Regulation of ABCG4 transporter expression by sterols and LXR ligands.

BACKGROUND: Oxysterols, which are derivatives of cholesterol produced by enzymic or non-enzymic pathways, are potent regulators of cellular lipid homeostasis. Sterol homeostasis in the brain is an important area of interest with regards to neurodegenerative conditions like Alzheimer's disease (AD). Brain cells including neurons and astrocytes express sterol transporters belonging to the ABC transporter family of proteins, including ABCA1, ABCG1 and ABCG4, and these transporters are considered of interest as therapeutic targets. Although regulation of ABCA1 and ABCG1 is well established, regulation of ABCG4 is still controversial, in particular whether the transporter is an Liver X receptor (LXR) target. ABCG4 is thought to transport cholesterol, oxysterols and cholesterol synthesis intermediates, and was recently found on the blood brain barrier (BBB), implicated in amyloid-beta export. In this study, we investigate the regulation of ABCG4 by oxysterols, cholesterol-synthesis intermediates and cholesterol itself. METHODS: ABC transporter expression was measured in neuroblastoma and gliablastoma cell lines and cells overexpressing ABCG4 in response to synthetic LXR ligands, oxysterols and cholesterol-synthesis intermediates. RESULTS: In contrast to previous reports, ABCG4 expression was induced by a synthetic LXR ligand in U87-MG astrocytes but not in neuroblastoma and BBB endothelial cell lines. In addition, ABCG4 protein was stabilized by cholesterol as was previously shown for ABCG1. ABCG4 protein was furthermore stabilized by cholesterol-synthesis intermediates, desmosterol, lathosterol and lanosterol. CONCLUSIONS: These results identify new aspects of the post-translational control of ABCG4 that warrant further exploration into the role of this transporter in the maintenance of sterol homeostasis in the brain.

New Evidence for P-gp-Mediated Export of Amyloid-ß PEPTIDES in Molecular, Blood-Brain Barrier and Neuronal Models.

Defective clearance mechanisms lead to the accumulation of amyloid-beta (Aß) peptides in the Alzheimer's brain. Though predominantly generated in neurons, little is known about how these hydrophobic, aggregation-prone, and tightly membrane-associated peptides exit into the extracellular space where they deposit and propagate neurotoxicity. The ability for P-glycoprotein (P-gp), an ATP-binding cassette (ABC) transporter, to export Aß across the blood-brain barrier (BBB) has previously been reported. However, controversies surrounding the P-gp-Aß interaction persist. Here, molecular data affirm that both Aß40 and Aß42 peptide isoforms directly interact with and are substrates of P-gp. This was reinforced ex vivo by the inhibition of Aß42 transport in brain capillaries from P-gp-knockout mice. Moreover, we explored whether P-gp could exert the same role in neurons. Comparison between non-neuronal CHO-APP and human neuroblastoma SK-N-SH cells revealed that P-gp is expressed and active in both cell types. Inhibiting P-gp activity using verapamil and nicardipine impaired Aß40 and Aß42 secretion from both cell types, as determined by ELISA. Collectively, these findings implicate P-gp in Aß export from neurons, as well as across the BBB endothelium, and suggest that restoring or enhancing P-gp function could be a viable therapeutic approach for removing excess Aß out of the brain in Alzheimer's disease.

The Adaptor Protein Alix is Involved in the Interaction Between the Ubiquitin Ligase NEDD4-1 and its Targets, ABCG1 and ABCG4.

Several ATP-Binding Cassette (ABC) transporters, including ABCG1 and the related ABCG4, are essential regulators of cellular lipid homeostasis. ABCG1 is expressed ubiquitously and is functional in the context of atherosclerosis. However, ABCG4 is expressed almost exclusively in brain and has been linked to Alzheimer's disease (AD). These transporters are highly regulated post-translationally by E3 ubiquitin ligases, with the ligase NEDD4-1 (Neural precursor cell-expressed developmentally downregulated gene 4) implicated in their protein stability. In this study, we investigated interacting partners of ABCG1 using peptide-mass spectrometry and identified the potential adaptor protein, Alix (apoptosis-linked gene 2-interacting protein X). In this paper, we hypothesized and investigated whether Alix could facilitate the interaction between NEDD4-1 and the ABC transporters. We showed that Alix and NEDD4-1 proteins were co-expressed in several commonly used cell lines. Knockdown of Alix in cells overexpressing ABCG1 or ABCG4 increased transporter protein expression while co-immunoprecipitation experiments showed interaction between NEDD4-1, Alix, and ABC transporters. In summary, we provide evidence that Alix serves as a co-factor for the interaction between the E3-ubiquitin ligase NEDD4-1 and the ABC transporter targets, ABCG1 and ABCG4.

The E3 ubiquitin ligase, HECTD1, is involved in ABCA1-mediated cholesterol export from macrophages.

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting ß-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated.

ABC-Transporter Mediated Sterol Export from Cells Using Radiolabeled Sterols.

Cholesterol export from cells to extracellular acceptors represents the first step of the reverse cholesterol transport process and is an essential part of the multifaceted pathway for cells to control their cholesterol levels. Malfunction of this pathway leads to cholesterol accumulation in cells such as macrophages, which can form the basis of conditions like atherosclerosis. A number of ATP-binding cassette (ABC) transporters, namely ABCA1, ABCA7, ABCG1, and ABCG4, play an essential role in this process. In this chapter, we describe methods utilizing radiolabeled sterols for measuring ABC-transporter mediated sterol export, utilizing endogenously expressed transporters as well as overexpression systems.

The E3 ubiquitin ligases, HUWE1 and NEDD4-1, are involved in the post-translational regulation of the ABCG1 and ABCG4 lipid transporters.

The ATP-binding cassette transporter ABCG1 has an essential role in cellular cholesterol homeostasis, and dysregulation has been associated with a number of high burden diseases. Previous studies reported that ABCG1 is ubiquitinated and degraded via the ubiquitin proteasome system. However, so far the molecular mechanism, including the identity of any of the rate-limiting ubiquitination enzymes, or E3 ligases, is unknown. Using liquid chromatography mass spectrometry, we identified two HECT domain E3 ligases associated with ABCG1, named HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase) and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), of which the latter is the founding member of the NEDD4 family of ubiquitin ligases. Silencing both HUWE1 and NEDD4-1 in cells overexpressing human ABCG1 significantly increased levels of the ABCG1 monomeric and dimeric protein forms, however ABCA1 protein expression was unaffected. In addition, ligase silencing increased ABCG1-mediated cholesterol export to HDL in cells overexpressing the transporter as well as in THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant reduction in protein levels of both the ABCG1 monomeric and dimeric forms. Like ABCG1, ABCG4 protein levels and cholesterol export activity were significantly increased after silencing both HUWE1 and NEDD4-1 in cells overexpressing this closely related ABC half-transporter. In summary, we have identified for the first time two E3 ligases that are fundamental enzymes in the post-translational regulation of ABCG1 and ABCG4 protein levels and cellular cholesterol export activity.