was observed in the spleen. The influence of PAR2 signalling on antigen transport to draining lymph nodes was assessed in the context of delayed-type hypersensitivity. PAR2 WT mice that were sensitized by skin-painting with fluorescein isothiocyanate (FITC) to induce delayed-type hypersensitivity possessed elevated proportion of FITC(+) DCs in draining lymph nodes 24 hr after FITC painting when compared with PAR2 KO mice (0.95% versus 0.47% of total lymph node cells). Collectively, these results demonstrate that PAR2 signalling promotes DC trafficking to the lymph nodes and subsequent T-cell activation, and thus provides an explanation for the pro-inflammatory effect of PAR2 in animal models of inflammation.

The IFN gamma receptor: a paradigm for cytokine receptor signaling.

During the last several years, the mechanism of IFN gamma-dependent signal transduction has been the focus of intense investigation. This research has recently culminated in the elucidation of a comprehensive molecular understanding of the events that underlie IFN gamma-induced cellular responses. The structure and function of the IFN gamma receptor have been defined. The mechanism of IFN gamma signal transduction has been largely elucidated, and the physiologic relevance of this process validated. Most recently, the molecular events that link receptor ligation to signal transduction have been established. Together these insights have produced a model of IFN gamma signaling that is nearly complete and that serves as a paradigm for signaling by other members of the cytokine receptor superfamily.

Hyaline fibromatosis syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors.

Hyaline Fibromatosis Syndrome (HFS) is a human genetic disease caused by mutations in the anthrax toxin receptor 2 (or cmg2) gene, which encodes a membrane protein thought to be involved in the homeostasis of the extracellular matrix. Little is known about the structure and function of the protein or the genotype-phenotype relationship of the disease. Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level. Altogether, we show that missense mutations that map to the extracellular von Willebrand domain or the here characterized Ig-like domain of CMG2 lead to folding defects and thereby to retention of the mutated protein in the endoplasmic reticulum (ER). Mutations in the Ig-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation. Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

Consequences of sleep deprivation on neurotransmitter receptor expression and function.

Several pieces of evidence suggest that sleep deprivation causes marked alterations in neurotransmitter receptor function in diverse neuronal cell types. To date, this has been studied mainly in wake- and sleep-promoting areas of the brain and in the hippocampus, which is implicated in learning and memory. This article reviews findings linking sleep deprivation to modifications in neurotransmitter receptor function, including changes in receptor subunit expression, ligand affinity and signal transduction mechanisms. We focus on studies using sleep deprivation procedures that control for side-effects such as stress. We classify the changes with respect to their functional consequences on the activity of wake-promoting and/or sleep-promoting systems. We suggest that elucidation of how sleep deprivation affects neurotransmitter receptor function will provide functional insight into the detrimental effects of sleep loss.

Malignant transformation of DMBA/TPA-induced papillomas and nevi in the skin of mice selectively lacking retinoid-X-receptor alpha in epidermal keratinocytes.

Retinoid-X-receptor alpha (RXRalpha), a member of the nuclear receptor (NR) superfamily, is a ligand-dependent transcriptional regulatory factor. It plays a crucial role in NR signalling through heterodimerization with some 15 NRs. We investigated the role of RXRalpha and its partners on mouse skin tumor formation and malignant progression upon topical DMBA/TPA treatment. In mutants selectively ablated for RXRalpha in keratinocytes, epidermal tumors increased in size and number, and frequently progressed to carcinomas. As keratinocyte-selective peroxisome proliferator-activated receptor gamma (PPARgamma) ablation had similar effects, RXRalpha/PPARgamma heterodimers most probably mediate epidermal tumor suppression. Keratinocyte-selective RXRalpha-null and vitamin-D-receptor null mice also exhibited more numerous dermal melanocytic growths (nevi) than control mice, but only nevi from RXRalpha mutant mice progressed to invasive human-melanoma-like tumors. Distinct RXRalpha-mediated molecular events appear therefore to be involved, in keratinocytes, in cell-autonomous suppression of epidermal tumorigenesis and malignant progression, and in non-cell-autonomous suppression of nevi formation and progression. Our study emphasizes the crucial role of keratinocytes in chemically induced epidermal and melanocytic tumorigenesis, and raises the possibility that they could play a similar role in UV-induced tumorigenesis, notably in nevi formation and progression to melanoma.

Chronic bradykinin receptor blockade modulates neonatal renal function.

Recent data indicate that bradykinin participates in the regulation of neonatal glomerular function and also acts as a growth regulator during renal development. The aim of the present study was to investigate the involvement of bradykinin in the maturation of renal function. Bradykinin beta2-receptors of newborn rabbits were inhibited for 4 days by Hoe 140. The animals were treated with 300 microg/kg s.c. Hoe 140 (group Hoe, n = 8) or 0.9% NaCl (group control, n = 8) twice daily. Clearance studies were performed in anesthetized rabbits at the age of 8-9 days. Bradykinin receptor blockade did not impair kidney growth, as demonstrated by similar kidney weights in the two groups, nor did it influence blood pressure. Renal blood flow was higher, while renal vascular resistance and filtration fraction were lower in Hoe 140-treated rabbits. No difference in glomerular filtration rate was observed. The unexpectedly higher renal perfusion observed in group Hoe cannot be explained by the blockade of the known vasodilator and trophic effect of bradykinin. Our results indicate that in intact kallikrein-kinin system is necessary for the normal functional development of the kidney.

Differential distribution of thyroid hormone receptor isoform in rat dorsal root ganglia and sciatic nerve in vivo and in vitro.

Using autoradiographic techniques carried out under precise conditions we previously demonstrated that both sensory neurons and peripheral glial cells in dorsal root ganglia (DRG) or sciatic nerve, possess specific [125I]-labeled T3 binding sites. Thyroid hormone receptors (TR) include several isoforms (TR alpha(1), TR alpha(2), TR beta(1), TR beta(2...)) The present study demonstrates that while sensory neurons and peripheral glial cells both possess functional TR, they express a differential expression of TR isoforms. Using a panel of antisera to specific for the TR alpha-common (alpha(1) and alpha(2)), TR alpha-1 or TR beta-1 isoforms, we detected TRs isoform localization at the cellular level during DRG and sciatic nerve development and regeneration. Immunohistochemical analysis revealed that during embryonic life, sensory neurons express TR alpha-common and TR beta-1 rather than TR alpha-1. The number of TR alpha-common and TR beta-1 positive neurons as well as the intensity of labeling increased during the first two postnatal weeks and remained more or less stable in adult life. TR alpha-1 immunoreactivity, which was undetectable in embryonic sensory neurons, became discreetly visible in neurons after birth. In developing DRG and sciatic nerves, Schwann cells exhibited TR alpha-common and TR alpha-1 rather than TR beta-1 immunolabeling. The appearance of TR alpha-common and alpha-1 isoform immunoreactivity in the sciatic nerve was restricted to a short period ranging from E17 up to two postnatal weeks. By comparing TR alpha-common and TR alpha-1 immunostaining we can deduce that Schwann cells primarily express TR alpha-1. Afterwards, in adult rat sciatic nerve TR alpha isoforms was no more detected. However transection of sciatic nerve caused a reexpression of TR alpha isoforms in degenerating nerve. The prevalence of TR alpha in Schwann cells in vivo was correlated with in vitro results. The differential expression of TR alpha and beta by sensory neurons and Schwann cells indicates that the feedback regulation of circulating thyroid hormone could occur by binding to either the alpha or beta TR isoforms. Moreover, the presence of multiple receptor isoforms in developing sensory neurons suggests that thyroid hormone uses multiple signaling pathways to regulate DRG and sciatic nerve development.

Severe childhood encephalopathy with dyskinesia and prolonged cognitive disturbances: evidence for anti-N-methyl-d-aspartate receptor encephalitis.

Aim We report four cases of acquired severe encephalopathy with massive hyperkinesia, marked neurological and cognitive regression, sleep disturbance, prolonged mutism, and a remarkably delayed recovery (time to full recovery between 5 and 18mo) with an overall good outcome, and its association with anti-N-methyl-d-aspartate (anti-NMDA) receptor antibodies. Method We reviewed the four cases retrospectively and we also reviewed the literature. Results Anti-NMDA receptor antibodies (without ovarian teratoma detected so far) were found in the two children tested in this study. Interpretation The clinical features are similar to those first reported in 1992 by Sebire et al.,(1) and rarely recognized since. Sleep disturbance was not emphasized as part of the disorder, but appears to be an important feature, whereas coma is less certain and difficult to evaluate in this setting. The combination of symptoms, evolution (mainly seizures at onset), severity, paucity of abnormal laboratory findings, very slow recovery, and difficult management justify its recognition as a specific entity. The neuropathological substrate may be anatomically close to that involved in encephalitis lethargica, in which the same target functions (sleep and movement) are affected but in reverse, with hypersomnolence and bradykinesia. This syndrome closely resembles anti-NMDA receptor encephalitis, which has been reported in adults and is often paraneoplastic.

Proline- and acidic amino acid-rich basic leucine zipper proteins modulate peroxisome proliferator-activated receptor alpha (PPAR alpha) activity.

In mammals, many aspects of metabolism are under circadian control. At least in part, this regulation is achieved by core-clock or clock-controlled transcription factors whose abundance and/or activity oscillate during the day. The clock-controlled proline- and acidic amino acid-rich domain basic leucine zipper proteins D-site-binding protein, thyrotroph embryonic factor, and hepatic leukemia factor have previously been shown to participate in the circadian control of xenobiotic detoxification in liver and other peripheral organs. Here we present genetic and biochemical evidence that the three proline- and acidic amino acid-rich basic leucine zipper proteins also play a key role in circadian lipid metabolism by influencing the rhythmic expression and activity of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα). Our results suggest that, in liver, D-site-binding protein, hepatic leukemia factor, and thyrotroph embryonic factor contribute to the circadian transcription of genes specifying acyl-CoA thioesterases, leading to a cyclic release of fatty acids from thioesters. In turn, the fatty acids act as ligands for PPARα, and the activated PPARα receptor then stimulates the transcription of genes encoding proteins involved in the uptake and/or metabolism of lipids, cholesterol, and glucose metabolism.

Expression and developmental regulation of Ehk-1, a neuronal Elk-like receptor tyrosine kinase in brain.

Protein tyrosine kinases are pivotal in central nervous tissue development and maintenance. Here we focus on the expression of Ehk-1, a novel Elk-related receptor tyrosine kinase. Ehk-1 gene expression is observed in the developing and adult central nervous system and is highly regulated throughout development at both the messenger RNA and protein levels. Three messenger RNA transcripts of 8.5, 5.9 and 5.1 kb are detectable in the rat brain and a variety of splice possibilities have been identified. However, a major protein species of around M(r) 120,000 predominates throughout development. Ehk-1 messenger RNA and protein levels are highest in the first postnatal week. By in situ messenger RNA hybridization the gene is expressed by all neurons of the adult brain, but mostly in the hippocampus, cerebral cortex and large neurons of the deep cerebellar nuclei, as well as the Purkinje and granular cells of the cerebellum. At earlier stages of development, transcripts are most prominent in the periventricular germinal layers of the brain. Immunohistochemistry reveals a pronounced membrane associated protein expression in immature neurons. In the adult animal, peak reactivity was found in the neuropil with sparing of most perikarya. The spatial and temporal pattern of ehk-1 gene expression suggests a role in both the development and maintenance of differentiated neurons of the central nervous system.

Ampakine CX546 bolsters energetic response of astrocytes: a novel target for cognitive-enhancing drugs acting as alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor modulators.

Glutamate was previously shown to enhance aerobic glycolysis i.e. increase glucose utilization and lactate production with no change in oxygen levels, in mouse cortical astrocytes by a mechanism involving glutamate uptake. It is reported here that a similar response is produced in both hippocampal and cerebellar astrocytes. Application of the cognitive-enhancing drug CX546 promoted further enhancement of glucose utilization by astrocytes from each brain area following glutamate exposure. alpha-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors represent the purported molecular target of cognitive-enhancing drugs such as CX546, and the presence of AMPA receptor subunits GluR1-4 was evidenced in astrocytes from all three regions by immunocytochemistry. AMPA itself did not stimulate aerobic glycolysis, but in the presence of CX546, a strong enhancement of glucose utilization and lactate production was obtained in cortical, hippocampal and cerebellar astrocytes. The effect of CX546 was concentration-dependent, with an EC(50) of 93.2 microm in cortical astrocytes. AMPA-induced glucose utilization in the presence of CX546 was prevented by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and the negative modulator GYKI 52466. In addition, the metabolic effect of CX546 in the presence of AMPA was mimicked by the AMPA receptor modulator cyclothiazide. Our data suggest that astrocyte energetics represents a novel target for cognitive-enhancing drugs acting as AMPA receptor modulators.

Amino acids of the alpha1B-adrenergic receptor involved in agonist binding: differences in docking catecholamines to receptor subtypes.

Site-directed mutagenesis and molecular dynamics analysis of the 3-D model of the alpha1B-adrenergic receptor (AR) were combined to identify the molecular determinants of the receptor involved in catecholamine binding. Our results indicate that the three conserved serines in the fifth transmembrane domain (TMD) of the alpha1B-AR play a distinct role in catecholamine binding versus receptor activation. In addition to the amino acids D125 in TMDIII and S207 in TMDV directly involved in ligand binding, our findings identify a large number of polar residues playing an important role in the activation process of the alpha1B-AR thus providing new insights into the structure/function relationship of G protein-coupled receptors.