Structural Perspective of NR4A Nuclear Receptor Family and Their Potential Endogenous Ligands.

There are 48 nuclear receptors in the human genome, and many members of this superfamily have been implicated in human diseases. The NR4A nuclear receptor family consisting of three members, NR4A1, NR4A2, and NR4A3 (formerly annotated as Nur77, Nurr1, and NOR1, respectively), are still orphan receptors but exert pathological effects on immune-related and neurological diseases. We previously reported that prostaglandin A1 (PGA1) and prostaglandin A2 (PGA2) are potent activators of NR4A3, which bind directly to the ligand-binding domain (LBD) of the receptor. Recently, the co-crystallographic structures of NR4A2-LBD bound to PGA1 and PGA2 were reported, followed by reports of the neuroprotective effects of these possible endogenous ligands in mouse models of Parkinson's disease. Based on these structures, we modeled the binding structures of the other two members (NR4A1 and NR4A3) with these potential endogenous ligands using a template-based modeling method, and reviewed the similarity and diversity of ligand-binding mechanisms in the nuclear receptor family.

New Studies of Pathogenesis of Rheumatoid Arthritis with Collagen-Induced and Collagen Antibody-Induced Arthritis Models: New Insight Involving Bacteria Flora.

Much public research suggests that autoimmune diseases such as rheumatoid arthritis (RA) are induced by aberrant "self" immune responses attacking autologous tissues and organ components. However, recent studies have reported that autoimmune diseases may be triggered by dysbiotic composition changes of the intestinal bacteria and an imbalance between these bacteria and intestinal immune systems. However, there are a few solid concepts or methods to study the putative involvement and relationship of these inner environmental factors in RA pathogenesis. Fortunately, Collagen-Induced Arthritis (CIA) and Collagen Antibody-Induced Arthritis (CAIA) models have been widely used as animal models for studying the pathogenesis of RA. In addition to RA, these models can be extensively used as animal models for studying complicated hypotheses in many diseases. In this review, we introduce some basic information about the CIA and CAIA models as well as how to apply these models effectively to investigate relationships between the pathogenesis of autoimmune diseases, especially RA, and the dysbiosis of intestinal bacterial flora.

Chemotactic responses of peripheral blood eosinophils to prostaglandin D2 in atopic keratoconjunctivitis.

BACKGROUND: Eosinophils appear to be key cells in the pathogenesis of conjunctival inflammation in atopic keratoconjunctivitis (AKC). Chemoattractant receptor homologous molecule expressed on TH2 cells (CRTH2) mediates prostaglandin D2 (PGD2)-dependent migration of eosinophils. However, it is unclear whether CRTH2/PGD2-dependent eosinophil migration is upregulated in allergic diseases. OBJECTIVE: To compare the chemotactic responses of peripheral blood eosinophils to prostaglandin D2 in patients with severe AKC and healthy individuals. METHODS: We used an enzyme immunoassay system to measure PGD2 levels in tears and blood samples from healthy individuals and patients with AKC. CRTH2 expression on peripheral blood eosinophils was determined using reverse-transcriptase polymerase chain reaction (RT-PCR), flow cytometry, and an oligonucleotide array system. Chemotaxis experiments were performed using a modified Boyden chamber technique and an optical assay system. RESULTS: The PGD2 concentrations were higher in tears from patients with severe AKC compared with healthy individuals. RT-PCR (severe and mild cases), flow cytometry (mild cases), and GeneChip analyses revealed a significantly higher expression of CRTH2 on peripheral blood eosinophils in patients with AKC than in healthy individuals. PGD2 and its stable metabolite 13,14-dihydro-15-keto-PGD2, a CRTH2 agonist, induced chemotaxis of eosinophils from patients with AKC; chemotaxis was significantly enhanced in eosinophils from patients with severe AKC compared with those from healthy individuals. CONCLUSION: CRTH2 is more abundantly expressed on eosinophils from patients with AKC and promoted PGD2-dependent migration to a greater extent than in healthy individuals.

The NR4A nuclear receptor family in eosinophils.

It is well-known that many members of the family of nuclear receptors have been implicated in human diseases, and metabolic disorders in particular. The NR4A nuclear receptor family consists of three members, Nur77, Nurr1, and NOR1. All of these are orphan receptors, and Nur77 and NOR1 exert possible pathological roles in immune diseases through the modulation of leukocyte functions. CD30 stimulation, which induces eosinophil-specific apoptosis, markedly enhances expression of Nur77 and NOR1 in eosinophils. This suggests the possibility of pharmacological modulation of Nur77- or NOR1-specific apoptotic pathways via receptor-dependent transactivation. In this review, we discuss treatment of allergic diseases by low molecular weight compounds acting through the NR4A receptor family to cause eosinophil apoptosis. NR4A nuclear receptor genes were selected following comprehensive analysis of differentially expressed genes in eosinophils of atopic dermatitis patients compared with healthy volunteers.

Prostaglandin A2 acts as a transactivator for NOR1 (NR4A3) within the nuclear receptor superfamily.

Within the nuclear receptor superfamily, Nur77, Nurr1, and NOR1 constitute the nuclear receptor subfamily 4 group A. Modulation of NOR1 function would be therapeutic potential for diseases related to dysfunction of NOR1, including extraskeletal myxoid chondrosarcoma and autoimmune diseases. By screening arachidonate metabolites for their capacity of transcriptional activation, we have identified prostaglandin (PG) A2 as a transactivator for NOR1. PGA2 acted as a potent activator of NOR1-dependent transcription through the GAL4-based reporter system. The putative ligand-binding domain (LBD) of the receptor directly bound PGA2, and LBD-deleted receptor showed little transcriptional activation by PGA2. Primary cultured spleen cells derived from transgenic mice overexpressing NOR1, showed higher sensitivity to PGA2 compared to those from wild-type mice. These observations suggest that PGA2 can serve as a transactivator of NOR1, and thus suggest a possibility of pharmacological modulation of the NOR1 pathways by PGA2-related compounds.

NR4A orphan nuclear receptor family in peripheral blood eosinophils from patients with atopic dermatitis and apoptotic eosinophils in vitro.

To identify novel genes related to the clinical signs of atopic dermatitis (AD), differentially expressed genes were sought in peripheral blood eosinophils from both AD patients and healthy volunteers. RNA was prepared from eosinophils, expression of various genes was monitored using the Affymetrix GeneChip, and expression was quantified by real-time RT-PCR. Two genes, Nur77 and NOR1, members of NR4A orphan nuclear receptor family, were expressed at a significantly higher level in AD patients than in healthy volunteers. Expression of another gene in the NR4A receptor family, Nurr1, was also higher in AD patients than in healthy volunteers. When peripheral blood leukocytes from healthy volunteers were fractionated, NOR1 expression was highest in eosinophils, but expression of Nur77 and Nurr1 genes was not eosinophil-specific. Extremely intense apoptosis was induced in both eosinophils and an eosinophil cell line, AML14.3D10, by treatment with antibody (Ab) to both CD30 and Fas. Rapid expression of the genes for the NR4A receptor family was observed with anti-CD30 Ab treatment but not with anti-Fas Ab. The NR4A orphan nuclear receptor family gene expression and the subsequent eosinophil apoptosis were downregulated by the MAPK inhibitor, U0126. These results suggest that the expression of the NR4A receptor family genes through CD30 signaling may regulate eosinophil apoptosis in allergic conditions such as AD.

Expression of a human SOCS protein, HSOCP-1, in peripheral blood eosinophils from patients with atopic dermatitis.

To identify new genes related to atopic dermatitis (AD), we screened for differentially expressed genes in peripheral blood eosinophils derived from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils obtained from both AD patients and healthy volunteers, and the expression of various genes was monitored using fluorescent differential display and real-time RT-PCR. One of the expressed sequence tags (ESTs) was expressed at a significantly higher level in AD patients than in healthy volunteers. A full-length cDNA was identified that encoded a human suppressor of cytokine signaling (SOCS) protein, HSOCP-1, also named hASB-8. The expression of HSOCP-1 was increased in cultured peripheral blood eosinophils after IL-4 stimulation, and overexpression of HSOCP-1 caused cell death in an eosinophil cell line, AML14.3D10. p34(SEI-1) was identified as a HSOCP-1-interacting protein by a yeast two-hybrid system. It is a protein that also interacts with the cyclin-dependent kinase inhibitor p16(INK4), suggesting that HSOCP-1 is involved in cell cycle control and apoptosis.

Analysis of gene expression in peripheral blood eosinophils from patients with atopic dermatitis by differential display.

To identify the genes related to atopic dermatitis (AD), we compared gene expression in eosinophils from AD patients and healthy volunteers. RNA was prepared from peripheral blood eosinophils. Gene expression was monitored by fluorescent differential display (DD) and real-time RT-PCR. Eighteen new sequences, including expressed sequence tags (ESTs), were expressed at higher levels in eosinophils from AD patients than in those from healthy volunteers. The functions of most of these genes are unknown. We found no correlation between the expression of a particular gene and clinical markers such as the number of eosinophils and the amount of IgE. Multivariate analysis of the gene expression data in each sample showed a very high coefficient of correlation among the copy numbers of each gene. The genes under investigation were also expressed in cultured blood eosinophils after IL-4, IL-5 and IFN-gamma stimulation. We were able to predict the function of some of the sequences by scanning for homologies within either the human or mouse genome databases. The mouse counterpart of one of these genes, intersectin 2, was expressed dramatically, as measured by ear edema, in 1-fluoro-2,4-dinitrobenzene-induced mouse contact dermatitis and in NC/Nga mouse dermatitis.

Gene expression analysis of atopic dermatitis-like skin lesions induced in NC/Nga mice by mite antigen stimulation under specific pathogen-free conditions.

BACKGROUND: Atopic dermatitis (AD) is a chronic relapsing inflammation characterized by pruritic and eczematous skin lesions usually observed in patients with a familial history of atopic diseases, but its exact etiology is unclear. An animal model is indispensable for the analysis of the pathogenesis and the development of new drugs to treat this disease. Here, we compare changes in gene expression profiles in the AD-like skin lesions of NC/Nga or BALB/c mice stimulated intradermally by mite antigen under specific pathogen-free (SPF) conditions. METHODS: Mite Extract-Dp was injected intradermally into the right and left pinnae and into the skin of the back of NC/Nga or BALB/c mice in 2 places once per 3 days, and the clinical symptoms and the ear thickness were measured. On day 14 or day 28 after starting mite extract injection, we collected plasma and pinnae from NC/Nga or BALB/c mice. The amount of total immunoglobulin E (IgE) in plasma was assayed. We analyzed mRNA transcripts in pinnae using real-time quantitative PCR for the murine counterparts of several known allergy-related genes. Moreover, genome-wide gene expression in pinnae from NC/Nga mice was analyzed using high-density oligonucleotide arrays (GeneChip, Affymetrix). RESULTS: From 2 weeks after stimulation, marked skin inflammation was induced in the pinnae of NC/Nga but not BALB/c mice. However, IgE levels in sera rose equally in both strains. Quantitative PCR analysis and comprehensive GeneChip analysis of the AD-like pinna skin lesions revealed that their development was accompanied by changes in expression of more than 1,000 genes. These included cytokines, cytokine receptors, proteases, and adhesion molecules. Furthermore, genes thus far not reported in association with AD were also affected. CONCLUSION: From these results, the NC/Nga mouse model using mite sensitization under SPF conditions could be useful for elucidating the mechanisms of AD pathogenesis and developing more effective therapy for AD.

Cloning and characterization of the highly expressed ETEA gene from blood cells of atopic dermatitis patients.

Analysis of patients with atopic dermatitis (AD) for differential expression of genes, as compared to normal individuals, will be useful for understanding the molecular pathogenesis of AD. We found that the expression of the gene ETEA in human peripheral blood CD3-positive cells from patients with atopic dermatitis was significantly higher than in normal individuals. Eosinophils from AD patients expressed ETEA at a significantly higher level than the healthy controls. The overall sequence of the 445 aa deduced polypeptide from the cloned ETEA cDNA showed homology to human Fas-associated factor 1 (FAF1), which is involved in Fas-mediated apoptosis. However, the interaction of ETEA with the Fas death domain was weaker than that of FAF1, as studied in yeast two-hybrid experiments. The ETEA-EGFP fusion protein was expressed in cytoplasm. During the course of activation-induced cell death of primary T cells, transcription levels of ETEA and FAF1 were upregulated with similar kinetics. The enhanced expression of ETEA may play a role in the regulating the resistance to apoptosis that is observed in T cells and eosinophils of AD patients.