Hepatic cholesterol metabolism and resistance to dietary cholesterol in LXRbeta-deficient mice.

The nuclear oxysterol-receptor paralogues LXRalpha and LXRbeta share a high degree of amino acid identity and bind endogenous oxysterol ligands with similar affinities. While LXRalpha has been established as an important regulator of cholesterol catabolism in cholesterol-fed mice, little is known about the function of LXRbeta in vivo. We have generated mouse lines with targeted disruptions of each of these LXR receptors and have compared their responses to dietary cholesterol. Serum and hepatic cholesterol levels and lipoprotein profiles of cholesterol-fed animals revealed no significant differences between LXRbeta(-/-) and wild-type mice. Steady-state mRNA levels of 3-hydroxy-3-methylglutaryl coenzyme A reductase, farnesyl diphosphate synthase, and squalene synthase were increased in LXRbeta(-/-) mice compared with LXRbeta(+/+) mice, when fed standard chow. The mRNA levels for cholesterol 7alpha-hydroxylase, oxysterol 7alpha-hydroxylase, sterol 12alpha-hydroxylase, and sterol 27-hydroxylase, respectively, were comparable in these strains, both on standard and 2% cholesterol chow. Our results indicate that LXRbeta(-/-) mice - in contrast to LXRalpha(-/-) mice - maintain their resistance to dietary cholesterol, despite subtle effects on the expression of genes coding for enzymes involved in lipid metabolism. Thus, our data indicate that LXRbeta has no complete overlapping function compared with LXRalpha in the liver.

Rat peroxisome proliferator-activated receptors and brown adipose tissue function during cold acclimatization.

Brown adipose tissue (BAT) hyperplasia is a fundamental physiological response to cold; it involves an acute phase of mitotic cell growth followed by a prolonged differentiation phase. Peroxisome proliferator-activated receptors (PPARs) are key regulators of fatty acid metabolism and adipocyte differentiation and may therefore mediate important metabolic changes during non-shivering thermogenesis. In the present study we have investigated PPAR mRNA expression in relation to peroxisome proliferation in rat BAT during cold acclimatization. By immunoelectron microscopy we show that the number of peroxisomes per cytoplasmic volume and acyl-CoA oxidase immunolabeling density remained constant (thus increasing in parallel with tissue mass and cell number) during the initial proliferative phase and the acute thermogenic response but increased after 14 days of cold exposure, correlating with terminal differentiation of BAT. A pronounced decrease in BAT PPARalpha and PPARgamma mRNA levels was found within hours of exposure to cold, which was reversed after 14 days, suggesting a role for either or both of these subtypes in the proliferation and induction of peroxisomes and peroxisomal beta-oxidation enzymes. In contrast, PPARdelta mRNA levels increased progressively during cold exposure. Transactivation assays in HIB 1B and HEK-293 cells demonstrated an adrenergic stimulation of peroxisome proliferator response element reporter activity via PPAR, establishing a role for these nuclear receptors in hormonal regulation of gene transcription in BAT.

Ligand-independent coregulator recruitment by the triply activatable OR1/retinoid X receptor-alpha nuclear receptor heterodimer.

OR1 is a member of the superfamily of steroid/thyroid hormone nuclear receptors and recognizes DNA as a heterodimer with the 9-cis-retinoic acid receptor RXR (retinoid X receptor). The heterodimeric complex has been shown to be transcriptionally activatable by the RXR ligand as well as certain oxysterols via OR1, but to date uniquely also by heterodimerization itself. Recent studies on other members of the superfamily of nuclear receptors have led to the identification of a number of nuclear receptor-interacting proteins that mediate their regulatory effects on transcription. Here, we address the question of involvement of some of these cofactors in the three modes of activation by the OR1/RXRalpha complex. We show that in vitro the steroid receptor coactivator SRC-1 can be recruited by RXRalpha upon addition of its ligand, and to OR1 upon addition of 22(R)-OH-cholesterol, demonstrating that the latter can act as a direct ligand to OR1. Additionally, heterodimerization is sufficient to recruit SRC-1 to OR1/RXRalpha, indicating SRC-1 as a molecular mediator of dimerization-induced activation. In transfection experiments, coexpression of a nuclear receptor-interacting fragment of SRC-1 abolishes constitutive activation by OR1/RXRalpha, which can be restored by over-expression of full-length SRC-1. This constitutes evidence for an in vivo role of SRC-1 in dimerization-induced activation by OR1/RXRalpha. Additionally, we show that the nuclear receptor-interacting protein RIP140 binds in vitro to OR1 and RXRalpha with requirements distinct from those of SRC-1, and that binding of the two cofactors is competitive. Taken together, our results suggest a complex modulation of differentially induced transactivation by OR1/RXR coregulatory molecules.

Identification of a novel DNA binding site for nuclear orphan receptor OR1.

The nuclear orphan receptor OR1 has been shown to bind as a heterodimer with retinoid X receptor (RXR) to direct repeat 4 (DR4) response elements. It remained unclear, however, whether this represents the only or the optimal binding site for this receptor. Therefore, we performed a DNA binding site selection assay that allows the identification of novel DNA binding sites for OR1 in an unbiased manner. While OR1 alone was not able to select a specific sequence from the pool of oligonucleotides, the OR1/RXR heterodimer selected a highly conserved DR1 element, termed DR1s, with two AGGTCA motifs spaced by one adenosine. The functional activity of the consensus binding site was verified in transient transfection assays and corroborated by in vitro studies. Based on the sequence of the consensus DR1s, we located putative natural binding sites in the 5'-promoter flanking regions of the rat S14 gene and the rat cholecystokinin type A receptor gene. Furthermore, we could show that although the OR1/RXR heterodimer has a distinct binding orientation on a DR4 element, it is able to bind in both orientations to the DR1s element. The OR1 paralog LXRalpha does not bind as a heterodimer with RXR to the DR1s element, indicating that these receptors, despite their homology, are involved in the regulation of different sets of genes.

Synergistic activation of RLD-1 by agents triggering PKA and PKC dependent signalling.

RLD-1 and OR-1 are closely related orphan nuclear receptors that can be activated by certain oxysterols. To obtain cells stably expressing RLD-1 or OR-1, CHOK1 cells were successively transfected with a DGRE2-ALP reporter and GR-RLD-1 or GR-OR-1 chimeric constructs. The selected cell clones that showed low background activity of the reporter and maximum fold induction by 22R(OH)cholesterol were used for subsequent experiments. Treatment of the cells with PGE2, TPA, or 8-bromo-cAMP alone did not transactivate the reporter. However, the induction of the reporter by 22R(OH)cholesterol was markedly enhanced in the presence of PGE2, TPA, 8-bromo-cAMP, or forskolin in cells expressing GR-RLD-1. The enhancement was inhibited by H-89 and bisindolylmaleimide, both inhibitors of protein kinases. These results suggest that transactivation by ligand-activated RLD-1 may be further modulated/regulated through other signal transduction pathways involving phosphorylation catalyzed by protein kinases.

Inefficient expression of the DNA-binding domain of GAL4 in transgenic plants.

The DNA-binding domain of the yeast transcriptional activator GAL4 was expressed in transgenic tobacco plants in order to attempt to obtain specific repression of reporter genes. Expression of the GUS and the NPTII gene was essentially the same regardless of the presence of a gene encoding a functional GAL4 DNA-binding domain or the presence of GAL4 recognition sequences in the promoter region of the reporter genes. Despite high levels of GAL4 mRNA, no translation product was detectable in transgenic plants indicating that the failure to detect functional GAL4 in plants is due to the inefficiency of GAL4 mRNA translation in plants.

Analysis of the involvement of ocs-like bZip-binding elements in the differential strength of the bidirectional mas1'2' promoter.

The ocs-like elements of the bidirectional mas1'2' promoter of Agrobacterium tumefaciens, mas1' and mas2', were analyzed to elucidate their role in the expression conferred by this promoter. Tetramers of the elements were cloned upstream of the beta-glucuronidase-coding region linked to the 35S promoter deleted at -54. Transient expression assays with tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) protoplasts showed that tetramers of the mas1' element had 3- to 8-fold enhancing activity, respectively. Enhancement obtained by tetramers of the mas2' element was higher, suggesting that this element plays a role in the stronger promoter activity from the 2' side. Three cDNA clones with higher homology to the tobacco transcription factor TGA1a were isolated from a potato root expression library. Overexpression of the proteins encoded by these cDNA clones in Escherichia coli and analysis of DNA-binding activity in bacterial extracts showed that all three factors could bind strongly to the mas1' ocs-like element. In contrast, only two of the mas-binding factors exhibited significant binding to the mas2' element. Southern analysis revealed the presence of a small, multigene family encoding the mas-binding factors in potato.

Expression of lipoxygenase in wounded tubers of Solanum tuberosum L.

A lipoxygenase cDNA clone from Solanum tuberosum L. was analyzed to study the role of lipoxygenases in potato development and wound response. Sequence analysis and comparison of the deduced amino acid sequence revealed high homology to other plant lipoxygenases. Expression of the cDNA sequences in Escherichia coli and subsequent analysis of bacterial protein extracts showed lipoxygenase activity using linoleic, linolenic, or arachidonic acid as substrates. Transcripts encoding the potato lipoxygenase were most abundant in tuber tissue, lower in roots, and hardly detectable in leaves, petioles, and stems. The induction of lipoxygenase expression in tubers by wounding was dependent on various parameters. Whereas lipoxygenase transcript levels increased in discs from stored tubers incubated under aerobic conditions, tubers taken from a growing plant did not accumulate lipoxygenase transcripts in response to wounding. Incubation of tuber discs in buffer did not lead to an increase in lipoxygenase RNA levels; however, methyl jasmonate stimulated lipoxygenase expression after 24 h in stored tubers. Proteinase inhibitor II mRNAs decreased in stored tubers as well as in discs from growing tubers.