ERβ Binds N-CoR in the Presence of Estrogens via an LXXLL-like Motif in the N-CoR C-terminus

Nuclear receptors (NRs) usually bind the corepressors N-CoR and SMRT in the absence of ligand or in the presence of antagonists. Agonist binding leads to corepressor release and recruitment of coactivators. Here, we report that estrogen receptor beta (ERbeta) binds N-CoR and SMRT in the presence of agonists, but not antagonists, in vitro and in vivo. This ligand preference differs from that of ERalpha interactions with corepressors, which are inhibited by estradiol, and resembles that of ERbeta interactions with coactivators. ERbeta /N-CoR interactions involve ERbeta AF-2, which also mediates coactivator recognition. Moreover, ERbeta recognizes a sequence (PLTIRML) in the N-CoR C-terminus that resembles coactivator LXXLL motifs. Inhibition of histone deacetylase activity specifically potentiates ERbeta LBD activity, suggesting that corepressors restrict the activity of AF-2. We conclude that the ER isoforms show completely distinct modes of interaction with a physiologically important corepressor and discuss our results in terms of ER isoform specificity in vivo.     

X-Ray Structures of the LXRα LBD in Its Homodimeric Form and Implications for Heterodimer Signaling

Liver X receptors (LXRs) are nuclear receptors that are central regulators of cholesterol homeostasis, and synthetic LXR agonists have shown promise as promoters of reverse cholesterol transport and anti-inflammatory agents. Here, we present three X-ray structures of three different agonists bound to the ligand binding domain of LXRα. These compounds are GW3965, F₃methylAA, and a benzisoxazole urea, and we show that these diverse chemical scaffolds address common structural themes, leading to high binding affinity for LXR. Our structures show the LXRα ligand binding domain in its homodimeric form, an arrangement previously thought to be stereochemically difficult. A comparison with existing structures of the LXRβ homodimer and LXRα:RXR (retinoid X receptor) heterodimers explains differences in dimer affinity and leads us to propose a model for allosteric activation in nuclear receptor dimers, in which an unactivated RXR partner provides an inhibitory tail wrap to the cofactor binding pocket of LXR.     

LXR and TSPO as new therapeutic targets to increase the levels of neuroactive steroids in the central nervous system of diabetic animals

Neuroactive steroid levels are decreased in the central nervous system (CNS) of streptozotocin (STZ) diabetic rats. In agreement, they exert protective effects in this experimental model, counteracting degenerative events occurring in the CNS. Therefore, an interesting therapeutic strategy could be to increase their levels directly in the CNS. In this study we have evaluated whether activation of translocator protein-18kDa (TSPO) or liver X receptors (LXRs) may affect the levels of neuroactive steroids present in the CNS of diabetic and non-diabetic animals. We observed that the treatment with either Ro5-4864 (i.e., a ligand of TSPO) or with GW3965 (i.e., a ligand of LXRs) induced an increase of neuroactive steroids in the spinal cord, the cerebellum and the cerebral cortex of STZ-rats, but not in the CNS of non-pathological animals. Interestingly, the pattern of induction was different among the three CNS areas analyzed and between the two pharmacological tools. In particular, the activation of LXRs might represent a promising neuroprotective strategy, because the treatment with GW3965, at variance to Ro5-4864 treatment, did not induce significant changes in the plasma levels of neuroactive steroids. This suggests that activation of LXRs may selectively increase the CNS levels of neuroactive steroids avoiding possible endocrine side effects exerted by the systemic treatment with these molecules. Interestingly GW3965 treatment induced an increase of dihydroprogesterone in the spinal cord of diabetic animals in association with an increase of myelin basic protein expression. Thus we demonstrated that LXR activation was able to rescue CNS symptoms of diabetes.     

The ligand-dependent interaction of mineralocorticoid receptor with coactivator and corepressor peptides suggests multiple activation mechanisms

We investigated the coregulator (coactivator and corepressor) interactions with the mineralocorticoid receptor (MR) that lead to activation and inhibition of the receptor in the presence of agonist and/or antagonist. Our results indicate that MR ligand binding domain (LBD) interacts strongly with only a few specific coactivator peptides in the presence of the agonist aldosterone and that these interactions are blocked by the antagonist eplerenone. We also discovered that cortisol, the preferred physiological ligand for the glucocorticoid receptor in humans, is a partial MR agonist/antagonist, providing a possible molecular explanation of the tissue-selective effects of glucocorticoids on MR. However, when we examined the coactivator and corepressor peptide interactions in the presence of cortisol, we found that MR bound with cortisol or aldosterone interacted with the same set of peptides. Thus, the partial agonism shown by cortisol is unlikely to be the result of differential interaction with known coactivators and corepressors. On the other hand, we have identified coactivator binding groove mutations that are critical for cortisol activation but not for aldosterone activation, suggesting that the two steroids induce different MR LBD conformations. In addition, we also show that cortisol becomes full agonist when S810L mutation is introduced in the LBD of MR. Interestingly, MR antagonists, such as eplerenone and progesterone, become partial agonist/antagonist of S810L but are still able to recruit LXXLL peptides to the mutant receptor. Together, these findings suggest a model to explain the MR activation by various ligands.     

T0901317 is a potent PXR ligand: implications for the biology ascribed to LXR

The liver X receptors (LXRalpha and beta) are nuclear receptors that coordinate carbohydrate and lipid metabolism. Insight into the physiologic roles of the LXRs has been greatly facilitated by the discovery of potent synthetic agonists. Here we show that one of these compounds, T0901317, is also a high-affinity ligand for the xenobiotic receptor pregnane X receptor (PXR). T0901317 binds and activates PXR with the same nanomolar potency with which it stimulates LXR activity. T0901317 induces expression not only of LXR target genes, but also of PXR target genes in cells and animals, including the scavenger receptor CD36, a property not shared by more specific LXR ligands, such as GW3965. Activation of PXR targets may explain why T0901317 induces dramatic liver steatosis, while GW3965 has a milder effect. These results suggest that many of the biological activities heretofore associated with LXR activation may be mediated by PXR, not LXR. Since T0901317 has been widely used in animals to study LXR function, the in vivo effects of this compound ascribed to LXR activation should be re-examined.     

Liver X receptor activation downregulates organic anion transporter 1 (OAT1) in the renal proximal tubule

Liver X receptors (LXRs) play an important role in the regulation of cholesterol by regulating several transporters. In this study, we investigated the role of LXRs in the regulation of human organic anion transporter 1 (hOAT1), a major transporter localized in the basolateral membrane of the renal proximal tubule. Exposure of renal S2 cells expressing hOAT1 to LXR agonists (TO901317 and GW3965) and their endogenous ligand [22(R)-hydroxycholesterol] led to the inhibition of hOAT1-mediated [(14)C]PAH uptake. This inhibition was abolished by coincubation of the above agonists with 22(S)-hydroxycholesterol, an LXR antagonist. Moreover, it was found that the effect of LXR agonists was not mediated by changes in intracellular cholesterol levels. Interestingly, the inhibitory effect of LXRs was enhanced in the presence of 9-cis retinoic acid, a retinoic X receptor agonist. Kinetic analysis revealed that LXR activation decreased the maximum rate of PAH transport (J(max)) but had no effect on the affinity of the transporter (K(t)). This result correlated well with data from Western blot analysis, which showed the decrease in hOAT1 expression following LXR activation. Similarly, TO901317 inhibited [(14)C]PAH uptake by the renal cortical slices as well as decreasing mOAT1 protein expression in mouse kidney. Our findings indicated for the first time that hOAT1 was downregulated by LXR activation in the renal proximal tubule.     

Ligand specificity and evolution of liver X receptors

Liver X receptors (LXRs) are key regulators of lipid and cholesterol metabolism in mammals. Little is known, however, about the function and evolution of LXRs in non-mammalian species. The present study reports the cloning of LXRs from African clawed frog (Xenopus laevis), Western clawed frog (Xenopus tropicalis), and zebrafish (Danio rerio), and their functional characterization and comparison with human and mouse LXRs. Additionally, an ortholog of LXR in the chordate invertebrate Ciona intestinalis was cloned and functionally characterized. Ligand specificities of the frog and zebrafish LXRs were very similar to LXRalpha and LXRbeta from human and mouse. All vertebrate LXRs studied were activated robustly by the synthetic ligands T-0901317 and GW3965 and by a variety of oxysterols. In contrast, Ciona LXR was not activated by T-0901317 or GW3965 but was activated by a limited number of oxysterols, as well as some androstane and pregnane steroids. Pharmacophore analysis, homology modeling, and docking studies of Ciona LXR predict a receptor with a more restricted ligand-binding pocket and less intrinsic disorder in the ligand-binding domain compared to vertebrate LXRs. The results suggest that LXRs have a long evolutionary history, with vertebrate LXRs diverging from invertebrate LXRs in ligand specificity.