Desmosterol as the Major Sterol in L-Cell Mouse Fibroblasts Grown in Sterol-Free Culture Medium

Rothblat, George H.; Burns, C. H.; Conner, Robert L.; Landrey, Josephine R.

doi:10.1126/science.169.3948.880

Cited by 69 publications

(24 citation statements)

References 13 publications

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“…Alternatively, embryonic development and the short postnatal survival in the seladin-1 knockout mice may depend on the substitution of cholesterol by desmosterol. In agreement with this possibility, one study indicated that desmosterol could replace cholesterol without deleterious effects in fibroblasts (Rothblat et al, 1970). Although this last scenario would imply a non-essential role of cholesterol in early developmental processes, it also indicates an essential requirement of cholesterol for full maturation.…”

Section: Discussionmentioning

confidence: 81%

The role of seladin-1/DHCR24 in cholesterol biosynthesis, APP processing and Aβ generation in vivo

Crameri

Biondi

Kuehnle

et al. 2006

EMBO J

162

179

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The cholesterol-synthesizing enzyme seladin-1, encoded by the Dhcr24 gene, is a flavin adenine dinucleotidedependent oxidoreductase and regulates responses to oncogenic and oxidative stimuli. It has a role in neuroprotection and is downregulated in affected neurons in Alzheimer's disease (AD). Here we show that seladin-1-deficient mouse brains had reduced levels of cholesterol and disorganized cholesterol-rich detergent-resistant membrane domains (DRMs). This was associated with inefficient plasminogen binding and plasmin activation, the displacement of b-secretase (BACE) from DRMs to APP-containing membrane fractions, increased b-cleavage of APP and high levels of Ab peptides. In contrast, overexpression of seladin-1 increased both cholesterol and the recruitment of DRM components into DRM fractions, induced plasmin activation and reduced both BACE processing of APP and Ab formation. These results establish a role of seladin-1 in the formation of DRMs and suggest that seladin-1-dependent cholesterol synthesis is involved in lowering Ab levels. Pharmacological enhancement of seladin-1 activity may be a novel Ab-lowering approach for the treatment of AD.

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Section: Discussionmentioning

confidence: 81%

The role of seladin-1/DHCR24 in cholesterol biosynthesis, APP processing and Aβ generation in vivo

Crameri

Biondi

Kuehnle

et al. 2006

EMBO J

162

179

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“…LM cells were mutagenized by incubating cells with N-methyl-N'-nitro-N-nitrosoguanidine (20 ,uM) To explore this approach we prepared cells with decreased membrane sterol content by utilizing oxygenated derivatives of cholesterol, such as 25-hydroxycholesterol, which are known to inhibit sterol synthesis specifically but not to substitute for the normal membrane sterol (13 (14)] decreased to 14.0, 10.9, 9.6, and 6.8 ,ug/mg of protein after 0, 5, 10,Iand 24 hr (slightly more than one generation), respectively. The concentration of filipin required for 50% inhibition of thymidine incorporation increased from 4.5 to more than 9 ,g/ml from the beginning to the end of this growth period.…”

Section: Methodsmentioning

confidence: 99%

Animal cell mutants defective in sterol metabolism: A specific selection procedure and partial characterization of defects

Saito

Chou

Silbert

1977

Proc. Natl. Acad. Sci. U.S.A.

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By using a chemically defined medium, a general and highly specific procedure was devised to select for mutant cells with less abundant or structurally altered sterol in their surface membranes. Within a certain concentration range, the polyene antibiotic filipin was shown to kill only cells with normal (as opposed to decreased) membrane sterol levels. Sterol-requiring derivatives of LM cells were isolated by chemical mutagenesis, filipin treatment, and cloning followed by replica plating in soft agar. Mutants (SI and S2) Cell Growth. LM cells (mouse fibroblasts) were purchased from the American Type Culture Collection (CCL 1.2) and were propagated in suspension culture or in monolayers. Minimal growth medium was Higuchi's medium (6) modified by the addition of 20 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes) buffer (final pH 7.4), 0.5% bovine serum albumin, and 0.015% Darvan no. 2 (R. T. Vanderbilt Co.) (to minimize cell clumping or attachment to the walls of the culture bottle). The suspension culture (25 ml in a 125-ml Wheaton screw-cap bottle) was agitated on a gyratory shaker (140 rpm) at 370. Growth was monitored by measuring cell number in a hemocytometer, and cell densities ranged generally from 0.5 to 3 X 106 cells per ml. Generation time in the minimal medium was about 24 hr. When cells were grown as monolayers or cloned in soft agar (7), a modified Eagle's medium (Flow Laboratories) supplemented with penicillin (50 units/ml) and streptomycin (50,ug/ml) was used and the cultures were grown in a CO2 incubator.Isolation of Mutants with Decreased Membrane Desmosterol Levels. LM cells were mutagenized by incubating cells with N-methyl-N'-nitro-N-nitrosoguanidine (20 ,uM)

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“…1A); (ii). DHE completely replaces the requirement for cholesterol as the only sterol source in the diet of the worm C. elegans (19); (iii) Mouse L-cell fibroblasts lack the enzyme 3β-hydroxysteroid-Δ 24 -reductase (desmosterol reductase, Dhcr24) (22). Desmosterol differs from cholesterol in having an extra double bound in the side chain.…”

Section: Historical Perspectivementioning

confidence: 99%

“…Desmosterol differs from cholesterol in having an extra double bound in the side chain. When cultured in chemically-defined medium the L-cells synthesize desmosterol, replace membrane cholesterol with desmosterol, and grow normally despite the absence of cholesterol (22,23); (iv). Mouse L-cells cultured in chemically-defined medium containing dehydroergosterol (DHE) accumulate DHE which replaces as much as 90% of endogenous membrane sterol without adverse effects on membrane phospholipid or fatty acid composition, sterol/phospholipid ratio, activity of cholesterol sensitive enzymes in the plasma membrane, or cell growth (24).…”

Section: Historical Perspectivementioning

confidence: 99%

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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Desmosterol as the Major Sterol in L-Cell Mouse Fibroblasts Grown in Sterol-Free Culture Medium

Cited by 69 publications

References 13 publications

The role of seladin-1/DHCR24 in cholesterol biosynthesis, APP processing and Aβ generation in vivo

The role of seladin-1/DHCR24 in cholesterol biosynthesis, APP processing and Aβ generation in vivo

Animal cell mutants defective in sterol metabolism: A specific selection procedure and partial characterization of defects

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

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