Regulation Of Cholesterol Trafficking Research Articles

Abstract C047: Phospholipid scramblase TMEM16F in cancer associated fibroblasts regulates trogocytosis to sustain critical dependency of pancreatic cancer cells on exogenous lipids

Abstract Pancreatic ductal adenocarcinoma (PDAC) cells derive their resistance to therapy and aggressive clinical course from the symbiotic signaling and metabolic interactions with cancer-associated fibroblasts (CAFs). Trogocytosis is a process of “nibbling” of plasma membranes between two living cells. Here, we demonstrate CAFs are the primary recipients of exogenous lipids which they transfer to metabolically “parasitic” PDAC cells via a contact-mediated trogocytosis. Whereas trogocytosis has been described in immune system and in normal development, the biochemical and signaling regulators of trogocytosis between CAFs and PDAC cells have not been defined. Results: We determine that trogocytosis occurs through heterotypic CAF-PDAC cell contacts: membrane blebbing (readily observed in activated CAFs and exaggerated by release of Ca2+ from the ER stores) results in the blebs uptake by the PDAC cells. The CAF blebs express externalized phosphatidylserine (PtdSer), and blockade of PtdSer in vitro transiently deters trogocytic uptake of CAF membranes. Probing a short list of candidate targets involved in regulation of cholesterol trafficking, membrane fusion and membrane protrusions using CRISPRi, has shown that CAFs deficient in CD81, ARF6 or TMEM16F exhibit markedly reduced ability to support the viability of cholesterol-auxotrophic PDAC cells in lipid-poor media. As a promising therapy target, TMEM16F is a Ca2+-regulated scramblase increasing PtdSer on the outer leaflet of the plasma membrane. TMEM16F protein is highly expressed in human PDAC CAFs compared to fibroblasts isolated from the adjacent non-malignant pancreatic tissues. The TMEM16F-null CAFs are unable to sustain of cholesterol-auxotrophic PDAC cells in lipid-poor co-cultures, and do not support the growth of PDAC cells in orthotopic co-implantation model. Furthermore, several clinically available antibiotics used to treat tapeworms are selective TMEM16F inhibitors. One of the widely available TMEM16F inhibitors, niclozamide, is effective in blocking cholesterol transfer from CAFs to PDAC cells in vivo. Conclusion: Our overall model is that activated CAFs initiate trogocytosis by expressing PtdSer as “eat me” signals on the surfaces of their membrane blebs. This process is regulated by Ca2+-dependent phospholipid scramblase TMEM16F which is an attractive drug-amenable target to dismantle the critical metabolic dependency in PDAC on the exogenous lipids. Re-purposing of clinically available TMEM16 inhibitors will make a tangible impact on treatment of PDAC patients in the near term. Citation Format: Charline Ogier, Alena Klochkova, Linara Gabitova-Cornell, Battuya Bayarmagnai, Diana Restifo, Aizhan Surumbayeva, Debora Barbosa Vendramini-Costa, Ralph Francescone, Janusz Franco-Barraza, Jaye Gardiner, Emmanuelle Nicolas, Andrei Efimov, Elizabeth A. Handorf, Kathy Q. Cai, Bojana Gligorijevic, Edna Cukierman, Igor Astsaturov. Phospholipid scramblase TMEM16F in cancer associated fibroblasts regulates trogocytosis to sustain critical dependency of pancreatic cancer cells on exogenous lipids [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C047.

Regulation of lipid rafts, angiogenesis and inflammation by AIBP.

Recent studies demonstrate an important role of the secreted apolipoprotein A-I binding protein (AIBP) in regulation of cholesterol efflux and lipid rafts. The article discusses these findings in the context of angiogenesis and inflammation. Lipid rafts are cholesterol-rich and sphingomyelin-rich membrane domains in which many receptor complexes assemble upon activation. AIBP mediates selective cholesterol efflux, in part via binding to toll-like receptor-4 (TLR4) in activated macrophages and microglia, and thus reverses lipid raft increases in activated cells. Recent articles report AIBP regulation of vascular endothelial growth factor receptor-2, Notch1 and TLR4 function. In zebrafish and mouse animal models, AIBP deficiency results in accelerated angiogenesis, increased inflammation and exacerbated atherosclerosis. Spinal delivery of recombinant AIBP reduces neuraxial inflammation and reverses persistent pain state in a mouse model of chemotherapy-induced polyneuropathy. Inhalation of recombinant AIBP reduces lipopolysaccharide-induced acute lung injury in mice. These findings are discussed in the perspective of AIBP's proposed other function, as an NAD(P)H hydrate epimerase, evolving into a regulator of cholesterol trafficking and lipid rafts. Novel findings of AIBP regulatory circuitry affecting lipid rafts and related cellular processes may provide new therapeutic avenues for angiogenic and inflammatory diseases.

Abstract 402: miRNA Targeting of Oxysterol Binding Protein-like 6 (OSBPL6) Regulates Cholesterol Trafficking and Efflux

Cholesterol homeostasis is fundamental to human health, and is thus, tightly regulated. MicroRNAs exert potent effects on biological pathways, including cholesterol metabolism, by repressing genes with related functions. We reasoned that this mode of pathway regulation could be exploited to identify novel genes involved in cholesterol homeostasis. Here, we identify oxysterol binding protein-like 6 ( OSBPL6 ) as a novel target of 2 miRNA hubs regulating cholesterol homeostasis: miR-33 and miR-27b. Characterization of OSBPL6 revealed that it is transcriptionally regulated in macrophages and hepatocytes by LXR and in response to cholesterol loading, and in mice and non-human primates by western diet feeding. OSBPL6 encodes the OSBPL-related protein 6 (ORP6), which contains a pleckstrin homology membrane-targeting domain and a FFAT domain predicted to interact with VAP proteins in the ER. Indeed, subcellular localization studies showed that ORP6 is associated with the plasma membrane, early endosomes, lysosomes and ER, suggesting a putative role for ORP6 in cholesterol trafficking between these compartments. Consistent with this, siRNA-mediated knockdown of OSBPL6 results in aberrant clustering of endosomes and promotes the accumulation of free cholesterol in these structures. As a consequence, esterification of cholesterol at the ER is reduced, triggering activation of SREBP2. Conversely, ORP6 overexpression enhances cholesterol trafficking and efflux to apoA1 in macrophages and hepatocytes. Moreover, we show that hepatic expression of OSBPL6 is positively correlated with plasma levels of HDL cholesterol in a cohort of 200 healthy individuals, whereas its expression is reduced in human atherosclerotic plaques. Collectively, these studies identify ORP6 as a novel regulator of cholesterol trafficking that is part of the miR-33 and miR-27b target gene networks that contribute to the maintenance of cholesterol homeostasis.

MiRNA Targeting of Oxysterol-Binding Protein-Like 6 Regulates Cholesterol Trafficking and Efflux.

Cholesterol homeostasis is fundamental to human health and is, thus, tightly regulated. MicroRNAs exert potent effects on biological pathways, including cholesterol metabolism, by repressing genes with related functions. We reasoned that this mode of pathway regulation could be exploited to identify novel genes involved in cholesterol homeostasis. Here, we identify oxysterol-binding protein-like 6 (OSBPL6) as a novel target of 2 miRNA hubs regulating cholesterol homeostasis: miR-33 and miR-27b. Characterization of OSBPL6 revealed that it is transcriptionally regulated in macrophages and hepatocytes by liver X receptor and in response to cholesterol loading and in mice and nonhuman primates by Western diet feeding. OSBPL6 encodes the OSBPL-related protein 6 (ORP6), which contains dual membrane- and endoplasmic reticulum-targeting motifs. Subcellular localization studies showed that ORP6 is associated with the endolysosomal network and endoplasmic reticulum, suggesting a role for ORP6 in cholesterol trafficking between these compartments. Accordingly, knockdown of OSBPL6 results in aberrant clustering of endosomes and promotes the accumulation of free cholesterol in these structures, resulting in reduced cholesterol esterification at the endoplasmic reticulum. Conversely, ORP6 overexpression enhances cholesterol trafficking and efflux in macrophages and hepatocytes. Moreover, we show that hepatic expression of OSBPL6 is positively correlated with plasma levels of high-density lipoprotein cholesterol in a cohort of 200 healthy individuals, whereas its expression is reduced in human atherosclerotic plaques. These studies identify ORP6 as a novel regulator of cholesterol trafficking that is part of the miR-33 and miR-27b target gene networks that contribute to the maintenance of cholesterol homeostasis.

Genome-Wide Association Studies Complemented with Mechanistic Biological Studies Identify Sortilin 1 as a Novel Regulator of Cholesterol Trafficking

Genome-Wide Association Studies Complemented with Mechanistic Biological Studies Identify Sortilin 1 as a Novel Regulator of Cholesterol Trafficking

25-Hydroxycholesterol Increases the Availability of Cholesterol in Phospholipid Membranes

Side-chain oxysterols are enzymatically generated oxidation products of cholesterol that serve a central role in mediating cholesterol homeostasis. Recent work has shown that side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), alter membrane structure in very different ways from cholesterol, suggesting a possible mechanism for how these oxysterols regulate cholesterol homeostasis. Here we extend our previous work by using molecular-dynamics simulations of 25-HC and cholesterol mixtures in 1-palmitoyl-2-oleoyl-phosphatidylcholine bilayers to examine the combined effects of 25-HC and cholesterol in the same bilayer. 25-HC causes larger changes in membrane structure when added to cholesterol-containing membranes than when added to cholesterol-free membranes. We also find that the presence of 25-HC changes the position, orientation, and solvent accessibility of cholesterol, shifting it into the water interface and thus increasing its availability to external acceptors. This is consistent with experimental results showing that oxysterols can trigger cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. These effects provide a potential mechanism for 25-HC-mediated regulation of cholesterol trafficking and homeostasis through modulation of cholesterol availability.

Nature’s Genetic Gradients and the Clinical Phenotype

When God created the earth, he placed (modern) mankind in East Africa ≈200 000 years ago, as suggested by the phylogenetic and archeological studies.1,2 These hunter-gatherers populated the land and started to migrate, first to the west and southern tip of Africa and then north toward North Africa. Mankind gradually crossed the Arabian Peninsula to the Middle East and Eurasia. Then, over thousands of years of northerly movement, man crossed the Bering land bridge that connected Siberia to Alaska and migrated down to the Americas. This out-of-Africa migration took thousands of generations. It was interrupted by the Ice Age, which led to depopulation and isolation of the surviving population followed by the new waves of migrations, resulting in multiple founding populations.3 Articles see pages 552 and 558 At the genetic level, the tree of life was produced through replication of the genome. For the genome to replicate, cells are equipped with DNA polymerases to copy the parental DNA, which they do at blazing speeds.4 However, “no protein is perfect,” and neither are the DNA polymerases. So, the DNA polymerases commit occasional errors in copying the template (parental) DNA and incorporate wrong nucleotides into the new DNA strands. To correct the errors, nature afforded the cells with DNA repair enzymes, which are assigned the task of maintaining the integrity of the nucleotide pools before replication.5 Nevertheless, the process of DNA replication and editing, although exceedingly precise, is imperfect. The error rate of the DNA replication and editing machinery is estimated to be at 10−6 to 10−10 nucleotides incorporated into the new strand of DNA.6,7 The inheritance is forward. Therefore, the misincorporated nucleotides are passed on to the next generation, and with each generation, new errors in DNA replication are introduced. The occasional “errors of …

Ezetimibe interferes with cholesterol trafficking from the plasma membrane to the endoplasmic reticulum in CaCo-2 cells

Niemann-Pick C1-like 1 protein (NPC1L1) is the putative intestinal sterol transporter and the molecular target of ezetimibe, a potent inhibitor of cholesterol absorption. To address the role of NPC1L1 in cholesterol trafficking in intestine, the regulation of cholesterol trafficking by ezetimibe was studied in the human intestinal cell line, CaCo-2. Ezetimibe caused only a modest decrease in the uptake of micellar cholesterol, but markedly prevented its esterification. Cholesterol trafficking from the plasma membrane to the endoplasmic reticulum was profoundly disrupted by ezetimibe without altering the trafficking of cholesterol from the endoplasmic reticulum to the plasma membrane. Cholesterol oxidase-accessible cholesterol at the apical membrane was increased by ezetimibe. Cholesterol synthesis was modestly increased. Although the amount of cholesteryl esters secreted at the basolateral membrane was markedly decreased by ezetimibe, the transport of lipids and the number of lipoprotein particles secreted were not altered. NPC1L1 gene and protein expression were decreased by sterol influx, whereas cholesterol depletion enhanced NPC1L1 gene and protein expression. These results suggest that NPC1L1 plays a role in cholesterol uptake and cholesterol trafficking from the plasma membrane to the endoplasmic reticulum. Interfering with its function will profoundly decrease the amount of cholesterol transported into lymph.

The multidrug transporter, P-glycoprotein, actively mediates cholesterol redistribution in the cell membrane.

P-glycoprotein (P-gp) is a plasma membrane ATP-binding cassette transporter, responsible for multidrug resistance in tumor cells. P-gp catalyzes the ATP hydrolysis-dependent efflux of numerous amphiphilic compounds of unrelated chemical structures. In the absence of any identified substrate, P-gp exhibits an apparently futile, basal ATPase activity. By using native membrane vesicles containing high amounts of P-gp, we show here that (i) this basal ATPase activity is tightly dependent on the presence of cholesterol in the membrane; (ii) the stimulation of P-gp ATPase activity by drugs transported by P-gp is higher in the absence than in the presence of cholesterol and, conversely, the stimulation of P-gp ATPase activity by cholesterol is higher in the absence than in the presence of known P-gp substrates; (iii) P-gp mediates the ATP-dependent relocation of cholesterol from the cytosolic leaflet to the exoplasmic leaflet of the plasma membrane; and (iv) the decrease of the cholesterol dependence of P-gp ATPase activity induced by known P-gp substrates is correlated with the inhibition of the ATP-dependent cholesterol redistribution within the membrane. These data are highly evocative of a coupling between the basal ATPase activity of P-gp and its intramembrane cholesterol-redistribution function, and they are fully consistent with the possibility that P-gp may actively translocate cholesterol in the membrane. Finally, this P-gp-mediated cholesterol redistribution in the cell membrane makes it likely that P-gp contributes in stabilizing the cholesterol-rich microdomains, rafts and caveolae, and that it is involved in the regulation of cholesterol trafficking in cells.