Gregory Brubaker

Cyclosporin A Traps ABCA1 at the Plasma Membrane and Inhibits ABCA1-Mediated Lipid Efflux to Apolipoprotein A-I

Objective—ABCA1 mediates cellular cholesterol and phospholipid efflux to apolipoprotein A-I and other apolipoprotein acceptors. In this study, we analyzed the effect of the immunosuppressant cyclosporin A on the ABCA1-mediated lipid effluxes reactions. Methods and Results—Cyclosporin A acted as a potent inhibitor of ABCA1 activity in several cell lines. Using the RAW264.7 mouse macrophage cell line, in which ABCA1 and its associated cholesterol efflux activity are inducible by cAMP analogues, cyclosporin A inhibition of cholesterol efflux to apolipoprotein A-I was rapidly reversible after its removal from the culture media, implying that ABCA1 levels were not drastically reduced by cyclosporin A. In fact, cyclosporin A treatment decreased ABCA1 turnover and yielded a 2-fold increase in cell-surface ABCA1. Despite the increase in cell-surface ABCA1, cyclosporin A decreased apolipoprotein A-I uptake, resecretion, and degradation in RAW cells. Finally, consistent with the inhibition of ABCA1 in vitro, cyclosporin A treatment induced a 33% reduction of high-density lipoprotein (HDL) levels in mice. Conclusion—ABCA1 inhibition by cyclosporin A supports a role for ABCA1 endocytic trafficking in ABCA1-mediated lipid efflux and could explain in part the low HDL levels observed in some patients with transplants.

Identification of the cAMP-Responsive Enhancer of the Murine ABCA1 Gene Requirement for CREB1 and STAT3/4 Elements

Objective—To determine the mechanism by which expression of the murine ABCA1 gene is highly induced by cAMP analogues. Methods and Results—ABCA1 mRNA turnover cannot account for its induction by cAMP. Thus cAMP induction of ABCA1 mRNA occurs at a transcriptional level. Shotgun cloning DNA fragments from the murine ABCA1 locus identified a strong cAMP responsive enhancer located in the first intron, which led to 25- to 100-fold cAMP-mediated induction of reporter gene activity. Deletions and mutations of this enhancer led to the identification a cAMP-responsive element (CRE) that was essential for the cAMP induction. Furthermore, the capacity of this CRE site to mediate the cAMP induction required the presence of a STAT3/4 element located 81 bp away. A dominant-negative CREB expression vector inhibited the cAMP induction of ABCA1, demonstrating that CREB was required for cAMP induction of ABCA1 expression in RAW264.7 cells. Conclusion—Phospho-CREB1 controls the cAMP-mediated induction of murine ABCA1 gene expression through a CRE site acting in cooperation with a nearby STAT element. This CRE site is not conserved in the human ABCA1 gene, explaining why human ABCA1 is not strongly stimulated by cAMP analogs.

Sphingomyelin Depletion Impairs Anionic Phospholipid Inward Translocation and Induces Cholesterol Efflux

Background: Phosphatidylserine floppase activity of ABCA1 is required for optimal cholesterol efflux, as demonstrated via a floppase-impaired ABCA1 mutation. Results: Sphingomyelin depletion compensates for floppase-impaired ABCA1 and increases cell surface phosphatidylserine. Conclusion: Sphingomyelin depletion inhibits flip of anionic phospholipids and thus promotes cholesterol efflux. Significance: Flippase inhibition may serve as a novel drug target to increase cholesterol efflux. The phosphatidylserine (PS) floppase activity (outward translocation) of ABCA1 leads to plasma membrane remodeling that plays a role in lipid efflux to apolipoprotein A-I (apoAI) generating nascent high density lipoprotein. The Tangier disease W590S ABCA1 mutation has defective PS floppase activity and diminished cholesterol efflux activity. Here, we report that depletion of sphingomyelin by inhibitors or sphingomyelinase caused plasma membrane remodeling, leading to defective flip (inward translocation) of PS, higher PS exposure, and higher cholesterol efflux from cells by both ABCA1-dependent and ABCA1-independent mechanisms. Mechanistically, sphingomyelin was connected to PS translocation in cell-free liposome studies that showed that sphingomyelin increased the rate of spontaneous PS flipping. Depletion of sphingomyelin in stably transfected HEK293 cells expressing the Tangier disease W590S mutant ABCA1 isoform rescued the defect in PS exposure and restored cholesterol efflux to apoAI. Liposome studies showed that PS directly increased cholesterol accessibility to extraction by cyclodextrin, providing the mechanistic link between cell surface PS and cholesterol efflux. We conclude that altered plasma membrane environment conferred by depleting sphingomyelin impairs PS flip and promotes cholesterol efflux in ABCA1-dependent and -independent manners.

PI(4,5)P2 Is Translocated by ABCA1 to the Cell Surface Where It Mediates Apolipoprotein A1 Binding and Nascent HDL Assembly

RATIONALE The molecular mechanism by which ATP-binding cassette transporter A1 (ABCA1) mediates cellular binding of apolipoprotein A-I (apoA1) and nascent high-density lipoprotein (HDL) assembly is not well understood. OBJECTIVE To determine the cell surface lipid that mediates apoA1 binding to ABCA1-expressing cells and the role it plays in nascent HDL assembly. METHODS AND RESULTS Using multiple biochemical and biophysical methods, we found that apoA1 binds specifically to phosphatidylinositol (4,5) bis-phosphate (PIP2). Flow cytometry and PIP2 reporter-binding assays demonstrated that ABCA1 led to PIP2 redistribution from the inner to the outer leaflet of the plasma membrane. Enzymatic cleavage of cell surface PIP2 or decreased cellular PIP2 by knockdown of phosphatidylinositol-5-phosphate 4-kinase impaired apoA1 binding and cholesterol efflux to apoA1. PIP2 also increased the spontaneous solubilization of phospholipid liposomes by apoA1. Using site-directed mutagenesis, we found that ABCA1s PIP2 and phosphatidylserine translocase activities are independent from each other. Furthermore, we discovered that PIP2 is effluxed from cells to apoA1, where it is associated with HDL in plasma, and that PIP2 on HDL is taken up by target cells in a scavenger receptor-BI-dependent manner. Mouse plasma PIP2 levels are apoA1 gene dosage-dependent and are >1 μM in apoA1 transgenic mice. CONCLUSIONS ABCA1 has PIP2 floppase activity, which increases cell surface PIP2 levels that mediate apoA1 binding and lipid efflux during nascent HDL assembly. We found that PIP2 itself is effluxed to apoA1 and it circulates on plasma HDL, where it can be taken up via the HDL receptor scavenger receptor-BI.

A Novel Compound Inhibits Reconstituted High-Density Lipoprotein Assembly and Blocks Nascent High-Density Lipoprotein Biogenesis Downstream of Apolipoprotein AI Binding to ATP-Binding Cassette Transporter A1–Expressing Cells

Objective—Nascent high-density lipoprotein (HDL) particles form from cellular lipids and extracellular lipid-free apolipoprotein AI (apoAI) in a process mediated by ATP-binding cassette transporter A1 (ABCA1). We have sought out compounds that inhibit nascent HDL biogenesis without affecting ABCA1 activity. Methods and Results—Reconstituted HDL (rHDL) formation and cellular cholesterol efflux assays were used to show that 2 compounds that bond via hydrogen with phospholipids inhibit rHDL and nascent HDL production. In rHDL formation assays, the inhibitory effect of compound 1 (methyl 3&agr;-acetoxy-7&agr;,12&agr;-di[(phenylaminocarbonyl)amino]-5&bgr;-cholan-24-oate), the more active of the 2, depended on its ability to associate with phospholipids. In cell assays, compound 1 suppressed ABCA1-mediated cholesterol efflux to apoAI, the 18A peptide, and taurocholate with high specificity, without affecting ABCA1-independent cellular cholesterol efflux to HDL and endocytosis of acetylated low-density lipoprotein and transferrin. Furthermore, compound 1 did not affect ABCA1 activity adversely, as ABCA1-mediated shedding of microparticles proceeded unabated and apoAI binding to ABCA1-expressing cells increased in its presence. Conclusion—The inhibitory effects of compound 1 support a 3-step model of nascent HDL biogenesis: plasma membrane remodeling by ABCA1, apoAI binding to ABCA1, and lipoprotein particle assembly. The compound inhibits the final step, causing accumulation of apoAI in ABCA1-expressing cells.

Miltefosine increased cholesterol efflux, induced autophagy and inhibited NLRP3-inflammasome assembly and IL-1β release.

In advanced human plaques and aged patients, athero-protective pathways such as autophagy and reverse cholesterol transport (RCT) become dysfunctional, while atherogenic pathways such as NLRP3 inflammasome and TLR2/4 are induced. Here, we report that Miltefosine, an FDA approved drug for treating leishmaniasis, increased cholesterol efflux, induced membrane remodeling, and induced autophagy in macrophages. Macrophages treated with Miltefosine exhibited markedly increased ABCA1 mediated cholesterol efflux and decreased phosphatidylserine flip from the cell-surface. Miltefosine treatment of macrophages led to redistribution of phosphatidylinositol 4,5-bisphosphate (PIP2) from plasma membrane to actin rich regions of the cell. RAW264.7 macrophages treated with Miltefosine showed marked increase in p62 and LC3 puncta staining vs. control cells. The Lipid droplet degradation was induced by Miltefosine leading to ~ 50% decrease in the CE:FC (cholesterol ester: free cholesterol) ratio. The TLR4 signaling pathway in LPS primed bone marrow derived macrophages was blunted by Miltefosine treatment, leading to ~75% reduction in pro-IL-1β mRNA levels. Miltefosine pretreatment of macrophages potently inhibited NLRP3 inflammasome assembly induced by LPS/ATP treatment, exhibiting ~70% reduction in ASC1 speck forming cells. Gasdermin D mediated release of mature IL-1β was reduced by ~80% in Miltefosine treated vs. control cells. The qRT-PCR and western blot analysis showed no changes in basal or LPS induced levels of inflammasome components (NLRP3, ASC1, procaspase1), while the LPS mediated induction in ROS levels was significantly blunted in Miltefosine treated vs. control macrophages. Miltefosine did not alter the AIM2 inflammasome activity indicating specific targeting of Nlrp3 inflammasome pathway. Overall, this study showed that Miltefosine targets lipid trafficking, cell migration, autophagy, and Nlrp3 inflammasome activity in macrophages. Significance Statement Atherosclerosis is a progressive inflammatory disease. In advanced human plaques, the athero-protective pathways such as reverse cholesterol transport (RCT) and autophagy become increasingly dysfunctional while atherogenic pathways such as NLRP3 inflammasome are aberrantly induced. The cholesterol efflux via RCT prevents atherosclerosis and inflammation by reducing lipid loads in foam cells. Autophagy, in addition to playing a pivotal role in removing stored cholesterol from macrophages, promotes removal of inflammasome activating stimuli from cytosol (such as damaged mitochondria), thus helping in sequestering IL-1β. Failure of foam cells to egress from plaques and prolific engulfment of modified lipids results in formation of cholesterol crystals, leading to induction of NLRP3 inflammasome assembly. The release of IL-1β from foam cells further amplify the inflammation and promote further infiltration of immune cells to plaque. How RCT, autophagy, and inflammation pathways coordinate with each other to maintain cellular homeostasis is not clear; thus, the interplay between these pathways needs to be investigated thoroughly. Increased cholesterol efflux and induced autophagy with simultaneous dampening of Nlrp3 inflammasome can prevent atherosclerosis. Here, we show that Miltefosine can target multiple pathways involved in lipid homeostasis and inflammation. The detailed investigation of mechanisms involved in Miltefosine’s action may led to novel therapeutic targets for preventing and treating atherosclerosis and CVD.

Th-P15:220 Identification of the camp-responsive enhancer of the murine ABCA1 gene: Requirement for CREB1 and STAT3/4 elements

OBJECTIVE To determine the mechanism by which expression of the murine ABCA1 gene is highly induced by cAMP analogues. METHODS AND RESULTS ABCA1 mRNA turnover cannot account for its induction by cAMP. Thus cAMP induction of ABCA1 mRNA occurs at a transcriptional level. Shotgun cloning DNA fragments from the murine ABCA1 locus identified a strong cAMP responsive enhancer located in the first intron, which led to 25- to 100-fold cAMP-mediated induction of reporter gene activity. Deletions and mutations of this enhancer led to the identification a cAMP-responsive element (CRE) that was essential for the cAMP induction. Furthermore, the capacity of this CRE site to mediate the cAMP induction required the presence of a STAT3/4 element located 81 bp away. A dominant-negative CREB expression vector inhibited the cAMP induction of ABCA1, demonstrating that CREB was required for cAMP induction of ABCA1 expression in RAW264.7 cells. CONCLUSIONS Phospho-CREB1 controls the cAMP-mediated induction of murine ABCA1 gene expression through a CRE site acting in cooperation with a nearby STAT element. This CRE site is not conserved in the human ABCA1 gene, explaining why human ABCA1 is not strongly stimulated by cAMP analogs.