Different transport routes for high density lipoprotein and its associated free sterol in polarized hepatic cells

Wüstner, Daniel; Mondal, Mousumi; Huang, Amy Y.; Maxfield, Frederick R.

doi:10.1194/jlr.m300440-jlr200

Cited by 74 publications

(122 citation statements)

References 46 publications

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“…The significance of the above findings in living cells was confirmed with DHE and other fluorescent sterols in a variety of cultured cells including L-cells, CHO cells, macrophages, and hepatocyte derived cell lines (39,43,45,160,(174)(175)(176). Studies with transfected L-cells showed that uptake and efflux of fluorescent sterols was highly dependent on the expression of cholesterol binding proteins (e.g.…”

Section: Physiological Significance Of In Vitro Studies With Dhe and mentioning

confidence: 65%

“…Recent improvements in imaging microscopy allowed the direct, real-time visualization of DHE in living cells by conventional (DHE delivered as micro-crystals) or video (DHE delivered as cyclodextrin complexes) UV fluorescence microscopy (39,160,(174)(175)(176) and by MPLSM (DHE delivered as LUV or micro-crystals) multiphoton laser scanning microscopy (71,82,203,204). Analysis of intracellular localization and distribution of DHE at the plasma membrane can be accomplished after segmentation of specific regions of interest such as cellular organelles and performed on a cell by cell basis (70,203 stastic-based technique, two small windows are created in the image and the pixel intensities in each window ranked from the lowest to highest; then a comparison is made by using Miller's rank statistic.…”

Section: Plasma Membrane Sterol-rich Microdomains: Real-time Imaging mentioning

confidence: 99%

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Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

McIntosh

Atshaves

Huang

et al. 2008

Lipids

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Cholesterol itself has very few structural/chemical features suitable for real-time imaging in living cells. Thus, the advent of dehydroergosterol [ergosta-5,7,9(11),22-tetraen-3β-ol, DHE] the fluorescent sterol most structurally and functionally similar to cholesterol to date, has proven to be a major asset for real-time probing/elucidating the sterol environment and intracellular sterol trafficking in living organisms. DHE is a naturally-occurring, fluorescent sterol analog that faithfully mimics many of the properties of cholesterol. Because these properties are very sensitive to sterol structure and degradation, such studies require the use of extremely pure (>98%) quantities of fluorescent sterol. DHE is readily bound by cholesterol-binding proteins, is incorporated into lipoproteins (from the diet of animals or by exchange in vitro), and for real-time imaging studies is easily incorporated into cultured cells where it co-distributes with endogenous sterol. Incorporation from an ethanolic stock solution to cell culture media is effective, but this process forms an aqueous dispersion of dehydroergosterol crystals which can result in endocytic cellular uptake and distribution into lysosomes which is problematic in imaging DHE at the plasma membrane of living cells. In contrast, monomeric DHE can be incorporated from unilamellar vesicles by exchange/fusion with the plasma membrane or from DHE-methyl-β-cyclodextrin (DHE-MβCD) complexes by exchange with the plasma membrane. Both of the latter techniques can deliver large quantities of monomeric dehydroergosterol with significant distribution into the plasma membrane. The properties and behavior of DHE in protein-binding, lipoproteins, model membranes, biological membranes, lipid rafts/caveolae, and real-time imaging in living cells indicate that this naturally-occurring fluorescent sterol is a useful mimic for probing the properties of cholesterol in these systems. Keywordsdehydroergosterol; cholesterol; ergosterol; fluorescent sterols; microscopy Historical PerspectiveIn order to resolve the dynamics and factors governing cholesterol trafficking within cells, it is important to recognize that the cholesterol molecule has very few polar constituents (Fig. 1A). Consequently, cholesterol is poorly soluble in aqueous environments such as cytosol as evidenced by critical micellar concentrations of cholesterol and other sterols being very low, *To whom correspondence should be addressed: Department of Physiology and Pharmacology, Texas A&M University, TVMC, College Station, TX 862-1433, FAX: (979) NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript near 20-30 nM (1-5). The physiological impact of cholesterol's low aqueous solubility is that spontaneous cholesterol desorption from the cytofacial leaflet of the plasma membrane or lysosomal membrane into the cytosol for transfer to intracellular sites for esterification, oxidation, or secretion is extremely slow (t 1/2 3 hours-days) (6-9). Therefore, it is important to resolve factors...

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Section: Physiological Significance Of In Vitro Studies With Dhe and mentioning

confidence: 65%

Section: Plasma Membrane Sterol-rich Microdomains: Real-time Imaging mentioning

confidence: 99%

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

McIntosh

Atshaves

Huang

et al. 2008

Lipids

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“…Thus, the extent of HDL particle uptake by SR-BI and the role of particle uptake in the process of selective lipid uptake has not been defined (14,15). SR-BI-mediated internalization of apoA-I from HDL has been shown in both murine and human hepatocytes (33) and Madin-Darby canine kidney cells (34). However, little if any SR-BI-mediated uptake of HDL particles or apolipoproteins is observed in CHO cells (25,26) or steroidogenic cells (25,35), in which SR-BI levels and rates of HDL-selective uptake are high.…”

Section: Discussionmentioning

confidence: 99%

Serum Amyloid A Is a Ligand for Scavenger Receptor Class B Type I and Inhibits High Density Lipoprotein Binding and Selective Lipid Uptake

Cai

Beer

et al. 2005

Journal of Biological Chemistry

161

141

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Serum amyloid A is an acute phase protein that is carried in the plasma largely as an apolipoprotein of high density lipoprotein (HDL). In this study we investigated whether SAA is a ligand for the HDL receptor, scavenger receptor class B type I (SR-BI), and how SAA may influence SR-BI-mediated HDL binding and selective cholesteryl ester uptake. Studies using Chinese hamster ovary cells expressing SR-BI showed that 125 Ilabeled SAA, both in lipid-free form and in reconstituted HDL particles, functions as a high affinity ligand for SR-BI. SAA also bound with high affinity to the hepatocyte cell line, HepG2. Alexa-labeled SAA was shown by fluorescence confocal microscopy to be internalized by cells in a SR-BI-dependent manner. To assess how SAA association with HDL influences HDL interaction with SR-BI, SAA-containing HDL was isolated from mice overexpressing SAA through adenoviral gene transfer. SAA presence on HDL had little effect on HDL binding to SR-BI but decreased (30 -50%) selective cholesteryl ester uptake. Lipid-free SAA, unlike lipid-free apoA-I, was an effective inhibitor of both SR-BI-dependent binding and selective cholesteryl ester uptake of HDL. We have concluded that SR-BI plays a key role in SAA metabolism through its ability to interact with and internalize SAA and, further, that SAA influences HDL cholesterol metabolism through its inhibitory effects on SR-BI-mediated selective lipid uptake.

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“…We interpret these results to suggest that localization of the receptor to the cell surface is optimal for selective uptake. SR-BI-dependent endocytosis of HDL has been reported by various groups (6,7,8,13), but increasing evidence points against a role for HDL endocytosis in selective uptake. As recently shown by Nieland et al (13), for example, blocking of SR-BI-dependent HDL endocytosis appeared not to inhibit selective uptake, and blocking of selective uptake by specific inhibitors did not affect endocytosis of HDL.…”

Section: Discussionmentioning

confidence: 99%

High Density Lipoprotein Endocytosis by Scavenger Receptor SR-BII Is Clathrin-dependent and Requires a Carboxyl-terminal Dileucine Motif

Eckhardt

Cai

Shetty

et al. 2006

Journal of Biological Chemistry

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The high density lipoprotein (HDL) receptor Scavenger Receptor BII (SR-BII) is encoded by an alternatively spliced mRNA from the SR-BI gene and is expressed in various tissues. SR-BII protein differs from SR-BI only in the carboxyl-terminal cytoplasmic tail, which, as we showed previously, must contain a signal that confers predominant intracellular expression and rapid endocytosis of HDL. We haveshownthatSR-BIImediatesHDLendocytosisthroughaclathrindependent, caveolae-independent pathway. Two candidate amino acid motifs were identified in the tail that could mediate association with clathrin-containing endocytic vesicles: a putative dileucine motif at position 492-493 and an overlapping tyrosine-based YXXZ motif starting at position 489. Although substitution of tyrosine at position 489 with alanine or histidine did not affect endocytosis, substitution L492A resulted in increased surface binding of HDL and reduced HDL particle endocytosis. Substitution L493A had a less dramatic effect. No other regions in the carboxyl-terminal tail appeared to contain motifs required for HDL endocytosis. Substitutions of leucine at position 492 with the hydrophobic amino acids valine or phenylalanine also reduced HDL endocytosis, stressing the importance of leucine at this position. Introducing the SR-BII YTPLL motif into the carboxyl-terminal cytoplasmic tail of SR-BI converted SR-BI into an endocytic receptor resembling SR-BII. These results demonstrated that SR-BII differs from SR-BI in subcellular localization and trafficking and suggest that the two isoforms differ in the manner in which they target ligands intracellularly.

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Different transport routes for high density lipoprotein and its associated free sterol in polarized hepatic cells

Cited by 74 publications

References 46 publications

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

Serum Amyloid A Is a Ligand for Scavenger Receptor Class B Type I and Inhibits High Density Lipoprotein Binding and Selective Lipid Uptake

High Density Lipoprotein Endocytosis by Scavenger Receptor SR-BII Is Clathrin-dependent and Requires a Carboxyl-terminal Dileucine Motif

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