ABCG1 regulates mouse adipose tissue macrophage cholesterol levels and ratio of M1 to M2 cells in obesity and caloric restriction

In addition to triacylglycerols, adipocytes contain a large reserve of unesterified cholesterol. During adipocyte lipolysis and cell death seen during severe obesity and weight loss, free fatty acids and cholesterol become available for uptake and processing by adipose tissue macrophages (ATMs). We hypothesize that ATMs become cholesterol enriched and participate in cholesterol clearance from adipose tissue. We previously showed that ABCG1 is robustly upregulated in ATMs taken from obese mice and further enhanced by caloric restriction. Here, we found that ATMs taken from obese and calorie-restricted mice derived from transplantation of WT or Abcg1-deficient bone marrow are cholesterol enriched. ABCG1 levels regulate the ratio of classically activated (M1) to alternatively activated (M2) ATMs and their cellular cholesterol content. Using WT and Abcg1^−/− cultured macrophages, we found that Abcg1 is most highly expressed by M2 macrophages and that ABCG1 deficiency is sufficient to retard macrophage chemotaxis. However, changes in myeloid expression of Abcg1 did not protect mice from obesity or impaired glucose homeostasis. Overall, ABCG1 modulates ATM cholesterol content in obesity and weight loss regimes leading to an alteration in M1 to M2 ratio that we suggest is due to the extent of macrophage egress from adipose tissue.     

Regulation of ABCG1 expression in human keratinocytes and murine epidermis

ABCG1, a member of the ATP binding cassette superfamily, facilitates the efflux of cholesterol from cells to HDL. In this study, we demonstrate that ABCG1 is expressed in cultured human keratinocytes and murine epidermis, and induced during keratinocyte differentiation, with increased levels in the outer epidermis. ABCG1 is regulated by liver X receptor (LXR) and peroxisome proliferator-activated receptor-δ (PPAR-δ) activators, cellular sterol levels, and acute barrier disruption. Both LXR and PPAR-δ activators markedly stimulate ABCG1 expression in a dose- and time-dependent fashion. PPAR-γ activators also increase ABCG1 expression, but to a lesser degree. In contrast, activators of PPAR-α, retinoic acid receptor, retinoid X receptor, and vitamin D receptor do not alter ABCG1 expression. In response to increased intracellular sterol levels, ABCG1 expression increases, whereas inhibition of cholesterol biosynthesis decreases ABCG1 expression. In vivo, ABCG1 is stimulated 3–6 h after acute barrier disruption by either tape stripping or acetone treatment, an increase that can be inhibited by occlusion, suggesting a potential role of ABCG1 in permeability barrier homeostasis. Although Abcg1-null mice display normal epidermal permeability barrier function and gross morphology, abnormal lamellar body (LB) contents and secretion leading to impaired lamellar bilayer formation could be demonstrated by electron microscopy, indicating a potential role of ABCG1 in normal LB formation and secretion.     

EZN-2208 (PEG-SN38) Overcomes ABCG2-Mediated Topotecan Resistance in BRCA1-Deficient Mouse Mammary Tumors

BRCA1 dysfunction in hereditary breast cancer causes defective homology-directed DNA repair and sensitivity towards DNA damaging agents like the clinically used topoisomerase I inhibitors topotecan and irinotecan. Using our conditional K14cre;Brca1^F/F;p53^F/F mouse model, we showed previously that BRCA1;p53-deficient mammary tumors initially respond to topotecan, but frequently acquire resistance by overexpression of the efflux transporter ABCG2. Here, we tested the pegylated SN38 compound EZN-2208 as a novel approach to treat BRCA1-mutated tumors that express ABCG2. We found that EZN-2208 therapy resulted in more pronounced and durable responses of ABCG2-positive tumors than topotecan or irinotecan therapy. We also evaluated tumor-specific ABCG2 inhibition by Ko143 in Abcg2^−/− host animals that carried tumors with topotecan-induced ABCG2 expression. Addition of Ko143 moderately increased overall survival of these animals, but did not yield tumor responses like those seen after EZN-2208 therapy. Our results suggest that pegylation of Top1 inhibitors may be a useful strategy to circumvent efflux transporter-mediated resistance and to improve their efficacy in the clinic.     

Loss of ABCG1 results in chronic pulmonary inflammation

ABCG1, a member of the ATP-binding cassette transporter superfamily, is highly expressed in multiple cells of the lung. Loss of ABCG1 results in severe pulmonary lipidosis in mice, with massive deposition of cholesterol in both alveolar macrophages and type 2 cells and the accumulation of excessive surfactant phospholipids. These observations are consistent with ABCG1 controlling cellular sterol metabolism. Herein, we report on the progressive and chronic inflammatory process that accompanies the lipidosis in the lungs of Abcg1-/- mice. Compared with wild-type animals, the lungs of aged chow-fed mice deficient in ABCG1 show distinctive signs of inflammation that include macrophage accumulation, lymphocytic infiltration, hemorrhage, eosinophilic crystals, and elevated levels of numerous cytokines and cytokine receptors. Analysis of bronchoalveolar lavages obtained from Abcg1-/- mice revealed elevated numbers of foamy macrophages and leukocytes and the presence of multiple markers of inflammation including crystals of chitinase-3-like proteins. These data suggest that cholesterol and/or cholesterol metabolites that accumulate in Abcg1-/- lungs can trigger inflammatory signaling pathways. Consistent with this hypothesis, the expression of a number of cytokines was found to be significantly increased following increased cholesterol delivery to either primary peritoneal macrophages or Raw264.7 cells. Finally, cholesterol loading of primary mouse macrophages induced cytokine mRNAs to higher levels in Abcg1-/-, as compared with wild-type cells. These results demonstrate that ABCG1 plays critical roles in pulmonary homeostasis, balancing both lipid/cholesterol metabolism and inflammatory responses.     

Deletion of the transmembrane transporter ABCG1 results in progressive pulmonary lipidosis

We show that mice lacking the ATP-binding cassette transmembrane transporter ABCG1 show progressive and age-dependent severe pulmonary lipidosis that recapitulates the phenotypes of different respiratory syndromes in both humans and mice. The lungs of chow-fed Abcg1(-/-) mice, >6-months old, exhibit extensive subpleural cellular accumulation, macrophage, and pneumocyte type 2 hypertrophy, massive lipid deposition in both macrophages and pneumocytes and increased levels of surfactant. No such abnormalities are observed at 3 months of age. However, gene expression profiling reveals significant changes in the levels of mRNAs encoding key genes involved in lipid metabolism in both 3- and 8-month-old Abcg1(-/-) mice. These data suggest that the lungs of young Abcg1(-/-) mice maintain normal lipid levels by repressing lipid biosynthetic pathways and that such compensation is inadequate as the mice mature. Studies with A-549 cells, a model for pneumocytes type 2, demonstrate that overexpression of ABCG1 specifically stimulates the efflux of cellular cholesterol by a process that is dependent upon phospholipid secretion. In addition, we demonstrate that Abcg1(-/-), but not wild-type macrophages, accumulate cholesterol ester droplets when incubated with surfactant. Together, these data provide a mechanism to explain the lipid accumulation in the lungs of Abcg1(-/-)mice. In summary, our results demonstrate that ABCG1 plays essential roles in pulmonary lipid homeostasis.     

Genetic ablation of caveolin-2 sensitizes mice to bleomycin-induced injury

Caveolar domains act as platforms for the organization of molecular complexes involved in signal transduction. Caveolin proteins, the principal structural components of caveolae, have been involved in many cellular processes. Caveolin-1 (Cav-1) and caveolin-2 (Cav-2) are highly expressed in the lung. Cav-1-deficient mice (Cav-1^−/−) and Cav-2-deficient mice (Cav-2^−/−) exhibit severe lung dysfunction attributed to a lack of Cav-2 expression. Recently, Cav-1 has been shown to regulate lung fibrosis in different models. Here, we show that Cav-2 is also involved in modulation of the fibrotic response, but through distinct mechanisms. Treatment of wild-type mice with the pulmonary fibrosis-inducer bleomycin reduced the expression of Cav-2 and its phosphorylation at tyrosine 19. Importantly, Cav-2^−/− mice, but not Cav-1^−/− mice, were more sensitive to bleomycin-induced lung injury in comparison to wild-type mice. Bleomycin-induced lung injury was characterized by alveolar thickening, increase in cell density, and extracellular matrix deposition. The lung injury observed in bleomycin-treated Cav-2^−/− mice was not associated with alterations in the TGF-β signaling pathway and/or in the ability to produce collagen. However, apoptosis and proliferation were more prominent in lungs of bleomycin-treated Cav-2^−/− mice. Since Cav-1^−/− mice also lack Cav-2 expression and show a different outcome after bleomycin treatment, we conclude that Cav-1 and Cav-2 have distinct roles in bleomycin induced-lung fibrosis, and that the balance of both proteins determines the development of the fibrotic process.     

ABCG1 is involved in vitamin E efflux

Vitamin E membrane transport has been shown to involve the cholesterol transporters SR-BI, ABCA1 and NPC1L1. Our aim was to investigate the possible participation of another cholesterol transporter in cellular vitamin E efflux: ABCG1. In Abcg1-deficient mice, vitamin E concentration was reduced in plasma lipoproteins whereas most tissues displayed a higher vitamin E content compared to wild-type mice. α- and γ-tocopherol efflux was increased in CHO cells overexpressing human ABCG1 compared to control cells. Conversely, α- and γ-tocopherol efflux was decreased in ABCG1-knockdown human cells (Hep3B hepatocytes and THP-1 macrophages). Interestingly, α- and γ-tocopherol significantly downregulated ABCG1 and ABCA1 expression levels in Hep3B and THP-1, an effect confirmed in vivo in rats given vitamin E for 5 days. This was likely due to reduced LXR activation by oxysterols, as Hep3B cells and rat liver treated with vitamin E displayed a significantly reduced content in oxysterols compared to their respective controls. Overall, the present study reveals for the first time that ABCG1 is involved in cellular vitamin E efflux.     

Cooperative functions of Chk1 and Chk2 reduce tumour susceptibility in vivo

Although the linkage of Chk1 and Chk2 to important cancer signalling suggests that these kinases have functions as tumour suppressors, neither Chk1^+/− nor Chk2^−/− mice show a predisposition to cancer under unperturbed conditions. We show here that Chk1^+/−Chk2^−/− and Chk1^+/−Chk2^+/− mice have a progressive cancer-prone phenotype. Deletion of a single Chk1 allele compromises G2/M checkpoint function that is not further affected by Chk2 depletion, whereas Chk1 and Chk2 cooperatively affect G1/S and intra-S phase checkpoints. Either or both of the kinases are required for DNA repair depending on the type of DNA damage. Mouse embryonic fibroblasts from the double-mutant mice showed a higher level of p53 with spontaneous DNA damage under unperturbed conditions, but failed to phosphorylate p53 at S23 and further induce p53 expression upon additional DNA damage. Neither Chk1 nor Chk2 is apparently essential for p53- or Rb-dependent oncogene-induced senescence. Our results suggest that the double Chk mutation leads to a high level of spontaneous DNA damage, but fails to eliminate cells with damaged DNA, which may ultimately increase cancer susceptibility independently of senescence.     

SUMO2 is essential while SUMO3 is dispensable for mouse embryonic development

Small ubiquitin-like modifier (SUMO1–3) conjugation plays a critical role in embryogenesis. Embryos deficient in the SUMO-conjugating enzyme Ubc9 die at the early postimplantation stage. Sumo1^−/− mice are viable, as SUMO2/3 can compensate for most SUMO1 functions. To uncover the role of SUMO2/3 in embryogenesis, we generated Sumo2- and Sumo3-null mutant mice. Here, we report that Sumo3^−/− mice were viable, while Sumo2^−/− embryos exhibited severe developmental delay and died at approximately embryonic day 10.5 (E10.5). We also provide evidence that SUMO2 is the predominantly expressed SUMO isoform. Furthermore, although Sumo2^+/− and Sumo2^+/−;Sumo3^+/− mice lacked any overt phenotype, only 2 Sumo2^+/−;Sumo3^−/− mice were found at birth in 35 litters after crossing Sumo2^+/−;Sumo3^+/− with Sumo3^−/− mice, and these rare mice were considerably smaller than littermates of the other genotypes. Thus, our findings suggest that expression levels and not functional differences between SUMO2 and SUMO3 are critical for normal embryogenesis.     

StarD7 Knockdown Modulates ABCG2 Expression, Cell Migration, Proliferation, and Differentiation of Human Choriocarcinoma JEG-3 Cells

Background

StAR-related lipid transfer domain containing 7 (StarD7) is a member of the START-domain protein family whose function still remains unclear. Our data from an explorative microarray assay performed with mRNAs from StarD7 siRNA-transfected JEG-3 cells indicated that ABCG2 (ATP-binding cassette sub-family G member 2) was one of the most abundantly downregulated mRNAs.

Methodology/Principal Findings

Here, we have confirmed that knocking down StarD7 mRNA lead to a decrease in the xenobiotic/lipid transporter ABCG2 at both the mRNA and protein levels (−26.4% and −41%, p<0.05, at 48 h of culture, respectively). Also a concomitant reduction in phospholipid synthesis, bromodeoxyuridine (BrdU) uptake and ³H-thymidine incorporation was detected. Wound healing and transwell assays revealed that JEG-3 cell migration was significantly diminished (p<0.05). Conversely, biochemical differentiation markers such as human chorionic gonadotrophin β-subunit (βhCG) protein synthesis and secretion as well as βhCG and syncytin-1 mRNAs were increased approximately 2-fold. In addition, desmoplakin immunostaining suggested that there was a reduction of intercellular desmosomes between adjacent JEG-3 cells after knocking down StarD7.

Conclusions/Significance

Altogether these findings provide evidence for a role of StarD7 in cell physiology indicating that StarD7 modulates ABCG2 multidrug transporter level, cell migration, proliferation, and biochemical and morphological differentiation marker expression in a human trophoblast cell model.     

ABCG1 regulates pulmonary surfactant metabolism in mice and men

Idiopathic pulmonary alveolar proteinosis (PAP) is a rare lung disease characterized by accumulation of surfactant. Surfactant synthesis and secretion are restricted to epithelial type 2 (T2) pneumocytes (also called T2 cells). Clearance of surfactant is dependent upon T2 cells and macrophages. ABCG1 is highly expressed in both T2 cells and macrophages. ABCG1-deficient mice accumulate surfactant, lamellar body-loaded T2 cells, lipid-loaded macrophages, B-1 lymphocytes, and immunoglobulins, clearly demonstrating that ABCG1 has a critical role in pulmonary homeostasis. We identify a variant in the ABCG1 promoter in patients with PAP that results in impaired activation of ABCG1 by the liver X receptor α, suggesting that ABCG1 basal expression and/or induction in response to sterol/lipid loading is essential for normal lung function. We generated mice lacking ABCG1 specifically in either T2 cells or macrophages to determine the relative contribution of these cell types on surfactant lipid homeostasis. These results establish a critical role for T2 cell ABCG1 in controlling surfactant and overall lipid homeostasis in the lung and in the pathogenesis of human lung disease.     

Reduced bone mass and muscle strength in male 5α-reductase type 1 inactivated mice

Androgens are important regulators of bone mass but the relative importance of testosterone (T) versus dihydrotestosterone (DHT) for the activation of the androgen receptor (AR) in bone is unknown. 5α-reductase is responsible for the irreversible conversion of T to the more potent AR activator DHT. There are two well established isoenzymes of 5α-reductase (type 1 and type 2), encoded by separate genes (Srd5a1 and Srd5a2). 5α-reductase type 2 is predominantly expressed in male reproductive tissues whereas 5α-reductase type 1 is highly expressed in liver and moderately expressed in several other tissues including bone. The aim of the present study was to investigate the role of 5α-reductase type 1 for bone mass using Srd5a1^−/− mice. Four-month-old male Srd5a1 ^−/− mice had reduced trabecular bone mineral density (−36%, p<0.05) and cortical bone mineral content (−15%, p<0.05) but unchanged serum androgen levels compared with wild type (WT) mice. The cortical bone dimensions were reduced in the male Srd5a1 ^−/− mice as a result of a reduced cortical periosteal circumference compared with WT mice. T treatment increased the cortical periosteal circumference (p<0.05) in orchidectomized WT mice but not in orchidectomized Srd5a1 ^−/− mice. Male Srd5a1 ^−/− mice demonstrated a reduced forelimb muscle grip strength compared with WT mice (p<0.05). Female Srd5a1 ^−/− mice had slightly increased cortical bone mass associated with elevated circulating levels of androgens. In conclusion, 5α-reductase type 1 inactivated male mice have reduced bone mass and forelimb muscle grip strength and we propose that these effects are due to lack of 5α-reductase type 1 expression in bone and muscle. In contrast, the increased cortical bone mass in female Srd5a1 ^−/− mice, is an indirect effect mediated by elevated circulating androgen levels.     

Activation of the Liver X Receptor Stimulates Trans-intestinal Excretion of Plasma Cholesterol

Recent studies have indicated that direct intestinal secretion of plasma cholesterol significantly contributes to fecal neutral sterol loss in mice. The physiological relevance of this novel route, which represents a part of the reverse cholesterol transport pathway, has not been directly established in vivo as yet. We have developed a method to quantify the fractional and absolute contributions of several cholesterol fluxes to total fecal neutral sterol loss in vivo in mice, by assessing the kinetics of orally and intravenously administered stable isotopically labeled cholesterol combined with an isotopic approach to assess the fate of de novo synthesized cholesterol. Our results show that trans-intestinal cholesterol excretion significantly contributes to removal of blood-derived free cholesterol in C57Bl6/J mice (33% of 231 μmol/kg/day) and that pharmacological activation of LXR with T0901317 strongly stimulates this pathway (63% of 706 μmol/kg/day). Trans-intestinal cholesterol excretion is impaired in mice lacking Abcg5 (−4%), suggesting that the cholesterol transporting Abcg5/Abcg8 heterodimer is involved in this pathway. Our data demonstrate that intestinal excretion represents a quantitatively important route for fecal removal of neutral sterols independent of biliary secretion in mice. This pathway is sensitive to pharmacological activation of the LXR system. These data support the concept that the intestine substantially contributes to reverse cholesterol transport.Reverse cholesterol transport (RCT)³ is defined as the flux of excess cholesterol from peripheral tissues toward the liver followed by biliary secretion and subsequent disposal via the feces (1). Accumulation of cholesterol in macrophages in the vessel wall is considered a primary event in the development of atherosclerosis and, therefore, removal of excess cholesterol from these cells is of crucial importance for prevention and/or treatment of atherosclerotic cardiovascular diseases. It is generally accepted that HDL is the obligate transport vehicle in RCT and that plasma HDL levels reflect the capacity to accommodate this flux. In line herewith, HDL-raising therapies are currently considered as a promising strategy for prevention and treatment of atherosclerotic cardiovascular diseases (2). In the “classical” scenario, the liver has a central role in RCT (3). Biliary secretion of free cholesterol, facilitated by the heterodimeric ABC-transporter ABCG5/ABCG8 (4), and hepatic conversion of cholesterol into bile acids followed by fecal excretion are referred to as the main routes for quantitatively important elimination of cholesterol from the body. Fecal excretion of sterols is stimulated upon whole body activation of the liver X receptor (LXR, NR1H2/3), a member of the nuclear receptor family for which oxysterols have been identified as natural ligands (5). LXR regulates expression of several genes involved in RCT and activation of LXR by synthetic agonists leads to elevated plasma HDL-cholesterol levels, increased hepatobiliary cholesterol secretion, reduced fractional intestinal cholesterol absorption and increased fecal sterol loss (6). LXR is thus considered an attractive target for therapeutic strategies aimed at stimulation of RCT, which, however, will require approaches to circumvent potential detrimental consequences of LXR activation such as induction of lipogenesis.Recent studies indicate that the classical concept of RCT may require reconsideration. Studies in apoA-I-deficient mice revealed that the magnitude of the centripetal cholesterol flux from the periphery to the liver is not related to the concentration of HDL-cholesterol or apoA-I in plasma (7). Furthermore, Abca1^−/− mice that completely lack plasma HDL show unaffected rates of hepatobiliary cholesterol secretion and fecal sterol loss (8). Additionally, mice lacking both Abcg5 and Abcg8 do not show a reduction in fecal neutral sterol excretion to the extent expected on the basis of their strongly reduced hepatobiliary cholesterol secretion (9). Recent studies by Plösch et al. (6) have revealed that increased fecal neutral sterol loss upon general LXR activation cannot be attributed to the increased hepatobiliary cholesterol secretion only, suggesting a major contribution of the intestine in excretion of cholesterol. This potential role of the intestine in cholesterol removal from the body has been corroborated by Kruit et al. (10), who showed that fecal sterol loss is not affected in Mdr2^−/− (Abcb4^−/−) mice that have a dramatic reduction in biliary cholesterol secretion (11). Moreover, intravenously administered [³H]cholesterol could be recovered in the neutral sterol fraction of the feces in these mice and fecal excretion of neutral sterols was stimulated upon treatment with an LXR agonist (10). However, the exact quantitative contribution of the direct intestinal pathway under physiological conditions has not directly been determined so far. Very recently, intestinal perfusion studies in mice revealed that, in the presence of mixed micelles as cholesterol acceptors in the intestinal lumen, murine enterocytes indeed have a high capacity to secrete cholesterol via a specific process that is most active in the proximal part of the small intestine (12). In addition, it was shown that direct trans-intestinal cholesterol excretion (TICE) could be stimulated by a high fat diet. The existence of a non-biliary route for fecal neutral sterol excretion is further supported by very recent studies by Brown et al. (13) in mice with targeted deletion of hepatic ACAT2.The present study provides insight into the relative and absolute contributions of several cholesterol fluxes relevant to total fecal sterol loss in mice, making use of a panel of stable isotope tracers. Our results show that TICE is a major route for removal of blood-derived free cholesterol and that pharmacological LXR activation strongly stimulates this arm of the reverse cholesterol transport pathway.     

GPIHBP1 stabilizes lipoprotein lipase and prevents its inhibition by angiopoietin-like 3 and angiopoietin-like 4

Glycosylphosphatidylinositol-anchored HDL-binding protein (GPIHBP1) binds both LPL and chylomicrons, suggesting that GPIHBP1 is a platform for LPL-dependent processing of triglyceride (TG)-rich lipoproteins. Here, we investigated whether GPIHBP1 affects LPL activity in the absence and presence of LPL inhibitors angiopoietin-like (ANGPTL)3 and ANGPTL4. Like heparin, GPIHBP1 stabilized but did not activate LPL. ANGPTL4 potently inhibited nonstabilized LPL as well as heparin-stabilized LPL but not GPIHBP1-stabilized LPL. Like ANGPTL4, ANGPTL3 inhibited nonstabilized LPL but not GPIHBP1-stabilized LPL. ANGPTL3 also inhibited heparin-stabilized LPL but with less potency than nonstabilized LPL. Consistent with these in vitro findings, fasting serum TGs of Angptl4^−/−/Gpihbp1^−/− mice were lower than those of Gpihbp1^−/− mice and approached those of wild-type littermates. In contrast, serum TGs of Angptl3^−/−/Gpihbp1^−/− mice were only slightly lower than those of Gpihbp1^−/− mice. Treating Gpihbp1^−/− mice with ANGPTL4- or ANGPTL3-neutralizing antibodies recapitulated the double knockout phenotypes. These data suggest that GPIHBP1 functions as an LPL stabilizer. Moreover, therapeutic agents that prevent LPL inhibition by ANGPTL4 or, to a lesser extent, ANGPTL3, may benefit individuals with hyperlipidemia caused by gene mutations associated with decreased LPL stability.