LSDP5 is a PAT protein specifically expressed in fatty acid oxidizing tissues.

The PAT family (originally named for Perilipin, ADFP and TIP47) now includes four members: Perilipins, ADFP, TIP47 and S3-12. Significant primary sequence homology and the ability to associate with lipid storage droplets (LSDs) are well conserved within this family and across species. In this study, we have characterized a novel PAT protein, lipid storage droplet protein 5 (LSDP5) of 463 residues. A detailed sequence analysis of all murine PAT proteins reveals that LSDP5, TIP47 and ADFP share the highest order of sequence similarity, whereas perilipin and S3-12 have more divergent carboxyl- and amino-termini, respectively. Ectopically-expressed YFP-LSDP5 or flag-LSDP5 fusion proteins associate with LSDs. In accord with recent published data for perilipin, forced expression of LSDP5 in CHO cells inhibits lipolysis of intracellular LSDs. The LSDP5 gene is primarily transcribed in cells that actively oxidize fatty acids, such as heart, red muscle and liver. Expression of LSDP5 is stimulated by ligand activation of peroxisomal proliferator-activated receptor alpha (PPARalpha), and significantly reduced in liver and heart in the absence of this transcription factor. PPARalpha is generally required for regulation of fatty acid metabolism during fasting, but fasting induces LSDP5 mRNA in liver even in the absence of PPARalpha.

Functional conservation of interactions between a homeodomain cofactor and a mammalian FTZ-F1 homologue.

Nuclear receptors are master regulators of metazoan gene expression with crucial roles during development and in adult physiology. Fushi tarazu factor 1 (FTZ-F1) subfamily members are ancient orphan receptors with homologues from Drosophila to human that regulate diverse gene expression programs important for developmental processes, reproduction and cholesterol homeostasis in an apparently ligand-independent manner. Thus, developmental and tissue-specific cofactors may be particularly important in modulating the transcriptional activities of FTZ-F1 receptors. In Drosophila, the homeodomain protein Fushi tarazu acts as a cofactor for FTZ-F1 (NR5A3), leading to the hypothesis that a similar type of homeodomain cofactor-nuclear receptor relationship might exist in vertebrates. In this study, we have identified and characterized the homeodomain protein Prox1 as a co-repressor for liver receptor homologue 1 (LRH1/NR5A2), a master regulator of cholesterol homeostasis in mammals. Our study suggests that interactions between LRH1 and Prox1 may fulfil roles both during development of the enterohepatic system and in adult physiology of the liver.

Glucocorticoid response and promoter occupancy of the mouse LXRalpha gene.

The liver X receptors alpha and beta (LXRalpha and LXRbeta) are members of the nuclear receptor superfamily of proteins which are highly expressed in metabolically active tissues. They regulate gene expression of critical genes involved in cholesterol catabolism and transport, lipid and triglyceride biosynthesis, and carbohydrate metabolism in response to distinct oxysterol intermediates in the cholesterol metabolic pathway. Several LXR target genes have been identified, but there is limited information on how expression of the LXRs themselves is controlled. In this study we have characterized the upstream flanking region of the mouse LXRalpha gene. Transient transfections show that the LXRalpha promoter is able to drive transcription of a luciferase reporter gene, however, the transcriptional potential of the promoter in the cell lines used was low. The -2143 to -1513 region of the promoter mediates repression of reporter gene activity in all cells analyzed and multiple DNA-protein interactions were detected in this region by DNase I footprinting. The Zta, Ets, and Hes1 transcription factors were all shown to mediate alterations in reporter gene activity driven by LXRalpha promoter deletion constructs. These factors have been linked to cell cycle and differentiation processes suggesting that expression of LXRalpha might be under control of signalling mechanisms regulating cell proliferation. Several putative binding sites of the glucocorticoid receptor (GR) were identified in the LXRalpha promoter and transient cotransfections of the GR and LXRalpha promoter deletion constructs induced reporter gene activity. Addition of dexamethasone, a GR agonist, abolished this effect suggesting cross talk between GR and LXR signalling.

Inactivation of the nuclear receptor coactivator RAP250 in mice results in placental vascular dysfunction.

Coactivators constitute a diverse group of proteins that are essential for optimal transcriptional activity of nuclear receptors. In the past few years many coactivators have been identified but it is still unclear whether these proteins interact indiscriminately with all nuclear receptors and whether there is some redundancy in their functions. We have previously cloned and characterized RAP250 (ASC-2/PRIP/TRBP/NRC), an LXXLL-containing coactivator for nuclear receptors. In order to study its biological role, Rap250 null mice were generated by gene targeting. Here we show that genetic disruption of Rap250 results in embryonic lethality at embryonic day (E) 13.5. Histological examination of placentas revealed a dramatically reduced spongiotrophoblast layer, a collapse of blood vessels in the region bordering the spongiotrophoblast, and labyrinthine layers in placentas from Rap250(-/-) embryos. These findings suggest that the lethality of Rap250(-/-) embryos is the result of obstructed placental blood circulation. Moreover, the transcriptional activity of PPAR gamma is reduced in fibroblasts derived from Rap250(-/-) embryos, suggesting that RAP250 is an essential coactivator for this nuclear receptor in the placenta. Our results demonstrate that RAP250 is necessary for placental development and thus essential for embryonic development.

Different regulation of the LXRalpha promoter activity by isoforms of CCAAT/enhancer-binding proteins.

LXRs have recently been shown to regulate key enzymes in cholesterol degradation, reverse transport of cholesterol from peripheral cells, cholesterol uptake and lipogenesis. The LXRalpha promoter was thus studied to investigate if LXRalpha gene expression is under the regulation of transcription factors involved in adipogenesis. We report that the C/EBP transcription factor interacts with the promoter of the LXRalpha gene. In in vitro footprinting experiments, protein extracts from several tissues gave footprints covering a putative C/EBP recognition site. Transfection experiments and EMSA showed a direct effect of these transcription factors on the LXRalpha promoter. C/EBPalpha upregulated expression of the reporter gene in an NIH 3T3-L1 preadipocyte cell line, while C/EBPbeta and C/EBPdelta had no effect. In liver hepatoma Fao II and Cos-7 kidney cells, both C/EBPalpha and C/EBPbeta downregulated expression of the reporter gene while C/EBPdelta induced activity, indicating that the functional consequences of C/EBP isoform interactions with the LXRalpha promoter are dependent on the cellular context. Monitoring of the LXR mRNA levels during adipose tissue differentiation showed that LXRbeta is constitutively expressed during the entire differentiation process while LXRalpha is induced upon addition of differentiation mix.

Inhibition of androgen receptor (AR) function by the reproductive orphan nuclear receptor DAX-1.

DAX-1 (NROB1) is an atypical member of the nuclear receptor family that is predominantly expressed in mammalian reproductive tissues. While a receptor function of DAX-1 remains enigmatic, previous work has indicated that DAX-1 inhibits the activity of the orphan receptor steroidogenic factor 1 and the estrogen receptors (ERs), presumably via direct occupation of the coactivator-binding surface and subsequent recruitment of additional corepressors. In vivo evidence points at a particular role of DAX-1 for the development and maintenance of male reproductive functions. In this study, we have identified the androgen receptor (AR) NR3C4 as a novel target for DAX-1. We show that DAX-1 potently inhibits ligand-dependent transcriptional activation as well as the interaction between the N- and C-terminal activation domains of AR. We provide evidence for direct interactions of the two receptors that involve the N-terminal repeat domain of DAX-1 and the C-terminal ligand-binding and activation domain of AR. Moreover, DAX-1, known to shuttle between the cytoplasm and the nucleus, is capable of relocalizing AR in both cellular compartments, suggesting that intracellular tethering is associated with DAX-1 inhibition. These results implicate novel inhibitory mechanisms of DAX-1 action with particular relevance for the modulation of androgen-dependent gene transcription in the male reproductive system.