Identification of a novel DNA binding site for nuclear orphan receptor OR1.
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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. (+info)
Retina-specific nuclear receptor: A potential regulator of cellular retinaldehyde-binding protein expressed in retinal pigment epithelium and Muller glial cells.
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In an effort to identify nuclear receptors important in retinal disease, we screened a retina cDNA library for nuclear receptors. Here we describe the identification of a retina-specific nuclear receptor (RNR) from both human and mouse. Human RNR is a splice variant of the recently published photoreceptor cell-specific nuclear receptor [Kobayashi, M., Takezawa, S., Hara, K., Yu, R. T., Umesono, Y., Agata, K., Taniwaki, M., Yasuda, K. & Umesono, K. (1999) Proc. Natl. Acad. Sci. USA 96, 4814-4819] whereas the mouse RNR is a mouse ortholog. Northern blot and reverse transcription-PCR analyses of human mRNA samples demonstrate that RNR is expressed exclusively in the retina, with transcripts of approximately 7.5 kb, approximately 3.0 kb, and approximately 2.3 kb by Northern blot analysis. In situ hybridization with multiple probes on both primate and mouse eye sections demonstrates that RNR is expressed in the retinal pigment epithelium and in Muller glial cells. By using the Gal4 chimeric receptor/reporter cotransfection system, the ligand binding domain of RNR was found to repress transcriptional activity in the absence of exogenous ligand. Gel mobility shift assays revealed that RNR can interact with the promoter of the cellular retinaldehyde binding protein gene in the presence of retinoic acid receptor (RAR) and/or retinoid X receptor (RXR). These data raise the possibility that RNR acts to regulate the visual cycle through its interaction with cellular retinaldehyde binding protein and therefore may be a target for retinal diseases such as retinitis pigmentosa and age-related macular degeneration. (+info)
Sterol upregulation of human CETP expression in vitro and in transgenic mice by an LXR element.
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The cholesterol ester transfer protein (CETP) facilitates the transfer of HDL cholesterol esters from plasma to the liver. Transgenic mice expressing human CETP, controlled by its natural flanking region, increase expression of this gene in response to hypercholesterolemia. We established a CETP promoter-luciferase reporter assay in differentiated 3T3-L1 adipocytes to map the sterol upregulatory element. Promoter mutagenesis suggested that a direct repeat of a nuclear receptor binding sequence separated by 4 nucleotides (DR4 element, -384 to -399) was responsible for this activity. Using mice carrying normal or mutated promoter sequences, we confirmed the importance of this element for gene induction by dietary sterol. A gel retardation complex containing LXR/RXR was identified using the CETP DR4 element and adipocyte nuclear extracts. Both LXRalpha/RXRalpha and LXRbeta/RXRalpha transactivated the CETP promoter via its DR4 element in a sterol-responsive fashion. Thus, the positive sterol response of the CETP gene is mediated by a nuclear receptor binding site that is activated by LXRs. That Cyp7a, the rate-limiting enzyme for conversion of cholesterol into bile acids in the liver, is also regulated by LXRalpha suggests that this class of nuclear receptor coordinates the regulation of HDL cholesterol ester catabolism and bile acid synthesis in the liver. (+info)
Oxysterols induce differentiation in human keratinocytes and increase Ap-1-dependent involucrin transcription.
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Ligands and activators of the nuclear hormone receptor superfamily are important in the regulation of epidermal development and differentiation. Previously, we showed that naturally occurring fatty acids, as well as synthetic ligands for the peroxisome proliferator-activated receptor, induce keratinocyte differentiation in vitro. Here we asked whether oxysterols, another class of lipids formed de novo in the epidermis and that activate liver X-activated receptor, regulate keratinocyte differentiation. mRNA and protein levels of involucrin and transglutaminase 1, markers of differentiation, increased 2- to 3-fold in normal human keratinocytes incubated in the presence of 25- or 22R-hydroxycholesterol in low calcium. In high calcium, which alone induces differentiation, mRNA levels were further increased by oxysterols. Rates of cornified envelope formation, an indicator of terminal differentiation, also increased 2-fold with oxysterol treatment. In contrast, the rate of DNA synthesis was inhibited approximately 50% by oxysterols. Transcriptional regulation was assessed in keratinocytes transfected with either transglutaminase 1 or involucrin promoter-luciferase constructs. 22R-hydroxycholesterol increased transglutaminase 1 and involucrin promoter activity 2- to 3-fold. Either deletion of the -2452 bp to -1880 bp region of the involucrin promoter, or mutation of the AP-1 site within this region, abolished oxysterol responsiveness. Moreover, increased AP-1 DNA binding was observed in oxysterol-treated keratinocytes by gel shift analyses. Finally, we demonstrated the presence of liver X-activated receptor alpha and beta mRNAs, and showed that oxysterols stimulate a liver X-activated receptor response element transfected into keratinocytes. These data suggest that oxysterols induce keratinocyte differentiation, in part through increased AP-1-dependent transcription of the involucrin gene, an effect that may be mediated by liver X-activated receptor. (+info)
The acute phase response is associated with retinoid X receptor repression in rodent liver.
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The acute phase response (APR) is associated with decreased hepatic expression of many proteins involved in lipid metabolism. The nuclear hormone receptors peroxisome proliferator-activated receptor alpha (PPARalpha) and liver X receptor (LXR) play key roles in regulation of hepatic lipid metabolism. Because heterodimerization with RXR is crucial for their action, we hypothesized that a decrease in RXR may be one mechanism to coordinately down-regulate gene expression during APR. We demonstrate that lipopolysaccharide (LPS) induces a rapid, dose-dependent decrease in RXRalpha, RXRbeta, and RXRgamma proteins in hamster liver. Maximum inhibition was observed at 4 h for RXRalpha (62%) and RXRbeta (50%) and at 2 h for RXRgamma (61%). These decreases were associated with a marked reduction in RXRalpha, RXRbeta, and RXRgamma mRNA levels. Increased RNA degradation is likely responsible for the repression of RXR, because LPS did not decrease RXRbeta and RXRgamma transcription and only marginally inhibited (38%) RXRalpha transcription. RXR repression was associated with decreased LXRalpha and PPARalpha mRNA levels and reduced RXR x RXR, RXR x PPAR and RXR x LXR binding activities in nuclear extracts. Furthermore, LPS markedly decreased both basal and Wy-14,643-induced expression of acyl-CoA synthetase, a well characterized PPARalpha target. The reduction in hepatic RXR levels alone or in association with other nuclear hormone receptors could be a mechanism for coordinately inhibiting the expression of multiple genes during the APR. (+info)
Human white/murine ABC8 mRNA levels are highly induced in lipid-loaded macrophages. A transcriptional role for specific oxysterols.
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To identify genes that are transcriptionally activated when human macrophages accumulate excess lipids, we employed the mRNA differential display technique using RNA isolated from human monocyte-macrophages incubated in the absence or presence of acetylated low density lipoprotein and sterols (cholesterol and 25-hydroxycholesterol). These studies identified a mRNA whose levels were highly induced in lipid-loaded macrophages. The mRNA encoded the human White protein, a member of the ATP-binding cassette (ABC) transporter superfamily of proteins. The mRNA levels of ABC8, the murine homolog of the human white gene, were also induced when a murine macrophage cell line, RAW264.7, was incubated with acetylated low density lipoprotein and sterols. Additional studies demonstrated that white/ABC8 mRNA levels were induced by specific oxysterols that included 25-, 20(S)-, and 22(R)-hydroxycholesterol, and by a retinoid X receptor-specific ligand. Furthermore, the oxysterol-mediated induction of ABC8 expression in mouse peritoneal macrophages was dependent on the presence of the nuclear oxysterol receptors, liver X receptors (LXRs). Macrophages derived from mice lacking both LXRalpha and LXRbeta failed to up-regulate the expression of ABC8 following incubation with 22(R)-hydroxycholesterol. Oxysterol-dependent induction of white/ABC8 mRNA was blocked by actinomycin D but not by cycloheximide treatment of cells. We conclude that the white and ABC8 genes are primary response genes that are transcriptionally activated by specific oxysterols and that this induction is mediated by the LXR subfamily of nuclear hormone receptors. These data strongly support the hypothesis that white/ABC8 has a role in cellular sterol homeostasis. (+info)
A deletion in a photoreceptor-specific nuclear receptor mRNA causes retinal degeneration in the rd7 mouse.
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The rd7 mouse, an animal model for hereditary retinal degeneration, has some characteristics similar to human flecked retinal disorders. Here we report the identification of a deletion in a photoreceptor-specific nuclear receptor (mPNR) mRNA that is responsible for hereditary retinal dysplasia and degeneration in the rd7 mouse. mPNR was isolated from a pool of photoreceptor-specific cDNAs originally created by subtractive hybridization of mRNAs from normal and photoreceptorless rd mouse retinas. Localization of the gene corresponding to mPNR to mouse Chr 9 near the rd7 locus made it a candidate for the site of the rd7 mutation. Northern analysis of total RNA isolated from rd7 mouse retinas revealed no detectable signal after hybridization with the mPNR cDNA probe. However, with reverse transcription-PCR, we were able to amplify different fragments of mPNR from rd7 retinal RNA and to sequence them directly. We found a 380-nt deletion in the coding region of the rd7 mPNR message that creates a frame shift and produces a premature stop codon. This deletion accounts for more than 32% of the normal protein and eliminates a portion of the DNA-binding domain. In addition, it may result in the rapid degradation of the rd7 mPNR message by the nonsense-mediated decay pathway, preventing the synthesis of the corresponding protein. Our findings demonstrate that mPNR expression is critical for the normal development and function of the photoreceptor cells. (+info)
Sterol-dependent transactivation of the ABC1 promoter by the liver X receptor/retinoid X receptor.
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Tangier disease, a condition characterized by low levels of high density lipoprotein and cholesterol accumulation in macrophages, is caused by mutations in the ATP-binding cassette transporter ABC1. In cultured macrophages, ABC1 mRNA was induced in an additive fashion by 22(R)-hydroxycholesterol and 9-cis-retinoic acid (9CRA), suggesting induction by nuclear hormone receptors of the liver X receptor (LXR) and retinoid X receptor (RXR) family. We cloned the 5'-end of the human ABC1 transcript from cholesterol-loaded THP1 macrophages. When transfected into RAW macrophages, the upstream promoter was induced 7-fold by 22(R)-hydroxycholesterol, 8-fold by 9CRA, and 37-fold by 9CRA and 22(R)-hydroxycholesterol. Furthermore, promoter activity was increased in a sterol-responsive fashion when cotransfected with LXRalpha/RXR or LXRbeta/RXR. Further experiments identified a direct repeat spaced by four nucleotides (from -70 to -55 base pairs) as a binding site for LXRalpha/RXR or LXRbeta/RXR. Mutations in this element abolished the sterol-mediated activation of the promoter. The results show sterol-dependent transactivation of the ABC1 promoter by LXR/RXR and suggest that small molecule agonists of LXR could be useful drugs to reverse foam cell formation and atherogenesis. (+info)