Induction Of ABCA1 Expression Research Articles

Liver X Receptor Agonists: A Potential Treatment for Blastic Plasmacytoid Dendritic Cell Neoplasm

Introduction: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is an aggressive hematologic malignancy derived from plasmacytoid dendritic cells (pDC). Today, no therapeutic consensus exists and only allogeneic hematopoietic cell transplantation provides a durable remission. BPDCN cells express high levels of the IL-3 receptor alpha chain (CD123) and exhibit an increased survival and growth in response to IL-3 (Chaperot et al. 2001). Recently, aberrant NF-κB activation has been reported in BPDCN (Sapienza et al. 2014), and we identified that liver X receptor (LXR) stimulation inhibits NF-κB activation in non-leukemic pDC. LXR represent nuclear receptors promoting cholesterol efflux via the ATP binding cassette A1 and G1 (ABCA1 and ABCG1, respectively) transporters and lipid acceptors, such as apolipoprotein A1 (ApoA1). This process has been shown to repress IL-3-induced proliferation of murine hematopoietic stem cells (Yvan-Charvet et al. 2010). The aim of this study was to investigate LXR activation in BPDCN, and determine whether LXR agonists possess therapeutic effects.Material and Methods: Cells from 13 BPDCN, 22 undifferentiated AML, 23 ALL patients and 4 normal pDC were evaluated for expression of LXR or cholesterol exchange related genes (lxra, lxrb, abca1, abcg1, apoe, srebp1c, ldlr, vldlr), plus nfkb1 gene by Affymetrix mRNA microarray. BPDCN cells (2 established cell lines [GEN2.2, CAL-1], a primary cell line [BES1] and 5 fresh BPDCN samples) were treated with two LXR agonists (T0901317 [T09] or GW3965 [GW] at 1 µM, 10 µM or 50 µM), NF-κB inhibitors (JSH 23, or Bortezomib [Bort]) and the cholesterol acceptor ApoA1 (10 µg/mL). Gene expression (abca1, abcg1, nfkb1, bcl2, bax and bak1) was assessed by qRT-PCR. Cell death was assessed by Annexin V/7AAD staining (flow cytometry) and caspase activation (Western blot). CAL-1 cell proliferation was assessed by dye dilution (cytometry). Cholesterol efflux was measured directly by H3 cholesterol efflux from cells and indirectly by membrane ABCA1 expression (confocal microscopy). NF-κB and IL-3 signaling pathway activation was explored by protein phosphorylation (phospho-RelA and phospho-STAT5, respectively; [confocal microscopy and Western blot]). A BPDCN xenograft model was used to evaluate LXR agonist effect in-vivo with blast cell infiltration attested by human CD45/CD123/BDCA4 staining (cytometry).Results: BPDCN cells showed a unique transcriptomic signature with LXR-related gene downregulation. LXR agonist treatment for 24h restored LXR target gene expression, including abca1 and abcg1, and induced a significant concentration-dependent killing (Fig. 1). Moreover, caspase 8 and 9 cleavage associated with bcl2 downregulation and bax and bak1 upregulation (0.78, 8 and 12.2 fold mRNA levels, respectively, for GW 50 µM, 6h, n=3) were observed. At a lower non cytotoxic concentration (10 µM), GW treatment inhibited cell proliferation (-25%, p<0.05, n=7) that was reinforced by addition of ApoA1 (-35%, n=2). This suggests a link with cholesterol efflux supported by induction of ABCA1 expression at BPDCN membrane and increase of H3 cholesterol efflux in response to LXR agonists. LXR agonists inhibited IL-3-induced STAT5 phosphorylation and NF-κB signaling pathway at transcriptional (nfkb1, 0.25 fold mRNA level, p<0.05, n=3 fresh BPDCN samples) and RelA phosphorylation levels. This suggests additional mechanisms by which LXR activation affects BPDCN. NF-kB inhibitor treatment (JSH23, 25 µM, 24h) reduced BPDCN survival (-35%, n=3) and proliferation (-20%, p<0.05, n=3). A synergic effect of NF-κB inhibitors (JSH23 25 µM or Bort 30 nM) and LXR agonists (GW or T09, 10 µM), was observed on cell death and inhibition of proliferation (Fig. 1). The in-vivo study revealed a reduced CAL-1 infiltration in the spleen and bone marrow of T09-treated mice (1.2 mg/2 days for 14 days) compared with control mice (62% vs. 33% and 17% vs. 9%, respectively). Effects of LXR plus NF-κB inhibitor treatment in vivo are under investigation.Conclusion: BPDCN exhibit a specific transcriptomic signature with LXR-related gene downregulation. LXR stimulation of BPDCN restores LXR-related gene expression and inhibits survival and proliferation. This may occur via several mechanisms: inhibition of IL-3 and NF-κB signaling, and/or increased cholesterol efflux. A cytotoxic or cytostatic effect was confirmed in-vivo by reduced BPDCN infiltration. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 5547: Can IL-6 Exert Atheroprotective Action on Human Macrophages via Activation of ABCA1 Through the Jak-2/Stat3 Pathway?

Cholesterol-laden macrophage foam cells constitute a hallmark of the inflammatory dimension of atherosclerosis. The ATP-binding cassette A1 transporter (ABCA1) promotes macrophage cholesterol efflux to apolipoprotein AI (apoAI), thereby attenuating atherosclerotic plaque progression. Interleukin-6 (IL-6) is a macrophage secretory product which may modulate intraplaque inflammation, but whose precise role in atherogenesis is unclear. We therefore evaluated the impact of IL-6 on cholesterol homeostasis in human monocyte-macrophages and THP-1 cells. ABCA1-mediated cholesterol efflux to apoAI was significantly increased (2.5-fold) by IL-6 (50 ng/mL) subsequent to the induction of ABCA1 mRNA and protein levels. Stimulation of ABCA1-mediated cholesterol efflux was however abolished by a specific inhibitor of Jak-2 or the Jak-2/Stat3 signalling pathway. The implication of Stat3 in the stimulatory effect of IL-6 was confirmed by the use of a Stat3 inhibitory peptide. In addition, ABCA1-mediated efflux of cholesterol, driven by the ingestion of apoptotic, free-cholesterol-loaded THP-1 cells, was equally induced by IL-6 in THP-1 phagocytes. Finally, IL-6 enhanced phagocytosis of apoptotic Jurkat cells in THP-1 macrophages, an effect abolished by either a Jak-2 inhibitor or glyburide, an inhibitor of ABCA1 activity. Thus, induction of ABCA1 expression by IL-6 in human monocyte-macrophages through the Jak-2/Stat3 pathway may favour reduction in both foam cell formation and growth of the necrotic plaque core, potentially contributing to enhanced plaque stability.

Old Drug, New Tricks

See related article, pages 156–165 Ironically, even though there are currently only a few drugs to specifically treat low high-density lipoprotein (HDL), numerous drugs have been observed to inadvertently alter HDL levels.1 This has been particularly noted for some antihypertensive drugs. Because they are used long-term, any positive or negative effect of antihypertensive drugs on lipids and lipoproteins can significantly impact on their effectiveness in preventing cardiovascular disease. Recently, advances in our understanding of HDL metabolism have helped us to unravel the mechanism of action of these and other drugs that unexpectedly alter HDL levels. This work has largely been led by the laboratory of Shinji Yokoyama and colleagues, who have previously identified the mechanism of action of three drugs on HDL metabolism. Probucol, which has been used to treat patients at risk for cardiovascular disease, is an antioxidant and lowers LDL, but it also lowers HDL. Probucol is now known to decrease HDL by inhibiting the activity of the ABCA1 transporter,2 a key protein involved in reverse cholesterol transport pathway and in HDL biogenesis.3 Verapamil, a calcium channel blocker, which can be used as antihypertensive and antiarrhythmic agent, and fenofibrate, a lipid-lowering drug, both raise HDL by increasing the gene expression of the ABCA1 transporter.4,5 In this issue of Circulation Research ,6 Iwamoto et al describe the mechanism of action of a fourth agent that unexpectedly alters HDL, namely the antihypertensive agent doxazosin. The results of this study raise the possibility of a new strategy for developing drugs to increase HDL. Doxazosin (Cardura) is a quinazoline based compound that is sold by Pfizer and is a long lasting selective inhibitor of the α-1a subtype of adrenergic receptors. It blocks the binding of norepinephrine to adrenergic receptors in the autonomic nervous system, which leads …

Physiologically regulated transgenic ABCA1 does not reduce amyloid burden or amyloid-β peptide levels in vivo

ABCA1-deficient mice have low levels of poorly lipidated apolipoprotein E (apoE) and exhibit increased amyloid load. To test whether excess ABCA1 protects from amyloid deposition, we crossed APP/PS1 mice to ABCA1 bacterial artificial chromosome (BAC) transgenic mice. Compared with wild-type animals, the ABCA1 BAC led to a 50% increase in cortical ABCA1 protein and a 15% increase in apoE abundance, demonstrating that this BAC supports modest ABCA1 overexpression in brain. However, this was observed only in animals that do not deposit amyloid. Comparison of ABCA1/APP/PS1 mice with APP/PS1 controls revealed no differences in levels of brain ABCA1 protein, amyloid, Abeta, or apoE, despite clear retention of ABCA1 overexpression in the livers of these animals. To further investigate ABCA1 expression in the amyloid-containing brain, we then compared ABCA1 mRNA and protein levels in young and aged cortex and cerebellum of APP/PS1 and ABCA1/APP/PS1 animals. Compared with APP/PS1 controls, aged ABCA1/APP/PS1 mice exhibited increased ABCA1 mRNA, but not protein, selectively in cortex. Additionally, ABCA1 mRNA levels were not increased before amyloid deposition but were induced only in the presence of extensive Abeta and amyloid levels. These data suggest that an induction of ABCA1 expression may be associated with late-stage Alzheimer's neuropathology.

Apolipoprotein A-I activates Cdc42 signaling through the ABCA1 transporter

It has been suggested that the signal transduction initiated by apolipoprotein A-I (apoA-I) activates key proteins involved in cholesterol efflux. ABCA1 serves as a binding partner for apoA-I, but its participation in apoA-I-induced signaling remains uncertain. We show that the exposure of human fibroblasts to ABCA1 ligands (apolipoproteins and amphipathic helical peptides) results in the generation of intracellular signals, including activation of the small G-protein Cdc42, protein kinases (PAK-1 and p54JNK), and actin polymerization. ApoA-I-induced signaling was abrogated by glyburide, an inhibitor of the ABC transporter family, and in fibroblasts from patients with Tangier disease, which do not express ABCA1. Conversely, induction of ABCA1 expression with the liver X receptor agonist, T0901317, and the retinoid X receptor agonist, R0264456, potentiated apoA-I-induced signaling. Similar effects were observed in HEK293 cells overexpressing ABCA1-green fluorescent protein (GFP) fusion protein, but not ABCA1-GFP (K939M), which fails to hydrolyze ATP, or a nonfunctional ABCA1-GFP with a truncated C terminus. We further found that Cdc42 coimmunoprecipitates with ABCA1 in ABCA1-GFP-expressing HEK293 cells exposed to apoA-I but not in cells expressing ABCA1 mutants. We conclude that ABCA1 transduces signals from apoA-I by complexing and activating Cdc42 and downstream kinases and, therefore, acts as a full apoA-I receptor.

Induction of ABCA1 Expression by Liver X Receptor (LXR) agonist in Renal Glomerular Mesangial Cells

Liver X receptors (LXRs) are hormone activated nuclear receptor transcription factors, which play an important role in regulating genes involved in fatty acid, cholesterol and glucose homeostasis. It has been recently reported that LXRs are expressed in the kidney. The present studies were designed to determine the expression and potential role of LXRs in cholesterol metabolism in mouse mesangial cells (MCs). MCs were cultured from mouse glomeruli. RT-PCR and immunoblot assays demonstrated that both LXR[alpha] and LXR[beta] are expressed in the MCs. A LXRE-luciferase reporter assay indicated that TO901317 treatment markedly increased LXRE activity, suggesting endogenous LXR receptors were functional in MCs. To further elucidate the role of LXRs in MCs, genome-wide gene expression profile was studied using the Affymetrix gene chip. The results demonstrated that there were 43 transcripts significantly regulated in the MCs by treatment of TO901317, a specific LXR agonist (10 mM, 24 hours). Among them, ABCA1 was one of the genes markedly induced. Real-time PCR analysis revealed that ABCA1 mRNA levels were upregulated by 2-fold in MCs. Moreover, activation of LXRs by TO901317 markedly increased the activity of ABCA1 gene promoter activity. Taken together, LXR receptors are functionally expressed in mouse mesangial cells, where their activation results in enhanced gene expression of ABCA1. The findings of these studies suggest that the LXR receptor activators may increase cholesterol efflux via induction of ABCA1 in mouse mesangial cells.

Sterol efflux mediated by endogenous macrophage ApoE expression is independent of ABCA1.

Sterol efflux importantly contributes to preservation of cellular cholesterol homeostasis, and multiple pathways may be involved for mediating such efflux. Recently, an important role has been ascribed to ABCA1 in facilitating lipid efflux from cells, including macrophages, to extracellular lipid-free apolipoproteins. Macrophages are relatively unique among cells because they express apoprotein E (apoE) as a major protein product, and this endogenous expression of apoE increases sterol and phospholipid efflux from macrophages. The studies in this article were designed to test whether the sterol efflux mediated by the endogenous expression of apoE in macrophages was dependent on ABCA1 expression. These studies were facilitated by comparing apoE-expressing J774 cells (J774E(+)) with nonexpressing parental cells (J774E(-)). Sterol efflux was higher from J774E(+) cells compared with J774E(-) cells, but the increment in efflux between these cell types was not increased by induction of ABCA1 expression with cAMP. Induction of ABCA1 with cAMP, however, did increase sterol efflux to exogenously added apoA1 from both cell types. Inhibitors of ABCA1 activity significantly reduced (by 40% to 50%) sterol efflux from both J774E(+) and J774E(-) cells treated with cAMP and apoA1. This inhibitor did not, however, reduce the increment in sterol efflux due to the expression of endogenous apoE. The results of these studies indicate that the increment in sterol efflux mediated by the endogenous expression of apoE in macrophages does not depend on ABCA1 expression or activity.