The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development

The embryonic expression of COUP-TFII, an orphan nuclear receptor, suggests that it may participate in mesenchymal-epithelial interactions required for organogenesis. Targeted deletion of the COUP-TFII gene results in embryonic lethality with defects in angiogenesis and heart development. COUP-TFII mutants are defective in remodeling the primitive capillary plexus into large and small microcapillaries. In the COUP-TFII mutant heart, the atria and sinus venosus fail to develop past the primitive tube stage. Reciprocal interactions between the endothelium and the mesenchyme in the vascular system and heart are essential for normal development of these systems. In fact, the expression of Angiopoietin-1, a proangiogenic soluble factor thought to mediate the mesenchymal-endothelial interactions during heart development and vascular remodeling, is down-regulated in COUP-TFII mutants. This down-regulation suggests that COUP-TFII may be required for bidirectional signaling between the endothelial and mesenchymal compartments essential for proper angiogenesis and heart development.     

Novel domains of expression for orphan receptor tyrosine kinase Ror2 in the human and mouse reproductive system

Background: The noncanonical Wnt receptor and tyrosine kinase Ror2 has been associated with recessive Robinow syndrome (RRS) and dominant brachydactyly type B1. The phenotypes of mouse mutants implicate Ror2 in the development of the heart, lungs, bone, and craniofacial structures, which are affected in RRS. Following a recently identified role of Ror2 in the migration of mouse primordial germ cells, we extensively characterized its expression throughout the fetal internal reproductive system and the postnatal ductal system. Results: We show that Ror2 gene products are present in the germ cells and somatic cells of the testis and the ovary of both the mouse and human fetus. In reproductive tract structures, we find that Ror2 is expressed in the mesonephros, developing Wolffian and Müllerian ducts, and later in their derivatives, the epididymal epithelium and uterine epithelium. Conclusions: This study sets the stage to explore function for this tyrosine kinase receptor in novel regions of expression in the developing reproductive system in both mouse and human. Developmental Dynamics 243:1037–1045, 2014. © 2014 Wiley Periodicals, Inc.     

HepG2细胞中RORγ相互作用蛋白的筛选及鉴定

目的筛选HepG2细胞中转录因子RORγ(the retinoid-related orphan nuclear receptor gamma)相互作用蛋白,并对这些蛋白进行初步鉴定,为阐述RORγ介导调节HepG2细胞中各种生理学进程提供理论依据。方法从HepG2细胞中克隆RORγ基因并连接入载体pCeMM CTAP(SG)中形成RORγ-CTAP(SG)融合基因,再将其亚克隆入含嘌呤霉素抗性基因的载体质粒中,构建pMSCVpuro RORγ-CTAP(SG)质粒;稳定转染HepG2细胞并对RORγ-CTAP(SG)融合基因的表达和定位进行检测后,进行大规模扩增培养;用TAP(串联亲和纯化)方法捕获RORγ相互作用蛋白,通过银染找出差异性蛋白条带,质谱鉴定得到候选RORγ相互作用蛋白;用免疫共沉淀方法对候选蛋白RORγ相互作用蛋白进行验证鉴定。结果成功构建了质粒pMSCVpuro RORγ-CTAP(SG)并得到稳定转染HepG2细胞株,同时RORγ-CTAP(SG)融合基因能定位表达于细胞核内;通过TAP方法获得了RORγ蛋白复合物,然后通过串联质谱分析和数据库搜索从银染差异性显著蛋白条带中找到7个候选RORγ相互作用蛋白;用RORγ蛋白进行免疫共沉淀,对纯化出的7个RORγ相互作用蛋白分别检测,证实了RIP140,HSP90和RORγ在HepG2中有相互作用关系。结论筛选并鉴定了HepG2细胞中蛋白RORγ的相互作用蛋白RIP140和HSP90,这些研究发现支持了RORγ是共调解蛋白依赖的转录因子且以复合物形式行使功能的假说。其中,HSP90可能扮演分子伴侣角色,而RIP140在HepG2细胞中则可能充当RORγ蛋白的转录调控伙伴蛋白,具体机制有待进一步研究。     

Expression and retinoic acid regulation of the zebrafish nr2f orphan nuclear receptor genes

Background : The vertebrate nuclear receptor subfamily 2, group f (nr2f) genes encode orphan receptors that have the capacity to act as negative regulators of retinoic acid (RA) signaling. Results : We describe embryonic and larval expression of four of the six zebrafish nr2f genes, nr2f1a, nr2f1b, nr2f2, and nr2f5. These genes show highly regulated patterns of expression within the central nervous system, including in the developing hindbrain, as well as in the mesoderm and endoderm. We also investigated the role of RA and fibroblast growth factor (Fgf) signaling in regulating early nr2f gene expression. RA is not required for nr2f expression in the hindbrain; however, exogenous RA can repress this expression. Conversely, we find that RA positively regulates nr2f1a expression in trunk endoderm and mesoderm. Fgf signaling is not required for nr2f expression onset in the hindbrain; however, it may play a role in maintaining rhombomere‐specific expression. Conclusions : We report detailed expression analysis of four nr2f genes in all three germ layers. The onset of nr2f expression in the hindbrain does not require RA or Fgf signals. Our finding that RA positively regulates nr2f1a expression in the trunk supports the possibility that Nr2fs function in a negative feedback loop to modulate RA signaling in this region. Developmental Dynamics 241:1603–1615, 2012. © 2012 Wiley Periodicals, Inc.     

Fine mapping of a swine quantitative trait locus for number of vertebrae and analysis of an orphan nuclear receptor, germ cell nuclear factor (NR6A1)

The number of vertebrae in pigs varies and is associated with meat productivity. Wild boars, which are ancestors of domestic pigs, have 19 vertebrae. In comparison, European commercial breeds have 21-23 vertebrae, probably owing to selective breeding for enlargement of body size. We previously identified two quantitative trait loci (QTL) for the number of vertebrae on Sus scrofa chromosomes (SSC) 1 and 7. These QTL explained an increase of more than two vertebrae. Here, we performed a map-based study to define the QTL region on SSC1. By using three F2 experimental families, we performed interval mapping and recombination analyses and defined the QTL within a 1.9-cM interval. Then we analyzed the linkage disequilibrium of microsatellite markers in this interval and found that 10 adjacent markers in a 300-kb region were almost fixed in European commercial breeds. Genetic variation of the markers was observed in Asian local breeds or wild boars. This region encoded an orphan nuclear receptor, germ cell nuclear factor (NR6A1, formerly known as GCNF), which contained an amino acid substitution (Pro192Leu) coincident with the QTL. This substitution altered the binding activity of NR6A1 to its corepressors, nuclear receptor-associated protein 80 (RAP80) and nuclear receptor corepressor 1 (NCOR1). In addition, somites of mouse embryos demonstrated expression of NR6A1 protein. Together, these results suggest that NR6A1 is a strong candidate for one of the QTL that influence number of vertebrae in pigs.     

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.     

RORgammaT, a thymus-specific isoform of the orphan nuclear receptor RORgamma / TOR, is up-regulated by signaling through the pre-T cell receptor and binds to the TEA promoter

TEA (T early alpha) is a genetic element located upstream of the TCR-Jalpha cluster. Thymocytes from mice carrying a targeted deletion of TEA do not rearrange their TCRalpha locus on a window spanning the first nine Jalpha segments. This led us to the hypothesis of TEA having a "rearrangement focusing" activity on the 5' side of the TCR-Jalpha region. We analyzed DNAseI and "phylogenetic" footprints within the TEA promoter in an attempt to identify trans-acting factors that could account for its regulatory function on DNA accessibility. One of these footprints corresponded to a putative DNA-binding site for an orphan nuclear receptor of the ROR / RZR family. The RORgammaT cDNA clone was isolated from a thymus library using a probe corresponding to the DNA-binding domain of RORgamma / TOR. RORgammaT is a thymus-specific isoform of RORgamma, expressed almost exclusively in immature double-positive thymocytes. RORgammaT binds, to the TEA promoter in vitro. Lastly, the expression of RORgammaT is stimulated in two situations that mimic activation through the pre-TCR and in which the thymocytes have their TCR-alpha locus in an "open", yet unrearranged DNA configuration. We propose that the expression of RORgammaT may be part of the pre-TCR activation cascade leading to the maturation of alpha / beta T cells and may participate in the regulation of DNA accessibility in the TCR-Jalpha locus.     

News from the brain: the GPR124 orphan receptor directs brain-specific angiogenesis

During development, microvessels acquire specialized functions to meet the requirements of different tissues and organs. The vasculature of the brain constitutes one of the best examples of an organ-specific and highly specialized microvasculature, in which the endothelial cells that line blood vessels form an active permeability barrier and transport system called the blood-brain barrier (BBB); little is known, however, about the molecular mechanisms that instruct endothelial cells toward a BBB phenotype. Now Kuhnert et al. reveal that the orphan heterotrimeric GTP-binding protein-coupled receptor GPR124/TEM5 acts as an organ-specific regulator of brain angiogenesis, required for normal endothelial cell sprouting, migration, and expression of the BBB marker Glut-1 in the forebrain and neural tube. These findings add to our knowledge of brain vascularization and may open up possibilities for new therapeutic regimes to treat several diseases, including stroke, brain tumors, and vascular malformations.