© 1995 American Heart Association, Inc.
Articles |
In Vivo LDL Receptor and HMG-CoA Reductase Regulation in Human Lymphocytes and Its Alterations During Aging
From the Institute for General and Experimental Pathology, University of Innsbruck (Austria) (T.M.S., L.A.H., G.W.); the Clinic for Urology, University Clinics Innsbruck (Austria) (H.K.); the Department of Metabolic Diseases, Pfizer Central Research, Groton, Conn (H.J.H.); the Institute for Medical Biochemistry, University of Graz (Austria) (G.J.); the Blood Transfusion Unit (D.S.) and the Central Laboratory for Medical and Chemical Laboratory Diagnosis (E.J.), University Clinics Innsbruck and the Institute for Biomedical Aging Research, Austrian Academy of Sciences (A.A., G.W.), Innsbruck, Austria.
Correspondence to Prof Dr G. Wick, Institute for General and Experimental Pathology, University of Innsbruck, Fritz-Pregl-Str 3/4, A-6020 Innsbruck, Austria.
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Abstract |
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Abstract The LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase play primary roles in the regulation of cellular cholesterol metabolism. To investigate the transcriptional regulation of lipid metabolism under physiological conditions ex vivo and its alterations during aging, we analyzed both the activity and mRNA concentration of the LDL receptor and HMG-CoA reductase in freshly isolated lymphocytes from healthy young and elderly donors. Data from fluorescent reverse transcriptase–polymerase chain reaction indicated that not only plasma LDL but also plasma HDL downregulates lymphocyte LDL receptor mRNA. Downregulation by HDL was three times more effective than that by LDL and presumably involved specific HDL binding sites. There was coordinate regulation of HMG-CoA reductase mRNA with LDL receptor mRNA that was independent of plasma lipoprotein concentrations. Despite elevated plasma concentrations of LDL, lymphocytes from elderly donors paradoxically expressed increased levels of the LDL receptor (P=.030) and HMG-CoA reductase mRNA (P=.062). The age-related dysregulation of the LDL receptor was predominantly due to impaired downregulation by plasma LDL rather than by HDL. Thus, not only LDL but also HDL and age significantly influences the transcriptional regulation of the LDL receptor in extrahepatic cells in vivo.
Key Words: aging • hydroxymethylglutaryl coenzyme A reductase • transcriptional regulation • LDL receptor • HDL receptor
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Introduction |
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Cellular cholesterol homeostasis is maintained primarily through regulation of the LDL receptor and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which affect exogenous uptake and endogenous synthesis, respectively, of cholesterol.1 Cholesterol uptake by the LDL receptor is generally considered to be regulated via transcriptional control mechanisms,1 2 3 4 5 whereas HMG-CoA reductase activity is regulated at many levels, from transcriptional control to degradation of the enzyme.6 7 8 A large number of studies have elucidated the role of these regulatory mechanisms in vitro, in experimental animals, and in humans treated with experimental diets or drugs.1 2 7 However, very little is known about the transcriptional regulation of cholesterol metabolism in peripheral cells from healthy subjects in vivo and its inherent physiological interindividual variations, ie, without induction of specific alterations by diet or drugs. Such evaluations are important to estimate the significance of the various regulatory mechanisms under physiological conditions in vivo, where the high concentration of LDL cholesterol in human plasma induces a very strong downregulation of LDL receptor and HMG-CoA reductase expression.1 2 5 9 10 11 12 13 14 One reason for the lack of information regarding the physiological regulation of cholesterol metabolism may lie in the need for more highly sensitive and accurate methods by which minimal amounts of specific mRNAs can be reliably quantified. This shortage has recently been overcome by the development of the quantitative reverse transcriptase–polymerase chain reaction (RT-PCR; Reference 1515 ).
Alterations in cellular cholesterol homeostasis are related to the development of atherosclerosis.1 2 Lymphocytes are widely used for studying the cholesterol metabolism of human extrahepatic cells in vivo.5 9 10 11 12 16 Lymphocytes from elderly donors paradoxically express increased LDL receptor activity,11 17 although the elevated concentrations of serum LDL cholesterol in this population18 19 should theoretically lead to downregulation of the receptor. This combination of increased LDL receptor activity and elevated plasma LDL cholesterol concentration in the elderly results in overaccumulation of cholesterol in freshly isolated lymphocytes from aged donors, which produces an elevated membrane microviscosity that may impair cellular function.20 21 22 However, the underlying cause for the paradoxical upregulation of the LDL receptor in cells from the elderly has not been investigated until now. It is not known whether this alteration is based on transcriptional dysregulation or impaired feedback inhibition by plasma lipoproteins.
To elucidate the regulatory mechanisms controlling cholesterol metabolism in extrahepatic cells and their alterations during aging, we analyzed LDL receptor mRNA expression in freshly isolated peripheral blood lymphocytes from healthy young and elderly donors by quantitative nonradioactive RT-PCR. Subsequently, we compared the LDL receptor mRNA levels with plasma lipoprotein concentrations and lymphocyte HDL-uptake activity. Because lymphocytes from the elderly may additionally overproduce cholesterol, apart from its exogenous accumulation, we extended our studies to the regulation of endogenous cholesterol synthesis by evaluating HMG-CoA reductase protein concentration, activity, and mRNA levels.
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Methods |
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Study Population and Plasma Cholesterol Determination
Elderly individuals (65 to 77 years), who were apparently healthy, self-supporting, and living independently, were randomly chosen from registers of the Blood Transfusion Unit of the University Hospital, Innsbruck. The young control subjects (20 to 32 years) were volunteers recruited from the laboratory staff and medical school. All blood donors were male, and appropriate informed consent was obtained. The number of blood donors for each experiment is given in the respective tables. To exclude any influence of disease, all subjects were carefully evaluated according to rigorous selection criteria of the SENIEUR protocol, which were originally established for immunogerontological studies by EURAGE (Concerted Action Programme on Aging of the European Community; References 17, 19, 23, and 2417 19 23 24 ). For this purpose, a detailed medical history was taken, including information on infectious, inflammatory, or allergic manifestations and gastrointestinal and metabolic disorders.19 Additionally, none of the subjects had been treated with lipid-lowering agents, antidiabetics, antibiotics, or hormones (eg, corticosteroids or thyroid or sex hormones; cf Reference 1919 ). Furthermore, blood laboratory values were determined, including fasting glucose, urea nitrogen, protein, liver enzymes, hemoglobin, erythrocyte mean cell volume, total and differential leucocyte count, and erythrocyte sedimentation rate to exclude latent disease.19 23 Plasma concentrations of total, LDL, and HDL cholesterol and triglycerides from fasting donors were determined by commercial tests as described.19 25
Lymphocyte Preparation
Peripheral blood mononuclear cells were isolated
from fasting donors by gradient centrifugation
(LymphoPrep, Nyegaard) as detailed previously.11 Cell
viability after preparation was estimated by trypan blue exclusion to
be >98% in both age groups. Monocyte content was between 5% and
23%, as determined by flow-cytometric scatter analysis. The
number of monocytes did not differ significantly between young and
elderly donors and had no significant effect on specific mRNA levels.
For detection of microsomal HMG-CoA reductase protein concentration and
activity, cells were additionally depleted of monocytes to <3% by
plastic adherence, as detailed previously.11 All of these
cells are referred to as "freshly isolated
lymphocytes".11
LDL- and HDL-Uptake Assay for Determination of Receptor
Activities
LDL and apoE-free HDL were isolated and labeled with
3,3'-dioctadecylindocarbocyanine (DiI, Lambda Probes), as described
previously.11 26 27 28 In brief, EDTA-plasma was pooled from
fasting, normolipidemic donors who were only weakly Lp(a)-positive (<1
mg/dL), and lipoprotein fractions were prepared by differential
ultracentrifugation (LDL, 1.020 to 1.050 g/mL;
HDL3, 1.125 to 1.230 g/mL). HDL3 was
further purified twice on heparin-Sepharose affinity columns (Pharmacia
Fine Chemicals) to remove the apoE-containing fraction.28
Lipoproteins were labeled with DiI according to Pitas et
al,26 with slight modifications.28
Chloramphenicol (50 mg/L, Serva), kallikrein inactivator
(100 000 U/L, Trasylol, Bayer), 
-amino-n-caproic acid
(1.3 g/L, Sigma), and EDTA (1 g/L, Merck) were present during all
steps of preparation and labeling to prevent lipid peroxidation and
apoprotein cleavage. The concentration of lipid
peroxides29 was <5 nmol/mg LDL and did not increase
during DiI labeling. The electrophoretic mobility of each batch of
labeled lipoproteins was compared with that of unlabeled lipoproteins
on agarose gels (Lipidophor System, Immuno AG; References 11 and
2811 28 ).
Binding and uptake of LDL and HDL via lipoprotein receptors/binding sites were determined by incubating lymphocytes for 2 hours at 37°C with or without 50 µg cholesterol/mL DiI-LDL or 30 µg cholesterol/mL apoE-free DiI-HDL, respectively.11 28 After repeated washing in PBS (pH 7.2), fluorescence was determined on a fluorescence-activated cell sorter (FACS III, Becton-Dickinson) gated on lymphocytes. This procedure leads to binding and uptake of labeled LDL and HDL in lymphocytes.2 11 30 31 This approach has been proven to yield data analogous to those from classic binding experiments, but with higher sensitivity and accurate specificity for the particular lipoprotein receptor.11 28 32
HMG-CoA Reductase Protein Concentration and Activity
Determination
HMG-CoA reductase (EC 1.1.1.34) activity of the fully
dephosphorylated enzyme was determined in lymphocyte
microsomes by measuring the enzymatic conversion of
[3-14C]HMG-CoA to mevalonic acid, as
described,33 and expressed as picomoles of mevalonate
produced in 1 minute by 1 mg microsomal protein. Microsomal HMG-CoA
reductase protein concentration was quantified by a noncompetitive,
solid-phase, bridged biotin-avidin enzyme immunoassay, as detailed
previously,6 and expressed as micrograms of immunoreactive
protein per milligram microsomal protein. HMG-CoA reductase specific
activity was determined as the ratio of HMG-CoA reductase activity to
protein concentration.
Quantitation of mRNA by RT-PCR
RNA was prepared by the acid/guanidinium
thiocyanate/phenol/chloroform method.34 35 RNA samples
were quantified and controlled for purity by absorption spectroscopy at
260, 270, and 280 nm.35 Previous observations have
revealed that the ratio of A260nm to A270nm is
a valuable means for detecting contaminating phenol, which strongly
affects quantitation of RNA.35 Only
A260-to-A270 ratios 
1.2 were
accepted,35 and degradation of RNA was never detected by
agarose gel electrophoresis. All buffers and reagents for RT-PCR were
obtained from Promega unless otherwise stated. Routinely, random-primed
reverse transcription of 200 ng RNA was carried out with 50 U MMLV RT
along with 20 U RNase inhibitor and 5x104
copies of pAW109 cRNA as an internal standard (Perkin-Elmer; Reference
1515 ). PCR amplification of cDNA corresponding to the 20 ng RNA added
initially was performed in the presence of 1.25 U Taq DNA
polymerase, 2 mmol/L MgCl2, and 7.5 pmol of each
RNA-specific primer as published previously,15 but the
forward primers were 5'-labeled with fluorescein isothiocyanate
(Microsynth; Reference 3636 ). Amplification with the LDL receptor primer
pair resulted in a 258-bp (mRNA) and a 301-bp (cRNA) product,
whereas amplification with the HMG-CoA reductase primer pair resulted
in amplicons of 246 bp (mRNA) and 303 bp (cRNA). PCR reactions were run
for 27 (LDL receptor) or 25 (HMG-CoA reductase) cycles with
denaturation at 95°C for 60 seconds, annealing at 57°C for 30
seconds, and extension at 72°C for 60 seconds. Subsequently,
ethanol-precipitated PCR products were separated by electrophoresis
through a denaturating 6% polyacrylamide sequencing gel on a
laser-equipped fluorescence-automated DNA sequencer (Applied
Biosystems) and analyzed by GENESCAN 672 software
(Applied Biosystems). The number of RNA copies per microgram of total
cellular RNA was calculated from the ratio (r) of peak areas between
mRNA and cRNA by the following equation: mRNA/µg RNA=rx(copies cRNA
added)/(µg RNA added). The variability in the quantitation of the
original amount of mRNA was less than ±10% when the ratio of mRNA to
cRNA was between 0.2 to 3 (data not shown), and this range was accepted
for reliable, specific mRNA quantitation.
Statistics
Data were analyzed by using SPSS software.
Mean values±SE are given, followed by the number of subjects. Groups
were compared by the Mann-Whitney U test. The partial
regression coefficients, including the standardized coefficient ß,
were calculated by multiple regression analysis. One outlier
(LDL receptor mRNA >4 SD) was excluded in all parametric tests. A
probability of .05 or less was considered statistically
significant.
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Results |
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Evaluation of Quantitative RT-PCR
The RT-PCR system used here was adapted from methods published previously.13 15 36 As shown in Fig 1
,
exactly linear responses were obtained when various amounts of cellular
RNA were added at the beginning of the protocol. The number of
temperature cycles, even as many as 36, did not influence the
quantitation of specific mRNA (data not shown). Thus, both PCR
products were affected proportionally even during nongeometric
amplification, which begins at approximately cycle 25 in our
PCR.37 To assess reproducibility, peripheral
blood lymphocytes from a single isolation were divided and RNA
was prepared separately in three tubes. RNA samples were carefully
controlled for quality (see "Methods") and revealed only minimal
differences in the results of fluorescent RT-PCR (28.1, 29.9, and
26.7x104 copies of LDL receptor mRNA per microgram of RNA,
respectively; corresponding data for HMG-CoA reductase gave 36.6 and
38.6x104 copies of mRNA per microgram of RNA,
respectively).
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Analysis of Lymphocyte Cholesterol
Metabolism From Young Versus Elderly Donors
LDL receptor activity, ie, binding and uptake of
fluorescence-labeled LDL during a 2-hour incubation period, was
significantly greater in freshly prepared lymphocytes from healthy
elderly donors (Table 1), confirming results from
previous studies.11 17 Additionally, LDL receptor mRNA was
increased by 40% in lymphocytes from the aged donors versus young
control subjects (Table 1).
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Microsomal HMG-CoA reductase activity was similar in lymphocytes from
elderly and young donors, as was HMG-CoA reductase protein
concentration and en-zyme specific activity (Table 1
). The
concentration of HMG-CoA reductase mRNA, however, was increased in
lymphocytes from the elderly, by 31% (Table 1), although not to
significant levels.
Correlations Between LDL Receptor mRNA and In Vivo
Parameters of Cholesterol
Metabolism
To address the question of whether plasma lipoproteins might be
regulatory factors for expression of LDL receptor and HMG-CoA reductase
mRNA in lymphocytes under physiological conditions,
we compared these parameters within our study population.
To assess the influence of individual parameters and their
dependence on age on specific mRNA expression, we calculated regression
models including only these two independent factors (Table 2, models A, B, C, and E). Two more complex models were
built to determine the power of our analysis and to ensure that
all included factors were independent of each other (Table 2, models D
and F). There was a strong negative correlation between LDL receptor
mRNA and plasma LDL concentrations (Table 2, model A; Fig 2). The partial regression coefficient for plasma LDL
predicted that increasing LDL cholesterol concentration by
1 mmol/L (39 mg/dL) will induce a decrease in LDL receptor mRNA of
about 3.7x104 copies per microgram of RNA. Downregulation
of LDL receptor mRNA by plasma LDL was strikingly impaired in
lymphocytes from the elderly (Table 2, model A; Fig 2).
At equal concentrations of plasma LDL, lymphocytes from the elderly
expressed 8.8x104 copies per microgram of RNA in excess of
that in young control subjects (Table 2, model A). Thus, at an LDL
concentration of 3.8 mmol/L (147 mg/dL), which is the mean of both
groups combined (Table 1), lymphocytes from an aged donor would express
90% more LDL receptor mRNA than those from a young donor (18.4 versus
9.6x104 copies per microgram of RNA).
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, dashed line) and
14 elderly (
, solid line) donors, and the concentration of LDL
receptor mRNA was assessed by fluorescent reverse
transcriptase–polymerase chain reaction; plasma LDL concentration was
determined by standard methods.
HDL was also shown to downregulate LDL receptor expression in
vitro,28 and there was a strong negative correlation
between both parameters in our study population (Table 2,
model B; Fig 3) independent of plasma LDL concentration
(model D). Age-related differences (model B; Fig 3) were about half as
strong as that in the regression with LDL (cf model A) and of
borderline significance (P=.054). The predictive value of
the entire model B was about the same as that of model A (cf adjusted
r2). According to the data in Table 2
(model B), an increase of HDL cholesterol by 1 mmol/L (39
mg/dL) would induce downregulation of LDL receptor mRNA by
11.8x104 copies per microgram of RNA. There were no
independent effects of plasma total cholesterol or
triglyceride concentration on the regulation of LDL
receptor mRNA, and both parameters were removed from the
model in stepwise regression analysis (not shown).
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HDL-uptake activity of lymphocytes, as assessed by uptake of
fluorescent apoE-free HDL, was inversely correlated with LDL receptor
mRNA and was significantly influenced by age (Table 2, model C). Model
D of Table 2 combined all of the factors described above (models A
through C), showing that plasma LDL and HDL cholesterol
levels and lymphocyte HDL-uptake activity were independent predictors
of LDL receptor mRNA concentration (partial Ps
.004). This
is underlined by the fact that the partial regression coefficients (b),
which estimate the influence of individual factors on LDL receptor mRNA
concentration, were virtually unchanged in the multifactorial model D
compared with the more simple models A through C. Thus, the overall
predictive value was increased to more than 68% (adjusted
r2, model D).
There was a significant correlation between mRNA concentrations of LDL
receptor and HMG-CoA reductase on an individual level, with no
significant effect due to age (Table 2, model E). However, HMG-CoA
reductase mRNA was correlated with neither plasma LDL nor plasma HDL
concentrations (Table 2, model F). Nevertheless, HMG-CoA reductase mRNA
or factors regulating its expression independently contributed to the
multifactorial regression model on LDL receptor mRNA, raising its
predictive value to 74% (data not shown).
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Discussion |
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This study was designed to determine which factors might be important in transcriptional regulation of the LDL receptor and HMG-CoA reductase in a physiological situation in vivo and how this regulation may change with age. Apart from further evidence that plasma HDL is an important regulatory factor in the transcriptional regulation of the LDL receptor, we have shown that feedback inhibition of LDL receptor mRNA by plasma LDL strongly depends on the age of the donor. For the first time, this age-related dysregulation provides a possible explanation for the paradoxically enhanced expression of the LDL receptor in peripheral cells from the elderly in vivo.
Our study contrasts with previous investigations that have
analyzed the changes in cholesterol
metabolism of lymphocytes following changes in lipoprotein
patterns induced by diet or drugs. Because it is impossible to collect
data regarding the actual metabolic state and
simultaneously induce specific alterations, data on the
physiological regulation of cholesterol
metabolism in human lymphocytes rely on statistical
comparisons. Surprisingly, although the blood donors differed with
regard to diet and genetic background, almost 70% of the total
interindividual variance in LDL receptor mRNA could be explained solely
by plasma concentrations of LDL and HDL, HDL-uptake activity, and age
(Table 2, model D).
In addition to LDL, which was expected to regulate LDL receptor
expression on the basis of many previous observations,1
HDL also downregulated LDL receptor mRNA (Table 2, models B and D). In
contrast to the hypothesis of reverse cholesterol
transport,38 data from various laboratories, including
ours, have shown that HDL can provide cholesterol and
lipids to lymphocytes and other extrahepatic cells in
vitro.28 30 31 39 40 HDL1+2b and
HDL2a may deliver cholesterol to
lymphocytes via the LDL receptor pathway because these HDL species can
carry apoB and/or apoE.2 38 40 However, the interaction of
apoE-free HDL with cells probably involves HDL-specific binding
sites/receptors,2 28 41 42 which have been shown to be
expressed on lymphocytes and to be specific for
apo-AI.28 30 31 43 Internalization and degradation of
HDL apoproteins suggest not only binding but also uptake of HDL
particles.30 31 The specific HDL-uptake activity of
lymphocytes correlates negatively with LDL receptor mRNA in vivo (Table 2, model C), indicating that HDL binding sites may be involved in
transcriptional downregulation of the LDL receptor. However, the
regulatory effect of HDL must not be solely attributed to lipid
delivery, as HDL and apoA-I can directly induce signal transduction
events, eg, activation of protein kinase C.42 44 45 46
Correlations with both plasma HDL and lymphocyte apoE-free HDL-uptake
activity strongly argue for the importance of HDL in the
transcriptional regulation of the LDL receptor in
peripheral cells in vivo. However, because HDL in human
plasma includes apoE-containing particles that may bind not only to the
HDL binding site/receptor but also to the LDL receptor, it cannot be
estimated to which fraction the HDL-driven downregulation of the LDL
receptor mRNA involves the apoA-I–specific HDL binding site/receptor
in vivo.
Downregulation of LDL receptor mRNA by HDL is about three times more
effective than that by LDL (b in Table 2, model D). Therefore, it could
be hypothesized that HDL protects lymphocytes from
cholesterol overaccumulation via enhanced LDL receptor
downregulation, particularly because the proposed HDL-mediated reverse
cholesterol transport38 could not be proven
with this extrahepatic cell type.30 This regulatory
function of plasma HDL on the LDL receptor pathway could contribute to
the antiatherosclerotic action of HDL.
HMG-CoA reductase was shown to be coordinately regulated with the LDL
receptor at the transcriptional level (Table 2, model E; References 7,
47, and 487 47 48 ). During preparation of this manuscript, Powell and
Kroon49 published the results of their study, showing a
comparable coordinate transcriptional regulation of the LDL receptor
and HMG-CoA reductase in peripheral blood mononuclear cells
and liver samples from middle-aged donors. However, the factors
involved in transcriptional regulation of HMG-CoA reductase in vivo are
only partly identical to those for LDL receptor regulation, because in
contrast to HMG-CoA reductase (Table 2, model F; Reference 4949 ), LDL
receptor mRNA is regulated by plasma concentrations of LDL (Table 2,
models A and D) and HDL (Table 2, models B and D; Reference 4949 ).
Factors other than sterols have also been shown to be involved
differently in transcriptional regulation of the LDL receptor and
HMG-CoA reductase, eg, mitogenic signals12 and
intracellular calcium,50 respectively.
The increased cholesterol content in lymphocyte membranes
from elderly donors20 21 22 may be mediated by a paradoxical
increase in lymphocyte LDL receptor activity.11 17 18 22
We confirmed the age-related upregulation of LDL receptors at the
transcriptional level, indicating that the paradoxical increase in LDL
receptor expression in the elderly is due to transcriptional
dysregulation (Table 1). Moreover, enhanced expression of LDL receptor
mRNA seems to be predominantly due to impaired downregulation by serum
LDL, which is affected to twice the degree as is downregulation by
serum HDL (Table 2, models A and B). This difference may further
support the hypothesis that LDL and HDL use separate pathways to exert
their regulatory function on LDL receptor mRNA.
The age-related impairment of LDL-driven feedback inhibition may involve any step of LDL receptor regulation that occurs between LDL uptake and LDL receptor gene transcription. Thus, the apparent transcriptional dysregulation may be due to alterations in the transport of cholesterol from the plasma membrane to the intracellular regulatory pool or to the action of cholesterol (or its active metabolites) on the LDL receptor gene. If impaired transport of cholesterol to the regulatory pool is the underlying age-related defect, then the increased cholesterol content in cells from the elderly (reflecting mainly plasma membrane cholesterol) would not only be the consequence of increased expression of the LDL receptor but also would be necessary to sufficiently raise cholesterol concentration in the regulatory pool. Therefore, the age-related alterations in LDL receptor gene expression and cellular cholesterol concentration may reflect disturbed feedback regulation, but at this time it is not yet possible to pinpoint the exact location of the defect within this loop.
It is unlikely that increased endogenous sterol synthesis
contributes to cellular cholesterol enrichment with age, as
there was no measurable age-related difference in protein concentration
or activity of microsomal HMG-CoA reductase (Table 1). HMG-CoA
reductase is effectively regulated not only via transcription but also
at various posttranscriptional levels, eg, degradation of the
enzyme, phosphorylation, the redox state of thiol
groups, etc.6 7 8 Thus, lymphocytes from the elderly seem to
compensate for the moderate increase in HMG-CoA reductase mRNA by
posttranscriptional mechanisms.2 6 7 8 47 In conclusion,
aging is an additional factor that causes dissociation of LDL receptor
and HMG-CoA reductase activity despite coordinate transcriptional
regulation.47
The present study has evaluated influences on the transcriptional regulation of the LDL receptor within the physiological state and stresses HDL and age as relevant regulatory factors. While LDL-driven transcriptional downregulation of the LDL receptor is well documented,1 3 the cellular mechanisms of HDL-induced LDL receptor regulation have yet to be elucidated in detail. Because HDL is a major protective factor against coronary heart disease,51 information about its mechanisms of action may be of considerable importance for public health. If HDL also exerts its function via regulatory influences, then the development of drugs mimicking this action would be possible.
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Acknowledgments |
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This work was supported by the Austrian Ministry for Science and Research. We thank Dr Gene C. Ness (University of South Florida, Tampa) for supplying the anti–HMG-CoA reductase antiserum used in these studies for measuring HMG-CoA reductase protein concentration. We are grateful to Gerhard Ledinski for his excellent technical assistance and to Drs H. Dieplinger and H.G. Kraft for critically reading the manuscript.
Received December 5, 1994; accepted April 12, 1995.
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