The ABC transporter, Mrp4, transports the sulfated steroid DHEA-s, and sulfated bile acids interact with Mrp4 with high affinity. Hepatic Mrp4 levels are low, but increase under cholestatic conditions. We therefore inferred that up-regulation of Mrp4 during cholestasis is a compensatory mechanism to protect the liver from accumulation of hydrophobic bile acids. We determined that the nuclear receptor CAR is required to coordinately up-regulate hepatic expression of Mrp4 and an enzyme known to sulfate hydroxy-bile acids and steroids, Sult2a1. CAR activators increased Mrp4 and Sult2a1 expression in primary human hepatocytes and HepG2, a human liver cell line. Sult2a1 was down-regulated in Mrp4-null mice, further indicating an inter-relation between Mrp4 and Sult2a1 gene expression. Based on the hydrophilic nature of sulfated bile acids and the Mrp4 capability to transport sulfated steroids, our findings suggest that Mrp4 and Sult2a1 participate in an integrated pathway mediating elimination of sulfated steroid and bile acid metabolites from the liver.
The RING E3 ubiquitin ligase inducible degrader of the LDL receptor (IDOL, also known as MYLIP) promotes ubiquitylation and subsequent lysosomal degradation of the LDL receptor (LDLR), thus acting to limit uptake of lipoprotein-derived cholesterol into cells. Next to the LDLR, IDOL also promotes degradation of two related receptors, the very LDL receptor (VLDLR) and apolipoprotein E receptor 2 (APOER2), which have important signaling functions in the brain. We review here the emerging role of IDOL in lipoprotein and energy metabolism, neurodegenerative diseases, and the potential for therapeutic targeting of IDOL.Genetic studies suggest an association between IDOL and lipoprotein metabolism in humans. Studies in rodents and nonhuman primates support an in-vivo role for IDOL in lipoprotein metabolism, and also uncovered an unexpected role in whole-body energy metabolism. Recent evaluation of IDOL function in the brain revealed a role in memory formation and progression of Alzheimer's disease. The report of the first IDOL inhibitor may facilitate further investigations on therapeutic strategies to target IDOL.IDOL is emerging as an important determinant of lipid and energy metabolism in metabolic disease as well as in Alzheimer's disease. IDOL targeting may be beneficial in treating these conditions.
The endothelial monolayer forms a barrier between the lumen of blood vessels and the underlying tissues. Stable VE-cadherin-based adherens junctions are essential for maintaining this barrier, whereas their remodeling is required for angiogenesis in health and disease. Here, we position the ERAD-associated ubiquitin ligase MARCH6 as a determinant of angiogenic sprouting and barrier integrity through its ability to promote the degradation of the rate-limiting cholesterol biosynthetic enzyme squalene epoxidase (SQLE). Accordingly, MARCHF6 ablation in endothelial cells increases SQLE protein and cholesterol load. This leads to altered membrane order, disorganized adherens junctions, decreased endothelial barrier function, and impaired SQLE-dependent sprouting angiogenesis. Akin to MARCHF6 silencing, the overexpression of SQLE impairs angiogenesis. However, angiogenesis is also attenuated when SQLE is silenced, indicating that fine-tuning cholesterol biosynthesis is a determinant of healthy endothelial function. In summary, we propose a mechanistic link between regulation of cholesterol homeostasis by the MARCH6-SQLE axis and endothelial integrity and angiogenesis.
Inhibition of multidrug resistance protein 1 (MRP1) mediated cytostatic drug efflux might be useful in the treatment of drug resistant tumors. Because the glutathione (GSH) conjugate of ethacrynic acid (EA), GS-EA, is a good substrate of MRP1, GS-EA derivatives are expected to be good inhibitors of MRP1. To study structure-activity relationships of MRP1 inhibition, a series of novel GS-EA analogs was synthesized in which peptide bonds of the GSH backbone were replaced by isosteric groups [Bioorg Med Chem10:195–205, 2002]. Several of these compounds were effective inhibitors of MRP1-mediated [3H]GS-EA and [3H]E217βG transport, as studied in membrane vesicles prepared from MRP1-overproducing Sf9 cells. The modifications of the peptide backbone have distinct implications for recognition by MRP1: the γ-glutamyl-cysteine peptide bond is important for binding, whereas the cysteinyl-glycine amide does not seem essential. When the γ-glutamyl-cysteine peptide bond (C-CO-N) is replaced by a urethane isostere (O-CO-N), an effective competitive MRP1-inhibitor (Ki = 11 μM) is obtained. After esterification of this compound to improve its cellular uptake, it inhibited MRP1-mediated efflux of calcein from 2008 ovarian carcinoma cells overexpressing MRP1. This compound also partially reversed the resistance of these cells to methotrexate. Because the urethane isostere is stable toward γ-glutamyl transpeptidase-mediated breakdown, it is an interesting lead-compound for the development of in vivo active MRP1 inhibitors.
The antifolate drug methotrexate (MTX) is transported by breast cancer resistance protein (BCRP; ABCG2) and multidrug resistance-associated protein1-4 (MRP1-4; ABCC1-4). In cancer patients, coadministration of benzimidazoles and MTX can result in profound MTX-induced toxicity coinciding with an increase in the serum concentrations of MTX and its main metabolite 7-hydroxymethotrexate. We hypothesized that benzimidazoles interfere with the clearance of MTX and/or 7-hydroxymethotrexate by inhibition of the ATP-binding cassette drug transporters BCRP and/or MRP2, two transporters known to transport MTX and located in apical membranes of epithelia involved in drug disposition. First, we investigated the mechanism of interaction between benzimidazoles (pantoprazole and omeprazole) and MTX in vitro in membrane vesicles from Sf9 cells infected with a baculovirus containing human BCRP or human MRP2 cDNA. In Sf9-BCRP vesicles, pantoprazole and omeprazole inhibited MTX transport (IC50 13 microm and 36 microm, respectively). In Sf9-MRP2 vesicles, pantoprazole did not inhibit MTX transport and at high concentrations (1 mm), it even stimulated MTX transport 1.6-fold. Secondly, we studied the transport of pantoprazole in MDCKII monolayers transfected with mouse Bcrp1 or human MRP2. Pantoprazole was actively transported by Bcrp1 but not by MRP2. Finally, the mechanism of the interaction was studied in vivo using Bcrp1-/- mice and wild-type mice. Both in wild-type mice pretreated with pantoprazole to inhibit Bcrp1 and in Bcrp1-/- mice that lack Bcrp1, the clearance of i.v. MTX was decreased significantly 1.8- to 1.9-fold compared with the clearance of i.v. MTX in wild-type mice. The conclusion is as follows: benzimidazoles differentially affect transport of MTX mediated by BCRP and MRP2. Competition for BCRP may explain the clinical interaction between MTX and benzimidazoles.
Abstract The sterol regulatory element binding proteins (SREBPs) are transcription factors that govern cholesterol and fatty acid metabolism. We recently identified SPRING as a post-transcriptional regulator of SREBP activation. Constitutive or inducible global ablation of Spring in mice is not tolerated, and we therefore develop liver-specific Spring knockout mice (LKO). Transcriptomics and proteomics analysis reveal attenuated SREBP signaling in livers and hepatocytes of LKO mice. Total plasma cholesterol is reduced in male and female LKO mice in both the low-density lipoprotein and high-density lipoprotein fractions, while triglycerides are unaffected. Loss of Spring decreases hepatic cholesterol and triglyceride content due to diminished biosynthesis, which coincides with reduced very-low-density lipoprotein secretion. Accordingly, LKO mice are protected from fructose diet-induced hepatosteatosis. In humans, we find common genetic SPRING variants that associate with circulating high-density lipoprotein cholesterol and ApoA1 levels. This study positions SPRING as a core component of hepatic SREBP signaling and systemic lipid metabolism in mice and humans.
AimsRecent genome-wide association studies suggest that IDOL (also known as MYLIP) contributes to variation in circulating levels of low-density lipoprotein cholesterol (LDL-C). IDOL, an E3-ubiquitin ligase, is a recently identified post-transcriptional regulator of LDLR abundance. Briefly, IDOL promotes degradation of the LDLR thereby limiting LDL uptake. Yet the exact role of IDOL in human lipoprotein metabolism is unclear. Therefore, this study aimed at identifying and functionally characterizing IDOL variants in the Dutch population and to assess their contribution to circulating levels of LDL-C.
