Abstract
Cholesterol homeostasis is maintained by coordinate regulation of endogenous synthesis and exogenous uptake of lipoprotein cholesterol by low density lipoprotein (LDL) receptors. In the lymphocyte, limiting the availability of exogenous cholesterol is known to increase the rate of endogenous sterol biosynthesis. However, the effect of cholesterol deprivation on the expression and regulation of the LDL receptor gene has not been delineated in lymphocytes. Here, LDL receptor mRNA was detected in freshly isolated human peripheral mononuclear cells. LDL receptor mRNA levels increased by 3-fold during a one-h in vitro culture in lipoprotein-deficient medium and by 6-fold during a 2-h incubation. Actinomycin D blocked the synthesis of LDL receptor mRNA in these cultures. However, neither cycloheximide nor LDL or oxygenated sterols suppressed the increase in LDL receptor mRNA levels observed after a 2-h incubation. The increase in LDL receptor mRNA was maintained for 24 h of culture in the absence of LDL. Ongoing gene transcription and not mRNA stabilization accounted for this expression. Inhibition of protein synthesis with cycloheximide completely prevented the sustained increase in LDL receptor mRNA levels measured after 24 h. Low concentrations of LDL (5 micrograms of cholesterol/ml) and oxygenated sterols also suppressed the level of LDL receptor mRNA measured after a 24-h incubation. These data show that the initial upregulation of LDL receptor gene expression is independent of protein synthesis and not suppressed by either LDL or oxygenated sterols. In contrast, the continued transcription necessary for the maintenance of steady-state levels of LDL receptor mRNA requires synthesis of new protein and is regulated by LDL and oxygenated sterols.
Highlights
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The relative abundance of LDL receptor mRNA in each sample was determined by comparing the relative content of LDL receptor mRNA to that observed in peripheral blood mononuclear cells (PBM) incubated for 2-24 h in lipoprotein-deficient medium
Detection of LDL Receptor mRNA in Circulating Human PBM-Small amounts of LDL receptor mRNA were detected in total RNA obtained from freshly isolated PBM (Fig. 1)
Summary
Cell Preparation and Culture-PBM were isolated from anticoagulated venous blood obtained from normal adults asdescribed previously [3].For some experiments, PBM were separated into T cellenriched (>95% rosette-positive)and T cell-depleted populations ( ~ 5 %rosette-positive)by rosetting with neuraminidase-treated sheep red blood cells and passing rosette-positive cells over a nylon wool column as detailed(3,lO).Cells were cultured in RPMI1640 medium (Inland Laboratories, Austin, TX) containing L-glutamine (0.3 mg/ ml),gentamicin (10pg/ml), and penicillinG(200 units/ml),and supplemented with 1% lipoprotein-poor plasma Cultures were supplemented with 25-hydroxycholesteroI(Steraloids, Inc., Wilton, NH), the sodium salt of mevalonate, prepared as described previously [3] or human LDL (density = 1.020-1.050 g/ml) isolated mRNA. The relative content of LDL receptor mRNA underdifferent conditions,within each individual experiment was calculated by normalizing for the amount of ["'PIcDNA incorporated into the @actinband of thesame sample. Measurement of Rates of Sterol Synthesis-Sterol synthesis was determined by measuring the rate of incorporation of [1-"Cjacetate into digitonin-precipitable sterols using previously described techniques [3]. Freshly isolated cells were incubated for 2 or 4 h at 37 "C with 1 mM [l-''C]acetate, after which they were saponified, and the sterolswere extracted and precipitated with digitonin. Data are expressed as the rate of sterol synthesis (picomoles of acetate incorporated into digitonin-precipitable sterols/h/106 cells). Incubations were carried out in sterile tissue culture flasks for RNA isolation with 1-2 X lo cells/ml initially cultured
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