Cholesterol and its oxysterol congeners are important constituents of cell membranes and function as intermediates in several crucial biosynthetic pathways. These compounds autoregulate their metabolic fate by end-product repression and activation of downstream catabolism. Although end-product repression by oxysterols is relatively well understood, the mechanism by which these compounds act as positive transcription signalling molecules is unknown. Here we identify a specific group of endogenous oxysterols that activate transcription through the nuclear receptor LXR alpha. Transactivation of LXR alpha by oxysterols occurs at concentrations at which these compounds exist in vivo. The most potent activators also serve as intermediary substrates in the rate-limiting steps of three important metabolic pathways: steroid hormone biosynthesis, bile acid synthesis, and conversion of lanosterol to cholesterol. Our results demonstrate the existence of a nuclear receptor signalling pathway for oxysterols and suggest that LXR alpha may be important as a sensor of cholesterol metabolites.
We demonstrate that mice lacking the oxysterol receptor, LXR alpha, lose their ability to respond normally to dietary cholesterol and are unable to tolerate any amount of cholesterol in excess of that which they synthesize de novo. When fed diets containing cholesterol, LXR alpha (-/-) mice fail to induce transcription of the gene encoding cholesterol 7alpha-hydroxylase (Cyp7a), the rate-limiting enzyme in bile acid synthesis. This defect is associated with a rapid accumulation of large amounts of cholesterol in the liver that eventually leads to impaired hepatic function. The regulation of several other crucial lipid metabolizing genes is also altered in LXR alpha (-/-) mice. These results demonstrate the existence of a physiologically significant feed-forward regulatory pathway for sterol metabolism and establish the role of LXR alpha as the major sensor of dietary cholesterol.
LXR␣ and - are nuclear receptors that regulate the metabolism of several important lipids, including cholesterol and bile acids. Previously, we have proposed that LXRs regulate these pathways through their interaction with specific, naturally occurring oxysterols, including 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, and 24(S),25-epoxycholesterol. Using a ligand binding assay that incorporates scintillation proximity technology to circumvent many of the problems associated with assaying extremely hydrophobic ligands, we now demonstrate that these oxysterols bind directly to LXRs at concentrations that occur in vivo. To characterize further the structural determinants required for potent LXR ligands, we synthesized and tested a series of related compounds for binding to LXRs and activation of transcription. These studies revealed that position-specific monooxidation of the sterol side chain is requisite for LXR high-affinity binding and activation. Enhanced binding and activation can also be achieved through the use of 24-oxo ligands that act as hydrogen bond acceptors in the side chain. In addition, introduction of an oxygen on the sterol B-ring results in a ligand with LXR␣-subtype selectivity. These results support the hypothesis that naturally occurring oxysterols are physiological ligands for LXRs and show that a rational, structure-based approach can be used to design potent LXR ligands for pharmacologic use.Nonsteroidal nuclear hormone receptors are ligand-activated transcription factors that regulate an array of signaling pathways. Several members of this protein family, including the vitamin D receptor, retinoic acid receptor, and peroxisome proliferator-activated receptor, are primary regulators in nutrient metabolism and are known to be associated with a variety of metabolic disorders (1). These receptors function by forming obligate heterodimers with the retinoid X receptor (RXR) and binding specific DNA sequences (response elements) within the promoters of the genes they regulate. Upon binding ligand, the heterodimerized receptor undergoes a conformational change that results in enhanced transcription of the target gene. Thus far, all known native ligands for nuclear receptors are small hydrophobic compounds, many of which are the products of lipid metabolism (1).The liver X receptors (LXRs) were originally identified as orphan members of the nuclear receptor superfamily because their ligands were unknown. Like other receptors in the family, LXRs heterodimerize with RXR and bind to specific response elements (LXREs) characterized by direct repeats separated by four nucleotides (2-4). Two genes (␣ and ) are known to encode LXR proteins. LXR␣ is expressed most highly in the liver and to a lesser extent in the kidney, small intestine, spleen, and adrenal gland (2, 4). In contrast to the restricted expression pattern of LXR␣, LXR is ubiquitously expressed (5). In a comprehensive screen for LXR ligands we observed that the naturally occurring oxysterols 24(S)-hydroxycholesterol [24(S)-H...
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