Retinoic acid receptor-related orphan receptors (RORs) regulate a variety of physiological processes including hepatic gluconeogenesis, lipid metabolism, circadian rhythm, and immune function. Here we present the first high-affinity synthetic ligand for both ROR␣ and ROR␥. In a screen against all 48 human nuclear receptors, the benzenesulfonamide liver X receptor (LXR) -benzenesulfonamide (T0901317) inhibited transactivation activity of ROR␣ and ROR␥ but not ROR␤. T0901317 was found to directly bind to ROR␣ and ROR␥ with high affinity (K i ϭ 132 and 51 nM, respectively), resulting in the modulation of the receptor's ability to interact with transcriptional cofactor proteins. T0901317 repressed ROR␣/␥-dependent transactivation of ROR-responsive reporter genes and in HepG2 cells reduced recruitment of steroid receptor coactivator-2 by ROR␣ at an endogenous ROR target gene (G6Pase). Using small interference RNA, we demonstrate that repression of the gluconeogenic enzyme glucose-6-phosphatase in HepG2 cells by T0901317 is ROR-dependent and is not due to the compound's LXR activity. In summary, T0901317 represents a novel chemical probe to examine ROR␣/␥ function and an excellent starting point for the development of ROR selective modulators. More importantly, our results demonstrate that small molecules can be used to target the RORs for therapeutic intervention in metabolic and immune disorders.

/ajplung. 00302.2001.-The purpose of this study was to identify culture conditions for maintenance of isolated mouse type II cells with intact surfactant protein (SP) and phospholipid production. Type II cells were isolated from 6-wk-old mice and cultured on Matrigel matrix-rat tail collagen (70:30 vol/ vol) in bronchial epithelial cell growth medium minus hydrocortisone plus 5% charcoal-stripped FBS and 10 ng/ml keratinocyte growth factor. Under these conditions, type II cells actively produced surfactant phospholipids and proteins for at least 7 days. Synthesis and secretion of surfactant phospholipids and SP-A, -B, -C, and -D declined on day 1 of culture but recovered by day 3, reaching levels comparable to or exceeding freshly isolated cells by day 5. Abundant lamellar bodies were readily apparent in cells examined on days 5 and 7, and a surfactant pellet was recovered by centrifugation of media harvested on each day of culture. Secretion of SP-B, SP-C, and phosphatidylcholine was stimulated by phorbol 12-myristate 13-acetate and was inhibited by compound 48/80. When tested with a bubble surfactometer, surfactant secreted by type II cells on day 5 of culture lowered surface tension to 5.2 Ϯ 2.3 mN/m. This is the first description of the synthesis and secretion of a functional surfactant complex by mouse type II cells after 7 days in primary culture.surfactant; secretion; lung ISOLATED ALVEOLAR type II cells in primary culture have provided insight into the function of this important cell type in the lung. However, the rapid loss of the type II cell phenotype has limited the usefulness of this system. Manipulation of culture substratum and media has identified conditions that support the synthesis of surfactant proteins (SPs) and phospholipids in primary cultures of rat type II cells. Key substratum components include elements of extracellular matrix, such as the basement membrane extracted from EngelbrethHolm-Swarm (EHS) tumor (commercially available as Matrigel), which contains laminin, type IV collagen, and heparin sulfate proteoglycan (24, 26). Interaction of type II cells with extracellular matrix is thought to promote a native cuboidal cell shape, which is important for type II cell function in vitro (25,27). Keratinocyte growth factor (KGF; fibroblast growth factor-7) has been identified as a critical component of the culture medium, which likely reflects the importance of epithelial-mesenchymal interactions in vivo (28,36). In addition to effects of media and substratum, the culture of type II cells at an air-liquid interface (by limiting the amount of apical medium and rocking the culture dish) has also been shown to enhance maintenance of the rat type II cell phenotype in vitro (7,35).Although considerable advances have been made in optimizing culture conditions for rat type II cells, comparable progress for mouse type II cell culture is lacking. The development of such a culture system is important, since it would allow the study of type II cells from a large number of transgenic mouse lines in whi...

Signal transduction pathways often use a transcriptional component to mediate adaptive cellular responses. Coactivator proteins function prominently in these pathways as the conduit to the basic transcriptional machinery. Here we present a high-throughput cell-based screening strategy, termed the ''coactivator trap,'' to study the functional interactions of coactivators with transcription factors. We applied this strategy to the cAMP signaling pathway, which utilizes two families of coactivators, the cAMP response element binding protein (CREB) binding protein (CBP)/p300 family and the recently identified transducers of regulated CREB activity family (TORCs1-3). In addition to identifying numerous known interactions of these coactivators, this analysis identified NONO (p54 nrb ) as a TORC-interacting protein. RNA interference experiments demonstrate that NONO is necessary for cAMP-dependent activation of CREB target genes in vivo. Furthermore, TORC2 and NONO complex on cAMP-responsive promoters, and NONO acts as a bridge between the CREB/TORC complex and RNA polymerase II. These data demonstrate the utility of the coactivator trap by identification of a component of cAMP-mediated transcription.transcription ͉ signal transduction ͉ cell-based screen ͉ RNA polymerase II ͉ transducer of regulated cAMP response element-binding protein T ranscription is regulated by large multisubunit complexes that can be grouped into three general categories; DNA binding proteins, coregulators (coactivators and corepressors), and basal transcriptional components. DNA binding proteins recognize discrete sequences or response elements within promoters and in general function as scaffolds that direct the recruitment of coregulatory proteins. Coregulators in turn function as a conduit to the basic transcriptional machinery. Coregulators may also influence gene expression via intrinsic enzymatic activity or by recruitment of other enzyme activities (e.g., acetylation, methylation, poly ADPribosylation, ubiquitination, sumoylation, or ATP-dependent remodeling complexes) capable of modifying both transcriptional proteins and chromatin (1). Thus, determining the interaction networks of coregulators recruited by transcription factors and the accompanying enzymatic activities is necessary for understanding the complexities of gene expression.The cAMP signal-transduction pathway activates transcription by stimulating interactions between cAMP response element binding protein (CREB) and two coactivator families, CREBbinding protein (CBP)/p300 and transducers of regulated CREB (TORCs) (2-4). CREB-CBP/p300 interaction occurs when elevations in intracellular cAMP liberate protein kinase A (PKA) catalytic subunits (PKA c ) from PKA regulatory subunits. PKA c directly phosphorylates serine 133 in the kinase-inducible domain of CREB, increasing the affinity of CBP/p300 for CREB (5, 6). CBP/p300 interacts with components of the RNA polymerase II (RNA pol II) complex to facilitate transcription and contains intrinsic acetyltransferase activity specula...

Aneuploid genomes, characterized by unbalanced chromosome stoichiometry (karyotype), are associated with cancer malignancy and drug-resistance of pathogenic fungi. The phenotypic diversity resulting from karyotypic diversity endows the cell population superior adaptability. We show here, using a combination of experimental data and a general statistical model, that the degree of phenotypic variation, thus evolvability, escalates with the degree of overall growth suppression. Such scaling likely explains the challenge of treating aneuploidy diseases with a single stress-inducing agent. Instead, we propose the design of an “evolutionary trap” (ET) targeting both karyotypic diversity and fitness. This strategy entails a selective condition “channeling” a karyotypically divergent population into one with a predominant and predictably-drugable karyotypic feature. We provide a proof-of-principle case in budding yeast and demonstrate the potential efficacy of this strategy toward aneuploidy-based azole resistance in Candida albicans. By analyzing existing pharmacogenomics data, we propose the potential design of an ET against glioblastoma.

CC chemokine receptor-3 (CCR-3) is a major receptor involved in regulating eosinophil trafficking; therefore, elucidation of ligand-induced CCR-3 events has important implications in understanding the biological and pathological properties of eosinophils. Previous studies have demonstrated that unique receptor events occur in different cell types supporting investigation of CCR-3-mediated events in eosinophilic cells. We now report biochemical characterization of CCR-3 internalization following exposure of eosinophils to CCR-3 ligands. Treatment of freshly isolated human eosinophils with CCR-3 ligands resulted in marked and differential internalization of CCR-3 in a dose-dependent manner. Exposure to 100 ng/ml eotaxin reduced surface expression to 43, 43, and 76% at 15 min, 1 h, and 3 h, respectively. RANTES (reduced on activation T cell expressed and secreted) treatment induced more significant and prolonged internalization of CCR-3 than eotaxin; following 100 ng/ml of RANTES, 29, 24, and 47% of the receptor was expressed at 15 min, 3 h, and 18 h, respectively. Confocal microscopy demonstrated that receptor modulation involved receptor internalization by an endocytic pathway shared with the transferrin receptor. Receptor internalization was accompanied by partial degradation of CCR-3, and reexpression of CCR-3 was dependent in part upon de novo protein synthesis. Internalization was not blocked by pretreatment of eosinophils with pertussis toxin. Furthermore, staurosporine did not inhibit internalization although it blocked phorbol 12-myristate 13-acetate-induced CCR-3 down-modulation. These results demonstrate that CCR-3 ligands induce differential receptor internalization that is not dependent upon G i -protein coupling, calcium transients, or protein kinase C.