To identify the physiological functions of the retinoid-related orphan receptor ␥ (ROR␥), a member of the nuclear receptor superfamily, mice deficient in ROR␥ function were generated by targeted disruption. ROR␥ ؊/؊ mice lack peripheral and mesenteric lymph nodes and Peyer's patches, indicating that ROR␥ expression is indispensable for lymph node organogenesis. Although the spleen is enlarged, its architecture is normal. The number of peripheral blood CD3 ؉ and CD4 ؉ lymphocytes is reduced 6-and 10-fold, respectively, whereas the number of circulating B cells is normal. The thymus of ROR␥ ؊/؊ mice contains 74.4% ؎ 8.9% fewer thymocytes than that of wild-type mice. Flow cytometric analysis showed a decrease in the CD4 ؉ CD8 ؉ subpopulation. T he nuclear hormone receptor superfamily consists of structurally related, ligand-dependent transcription factors and a large number of orphan receptors for which the ligand has not yet been identified (1, 2). Nuclear receptors share a common modular structure composed of several domains that have functions in DNA binding, ligand binding, nuclear localization, dimerization, repression, and transactivation (3). Typically, the transactivation activity of nuclear receptors is controlled by small lipophilic molecules that bind to the receptor, thereby causing a conformational change in the receptor. This change in conformation causes dissociation of corepressor complexes and promotes interaction of the receptor with coactivators (4). The latter leads then to increased gene expression and consequently modulation of many physiological processes.The retinoid-related orphan receptors ROR␣, -␤, and -␥ constitute a subfamily of nuclear orphan receptors (5-11). Each of these receptors binds as a monomer to response elements (ROREs) consisting of the consensus core motif AGGTCA preceded by an A͞T-rich region (7, 12). RORs have been reported to play critical roles in a wide variety of biological processes (13,14). ROR␥ is highly expressed in thymus, kidney, liver, muscle, and brown fat but not in white fat tissue (6,7,10,15). In the thymus, two isoforms, ␥1 and ␥2 (also named ROR␥T), have been identified (15). The ␥2 differs from the ␥1 isoform in that it lacks the amino terminus of ␥1. The expression of the ␥2 isoform is highly restricted to the double-positive thymocytes, suggesting a regulatory role for ROR␥2 in these cells (15, 16). Recently, overexpression of ROR␥ in T-cell hybridomas has been shown to inhibit T cell antigen receptor (TCR)-activation-induced apoptosis by repressing the induction of Fas ligand (FasL) (15) (M.S., S.K., and A.M.J., unpublished observations).To investigate the biological role(s) of ROR␥ in vivo, we used homologous recombination in embryonic stem cells to generate mice in which the ROR␥ gene was disrupted. In this study, we show that ROR␥ Ϫ/Ϫ mice lack lymph nodes and Peyer's patches, suggesting that ROR␥ is essential for their development. In addition, we demonstrate that ROR␥ plays a critical role in thymopoiesis and T cell homeostasis. The rapid induct...

Objective-There has been accumulating evidence demonstrating that activators for peroxisome proliferator-activated receptor ␣ (PPAR␣) have antiinflammatory, antiatherogenic, and vasodilatory effects. We hypothesized that PPAR␣ activators can modulate endothelial nitric oxide synthase (eNOS) expression and its activity in cultured vascular endothelial cells. Methods and Results-Bovine aortic endothelial cells were treated with the PPAR␣ activator fenofibrate. The amount of eNOS activity and the expression of eNOS protein and its mRNA were determined. Our data show that treatment with fenofibrate for 48 hours resulted in an increase in eNOS activity. Fenofibrate failed to increase eNOS activity within 1 hour. Fenofibrate also increased eNOS protein as well as its mRNA levels. RU486, which has been shown to antagonize PPAR␣ action, inhibited the fenofibrate-induced upregulation of eNOS protein expression. WY14643 and bezafibrate also increased eNOS protein levels, whereas rosiglitazone did not. Transient transfection experiments using human eNOS promoter construct showed that fenofibrate failed to enhance eNOS promoter activity. Actinomycin D studies demonstrated that the half-life of eNOS mRNA increased with fenofibrate treatment. Conclusions-PPAR␣ activators upregulate eNOS expression, mainly through mechanisms of stabilizing eNOS mRNA. This is a new observation to explain one of the mechanisms of PPAR␣-mediated cardiovascular protection. Key Words: atherosclerosis Ⅲ endothelium Ⅲ nitric oxide Ⅲ vascular biology Ⅲ vasodilatation H ypolipidemic fibrates are pharmacological compounds that activate peroxisome proliferator-activated receptor ␣ (PPAR␣), a member of the nuclear hormone receptor superfamily. 1 These fibrates have been widely used as effective drugs lowering serum triglycerides and low-density lipoprotein cholesterol and raising high-density lipoprotein cholesterol. 2 There has been accumulating evidence showing that fibrates have favorable effects of slowing the progression of atherosclerosis and reducing the number of events of coronary heart diseases in high-risk patients. 3-5 PPAR␣ is known to be expressed in the liver, which is mainly involved in lipid and lipoprotein metabolism exerted by fibrates. 1 In addition, recent studies have shown that PPAR␣ is also expressed in the cardiovascular system, including heart and vascular wall component cells such as vascular endothelial, vascular smooth muscle, and monocyte cells, and performs a direct antiatherogenic and antiinflammatory action. 6 Staels et al have shown that PPAR␣ ligands inhibit interleukin (IL)-1-induced expression of IL-6, prostaglandin, and cyclooxygenase 2 in aortic smooth muscle cells. 7 These authors further showed that patients receiving fenofibrate, a potent fibrate, had lower plasma C-reactive protein, fibrinogen, and IL-6 concentrations. 7 Furthermore, it has been demonstrated that PPAR␣ activators inhibit cytokine-induced vascular cell adhesion molecule-1 (VCAM-1), 8,9 and thrombin-induced endothelin-1 expression 10 in vascular en...

In this study, we describe the characterization of a gene encoding a novel Krü ppel-like protein, named Glis2. Glis2 encodes a relatively proline-rich, basic 55.8-kDa protein. Its five tandem Cys 2 -His 2 zinc finger motifs exhibit the highest homology to those of members of the Gli and Zic subfamilies of Krü ppel-like proteins. Confocal microscopic analysis demonstrated that Glis2 localizes to the nucleus. Analysis of the genomic structure of the Glis2 gene showed that it is composed of 6 exons separated by 5 introns spanning a genomic region of more than 7.5 kb. Fluorescence in situ hybridization mapped the mouse Glis2 gene to chromosome 16A3-B1. Northern blot analysis showed that the Glis2 gene encodes a 3.8-kb transcript that is most abundant in adult mouse kidney. By in situ hybridization, expression was localized to somites and neural tube, and during metanephric development predominantly to the ureteric bud, precursor of the collecting duct, and inductor of nephronic tubule formation. One-hybrid analysis using Glis2 deletion mutants identified a novel activation function (AF) at the N terminus. The activation of transcription through this AF domain was totally suppressed by two repressor functions just downstream from the AF. One of the repressor functions is contained within the first zinc finger motif. The level of transcriptional activation and repression varied with the cell line tested, which might be due to differences in cell typespecific expression of co-activators and co-repressors. Our results suggest that Glis2 behaves as a bifunctional transcriptional regulator. Both the activation and repressor functions may play an important role in the regulation of gene expression during embryonic development.