Se-Hyung Park

Bisphenol A and its analogues activate human pregnane X receptor.

Background: Bisphenol A (BPA) is a base chemical used extensively in many consumer products. BPA and its analogues are present in environmental and human samples. Many endocrine-disrupting chemicals, including BPA, have been shown to activate the pregnane X receptor (PXR), a nuclear receptor that functions as a master regulator of xenobiotic metabolism. However, the detailed mechanism by which these chemicals activate PXR remains unknown. Objective: We investigated the mechanism by which BPA interacts with and activates PXR and examined selected BPA analogues to determine whether they bind to and activate PXR. Methods: Cell-based reporter assays, in silico ligand–PXR docking studies, and site-directed mutagenesis were combined to study the interaction between BPA and PXR. We also investigated the influence of BPA and its analogues on the regulation of PXR target genes in human LS180 cells. Results: We found that BPA and several of its analogues are potent agonists for human PXR (hPXR) but do not affect mouse PXR activity. We identified key residues within hPXR’s ligand-binding pocket that constitute points of interaction with BPA. We also deduced the structural requirements of BPA analogues that activate hPXR. BPA and its analogues can also induce PXR target gene expression in human LS180 cells. Conclusions: The present study advances our understanding of the mechanism by which BPA interacts with and activates human PXR. Activation of PXR by BPA may explain some of the adverse effects of BPA in humans.

Bisphenol A Increases Atherosclerosis in Pregnane X Receptor-Humanized ApoE Deficient Mice

Background Bisphenol A (BPA) is a base chemical used extensively in many consumer products. BPA has recently been associated with increased risk of cardiovascular disease (CVD) in multiple large‐scale human population studies, but the underlying mechanisms remain elusive. We previously reported that BPA activates the pregnane X receptor (PXR), which acts as a xenobiotic sensor to regulate xenobiotic metabolism and has pro‐atherogenic effects in animal models upon activation. Interestingly, BPA is a potent agonist of human PXR but does not activate mouse or rat PXR signaling, which confounds the use of rodent models to evaluate mechanisms of BPA‐mediated CVD risk. This study aimed to investigate the atherogenic mechanism of BPA using a PXR‐humanized mouse model. Methods and Results A PXR‐humanized ApoE deficient (huPXR•ApoE−/−) mouse line was generated that respond to human PXR ligands and feeding studies were performed to determine the effects of BPA exposure on atherosclerosis development. Exposure to BPA significantly increased atherosclerotic lesion area in the aortic root and brachiocephalic artery of huPXR•ApoE−/− mice by 104% (P<0.001) and 120% (P<0.05), respectively. By contrast, BPA did not affect atherosclerosis development in the control littermates without human PXR. BPA exposure did not affect plasma lipid levels but increased CD36 expression and lipid accumulation in macrophages of huPXR•ApoE−/− mice. Conclusion These findings identify a molecular mechanism that could link BPA exposure to increased risk of CVD in exposed individuals. PXR is therefore a relevant target for future risk assessment of BPA and related environmental chemicals in humans.

Myeloid-Specific IκB Kinase β Deficiency Decreases Atherosclerosis in Low-Density Lipoprotein Receptor–Deficient Mice

Objective—Inflammatory responses are the driving force of atherosclerosis development. I&kgr;B kinase &bgr; (IKK&bgr;), a central coordinator in inflammation through regulation of nuclear factor-&kgr;B, has been implicated in the pathogenesis of atherosclerosis. Macrophages play an essential role in the initiation and progression of atherosclerosis, yet the role of macrophage IKK&bgr; in atherosclerosis remains elusive and controversial. This study aims to investigate the impact of IKK&bgr; expression on macrophage functions and to assess the effect of myeloid-specific IKK&bgr; deletion on atherosclerosis development. Methods and Results—To explore the issue of macrophage IKK&bgr; involvement of atherogenesis, we generated myeloid-specific IKK&bgr;-deficient low-density lipoprotein receptor–deficient mice (IKK&bgr;&Dgr;MyeLDLR−/−). Deficiency of IKK&bgr; in myeloid cells did not affect plasma lipid levels but significantly decreased diet-induced atherosclerotic lesion areas in the aortic root, brachiocephalic artery, and aortic arch of low-density lipoprotein receptor–deficient mice. Ablation of myeloid IKK&bgr; attenuated macrophage inflammatory responses and decreased atherosclerotic lesional inflammation. Furthermore, deficiency of IKK&bgr; decreased adhesion, migration, and lipid uptake in macrophages. Conclusion—The present study demonstrates a pivotal role for myeloid IKK&bgr; expression in atherosclerosis by modulating macrophage functions involved in atherogenesis. These results suggest that inhibiting nuclear factor-&kgr;B activation in macrophages may represent a feasible approach to combat atherosclerosis.

Pregnane X receptor mediates dyslipidemia induced by the HIV protease inhibitor amprenavir in mice.

Human immunodeficiency virus (HIV) protease inhibitors (PIs) have been used successfully in extending the life span of people infected with HIV. The use of PIs has also been associated with dyslipidemia and an increased risk of cardiovascular disease, but the underlying mechanisms remain elusive. Several PIs have been implicated in activating the nuclear receptor pregnane X receptor (PXR), which acts as a xenobiotic sensor to regulate xenobiotic metabolism in the liver and intestine. Recent studies indicate that PXR may also play an important role in the regulation of lipid homeostasis. In the present study, we identified amprenavir, a widely used HIV PI, as a potent PXR-selective agonist. Computational docking studies combined with site-direct mutagenesis identified several key residues within the ligand-binding pocket of PXR that constitute points of interaction with amprenavir. Amprenavir efficiently activated PXR and induced PXR target gene expression in vitro and in vivo. Short-term exposure to amprenavir significantly increased plasma total cholesterol and atherogenic low-density lipoprotein cholesterol levels in wild-type mice, but not in PXR-deficient mice. Amprenavir-mediated PXR activation stimulated the expression of several key intestinal genes involved in lipid homeostasis. These findings provide critical mechanistic insight for understanding the impact of PIs on cardiovascular disease and demonstrate a potential role of PXR in mediating the adverse effects of HIV PIs in humans.

Intestinal Pregnane X Receptor Links Xenobiotic Exposure and Hypercholesterolemia

Recent studies have associated endocrine-disrupting chemical (EDC) exposure with the increased risk of cardiovascular disease in humans, but the underlying mechanisms responsible for these associations remain elusive. Many EDCs have been implicated in activation of the nuclear receptor pregnane X receptor (PXR), which acts as a xenobiotic sensor to regulate xenobiotic metabolism in the liver and intestine. Here we report an important role of intestinal PXR in linking xenobiotic exposure and hyperlipidemia. We identified tributyl citrate (TBC), one of a large group of Food and Drug Administration-approved plasticizers for pharmaceutical or food applications, as a potent and selective PXR agonist. TBC efficiently activated PXR and induced PXR target gene expression in vitro and in vivo. Interestingly, TBC activated intestinal PXR but did not affect hepatic PXR activity. Exposure to TBC increased plasma total cholesterol and atherogenic low-density lipoprotein cholesterol levels in wild-type mice, but not in PXR-deficient mice. TBC-mediated PXR activation stimulated the expression of an essential cholesterol transporter, Niemann-Pick C1-like 1 (NPC1L1), in the intestine. Promoter analysis revealed a DR-4 type of PXR response element in the human NPC1L1 promoter, and TBC promoted PXR recruitment onto the NPC1L1 promoter. Consistently, TBC treatment significantly increased lipid uptake by human and murine intestinal cells and deficiency of PXR inhibited TBC-elicited lipid uptake. These findings provide critical mechanistic insight for understanding the impact of EDC-mediated PXR activation on lipid homeostasis and demonstrate a potential role of PXR in mediating the adverse effects of EDCs on cardiovascular disease risk in humans.

IKKβ is essential for adipocyte survival and adaptive adipose remodeling in obesity

IκB kinase β (IKKβ), a central coordinator of inflammatory responses through activation of nuclear factor-κB (NF-κB), has been implicated as a critical molecular link between inflammation and metabolic disorders; however, the role of adipocyte IKKβ in obesity and related metabolic disorders remains elusive. Here we report an essential role of IKKβ in the regulation of adipose remodeling and adipocyte survival in diet-induced obesity. Targeted deletion of IKKβ in adipocytes does not affect body weight, food intake, and energy expenditure but results in an exaggerated diabetic phenotype when challenged with a high-fat diet (HFD). IKKβ-deficient mice have multiple histopathologies in visceral adipose tissue, including increased adipocyte death, amplified macrophage infiltration, and defective adaptive adipose remodeling. Deficiency of IKKβ also leads to increased adipose lipolysis, elevated plasma free fatty acid (FFA) levels, and impaired insulin signaling. Mechanistic studies demonstrated that IKKβ is a key adipocyte survival factor and that IKKβ protects murine and human adipocytes from HFD- or FFA-elicited cell death through NF-κB–dependent upregulation of antiapoptotic proteins and NF-κB–independent inactivation of proapoptotic BAD protein. Our findings establish IKKβ as critical for adipocyte survival and adaptive adipose remodeling in obesity.

Targeting IκB kinase β in Adipocyte Lineage Cells for Treatment of Obesity and Metabolic Dysfunctions.

IκB kinase β (IKKβ), a central coordinator of inflammation through activation of nuclear factor‐κB, has been identified as a potential therapeutic target for the treatment of obesity‐associated metabolic dysfunctions. In this study, we evaluated an antisense oligonucleotide (ASO) inhibitor of IKKβ and found that IKKβ ASO ameliorated diet‐induced metabolic dysfunctions in mice. Interestingly, IKKβ ASO also inhibited adipocyte differentiation and reduced adiposity in high‐fat (HF)‐fed mice, indicating an important role of IKKβ signaling in the regulation of adipocyte differentiation. Indeed, CRISPR/Cas9‐mediated genomic deletion of IKKβ in 3T3‐L1 preadipocytes blocked these cells differentiating into adipocytes. To further elucidate the role of adipose progenitor IKKβ signaling in diet‐induced obesity, we generated mice that selectively lack IKKβ in the white adipose lineage and confirmed the essential role of IKKβ in mediating adipocyte differentiation in vivo. Deficiency of IKKβ decreased HF‐elicited adipogenesis in addition to reducing inflammation and protected mice from diet‐induced obesity and insulin resistance. Further, pharmacological inhibition of IKKβ also blocked human adipose stem cell differentiation. Our findings establish IKKβ as a pivotal regulator of adipogenesis and suggest that overnutrition‐mediated IKKβ activation serves as an initial signal that triggers adipose progenitor cell differentiation in response to HF feeding. Inhibition of IKKβ with antisense therapy may represent as a novel therapeutic approach to combat obesity and metabolic dysfunctions. Stem Cells 2016;34:1883–1895

IKK β is a β -catenin kinase that regulates mesenchymal stem cell differentiation

Mesenchymal stem cells (MSCs) can give rise to both adipocytes and osteoblasts, but the molecular mechanisms underlying MSC fate determination remain poorly understood. IκB kinase β (IKKβ), a central coordinator of inflammation and immune responses through activation of NF-κB, has been implicated as a critical molecular link between obesity and metabolic disorders. Here, we show that IKKβ can reciprocally regulate adipocyte and osteoblast differentiation of murine and human MSCs through an NF-κB-independent mechanism. IKKβ is a β-catenin kinase that phosphorylates the conserved degron motif of β-catenin to prime it for β-TrCP-mediated ubiquitination and degradation, thereby increasing adipogenesis and inhibiting osteogenesis in MSCs. Animal studies demonstrated that deficiency of IKKβ in BM mesenchymal stromal cells increased bone mass and decreased BM adipocyte formation in adult mice. In humans, IKKβ expression in adipose tissue was also positively associated with increased adiposity and elevated β-catenin phosphorylation. These findings suggest IKKβ as a key molecular switch that regulates MSC fate, and they provide potentially novel mechanistic insights into the understanding of the cross-regulation between the evolutionarily conserved IKKβ and Wnt/β-catenin signaling pathways. The IKKβ-Wnt axis we uncovered may also have important implications for development, homeostasis, and disease pathogenesis.

Perinatal Bisphenol A Exposure Increases Atherosclerosis in Adult Male PXR-Humanized Mice

Bisphenol A (BPA) is a base chemical used extensively in numerous consumer products, and human exposure to BPA is ubiquitous. Higher BPA exposure has been associated with an increased risk of atherosclerosis and cardiovascular disease (CVD) in multiple human population-based studies. However, the underlying mechanisms responsible for the associations remain elusive. We previously reported that BPA activates the xenobiotic receptor pregnane X receptor (PXR), which has proatherogenic effects in animal models. Because BPA is a potent agonist for human PXR but does not affect rodent PXR activity, a suitable PXR-humanized apolipoprotein E-deficient (huPXR•ApoE-/-) mouse model was developed to study BPAs atherogenic effects. Chronic BPA exposure increased atherosclerosis in the huPXR•ApoE-/- mice. We report that BPA exposure can also activate human PXR signaling in the heart tubes of huPXR•ApoE-/- embryos, and perinatal BPA exposure exacerbated atherosclerosis in adult male huPXR•ApoE-/- offspring. However, atherosclerosis development in female offspring was not affected by perinatal BPA exposure. Perinatal BPA exposure did not affect plasma lipid levels but increased aortic and atherosclerotic lesional fatty acid transporter CD36 expression in male huPXR•ApoE-/- offspring. Mechanistically, PXR epigenetically regulated CD36 expression by increasing H3K4me3 levels and decreasing H3K27me3 levels in the CD36 promoter in response to perinatal BPA exposure. The findings from the present study contribute to our understanding of the association between BPA exposure and increased atherosclerosis or CVD risk in humans, and activation of human PXR should be considered for future BPA risk assessment.

Myeloid β-Catenin Deficiency Exacerbates Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient Mice

Objective— The Wnt/&bgr;-catenin signaling is an ancient and evolutionarily conserved pathway that regulates essential aspects of cell differentiation, proliferation, migration and polarity. Canonical Wnt/&bgr;-catenin signaling has also been implicated in the pathogenesis of atherosclerosis. Macrophage is one of the major cell types involved in the initiation and progression of atherosclerosis, but the role of macrophage &bgr;-catenin in atherosclerosis remains elusive. This study aims to investigate the impact of &bgr;-catenin expression on macrophage functions and atherosclerosis development. Approach and Results— To investigate the role of macrophage canonical Wnt/&bgr;-catenin signaling in atherogenesis, we generated &bgr;-catenin&Dgr;myeLDLR−/− mice (low-density lipoprotein receptor–deficient mice with myeloid-specific &bgr;-catenin deficiency). As expected, deletion of &bgr;-catenin decreased macrophage adhesion and migration properties in vitro. However, deficiency of &bgr;-catenin significantly increased atherosclerotic lesion areas in the aortic root of LDLR−/− (low-density lipoprotein receptor–deficient) mice without affecting the plasma lipid levels and atherosclerotic plaque composition. Mechanistic studies revealed that &bgr;-catenin can regulate activation of STAT (signal transducer and activator of transcription) pathway in macrophages, and ablation of &bgr;-catenin resulted in STAT3 downregulation and STAT1 activation, leading to elevated macrophage inflammatory responses and increased atherosclerosis. Conclusions— This study demonstrates a critical role of myeloid &bgr;-catenin expression in atherosclerosis by modulating macrophage inflammatory responses.