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AOP: 545
Title
Activation, Pregnane-X receptor, NR1l2 leads to increased plasma low-density lipoprotein (LDL) cholesterol via increased cholesterol synthesis
Short name
Graphical Representation
Point of Contact
Contributors
- John Frisch
Coaches
OECD Information Table
OECD Project # | OECD Status | Reviewer's Reports | Journal-format Article | OECD iLibrary Published Version |
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This AOP was last modified on February 10, 2025 15:37
Revision dates for related pages
Page | Revision Date/Time |
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Activation, Pregnane-X receptor, NR1l2 | February 10, 2025 15:38 |
Decreased, insulin-induced gene 1 (INSIG1) protein activity | February 10, 2025 15:52 |
Increased, Sterol Regulatory Element Binding Protein 2 (SREBP2) protein expression | February 10, 2025 15:53 |
Increased, cholesterol synthesis enzymes | February 10, 2025 15:54 |
Increased, plasma low-density lipoprotein (LDL) cholesterol | February 10, 2025 15:55 |
Activation, Pregnane-X receptor, NR1l2 leads to Decreased, INSIG1 protein activity | February 10, 2025 15:57 |
Decreased, INSIG1 protein activity leads to Increased, SREBP2 protein expression | February 10, 2025 15:58 |
Increased, SREBP2 protein expression leads to Increased, cholesterol synthesis enzymes | February 10, 2025 15:59 |
Increased, cholesterol synthesis enzymes leads to Increased, plasma LDL cholesterol | February 10, 2025 16:00 |
Abstract
Pregnane X receptor (PXR) is a nuclear receptor that, once activated, binds to Retinoid X receptor (RXR), resulting in regulation of target genes, with an important role in maintenance of lipid homeostasis by affecting rates of glucose synthesis, cholesterol synthesis, lipid synthesis, and lipid uptake (Lv et al. 2022). Increased Pregnane X Receptor (PXR) activation results in decreased insulin-induced gene 1 (INSIG1) protein activity, suggested through destabilization of the INSIG1 protein (Karpale et al. 2021; Itkonen et al. 2023), resulting in increased lipid synthesis.
Insulin-induced gene 1 (INSIG1) is a transmembrane protein in the endoplasmic reticulum membrane (Ouyang et al. 2020). INSIG1 has an important role in lipid synthesis regulation. At low cholesterol levels, INSIG1 has lower binding affinity for SREBP cleavage-activating protein (SCAP), allowing free SCAP to bind to Coat Protein Complex II (COPII; Ouyang et al. 2020). The SCAP-COPII complex enables Sterol Regulatory Element Binding Proteins (SREBPs) to move through the endoplasmic reticulum to the Golgi, where membrane-bound transcription factor site-1-protease (S1P) and site-2-protease (S2P) enable proteolytic processing that allows SREBPs to enter the nucleus (Yabe et al. 2002; Yang et al. 2002). In the nucleus, SREBP2 increases gene expression for enzymes involved in cholesterol synthesis (Ouyang et al. 2020; Itkonen et al., 2023). At high cholesterol levels, INSIG1 binds to SCAP, competitively inhibiting the ability of SCAP to bind to COPII (Ouyang et al. 2020). SREBPs are retained in the endoplasmic reticulum rather than being transferred to the Golgi, reducing levels of cholesterol synthesis (Ouyang et al. 2020; Itkonen et al., 2023).
Cholesterol has a variety of roles in organisms, including as a cellular membrane component that helps maintain structure and fluidity, and a precursor for steroid hormones (Sakakura et al. 2001; Horton et al. 2003; Howe et al. 2017). The cholesterol synthesis pathway involves a number of precursor molecules and enzymes (Sakakura et al. 2001; Itkonen et al. 2023), with Sterol regulatory element-binding proteins (SREBPs) regulating transcription rates of genes coding for cholesterol synthesis enzymes (Engelking et al. 2005; Karpale et al. 2021), and high cholesterol levels acting as a negative feedback on additional cholesterol synthesis (Itkonen et al. 2023; MacFarlaine et al. 2014). Individuals with high levels of plasma low-density lipoprotein (LDL) cholesterol have departed from lipid homeostasis in maintaining cholesterol levels needed for typical function, and in displaying hypercholesterolemia are at greater risk of cardiovascular events (Lalanne et al. 2005; Lambert et al. 2006).
AOP Development Strategy
Context
This Adverse Outcome Pathway (AOP) was developed as part of an Environmental Protection Agency effort to represent putative AOPs from peer-reviewed literature which were heretofore unrepresented in the AOP-Wiki. The originating work for this AOP was: Itkonen, A., Hakkola, J., and Rysa, J. 2023. Adverse outcome pathway for pregnane X receptor‑induced Hypercholesterolemia. Archives of Toxicology 97: 2861–2877. This publication, and the work cited within, were used create and support this AOP and its respective KE and KER pages.
The focus of the originating work was to use an AOP framework to investigate the mechanisms linking Pregnane X receptor (PXR) activation to increased plasma low-density lipoprotein (LDL) cholesterol. Itkonen et al. (2023) provided network analysis from a literature search to assess the empirical evidence, biological plausibility and domain of applicability for each developed key event and key event relationship. Available evidence provided support of mechanisms for increased plasma low-density lipoprotein (LDL) cholesterol through multiple pathways, including: 1. Increased, cholesterol synthesis enzymes; 2. Increased, protein expression of proprotein convertase subtilisin/kexin type 9 (PCSK9). In this AOP, we focus on the pathway through Increased, cholesterol synthesis enzymes. Empirical studies were primarily from laboratory rodents and humans.
Strategy
The originating authors conducted a literature search to develop a database of publications categorized by discipline or field of study: toxicology, epidemiology, exposure, and gene-environment interaction. The literature search relied on standard search engines such as Web of Science and Google Scholar, and the search strategy focused on mechanisms linking PXR activation to increased level of plasma LDL cholesterol in organisms. The originating authors reviewed references from individual citations to identify additional studies not captured through the literature search itself. They then included all relevant publications through 2023.
The scope of the aforementioned EPA project was limited to re-representing the AOP(s) as presented in the originating publication. The literature used to support this AOP and its constituent pages began with the originating publication and followed to the primary, secondary, and tertiary works cited therein. KE and KER page creation and re-use was determined using Handbook principles where page re-use was preferred.
Summary of the AOP
Events:
Molecular Initiating Events (MIE)
Key Events (KE)
Adverse Outcomes (AO)
Type | Event ID | Title | Short name |
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MIE | 239 | Activation, Pregnane-X receptor, NR1l2 | Activation, Pregnane-X receptor, NR1l2 |
KE | 2268 | Decreased, insulin-induced gene 1 (INSIG1) protein activity | Decreased, INSIG1 protein activity |
KE | 2269 | Increased, Sterol Regulatory Element Binding Protein 2 (SREBP2) protein expression | Increased, SREBP2 protein expression |
KE | 2270 | Increased, cholesterol synthesis enzymes | Increased, cholesterol synthesis enzymes |
AO | 2271 | Increased, plasma low-density lipoprotein (LDL) cholesterol | Increased, plasma LDL cholesterol |
Relationships Between Two Key Events (Including MIEs and AOs)
Title | Adjacency | Evidence | Quantitative Understanding |
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Network View
Prototypical Stressors
Life Stage Applicability
Life stage | Evidence |
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All life stages | Moderate |
Taxonomic Applicability
Term | Scientific Term | Evidence | Link |
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mammals | mammals | High | NCBI |
Sex Applicability
Sex | Evidence |
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Unspecific | High |
Overall Assessment of the AOP
1. Support for Biological Plausibility of Key Event Relationships: Is there a mechanistic relationship between KEup and KEdown consistent with established biological knowledge? |
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Key Event Relationship (KER) |
Level of Support Strong = Extensive understanding of the KER based on extensive previous documentation and broad acceptance. Moderate = Support of the relationship based on empirical studies, with some uncertainty of the direction of the response (decreased or increased) and the mechanism. |
Relationship 3365: Activation Pregnane-X receptor, NR1l2 leads to Decreased, INSIG1 protein activity |
Moderate support. The relationship between activation of pregnane-X receptor and INSIG1 protein activity is broadly accepted in humans and laboratory mammals, but there are some empirical studies where activation of Pregnane-X receptor leads to increased INSIG protein activity, the opposite direction from the mechanism of the AOP. Destabilization of the INSIG1 protein has been the suggested mechanism (Karpale et al. 2021; Itkonen et al. 2023). |
Relationship 3366: Decreased, INSIG1 protein activity leads to Increased, SREBP2 protein expression |
Strong support. The relationship between decreased INSIG protein activity and increased SREBP2 protein expression is broadly accepted and supported among humans and laboratory mammal data. |
Relationship 3367: Increased, SREBP2 protein expression leads to Increased, cholesterol synthesis enzymes |
Strong support. The relationship between increased SREBP2 protein expression and increased cholesterol synthesis enzymes is broadly accepted and supported among humans and laboratory mammal data. |
Relationship 3368: Increased, cholesterol synthesis enzymes leads to Increased, plasma LDL cholesterol |
Strong support. The relationship between increased cholesterol synthesis enzymes and increased plasma LDL cholesterol is broadly accepted and supported among humans and laboratory mammal data. |
Overall |
Moderate to strong support. Extensive understanding of the relationships between events from empirical studies from humans and laboratory mammals, with some inconsistency of the direction of the response in INSIG1 protein activity from Pregnane-X receptor activation. |
Domain of Applicability
Life Stage: All life stages.
Sex: Applies to both males and females; not sex-specific.
Taxonomic: Primarily studied in humans and laboratory rodents.
Essentiality of the Key Events
2. Essentiality of Key Events: Are downstream KEs and/or the AO prevented if an upstream KE is blocked? |
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Key Event (KE) |
Level of Support Strong = Direct evidence from specifically designed experimental studies illustrating essentiality and direct relationship between key events. Moderate = Indirect evidence from experimental studies inferring essentiality of relationship between key events due to difficulty in directly measuring at least one of key events, or some question of the direction of the response (decreased or increased). |
KE 239 Activation Pregnane-X receptor, NR1l2 |
Moderate support. Activation of Pregnane-X receptor is linked to INSIG1 protein uptake, but there are some empirical studies where activation of Pregnane-X receptor leads to increased INSIG protein activity, the opposite direction from the mechanism of the AOP. Evidence is available from toxicant, diet, and gene-knockout studies measuring gene expression and protein levels. Best evidence for essentiality of activation of Pregnane-X receptor is in toxicant and diet studies leading to decreased INSIG1 activity, with normal INSIG1 activity in the absence of stressor. Additional studies examining the conditions required for INSIG1 protein stability, and the relationship between INSIG1 gene and protein levels in the presence/absence of stressors, would be beneficial. |
KE 2268 Decreased, INSIG1 protein activity |
Strong support. Decreased INSIG 1 protein activity is linked to increased SREBP2 protein expression. Evidence is available from gene-knockout, gene transfection, and diet studies measuring gene expression and protein levels. Best evidence for essentiality of INSIG1 protein is in gene and diet studies showing INSIG1 protein levels and SREBP2 protein levels in the endoplasmic reticulum and nucleus, as inactive SREBP2 is known to be bound to the endoplasmic reticulum, and needs to be transferred to the Golgi by escort proteins for activation to regulate downstream loci. High levels of INSIG1 protein have been established to bind to SREBP cleavage-activating protein (SCAP) and promote retention of inactive SREBP2 in the endoplasmic reticulum; low levels of INSIG1 protein allow SCAP to bind to SREBP2 and increase transfer rates to the Golgi. |
KE 2269 Increased, SREBP2 protein expression |
Strong support. Increased SREBP2 protein activity is linked to increased cholesterol synthesis enzymes. Evidence is available from gene-knockout, gene transfection, and diet studies measuring gene expression and protein levels. Best evidence for essentiality of SREBP2 protein is in studies showing that SREBP2 protein binds to the promoter site of cholesterol synthesis enzymes to activate transcription, in gene knock-out studies where decreased SREBP2 protein expression led to decreased cholesterol synthesis enzymes, and in gene overexpression studies where increased SREBP2 protein expression led to increased cholesterol synthesis enzymes. |
KE 2270 Increased, cholesterol synthesis enzymes |
Strong support. Increased cholesterol synthesis enzymes is linked to increased plasma LDL cholesterol. Evidence is available from gene-knockout, gene transfection, and diet studies measuring protein levels. Best evidence for essentiality of cholesterol synthesis enzymes are studies showing that increases in cholesterol synthesis enzymes lead to increased plasma LDL cholesterol. However, increased plasma LDL cholesterol can be caused by additional mechanisms, including increased intake of cholesterol in diet, increased uptake of cholesterol, and decreased efflux and excretion. |
KE 2271 Increased, plasma LDL cholesterol |
This is the final event of the AOP. |
Overall |
Moderate to strong support. Direct evidence from empirical studies from laboratory mammals and human cell lines for most key events, with more inferential evidence for events occurring in cells and tissues. |
Evidence Assessment
3. Empirical Support for Key Event Relationship: Does empirical evidence support that a change in KEup leads to an appropriate change in KEdown? |
|
Key Event Relationship (KER) |
Level of Support Strong = Experimental evidence from exposure to toxicant shows consistent change in both events across taxa and study conditions. Moderate = Support of the relationship based on empirical studies, with some uncertainty of the direction of the response (decreased or increased). |
Relationship 3365: Activation Pregnane-X receptor, NR1l2 leads to Decreased, INSIG1 protein activity |
Moderate support. Activation of Pregnane-X receptor leads to decreased INSIG1 protein activity from toxicant, diet, and gene-knockout studies. Some studies show activation of Pregnane-X receptor leads to an increase in INSIG1 protein activity, the opposite direction from the mechanism of the AOP. Activation of Pregnane-X receptor occurred earlier in the time-course of exposure than decreased INSIG1 protein activity, and the concentrations that activated Pregnane-X receptor were equal to or lower than the concentrations that decreased INSIG1 protein activity. Therefore, the data support a causal relationship. |
Relationship 3366: Decreased, INSIG1 protein activity leads to Increased, SREBP2 protein expression |
Strong support. Decreased INSIG1 protein activity leads to increased SREBP2 protein expression from gene-knockout, gene transfection, and diet studies. Decreased INSIG1 protein activity occurred earlier in the time-course of exposure than increased SREBP2 protein expression, and the concentrations that decreased INSIG1 protein activity were equal to or lower than the concentrations that increased SREBP2 protein expression. Therefore, the data support a causal relationship. |
Relationship 3367: Increased, SREBP2 protein expression leads to Increased, cholesterol synthesis enzymes |
Strong support. Increased SREBP2 protein expression leads to increased cholesterol synthesis enzymes from gene-knockout, gene transfection, and diet studies. Increased SREBP2 protein expression occurred earlier in the time-course of exposure than increased cholesterol synthesis enzymes, and the concentrations that increased SREBP2 protein expression were equal to or lower than the concentrations that increased cholesterol synthesis enzymes. Therefore, the data support a causal relationship. |
Relationship 3368: Increased, cholesterol synthesis enzymes leads to Increased, plasma LDL cholesterol |
Strong support. Increased cholesterol synthesis enzymes leads to increased plasma LDL cholesterol from gene-knockout, gene transfection, and diet studies. Increased cholesterol synthesis enzymes occurred earlier in the time-course of exposure than increased plasma LDL cholesterol, and the concentrations that increased cholesterol synthesis enzymes were equal to or lower than the concentrations that increased plasma LDL cholesterol. Therefore, the data support a causal relationship. |
Overall |
Moderate to strong support. Evidence from empirical studies shows consistent change in both events including frequent testing in lab mammals, with upstream events occurring earlier in the time-course of exposure and at equal or lower concentrations than downstream events, supporting causal relationships. The relationship between Pregnane-X receptor and INSIG 1 protein activity is a possible exception, as there are some studies that show activation of Pregnane-X receptor leads to an increase in INSIG1 protein activity, the opposite direction from the mechanism of the AOP. |
Uncertainties and Inconsistencies
There is evidence from some studies that cholesterol synthesis enzyme squalene epoxidase (SQLE) protein expression can be increased by directly activation by binding by Pregnane X receptor (PXR) protein (Gwag et al. 2019; Jiang et al. 2021).
Known Modulating Factors
Quantitative Understanding
Considerations for Potential Applications of the AOP (optional)
References
Engelking, L.J., Liang, G., Hammer, R.E., Takaishi, K., Kuriyama, H., Evers, B.M., Li, W.-P., Horton, J.D., Goldstein, J.L., and Brown, M.S. 2005. Schoenheimer effect explained — feedback regulation of cholesterol synthesis in mice mediated by Insig proteins. The Journal of Clinical Investigation 115(9): 2489–2498.
Gwag, T., Meng, Z., Sui, Y., Helsley, R.N., Park, S.-H., Wang, S., Greenberg, R.N., and Zhou, C. 2019. Non-nucleoside reverse transcriptase inhibitor efavirenz activates PXR to induce hypercholesterolemia and hepatic steatosis. Journal of Hepatology 70: 930–940.
Horton, J.D., Shah, N.A., Warrington, J.A., Anderson, N.N., Park, S.W., Brown, M.S., and Goldstein, J.L. 2003. Combined analysis of oligonucleotide microarray data from transgenic and knockout mice identifies direct SREBP target genes. Proceedings of the National Academy of Sciences 100(21): 12027–12032.
Howe, V., Sharpe, L.J., Prabhu, A.V., and Brown, A.J. 2017. New insights into cellular cholesterol acquisition: promoter analysis of human HMGCR and SQLE, two key control enzymes in cholesterol synthesis. Biochim Biophys Acta 1862: 647–657.
Itkonen, A., Hakkola, J., and Rysa, J. 2023. Adverse outcome pathway for pregnane X receptor‑induced Hypercholesterolemia. Archives of Toxicology 97: 2861–2877.
Jiang, Y., Yao, X., Fan, S., Gao, Y., Zhang, H., Huang, M., and Bi, H. 2021. Lipidomic profiling reveals triacylglycerol accumulation in the liver during pregnane X receptor activation-induced hepatomegaly. Journal of Pharmaceutical and Biomedical Analysis 195: 113851.
Karpale, M. Karajamaki, A.J., Kummu, O., Gylling, H., Hyotylainen, T., Oresic, M., Tolonen, A., Hautajarvi, H., Savolainen, M.J., Ala-Korpela, M., Hukkanen, J., and Hakkola, J. 2021. Activation of pregnane X receptor induces atherogenic lipids and PCSK9 by a SREBP2-mediated mechanism. British Journal of Pharmacology 178: 2461–2481.
Lalanne, F., Lambert, G., Amar, M.J.A., Chetiveaux, M., Zair, Y., Jarnoux, A.-L., Ouguerram, K., Friburg, J., Seidah, N.G., Brewer, Jr., H.B., Krempf, M., and Costet, P. 2005. Wild-type PCSK9 inhibits LDL clearance but does not affect apoB-containing lipoprotein production in mouse and cultured cells. Journal of Lipid Research 46: 1312–1319.
Lambert, G., Jarnoux, A.-J., Pineau, T., Pape, O., Chetiveaux, M., Laboisse, C., Krempf, M., and Costet, P. 2006. Fasting induces hyperlipidemia in mice overexpressing Proprotein Convertase Subtilisin Kexin Type 9: Lack of modulation of very-low-density lipoprotein hepatic output by the low-density lipoprotein receptor. Endocrinology 147(10): 4985–4995.
Lv, Y., Luo, Y.-Y., Ren, H.-W., Li, C.-J., Xiang, Z.-X., and Luan, Z.L. 2022. The role of pregnane X receptor (PXR) in substance metabolism. Frontiers in Endocrinology 13: 959902.
MacFarlaine, M.R., Liang, G., and Engelking, L.J. 2014. Insig proteins mediate feedback inhibition of cholesterol synthesis in the intestine. The Journal of Biological Chemistry 289(4): 2148-2156.
Ouyang, S., Mo, Z., Sun, S., Yin, K., and Lv, Y. 2020. Emerging role of Insig-1 in lipid metabolism and lipid disorders. Clinica Chimica Acta 508: 206–212.
Sakakura, Y., Shimano, H., Sone, H., Takahashi, A., Inoue, N., Toyshima, H., Suzuki, S. and Yamada, N. 2001. Sterol regulatory element-binding proteins induce an entire pathway of cholesterol synthesis. Biochemical and Biophysical Research Communications 286: 176–183.
Yabe, D., Brown, M.S., and Goldstein, J.L. 2002. Insig-2, a second endoplasmic reticulum protein that binds SCAP and blocks export of sterol regulatory element-binding proteins. Proceedings of the National Academy of Sciences 99(20): 12753–12758.
Yang, T., Espenshade, P.J., Wright, M.E., Yabe, D., Gong, Y., Aebersold, R., Goldstein, J.L., and Brown, M.S. 2002. Crucial step in cholesterol homeostasis: sterols promote binding of SCAP to INSIG-1, a membrane protein that facilitates retention of SREBPs in ER. Cell 110: 489–500.