Activation, LXR

The LXR receptor

Liver X receptors (LXR) are ligand-activated transcription factors of the nuclear receptor superfamily first identified in 1994 in rat liver (Apfel et al. 1994, Song 1994). There are two LXR isoforms termed a and ß (NR1H3 and NR1H2) which upon activation form heterodimers with retinoid X receptor (RXR) and bind to the LXR response element found in the promoter region of the target genes (Baranowski 2008). LXRs were shown to function as sterol sensors protecting the cells from cholesterol overload by stimulating reverse cholesterol transport and activating its conversion to bile acids in the liver (Baranowski 2008).

LXRa expression is restricted to liver, kidney, intestine, fat tissue, macrophages, lung, and spleen and is highest in liver, hence the name liver X receptor a (LXRa). LXRβ is expressed in almost all tissues and organs, hence the early name UR (ubiquitous receptor) (Ory 2004). The different pattern of expression suggests that LXRa and LXRβ have different roles in regulating physiological function. This is also supported from the observation that LXRa deficient mice do not develop hepatic steatosis when treated with LXR agonist that activates both types (Lund et al. 2006) and consequently the role of the two isoforms in relation to adverse effects could be different.

The molecular initiating event

Generally speaking chemicals that are able to act through NRs are usually specific ligands. These chemicals are mainly lipophilic and they mimic the action of natural hormones. However, in some cases hydrophilic chemicals (like phthalates) are also capable to act as ligands in NRs due to the molecular structure of the proteins and the pocket sites of the receptors.

The molecular initiating event in the presented MoA is the binding to the LXR or the permissive RXR of the LXR-RXR dimer leading to activation. LXR activation can be achieved via a wide range of endogenous neutral and acidic ligands as shown by crystallographic analysis (Williams et al. 2003). There are known endogenous but also synthetic ligands that can act as agonists. Endogenous agonists for this receptor are the oxysterols (oxidized cholesterol derivatives like 22(R)-hydroxycholesterol, 24(S)-hydroxycholesterol, 27-hydroxycholesterol, and cholestenoic acid) mainly with similar affinity for the two isoforms (Baranowski 2008). Oxysterols bind directly to the typical hydrophobic pocket in the C-terminal domain (Williams et al. 2003). Other endogenous ligands are the D-glucose and D-Glucose-6-phosphate (Mitro 2007). However, the hydrophilic nature of glucose and its low affinity for LXR present a challenge to the central dogma about the nature of the NR-ligand interaction (Lazar & Wilson 2007). Unsaturated fatty acids have also been shown to bind and regulate LXRa activity in cells. However, in contrast to the role of oxysterols, the biological relevance of this observation has not been established in vivo (Pawar et al. 2003). The function of LXRs is also modulated by many currently used drugs such as statins, fibrates, and thazolidinedione derivatives (Jamroz-Wiśniewska et al. 2007). Some synthetic LXR agonists have been developed like the non-steroidal agonists T0901317 and GW3965 (Schultz et al 2000, Collins et al. 2002). LXR forms a permissive dimer with the RXR which means that chemicals that can activate this receptor can trigger the same pathway as the LXR agonists. The endogenous RXR agonist is 9-cis-retinoic acid (Heyman et al. 1992) while synthetic agonists include LGD1069 and LG100268 (Boehm et al. 1994 and 1995).

In addition to the agonist binding in the LXR there are other mechanisms for its control. LXRa gene promoter contains also functional peroxisome proliferator response element (PPRE) and peroxisome proliferator-activated receptor (PPAR) a and γ agonists were shown to stimulate LXRa expression in human and rodent (Baranowski 2008). Control of the LXRa expression is also dependent on insulin and post-translationally by protein kinase A that phosphorylates receptor protein at two sites thereby impairing its dimerization and DNA-binding (Baranowski 2008).

Identification of the site of action

As already mentioned above LXR isoforms are expressed in various tissues but in relation to the presented MoA we refer to LXRs that are expressed in the hepatocytes.

Nuclear receptors may be classified into two broad classes according to their sub-cellular distribution in the absence of ligand. Type I NRs (like ER and AhR) are located in the cytosol (and they are translocated into the nucleus after ligand binding) while type II NRs like LXRs (but also PXR, PPARa and PPARγ) are located in the nucleus of the cell.

The specific site of binding and the affinity of a ligand for the LXRs depend on the structure of the ligand.

Binding in the LXREs and target genes transcription

Upon ligand-induced activation both isoforms form obligate heterodimers with the retinoid X receptor (RXR) and regulate gene expression through binding to LXR response elements (LXREs) in the promoter regions of the target genes (Fig. 1). The LXRE consists of two idealized hexanucleotide sequences (AGGTCA) separated by four bases (DR-4 element).

Figure 1. Mechanism of transcriptional regulation mediated by LXRs. RXR - retinoid X receptor, LXRE - LXR response element (Baranowski 2008)

Target genes of LXRs are involved in cholesterol and lipid metabolism regulation (^[1], ^[2]) including:

• ABC - ATP Binding Cassette transporter isoforms A1, G1, G5, and G8
• ApoE - Apolipoprotein E
• CETP - Cholesterylester Transfer Protein
• CYP7A1 - Cytochrome P450 isoform 7A1 - cholesterol 7a-hydroxylase
• FAS - Fatty Acid Synthase
• LPL - Lipoprotein Lipase
• LXR-a - Liver X Receptor-a
• SREBP-1c - Sterol Response Element Binding Protein 1c
• ChREBP - Carbohydrate Response Element Binding Protein
• FAT/CD36 – Fatty acid uptake transporter (liver)

Auto-regulation of the LXRa

Human specific auto-regulated expression specifically of the LXRa has been demonstrated from several studies (Laffitte et al. 2001, Whitney et al. 2001, Li et al. 2002, Kase et al. 2007). Human LXRa gene promoter has a functional LXRE activated by both LXRa and β. In addition human liver LXRa expression is induced by both natural and synthetic LXR agonists.

Liver X receptor (LXR) activation is measured by changes in gene expression and protein levels.  Effects of LXR on expression of downstream genes can be investigating using metabolomics and RT-qPCR approaches.  In addition, targeted ToxCast assays using SeqAPASS evaluations can evaluate gene expression changes from chemical exposure for model species (e.g. Lalone et al. 2018).  Relevent ToxCast assays are ATG_LXRa_TRANS; ATG_LXRb_TRANS; ATG_DR4_LXR_CIS (U.S. EPA 2024).

Life Stage: Older individuals are more likely to manifest this adverse outcome pathway (adults > juveniles) due to increased opportunity to upregulate gene expression.

Sex: Applies to both males and females.

Taxonomic: Appears to be present broadly in vertebrates, with most representative studies in mammals (humans, lab mice, lab rats).

1. ↑ Peet 1998 - Peet D.J., Cholesterol and Bile Acid Metabolism Are Impaired in Mice Lacking the Nuclear Oxysterol Receptor LXRa in mammals, Cell, 93, 693–704, 1998
2. ↑ Edwardsa et al. 2002 - Edwardsa P.A., et al, LXRs; Oxysterol-activated nuclear receptors that regulate genes controlling lipid homeostasis, (Oxidized Lipids as Potential Mediators of Atherosclerosis), Vascular Pharmacology, 38 (No 4), 249–256, 2002
3. LaLone, C.A., Villeneuve, D.L., Doering, J.A., Blackwell, B.R., Transue, T.R., Simmons, C.W., Swintek, J., Degitz, S.J., Williams, A.J., and Ankley, G.T.  2018.  Evidence for Cross Species Extrapolation of Mammalian-Based High-Throughput Screening Assay Results.  Environmental Science and Technology 52: 13960−13971.
4. U.S. EPA. 2024. ToxCast & Tox21 Summary Files from invitrodb_v4. Retrieved from https://www.epa.gov/chemical-research/toxicity-forecaster-toxcasttm-data. 

NOTE: Italics symbolize edits from John Frisch