|
Volume 271, Number 22,
Issue of May 31, 1996
pp. 12724-12736
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
27-Hydroxylated Low Density Lipoprotein (LDL) Cholesterol Can
Be Converted to 7 ,27-Dihydroxy-4-cholesten-3-one (Cytosterone)
before Suppressing Cholesterol Production in Normal Human
Fibroblasts
EVIDENCE THAT AN ALTERED METABOLISM OF LDL CHOLESTEROL CAN
UNDERLIE A DEFECTIVE FEEDBACK CONTROL IN MALIGNANT CELLS*
(Received for publication, December 22, 1995, and in revised form, March 4, 1996)
Magnus
Axelson
§ and
Olle
Larsson
¶
From the Department of Clinical Chemistry and the
¶ Department of Tumor Pathology, Karolinska Hospital, S-171 76
Stockholm, Sweden
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
ABSTRACT
The formation of oxysterols in
cultured human fibroblasts and their physiological roles as
intracellular regulators of cholesterol production have been
investigated. In the presence of low density lipoproteins (LDL), normal
fibroblasts converted LDL cholesterol to 27hydroxycholesterol,
which was further metabolized to 7 ,27-dihydroxycholesterol,
7 ,27-dihydroxy-4-cholesten-3-one, and
7 -hydroxy-3-oxo-4-cholestenoic acid. Autooxidation products of
cholesterol contaminating the lipoproteins were also metabolized in the
cells. 7 -Hydroxycholesterol was converted to
7 -hydroxy-4-cholesten-3-one prior to 27-hydroxylation and further
oxidation to 7 -hydroxy-3-oxo-4-cholestenoic acid.
7 -Hydroxycholesterol and 7-oxocholesterol were 27-hydroxylated and
then oxidized to C27-acids. Oxidation of the 7 -hydroxy
group also occurred. 25-Hydroxycholesterol was 7 -hydroxylated and
further oxidized to 7 ,25-dihydroxy-4-cholesten-3-one.
25-Hydroxylation of sterols was observed only under specific
conditions. In contrast, only small amounts of oxysterols were formed
in virus-transformed human fibroblasts when incubated with
lipoproteins. This was due to very low activities of the 27- and
7 -hydroxylating enzymes. The rate of oxidation at C-3 was also
decreased moderately.
A defective suppression of 3-hydroxy-3-methylglutaryl coenzyme A
reductase by LDL and autooxidation products of cholesterol observed in
the transformed fibroblasts could be caused by the deficiencies of the
sterol-metabolizing enzymes, since these cells responded normally to
the sterol metabolites 7 ,27-dihydroxy-4-cholesten-3-one,
7 ,25-dihydroxy-4-cholesten-3-one, and 27-hydroxy-7-oxo-cholesterol.
These metabolites, which all possessed an oxo group with a conjugated
double bond in the steroid nucleus and a hydroxyl group in the side
chain, did not seem to require further metabolism in order to be
active. An impaired response to LDL was also seen in other human tumor
cells, including breast carcinoma, colonic carcinoma, and malignant
melanoma cells. Common to all the malignant cells was an intracellular
shortage of 7 ,27-dihydroxy-4-cholesten-3-one caused by a decreased
formation or an increased metabolism.
INTRODUCTION
Low density lipoprotein (LDL)1 is the
only regulator of cellular cholesterol production whose physiological
role has been established (1, 2, 3, 4, 5). Internalized LDL suppresses
3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the
rate-limiting enzyme of cholesterol synthesis, but the underlying
mechanisms are not fully understood. Recently, we reported that LDL
cholesterol is converted to 27-hydroxycholesterol in human fibroblasts
and that this oxysterol is an important intracellular mediator of the
LDL-induced suppression of HMG-CoA reductase (6). The metabolism of LDL
cholesterol and the biological effects of its metabolites in normal
human fibroblasts have now been investigated further. Virus-transformed
fibroblasts were included in the study, since a defective regulatory
response to LDL has been observed previously in several different
malignant cells (7, 8, 9, 10). This impaired cholesterol feedback system, the
causes of which are unknown, seems to appear early in the development
of cancer and has been considered to promote cell growth by increasing
the cellular supply of cholesterol and intermediates in the mevalonate
pathway (11). The two fibroblastic cell lines permitted us to make a
direct comparison between the handling of LDL cholesterol and the
suppression of HMG-CoA reductase in the normal and the corresponding
tumor-transformed cell. Here we report that in normal human fibroblasts
27-hydroxylated LDL cholesterol is converted to
7 ,27-dihydroxy-4-cholesten-3-one, which does not seem to be
metabolized further before suppressing HMG-CoA reductase. The study
also shows that the metabolism of LDL cholesterol is markedly changed
following transformation of fibroblasts and that these cells display a
defective regulatory response to LDL. An altered metabolism of LDL
cholesterol and an impaired suppression of HMG-CoA reductase were also
noted in other human neoplastic cells.
MATERIALS AND METHODS
Steroids, Chemicals, and Sera
Diosgenin
((25R)-5-spirosten-3 -ol) was from Sigma and was used as
the starting material for the synthesis of 27-oxygenated steroids
(12, 13, 14). In addition, 5-cholestene-3 ,7 ,25-triol
(7 ,25-dihydroxycholesterol) was prepared from the
3-acetate,25-trimethylsilyl ether derivative of 25-hydroxycholesterol,
and, after hydrolysis, this steroid was further oxidized to
7 ,25-dihydroxy-4-cholesten-3-one as described for the corresponding
27-hydroxysteroids (14). 25-Hydroxycholesterol was oxidized in the same
way to 25-hydroxy-4-cholesten-3-one. Other unlabeled steroids were
those used in a previous study (15).
[1 ,2 -3H]Cholesterol (44 Ci/mmol) and
[1 ,2 -3H]cholesteryl oleate (49 Ci/mmol) were
purchased from Amersham and
25-[26,27-3H]hydroxycholesterol (86 Ci/mmol) was from Du
Pont de Nemours NV, NEN Products (Brussels, Belgium). Radioactivity was
determined in an LKB/Wallac 1215 Rackbeta Scintillation Counter with
OptiPhase ``HiSafe'' (Wallac) as the scintillation liquid.
Cyclosporin A (CsA) and ketoconazole were kind gifts from Sandoz Pharma
Ltd. (Basel, Switzerland) and Janssen Pharmaceutica (Beerse, Belgium),
respectively. Fetal calf serum (FCS) having a total cholesterol
concentration of 1.2 mM (12% free and 88% esterified
cholesterol) was from Life Technologies, Inc. (Stockholm, Sweden).
About 70% of the total cholesterol was found in the LDL fraction. The
lipoprotein-deficient serum (LDS) was prepared by treating FCS with
Cab-O-Sil (16), and the cholesterol concentration after this treatment
was 0.1 mM. Cell growth was not affected negatively when
this serum was used. LDL particles containing 3H-labeled
cholesterol or cholesteryl oleate were obtained by incubating the
radioactive steroid with FCS overnight at 20 °C (6).
Cell Culture Conditions
Normal human fibroblasts (line GM
08333) were obtained from NiGMS, Corriell Institute for Medical
Research (Camden, NJ) and SV40 virus-transformed human fibroblasts
(90-VA IV) were a kind gift from Dr. Stein (University of Colorado,
Boulder, CO). Human colonic carcinoma (WiDr), breast carcinoma (MDA
231), and malignant melanoma (SK-MEL-2) cell lines were from American
Type Culture Collection
All cell lines were grown in monolayers in tissue culture flasks
maintained in a 95% air, 5% CO2 atmosphere at 37 °C in
a humidified incubator and were cultured in either Dulbecco's modified
Eagle's medium (MDA 231) or minimal Eagle's medium (the other cells)
supplemented with essential and nonessential amino acids and 10% FCS
(v/v). For experimental purposes, cells were cultured in dishes. Cells
were seeded at a density of 5,000 cells per cm2. The
experiments were started 48-72 h later, at which time a cell density
of approximately 20,000 per cm2 had been reached. The cells
were subconfluent also at the end of the incubations. When the
metabolism of cholesterol or oxysterols was studied, normal or
transformed fibroblasts (cell number 3-6 × 106 in
57-143-cm2 dishes) were first preincubated for 24 h in
medium containing 10% LDS and were then incubated for 3-68 h with
7-10 ml of medium containing 4-10% FCS (with or without
3H-labeled cholesterol or cholesteryl oleate) or were
incubated with the oxysterol in 10% LDS for 24-48 h. Control cells
were incubated in the same way, but only for 15 min. Effects of CsA,
ketoconazole, and oxysterols were tested on normal and transformed
fibroblasts at concentrations of 10-30 µM, 30
µM, and 0.12 µM, respectively, in cell
media containing 0-10% FCS and 10-0% LDS. The substances were added
to the incubation media in freshly prepared ethanol solutions, and the
ethanol concentrations of media became 0.1-0.5%. Control cells were
incubated in the same way, but without CsA, ketoconazole, or
oxysterols. The dish size and volume of media when HMG-CoA reductase
activity was to be determined were 20 cm2 and 5 ml,
respectively, and the incubations were carried out in duplicate for
3-24 h. Each oxysterol was tested in 2-5 separate experiments.
Determination of HMG-CoA reductase activity was then carried out as
described previously (6, 17, 18).
Analysis of Oxysterols and Steroid Acids
The procedure for
extraction and purification of oxysterols present in incubation media
and cells was essentially the same as described previously (6).
Following the collection of a neutral oxysterol fraction from the
lipophilic anion exchanger (6), a fraction containing steroids with a
free carboxyl group was eluted with 0.15 M acetic acid in
95% aqueous methanol prior to elution of a fraction containing
stronger acids (including steroid sulfates) with 0.5 M
potassium acetate/potassium hydroxide, apparent pH 10.0, in 72%
aqueous methanol (19).
Trimethylsilyl ethers of oxysterols and methyl ester trimethylsilyl
ether derivatives of steroid acids were prepared (19) and were
analyzed by gas chromatography-mass spectrometry (GC/MS) as described
previously (6).
High Performance Liquid Chromatography
(HPLC)
3H-Labeled cholesterol, cholesteryl oleate,
and 25-hydroxycholesterol and/or their radioactive metabolites were
analyzed by HPLC prior to or after group fractionation and purification
as described above. 3H-Labeled cholesterol and cholesteryl
esters were extracted from small aliquots of the incubation media with
mixtures of isopropyl alcohol and hexane and from cells with mixtures
of ethanol and water prior to separation by straight-phase HPLC using
hexane/isopropyl alcohol, 98:2 (v/v), as the mobile phase (6).
Appropriate fractions from the HPLC effluent were collected in vials,
and the radioactivity was then determined by scintillation counting.
Radioactive neutral and acidic metabolites of
[3H]cholesterol or [3H]cholesteryl oleate
were isolated from media and cells as described above and were then
characterized by HPLC. For this purpose, three HPLC systems were used
in the following order. Reversed-phase HPLC was carried out on a column
of LiChrospher (250 × 4 mm, Hibar, 100RP-18, 5 µm, Merck, Darmstadt,
Germany) using a pump (Constametric III) and a variable wavelength
detector (Spectra Monitor D from LDC/Milton Roy, Riviera Beach, FL) set
at 220 or 240 nm and a Rheodyne Model 7125 injector with a 100-µl
loop. The mobile phase first used for neutral metabolites was a mixture
of methanol/ethanol/water, 80:20:10 (by volume, flow rate 1 ml ×
min 1), and fractions were collected between 0 and 11 min
(fraction 1; containing polar metabolites, e.g.
7 ,27-dihydroxy-4-cholesten-3-one, retention time about 4.5 min) and
between 11 and 14 min (fraction 2; containing
7 -hydroxy-4-cholesten-3-one and 27-hydroxycholesterol having
retention times 11.5 and 12.5 min, respectively). The mobile phase was
then changed to 85% aqueous methanol (flow rate 1 ml ×
min 1) for separation of sterols in fraction 1 or for
separation of steroid acids (as methyl ester derivatives). In the
former case, a fraction of the effluent containing
7 ,27-dihydroxy-4-cholesten-3-one (retention time about 8.5 min) was
collected between 8.0 and 9.0 min, and in the latter case a fraction of
the effluent containing 7 -hydroxy-3-oxo-4-cholestenoic acid
(retention time about 12 min) was collected between 11.0 and 13.0 min.
These fractions and fraction 2 (containing
7 -hydroxy-4-cholesten-3-one and 27-hydroxycholesterol) were then
reanalyzed by straight-phase HPLC. The latter was carried out with an
instrument similar to that above, but with a column (250 × 4.5 mm) of
LiChrospher (Hibar, Si 100, 5 µM, Merck). The mobile
phase was hexane/isopropyl alcohol, 94:6 (v/v), when fraction 2 was
analyzed (retention times of 7 -hydroxy-4-cholesten-3-one and
27-hydroxycholesterol were about 8 and 9 min, respectively), and 90:10
(v/v), when the fractions containing
7 ,27-dihydroxy-4-cholesten-3-one or
7 -hydroxy-3-oxo-4-cholestenoic acid methyl ester were analyzed. The
flow rate was 1.0 ml × min 1 in all cases. The HPLC
effluent during the latter analyses was collected in scintillation
vials with 15-60-s intervals, and, after addition of scintillation
fluid, the radioactivity was determined.
25-[3H]Hydroxycholesterol and its metabolites in media
and cells were analyzed by HPLC following extraction. Medium was
extracted with ethanol, and, after centrifugation and removal of the
supernatant, the pellet was re-extracted with ethanol/isopropyl
alcohol, 1:1 (v/v). The extracts were combined and the solvent was then
evaporated. Nonpolar compounds were dissolved in hexane, and, after
removal, the solid residue was dissolved in 60% aqueous methanol,
which was passed through a column (1.5 × 0.8 cm) of
octadecylsilane-bonded silica (Preparative C18; Waters
Associates Inc., Milford, MA) and collected. The methanol in the eluate
was then removed in vacuo, and the aqueous solution was
re-extracted on the same column. After washing the column with water,
sorbed steroids (polar metabolites) were eluted with
methanol/chloroform, 1:1 (v/v), and were combined with the nonpolar
metabolites present in the hexane fraction. This combined extract was
evaporated to dryness and dissolved in methanol or hexane/isopropyl
alcohol, 90:10 (v/v), prior to analysis by reversed-phase HPLC (mobile
phase: methanol/ethanol/water, 80:20:10 (v/v) or straight-phase HPLC
(mobile phase: hexane/isopropyl alcohol, 97:3 (v/v)).
RESULTS
Formation of Oxygenated Cholesterol Derivatives in Human
Fibroblasts
The formation of oxygenated cholesterol derivatives
in normal and virus-transformed human fibroblasts has been investigated
in detail. A general method for the isolation of
C27-steroids from media and cells were used, so that no
major steroid was expected to escape detection. The final analysis was
based on GC/MS.
When normal fibroblasts were incubated for 48 h in the absence of
lipoproteins (media containing 10% LDS), only trace amounts (<1-10
pmol/mg of cell protein) of oxysterols could be detected in the medium
and cells, with the exception of 7 -hydroxycholesterol,
7 -hydroxycholesterol, and 7-oxocholesterol (amounts about 5-50
pmol/mg of cell protein). Most of these sterols were probably formed by
autooxidation of cholesterol during the purification of the samples,
since their amounts did not differ significantly from those of the
controls (15-min incubations).
In contrast, when normal fibroblasts were incubated for 24-48 h with
lipoproteins (media containing 10% FCS), 13 neutral and 5 acidic
C27-steroids were found in the media. Steroids identified
are listed in Table I. The identification was based on
the GC retention indices of the derivatives and mass spectra, which
were compared with those of the authentic steroids. Most of the
steroids had additional oxygen groups both at C-7 and in the side
chain. No additional steroids were identified in the cell extracts,
and, with the exception of the autooxidation products of cholesterol
(see above), the amounts of oxysteroids in the cell extracts were low
and barely detectable (<10-20% of those in media). Because of this,
oxysterols in the cells were usually not analyzed, unless otherwise
stated. Fig. 1 shows a GC/MS analysis of neutral
C27-steroids isolated from the medium after incubating
normal fibroblasts with lipoproteins.
Table I.
Oxysterols in medium after incubating normal fibroblasts with
lipoproteins
Oxygenated cholesterol derivatives identified in the neutral and acidic
fractions from media (containing 10% FCS) after incubation with normal
human fibroblasts and their gas chromatographic-mass spectrometric
characteristics as trimethylsilyl ethers and methyl estertrimethylsilyl
ether derivatives, respectively.
Oxygenated cholesterol derivatives identified in the neutral and acidic
fractions from media (containing 10% FCS) after incubation with normal
human fibroblasts and their gas chromatographic-mass spectrometric
characteristics as trimethylsilyl ethers and methyl estertrimethylsilyl
ether derivatives, respectively.
| No. |
Steroid
name |
Structurea |
Retention indexb |
Molecular and
significant ionsc
|
|
|
|
|
|
m/z
|
|
Neutral
C27-steroids
|
| 1 |
7 -Hydroxycholesterol |
C5-3 ,7 -ol |
3115 |
546,
456
|
| 2 |
7 -Hydroxy-4-cholesten-3-one |
C4-7 -ol-3-one |
3210 |
472,
457, 382, 269
|
| 3 |
7 -Hydroxycholesterol |
C5-3 ,7 -ol |
3235 |
546,
456
|
| 4 |
7-Oxocholesterol |
C5-3 -ol-7-one |
3375 |
472,
382, 367, 129 |
| 5 |
24-Hydroxycholesterol |
C5-3 ,24
(R/S)-ol |
3385 |
546, 413, 145, 129
|
| 6 |
25-Hydroxycholesterol |
C5-3 ,25-ol |
3405 |
546,
456, 131
|
| 7 |
7 ,25-Dihydroxycholesterol |
C5-3 ,7 ,25-ol |
3390 |
634,
544, 131
|
| 8 |
7 ,25-Dihydroxy-4-cholesten-3-one |
C4-7 ,25-ol-3-one |
3490 |
560,
545, 412, 131
|
| 9 |
27-Hydroxycholesterol |
C5-3 ,27-ol |
3455 |
546,
456, 417, 129
|
| 10 |
7 ,27-Dihydroxycholesterol |
C5-3 ,7 ,27-ol |
3445 |
634,
544
|
| 11 |
7 ,27-Dihydroxy-4-cholesten-3-one |
C4-7 ,27-ol-3-one |
3545 |
560,
545, 470, 269
|
| 12 |
7 ,27-Dihydroxycholesterol |
C5-3 ,7 ,27-ol |
3555 |
634,
544
|
| 13 |
27-Hydroxy-7-oxo-cholesterol |
C5-3 ,27-ol-7-one |
3710 |
560,
545, 470, 129 |
|
C27-steroid acids
|
| 14 |
3 -Hydroxy-5-cholestenoate |
CA5-3 -ol |
3425 |
502,
412, 373, 129
|
| 15 |
3 ,7 -Dihydroxy-5-cholestenoate |
CA5-3 ,7 -ol |
3415 |
590,
500
|
| 16 |
7 -Hydroxy-3-oxo-4-cholestenoate |
CA4-7 -ol-3-one |
3515 |
516,
501, 426, 269
|
| 17 |
3 ,7 -Dihydroxy-5-cholestenoate |
CA5-3 ,7 -old |
3530 |
590,
500
|
| 18 |
3 -Hydroxy-7-oxo-5-cholestenoate |
CA5-3 -ol-7-oned |
3680 |
516,
426, 411, 129 |
|
|
a
C, cholestane; CA, cholestanoate; superscript
indicates position of double bond; greek letters denote configuration
of hydroxyl groups.
|
|
b
Kovats, on a fused silica capillary column coated with
cross-linked methyl silicone.
|
|
c
Intensities of fragment ions with m/z values
above 200-300 were enhanced relative to those of lighter fragments;
base peak is shown in italics; m/z, mass/charge.
|
|
d
Tentative identification, reference compound not
available.
|
|
Fig. 1.
Gas chromatographic-mass spectrometric
analysis of neutral oxysterols isolated from the medium after
incubating normal fibroblasts with lipoproteins. Normal human
fibroblasts (protein content: 1.1 mg, dish size: 143 cm2)
were incubated with 10 ml of medium containing 10% FCS (cholesterol
concentration: 1.2 mM) for 24 h, and the medium was then
taken for analysis by GC/MS. Fragment ion current chromatograms
characteristic of the trimethylsilyl ethers of oxysterols were
constructed by the computer from mass spectra taken every 2 s during
the analysis and for the purpose of illustration the intensities of the
ions (m/z) were multiplied by appropriate
factors. The principal sterols indicated by the numbers are
listed in Table I. The equivalent of about 0.2 ml of medium was
injected onto a Finnigan SSQ 710 instrument housing a 25-m fused-silica
column coated with methyl silicone, and the oven temperature was
programmed from 185 to 280 °C at a rate of 5 °C ×
min 1.
The quantitative results after incubating normal and transformed
fibroblasts for 48 h with lipoproteins are shown in Table
II. In addition to 27-hydroxycholesterol which is formed
from LDL cholesterol (6), the amounts of three other
27-hydroxylated sterols also increased 10-20-fold when incubating
normal fibroblasts. These sterols were
7 ,27-dihydroxy-4-cholesten-3-one, 27-hydroxy-7-oxocholesterol, and
7 ,27-dihydroxycholesterol. In fact, the former of these was the
major oxysterol formed. The amounts of their corresponding
C27-acids also increased (about 5-10-fold). Also,
25-hydroxycholesterol and 7 ,25-dihydroxy-4-cholesten-3-one
increased, but the amounts of the former varied considerably, possibly
indicating that a part of this sterol had been produced by
autooxidation of cholesterol. A decrease of the amount of
7 -hydroxycholesterol was also noted indicating a consumption of this
sterol during the incubations. The amounts of the other steroids were
similar to those of the controls, suggesting that they were present in
FCS when added to the media.
Table II.
Production of oxysterols in normal and virus-transformed human
fibroblasts when incubated with lipoproteins
The amounts of neutral and acidic oxygenated cholesterol derivatives
were determined in the media (10 ml) containing 10% FCS (cholesterol
concentration: 1.2 mM) after incubation with fibroblasts
for 48 h. Cells incubated for 0.25 h served as controls.
The amounts of neutral and acidic oxygenated cholesterol derivatives
were determined in the media (10 ml) containing 10% FCS (cholesterol
concentration: 1.2 mM) after incubation with fibroblasts
for 48 h. Cells incubated for 0.25 h served as controls.
| No. |
Steroid
structurea |
Amountb of oxysteroid found
in media
|
Normal
fibroblastsc
|
Virus-transformed
fibroblastsc
|
| 0.25 h; n = 2d |
48 h;
n = 4d |
0.25 h; n = 2d |
48 h;
n = 4d
|
|
|
|
pmol
|
| 1 |
C5-3 ,7 -ole |
171: 117-225 |
54: 39-81 |
156: 81-228 |
60: 54-123
|
| 2 |
C4-7 -ol-3-one |
<6: <3-<6 |
<12: <6-<18 |
<9: <6-<9 |
69: 51-159
|
| 3 |
C5-3 ,7 -ole |
138: 108-165 |
114: 99-156 |
144: 81-207 |
129: 75-216
|
| 4 |
C5-3 -ol-7-onee |
879: 840-915 |
816: 609-1140 |
993: 633-1350 |
867: 657-1881
|
| 5 |
C5-3 ,24-ol |
6: <6-6 |
6: <6-9 |
3: <3-6 |
9: <6-18
|
| 6 |
C5-3 ,25-ole |
6: <3-12 |
45: 25-150 |
9: <3-18 |
27: 15-30
|
| 7 |
C5-3 ,7 ,25-ol |
<3: <3-3 |
<6: <3-<9 |
<3: <3-3 |
<3: <3-<3
|
| 8 |
C4-7 ,25-ol-3-one |
<6: <3-<6 |
21: <9-27 |
<3: <3-<3 |
<3: <3-<3
|
| 9 |
C5-3 ,27-ol |
12: 9-18 |
219: 153-267 |
12: 9-12 |
21: 15-33
|
| 10 |
C5-3 ,7 ,27-ol |
<3: <3-<3 |
<3: <3-<3 |
<3: <3-<3 |
<3: <3-<3
|
| 11 |
C4-7 ,27-ol-3-one |
<12: <12-<12 |
270: 144-321 |
<9: <3-<12 |
<12: <9-<15
|
| 12 |
C5-3 ,7 ,27-ol |
<3: <3-<3 |
42: 33-48 |
<3: <3-<3 |
<3: <3-<3
|
| 13 |
C5-3 ,27-ol-7-one |
<9: <6-<12 |
81: 33-108 |
<12: <3-<18 |
<9: <6-15
|
| 14 |
CA5-3 -ol |
21: 12-27 |
21: 18-27 |
15: 12-15 |
15: 12-18
|
| 15 |
CA5-3 ,7 -ol |
9: 6-9 |
6: 3-6 |
6: 6-6 |
6: 3-6
|
| 16 |
CA4-7 -ol-3-one |
21: 18-21 |
75: 63-117 |
18: 15-18 |
9: 6-15
|
| 17 |
CA5-3 ,7 -ol |
12: 9-12 |
51: 45-72 |
3: <3-3 |
<3: <3-<3
|
| 18 |
CA5-3 -ol-7-one |
<3: <3-<3 |
27: 18-30 |
<3: <3-<3 |
<3: <3-<3 |
|
|
a
For abbreviations and steroid names, see Table I.
|
|
b
Expressed as median: range; < = an amount at or below the
detection limit.
|
|
c
Protein contents of normal and virus-transformed fibroblasts
were 0.6 mg and 1.4 mg, respectively. The size of the incubation dishes
was 57 cm2. All cells had been preincubated for 24 h in media
containing 10% LDS.
|
|
d
n = number of incubations.
|
|
e
Can also be formed by autooxidation of cholesterol during
the incubations or during purification of samples.
|
|
Much smaller amounts of oxysterols were formed by the transformed
fibroblasts (Table II). For example, the amounts of
27-hydroxycholesterol only increased about 2-fold, and the other
27-hydroxylated sterols were barely detectable in the media. Only the
amounts of 7 -hydroxy-4-cholesten-3-one increased significantly (this
was not the case in normal fibroblasts) which may be related to a
decrease of the amount of its potential precursor
7 -hydroxycholesterol. These results indicated that 27-hydroxylation
of sterols may be obstructed in transformed fibroblasts (see
below).
The oxysterols were also studied with regard to the time course of
their cellular production. Incubation of normal human fibroblasts for
different lengths of time showed that the formation of 27-hydroxylated
sterols had started 3-8 h after exposure to lipoproteins (Table
III). No production of 25-hydroxylated sterols was
observed during the first 24 h. A decrease of the amounts of
7 -hydroxycholesterol was noted after 8 h of incubation. Since the
oxysterols having oxy groups both in the 7- and 27-positions could be
derived from either 27-hydroxylated LDL cholesterol or 7-oxygenated
cholesterol derivatives (autooxidation products present in the medium),
their origin was investigated.
Table III.
Time-response for the production of oxysterols in normal fibroblasts
when incubated with lipoproteins
The amounts of neutral oxygenated cholesterol derivatives were
determined in the media (15 ml) containing 10% FCS (cholesterol
concentration: 1.2 mM) after incubation for 0.25-24 h with
normal human fibroblasts (protein content 1.7 mg, size of dishes 143
cm2). All cells had been preincubated for 24 in media
containing 10% LDS.
The amounts of neutral oxygenated cholesterol derivatives were
determined in the media (15 ml) containing 10% FCS (cholesterol
concentration: 1.2 mM) after incubation for 0.25-24 h with
normal human fibroblasts (protein content 1.7 mg, size of dishes 143
cm2). All cells had been preincubated for 24 in media
containing 10% LDS.
| No. |
Steroid
structurea |
Amount of oxysterol found in media
after incubation for
|
| 0.25 h |
3 h |
8 h |
24 h
|
|
|
|
pmol
|
| 1 |
C5-3 ,7 -olb |
92 |
92 |
31 |
15
|
| 2 |
C4-7 -ol-3-one |
55 |
65 |
58 |
45
|
| 3 |
C5-3 ,7 -olb |
92 |
92 |
58 |
66
|
| 4 |
C5-3 -ol-7-oneb |
508 |
500 |
369 |
412
|
| 5 |
C5-3 ,24-ol |
15 |
15 |
10 |
10
|
| 6 |
C5-3 ,25-olb |
28 |
28 |
10 |
15
|
| 7 |
C5-3 ,7 ,25-ol |
25 |
20 |
5 |
5
|
| 8 |
C4-7 ,25-ol-3-one |
33 |
28 |
25 |
30
|
| 9 |
C5-3 ,27-ol |
23 |
30 |
70 |
240
|
| 10 |
C5-3 ,7 ,27-ol |
2 |
2 |
1 |
1
|
| 11 |
C4-7 ,27-ol-3-one |
65 |
58 |
138 |
308
|
| 12 |
C5-3 ,7 ,27-ol |
3 |
3 |
15 |
13
|
| 13 |
C5-3 ,27-ol-7-one |
8 |
5 |
43 |
108 |
|
|
a
For abbreviations and steroid names, see Table I.
|
|
b
Can also be formed by autooxidation of cholesterol during
the incubations or during purification of samples.
|
|
Metabolism of LDL Cholesterol and 7-Oxygenated Sterols in Normal
Fibroblasts
The metabolism of 7 -hydroxycholesterol,
7 -hydroxycholesterol, and 7-oxocholesterol was studied by incubating
these sterols (5 nmol) with normal human fibroblasts (protein content
about 0.5 mg, dish size 57 cm2) for 24 or 48 h in media (10
ml) containing 10% LDS. The values given in percent represent the
distribution of observed metabolites.
When 7 -hydroxycholesterol was incubated (24 h), the major
metabolites found were 7 -hydroxy-4-cholesten-3-one (57%) and
7 ,27-hydroxy-4-cholesten-3-one (43%) (acids were not analyzed).
Only a small amount of 7 ,27-dihydroxycholesterol (0.5%) was noted
suggesting that oxidation/isomerization of 7 -hydroxycholesterol to
7 -hydroxy-4-cholesten-3-one precedes 27-hydroxylation. The
corresponding enzyme activities have been found previously in
fibroblasts (20). When 7 -hydroxy-4-cholesten-3-one was incubated
with the fibroblasts for 48 h, the steroid was extensively converted to
7 ,27-dihydroxy-4-cholesten-3-one (37%) and
7 -hydroxy-3-oxo-4-cholestenoic acid (63%). A small amount of
25-hydroxylated 7 -hydroxy-4-cholesten-3-one (0.5%) was also found.
These results show that 7 -hydroxycholesterol can be converted to
7 -hydroxy-4-cholesten-3-one, 7 ,27-dihydroxy-4-cholesten-3-one,
and 7 -hydroxy-3-oxo-4-cholestenoic acid by normal fibroblasts and
this could explain the appearance of these metabolites and the
disappearance of 7 -hydroxycholesterol in media during the
incubations with FCS (Table II).
The metabolism of 7 -hydroxycholesterol differed from that of
7 -hydroxycholesterol. When this sterol was incubated for 48 h with
normal fibroblasts, the major metabolites were
7 ,27-dihydroxycholesterol (1%) and
3 ,7 -dihydroxy-5-cholestenoic acid (62%). In addition, a large
portion (about one-third) of 7 -hydroxycholesterol was converted to
7-oxocholesterol (20%), 27-hydroxy-7-oxocholesterol (1%), and
3 -hydroxy-7-oxo-5-cholestenoic acid (14%). Oxidation of the
7 -hydroxy group also occurred when 7 ,27-dihydroxycholesterol was
incubated. No conversion of 7 -hydroxycholesterol to the
corresponding 3-oxo- 4 derivative was observed, which was
consistent with the absence of 7 -hydroxylated 3-oxo- 4
steroids in media after incubating fibroblasts with FCS (Table I).
Incubation of 7-oxocholesterol with normal fibroblasts resulted in the
formation of 27-hydroxy-7-oxocholesterol (55%) and the corresponding
C27-acid (35%). A small amount was converted to
7 -hydroxycholesterol (10%), but a conversion to 7 -hydroxylated
products was not observed. Thus, the formation of
C27-steroids having oxygen groups in both the 7- and
27-positions by normal fibroblasts could be due to the presence of
autooxidation products of cholesterol in the medium. However, this did
not exclude the possibility that 7 ,27-dihydroxy-4-cholesten-3-one
and 7 -hydroxy-3-oxo-4-cholestenoic acid could also be derived from
27-hydroxycholesterol. 7 -Hydroxylation of 27-hydroxycholesterol in
human fibroblasts was first noted by us (15) and was later found to be
occurring generally in these cells (21). The product
7 ,27-dihydroxycholesterol is extensively converted to
7 ,27-dihydroxy-4-cholesten-3-one and the corresponding acid in the
cells (15, 21).
In order to determine whether LDL cholesterol (via
27-hydroxycholesterol) could be converted to
7 ,27-dihydroxy-4-cholesten-3-one and the acid, the contribution from
7 -hydroxycholesterol (which is always present when the medium
contains lipoproteins) had to be accounted for. This was made possible
by the following experiment. LDL and other lipoproteins in FCS were
first labeled with [3H]cholesteryl oleate and were then
incubated with normal fibroblasts in the presence and absence of
cyclosporin A (CsA), a selective inhibitor of the sterol 27-hydroxylase
(6, 22, 23). When lipoproteins are labeled in this way, the cellular
uptake of [3H]cholesteryl oleate is due solely to a LDL
receptor-dependent process (i.e. a physiological
uptake of LDL) (6). After the incubations, radioactive
27-hydroxycholesterol, 7 ,27-dihydroxy-4-cholesten-3-one, and
7 -hydroxy-3-oxo-4-cholestenoic acid were analyzed by HPLC.
3H-Labeled 7 -hydroxy-4-cholesten-3-one, the direct
metabolite of 7 -hydroxycholesterol, was also determined. If
3H-labeled 7 -hydroxycholesterol (free or esterified) was
present in the medium, its 3-oxidized metabolite was expected to
accumulate in the presence of CsA, since the drug prevented further
metabolism (see above). The amount of the metabolite would then reflect
the contribution to 7 ,27-dihydroxy-4-cholesten-3-one and the acid
from 7 -hydroxycholesterol in the absence of CsA. Obviously,
essentially no formation of 27-hydroxylated compounds was expected in
the presence of CsA (see also Fig. 5). For comparison, fibroblasts were
also incubated with lipoproteins labeled with
[3H]cholesterol, whose cellular uptake is not entirely
dependent on LDL receptors.
Fig. 5.
A simplified scheme of the metabolism of LDL
cholesterol and major autooxidation products of cholesterol in human
fibroblasts and the formation of potent HMG-CoA reductase
suppressors. The names of the steroids are listed in Table I. In
addition to hydrolysis and esterification (not shown), major reactions
of sterols were: I, 27-hydroxylation; II,
7 -hydroxylation; III, 3-oxidation with isomerization of
the 5-double bond; and IV, oxidation to a 27-carboxy group.
Hydrolyzed LDL cholesterol was shown to be metabolized via these
reactions (filled arrows). Oxidation of a 7 -hydroxy group
(V) to a ketone was also observed. Minor reactions noted are
shown by broken arrows. The formation of
7 ,25-dihydroxy-4-cholesten-3-one by 25-hydroxylation of sterols (not
shown) was observed under specific conditions. Reactions I, II, and III
were obstructed in virus-transformed fibroblasts displaying a defective
suppression of HMG-CoA reductase by LDL cholesterol and autooxidation
products of cholesterol. Sterol metabolites with an apparently normal
suppressive effect also in transformed cells are indicated
in-frame.
The results of these incubations are summarized in Table
IV. In addition to 27-hydroxycholesterol, both
7 ,27-dihydroxy-4-cholesten-3-one and
7 -hydroxy-3-oxo-4-chole-stenoic acid were found as
3H-labeled compounds after incubation with
[3H]cholesteryl oleate. The HPLC analyses of these
metabolites are shown in Fig. 2. Since no accumulation
of radioactive 7 -hydroxy-4-cholesten-3-one was observed in the
corresponding incubation with CsA (Table IV), autooxidation
(7 -hydroxylation) of [3H]cholesteryl oleate had not
occurred during the incubations. In the incubations with
[3H]cholesterol, much larger amounts of radioactive
7 ,27-dihydroxy-4-cholesten-3-one and the corresponding acid were
found, although the amount of 27-hydroxycholesterol was less than with
[3H]cholesteryl oleate. However, the incubation with
[3H]cholesterol and CsA resulted in a significant
accumulation of 3H-labeled 7 -hydroxy-4-cholesten-3-one
suggesting that most of the 27-oxygenated metabolites had been produced
from autooxidized [3H]cholesterol
(7 -hydroxycholesterol). The difference in chemical stability toward
oxygen between [3H]cholesteryl oleate and
[3H]cholesterol was surprising, but was confirmed by
exposing them to air and heat in an aqueous/methanolic environment for
24 h. No autooxidation products (<0.1%) of
[3H]cholesteryl oleate could be detected, whereas about
2% of [3H]cholesterol were autooxidized. Thus, the
results demonstrate that LDL cholesteryl esters are hydrolyzed and are
converted to 27-hydroxycholesterol, which is then 7 -hydroxylated and
oxidized to 7 ,27-dihydroxy-4-cholesten-3-one and
7 -hydroxy-3-oxo-4-cholestenoic acid in normal human fibroblasts. The
latter seems to be the major metabolic end product of this extended LDL
pathway in fibroblasts.
Table IV.
Formation of radioactive metabolites from LDL
[3H]cholesteryl oleate in normal fibroblasts
The amounts of 3H-labeled 27-hydroxycholesterol,
7 ,27-dihydroxy-4-cholesten-3-one and
7 -hydroxy-3-oxo-4-cholestenoic acid were determined in media and
cells after incubating normal human fibroblasts (protein content, 0.7
mg; size of dishes, 57 cm2) for 68 h with media (10 ml)
containing LDL (4% FCS; cholesterol concentration, 1.2 mM)
labeled with [3H]cholesteryl oleate or
[3H]cholesterol. When 27-hydroxylation of sterols was
obstructed by cyclosporin A (CsA, 10 µM), the
accumulation of 3H-labeled 7 -hydroxy-4-cholesten-3-one
indicated the presence of autooxidized [3H]cholesterol or
[3H]cholesteryl oleate (i.e. free or esterified
7 -hydroxycholesterol) in the media during the incubation.
The amounts of 3H-labeled 27-hydroxycholesterol,
7 ,27-dihydroxy-4-cholesten-3-one and
7 -hydroxy-3-oxo-4-cholestenoic acid were determined in media and
cells after incubating normal human fibroblasts (protein content, 0.7
mg; size of dishes, 57 cm2) for 68 h with media (10 ml)
containing LDL (4% FCS; cholesterol concentration, 1.2 mM)
labeled with [3H]cholesteryl oleate or
[3H]cholesterol. When 27-hydroxylation of sterols was
obstructed by cyclosporin A (CsA, 10 µM), the
accumulation of 3H-labeled 7 -hydroxy-4-cholesten-3-one
indicated the presence of autooxidized [3H]cholesterol or
[3H]cholesteryl oleate (i.e. free or esterified
7 -hydroxycholesterol) in the media during the incubation.
| Steroid structurea |
Amount of
3H-labeled oxysterol foundb
|
FCS +
[3H]cholesteryl oleatec
|
FCS +
[3H]cholesterolc
|
| Control |
+CsA |
Control |
+CsA
|
|
|
dpm
|
C5-3 ,27-ol |
12,000 |
<1,970 |
7,090 |
<120
|
C4-7 ,27-ol-3-one |
5,140 |
<1,140 |
88,800 |
12,800
|
CA4-7 -ol-3-one |
10,800 |
<120 |
26,300 |
1,690
|
C4-7 -ol-3-one |
<410 |
<410 |
1,450 |
25,300 |
|
|
a
For abbreviations and steroid names, see Table I.
|
|
b
Values represent the sum of the amounts found in medium and
cells; < = an amount at or below the detection limit.
|
|
c
FCS was preincubated for 16 h at 20 °C with
[3H]cholesteryl oleate (48 × 106 dpm) or
[3H]cholesterol (49 × 106 dpm). All cells had been
preincubated for 24 h in media containing 10% LDS.
|
|
Fig. 2.
HPLC analyses of 3H-labeled
7 ,27-dihydroxy-4-cholesten-3-one and
7 -hydroxy-3-oxo-4-cholestenoic acid isolated from the medium of
normal human fibroblasts. The cells (0.7 mg of protein) were
incubated for 68 h with LDL (4% FCS) labeled with
[3H]cholesteryl oleate, and the medium was then taken for
analysis by HPLC. For experimental details, see ``Materials and
Methods'' and Table IV. After the injections, fractions of the HPLC
column effluent were collected in scintillation vials with 15-60-s
intervals, and the radioactivity was then determined. For purpose of
illustration, unlabeled 7 ,27-dihydroxy-4-cholesten-3-one (results
shown in top chromatograms) or
7 -hydroxy-3-oxo-4-cholestenoic acid (as methyl ester derivative,
bottom chromatograms) were injected together with the
samples, and the peaks of these compounds are seen in the UV
chromatograms. A column of silica (LiChrospher) connected to a UV
detector was used with hexane/isopropyl alcohol (90:10) as mobile
phase, and the flow rate was 1.0 ml × min 1.
Metabolism of LDL Cholesterol and Side-chain Hydroxylated Sterols
in Transformed Fibroblasts
In contrast to normal fibroblasts,
only small amounts of 27-oxygenated sterols were detected in media
after incubating transformed human fibroblasts with lipoproteins (Table
II). Although the increased amounts of 7 -hydroxy-4-cholesten-3-one
could indicate that 27-hydroxylation of sterols was obstructed in these
cells (see above), the lack of 27-hydroxylated sterols in the media
could also be due to a decreased cellular uptake of LDL and
oxysterols, or to an increased formation of conjugates
(e.g. fatty acid esters or sulfate esters).
Table V shows the distribution of 3H-labeled
cholesterol and cholesteryl esters after incubating normal and
transformed fibroblasts with lipoproteins labeled with radioactive
cholesterol or cholesteryl oleate for 48 h. Results on the oxysterol
production from the same incubations are those shown in Table II. As
seen in Table V, the cellular uptake and retention (cell content) of
[3H]cholesterol in normal and transformed cells were
about 16 and 22%, respectively. About 4% had been esterified by both
cell types. After the incubations with [3H]cholesteryl
oleate, the retention of the compound was about 11% in the normal
cells and 15% in the transformed cells, although a major portion had
been hydrolyzed in the cells. About 9% and 35% of hydrolyzed
[3H]cholesterol were also present in media of the normal
and transformed fibroblasts, respectively, due to an efflux of
hydrolyzed LDL cholesterol from the cells (6, 24). When the cellular
content and the efflux of cholesterol in the incubations with
[3H]cholesteryl oleate were added together, the total
uptake of [3H]cholesteryl oleate in the normal and the
transformed cells was 19% (32%/mg of protein) and 50% (36%/mg of
protein), respectively. These results show that the uptake of LDL
(reflected by that of [3H]cholesteryl oleate) was not
decreased but possibly increased in the transformed cells also when the
protein content of the cells was taken into account. Thus, a possible
reduced formation of 27-hydroxylated sterols by transformed fibroblasts
(Table II) was not due to a decreased uptake of LDL.
Table V.
Uptake and handling of free and esterified [3H]cholesterol in
normal and transformed fibroblasts
Distribution of radioactivity after incubating normal and
virus-transformed human fibroblasts for 48 h with media containing
lipoproteins (10% FCS) labeled with [3H]cholesterol or
[3H]cholesteryl oleate. The total concentration of unlabeled
cholesterol in FCS was 1.2 mM. Incubations for 0.25 h
served as controls.
Distribution of radioactivity after incubating normal and
virus-transformed human fibroblasts for 48 h with media containing
lipoproteins (10% FCS) labeled with [3H]cholesterol or
[3H]cholesteryl oleate. The total concentration of unlabeled
cholesterol in FCS was 1.2 mM. Incubations for 0.25 h
served as controls.
Incubation
conditionsa
|
Distribution of free and esterified
[3H]cholesterol
|
| Time |
Additions to incubation
mediumb |
Medium
|
Cells
|
| Free |
Esters |
Total in medium |
Free |
Esters |
Total in
cells |
|
| h |
|
%
recovered radioactivityc
|
|
Normal cells
|
| 0.25 |
[3H]Cholesterol in
FCS |
95 |
4 |
99 |
1 |
<1 |
1
|
| 48 |
[3H]Cholesterol in
FCS |
82 |
3 |
85 |
11 |
4 |
15
|
| 48 |
[3H]Cholesterol in
FCS |
80 |
4 |
84 |
11 |
4 |
16
|
| 0.25 |
[3H]Cholesteryl oleate in
FCS |
2 |
98 |
100 |
<1 |
<1 |
<1
|
| 48 |
[3H]Cholesteryl oleate in
FCS |
10 |
81 |
91 |
6 |
4 |
9
|
| 48 |
[3H]Cholesteryl oleate in
FCS |
11 |
77 |
88 |
9 |
3 |
12 |
|
Transformed cells
|
| 0.25 |
[3H]Cholesterol in
FCS |
95 |
4 |
99 |
1 |
<1 |
1
|
| 48 |
[3H]Cholesterol in
FCS |
66 |
12 |
79 |
17 |
4 |
21
|
| 48 |
[3H]Cholesterol in
FCS |
73 |
5 |
78 |
18 |
4 |
22
|
| 0.25 |
[3H]Cholesteryl oleate in
FCS |
1 |
99 |
100 |
<1 |
<1 |
<1
|
| 48 |
[3H]Cholesteryl oleate in
FCS |
38 |
49 |
86 |
11 |
3 |
14
|
| 48 |
[3H]Cholesteryl oleate in
FCS |
34 |
51 |
84 |
12 |
3 |
16 |
|
|
a
Protein contents of normal and virus-transformed
fibroblasts were 0.6 mg and 1.4 mg, respectively. The size of the
incubation dishes was 57 cm2. All cells had been preincubated
for 24 h in media containing 10% LDS. Results from these incubations
are also shown in Table II.
|
|
b
The amounts of [3H]cholesterol and
[3H]cholesteryl oleate added to the medium were 22.8 ×
106 dpm and 18.9 × 106 dpm, respectively. The sterols
were preincubated with FCS for 16 h at 20 °C.
|
|
c
The total recovery was >90% of the radioactivity added.
|
|
In order to determine whether the shortage of 27-hydroxycholesterol and
other 27-hydroxylated sterols in media of transformed fibroblasts could
be due to an increased metabolism (other than formation of
C27-acids) or conjugation, the metabolism of
25-hydroxycholesterol was studied. The major reason for selecting this
sterol instead of 27-hydroxycholesterol was that 25-hydroxycholesterol
was available in a 3H-labeled form, so that major
metabolites or conjugates could be traced and would not escape
detection. Because of their similarities in structure (both having a
3 -hydroxy- 5 structure and one hydroxyl group in the
side chain), the cellular handling of the two sterols was expected to
be similar (except that the 25-hydroxy group could not be oxidized to a
carboxyl group). Table VI shows the distribution of
radioactivity when 3H-labeled 25-hydroxycholesterol (plus
unlabeled, 1.2 nmol) had been incubated with normal and transformed
fibroblasts for 48 h in 10% LDS. In addition to
25-[3H]hydroxycholesterol, two major radioactive
metabolites, one polar and one nonpolar, were found by HPLC. Other
metabolites constituted less than 1% each of the recovered
radioactivity. No radioactivity (<0.1%) corresponding to oxidized
25-[3H]hydroxycholesterol without a 7 -hydroxy group
(i.e. 25-hydroxy-4-cholesten-3-one, see below) was found.
Neither did we find any radioactivity (<1%) in fractions containing
weak acids (e.g. having a free carboxyl group) or stronger
acids (e.g. glucuronides or mono- or disulfates) which were
isolated from the extracts by anion exchange chromatography. After
collecting a fraction of the HPLC effluent containing the polar
metabolite and derivatization, it was identified by GC/MS as
7 ,25-dihydroxy-4-cholesten-3-one (21). The nonpolar metabolite(s)
had a retention time (3.7 min), which was similar to that of the
3-acetate derivative of 25-hydroxycholesterol (retention time 4.9 min)
by straight-phase HPLC (retention time of free 25-hydroxycholesterol
was 11.8 min). It was therefore tentatively characterized as being
fatty acid esters of 25-[3H]hydroxycholesterol. This was
supported further by the recovery of free
25-[3H]hydroxycholesterol after treating the nonpolar
metabolite(s) with mild alkali in a methanolic solution. Table VI
clearly reveals large differences in the handling of
25-[3H]hydroxycholesterol between the two cell lines.
Intact 25-[3H]hydroxycholesterol was found mainly in the
cells (32% in the normal and 50% in the transformed cells). A large
portion (about 43%; 71%/mg of protein) of
25-[3H]hydroxycholesterol had been converted to
7 ,25-dihydroxy-4-cholesten-3-one by normal cells (21) but much less
so (about 3%; 2%/mg of protein) by the transformed cells. This sterol
was recovered mainly in the media. On the other hand, esterification of
25-[3H]hydroxycholesterol was noted only in the
transformed cells, although the amount of esters was relatively small
(about 7%). These results show that 25-hydroxycholesterol is readily
taken up by both normal and transformed cells, but whereas the sterol
is extensively 7 -hydroxylated in normal cells, this reaction is
obstructed in transformed cells. The results also suggested that the
apparent shortage of 27-hydroxycholesterol in transformed cells was due
to a decreased formation rather than an increased conjugation, since
only a minor amount of the analogous sterol 25-hydroxycholesterol was
esterified and since no other conjugates were found.
After these observations, the rates of oxidation/isomerization of
3 ,7 -dihydroxy- 5 steroids to 3-oxo- 4
steroids in normal and transformed cells were also investigated.
7 ,27-Dihydroxycholesterol (1.2 nmol) was therefore incubated with
normal and transformed fibroblasts (protein contents 0.4 and 1.1 mg,
respectively, size of dishes 57 cm2) for 48 h in media (10
ml) containing 10% LDS, and the metabolites were then analyzed by
GC/MS. Incubations for 15 min served as controls. The analyses showed
that this sterol was readily taken up by the cells, since only about
1% remained in the media. In media from normal cells, about 26% and
39% were recovered as 7 ,27-dihydroxy-4-cholesten-3-one and
7 -hydroxy-3-oxo-4-cholestenoic acid, respectively. The corresponding
values for the transformed cells were 28% and 19%. Trace amounts
(about 1%) were converted to 3 ,7 -dihydroxy-5-cholestenoic acid
in the transformed cells. No 7-oxo-, 7 -hydroxy-, or other
metabolites were found. Thus, almost the same amounts of
7 ,27-dihydroxycholesterol were oxidized by the normal and
transformed fibroblasts. However, when the number of incubated cells
(cell protein content) were taken into account, the oxidation rate in
transformed cells was calculated to be about 25% of that of normal
cells. These studies show that the apparent activities of 27- and
7 -hydroxylating enzymes are much lower in transformed than in normal
fibroblasts (estimated to be <2% of the normal activity when
corrected for the cellular protein content or total uptake of LDL),
whereas the enzyme catalyzing oxidation of
3 -hydroxy- 5 sterols is affected to a lesser
extent.
Suppression of HMG-CoA Reductase by LDL and Oxysterols in
Fibroblasts
The suppression of HMG-CoA reductase by LDL in normal
and transformed fibroblasts was also studied and was related to the
production of different oxysterols. Fig. 3 shows that
the response to LDL was defective in the transformed cells when
compared with normal fibroblasts. In fact, almost 10 times higher
concentrations of FCS (4% versus 0.4%) were required to
cause a 50% suppression of the activity of HMG-CoA reductase in the
transformed cells.
Fig. 3.
Effects of LDL on HMG-CoA reductase in normal
( ) and virus-transformed ( ) human fibroblasts. Activities of
HMG-CoA reductase in the fibroblasts were determined after incubation
for 24 h with media containing different concentrations of FCS. In the
absence of FCS, media contained 10% LDS. All cells were preincubated
for 24 h in media containing 10% LDS. The concentrations of
cholesterol in FCS and LDS were 1.2 and 0.1 mM,
respectively. The control activities of HMG-CoA reductase in normal and
transformed cells were 72 and 101 pmol/min/mg of protein,
respectively.
The effects of the different metabolites of LDL cholesterol and other
oxysterols on HMG-CoA reductase in normal and transformed cells were
also tested. A low oxysterol concentration (0.12 µM) was
selected, so that a partial conversion into a more potent metabolite
might be discovered, at least when the reaction was obstructed. Also, a
0.06 µM concentration of the most potent sterols could
induce >50% suppression of HMG-CoA reductase in normal cells, but
0.03 µM had only a weak suppressive effect (<20%).
Table VII shows that oxysterols which were normally
27-hydroxylated or 7 -hydroxylated in fibroblasts, had much less
suppressive effects in transformed than in normal cells (mean: 32%
versus 68% suppression). Also, sterols that were normally
3-oxidized/isomerized had less suppressive effects in transformed than
in normal cells (mean: 46% versus 74% suppression). In
contrast, the sterol metabolites with a 3-oxo- 4
structure and a hydroxyl group in the side-chain
(7 ,27-dihydroxy-4-cholesten-3-one and
7 ,25-dihydroxy-4-cholesten-3-one) were potent suppressors also in
the transformed cells, and their effects were essentially the same as
in normal cells. This result was surprising. It implied that
27-hydroxycholesterol and 25-hydroxycholesterol had to be metabolized
in order to be active, since the transformed fibroblasts could respond
normally to their metabolites. Furthermore, the regulatory defect in
the transformed cells was evidently not beyond the formation of these
metabolites. We have previously reported that 27-hydroxylated
3-oxo- 4 sterols are potent suppressors in normal
fibroblasts (15), and, more recently, we have found that one of the
mechanisms by which they act is by inhibiting the synthesis of HMG-CoA
reductase. In agreement with our previous results (15), these compounds
were active also when they lacked a 7 -hydroxy group (Table VII).
Oxidation of the 27-hydroxy group to an acid seemed to decrease their
biological activity (15) at least in the transformed fibroblasts. In
addition to this group of sterols, 27-hydroxy-7-oxo-cholesterol was
also found to be a potent suppressor in the transformed fibroblasts,
although it seemed to have a stronger effect in normal cells (Table
VII).
Table VII.
Effects of LDL and oxysterols on HMG-CoA reductase in normal and
transformed fibroblasts
The effects of LDL (0.5-8% FCS; cholesterol concentration, 1.2
mM) and of oxysterols (0.12 µM in media
containing 10% LDS) on HMG-CoA reductase in normal and
virus-transformed human fibroblasts were tested. The activities of
HMG-CoA reductase were determined after incubating the fibroblasts for
24 h. All cells were preincubated for 24 h in media containing 10%
LDS.
The effects of LDL (0.5-8% FCS; cholesterol concentration, 1.2
mM) and of oxysterols (0.12 µM in media
containing 10% LDS) on HMG-CoA reductase in normal and
virus-transformed human fibroblasts were tested. The activities of
HMG-CoA reductase were determined after incubating the fibroblasts for
24 h. All cells were preincubated for 24 h in media containing 10%
LDS.
| No. |
Steroid
structurea |
Observed
metabolismb |
Activity of HMG-CoA
reductase
|
Suppressionc |
| Normal cells |
Transformed
cells |
|
|
|
|
%
controld |
% |
|
LDL
cholesterole |
27-Hydroxylation |
15-31 |
40-88 |
32-57
|
| 2 |
C4-7 -ol-3-one |
27-Hydroxylation |
26 |
50 |
24
|
| 3 |
C5-3 ,7 -ol |
7-Oxidation or
27-Hydroxylation |
39 |
60 |
21
|
| 4 |
C5-3 -ol-7-one |
27-Hydroxylation |
38 |
100 |
62
|
|
5 -C8(14)>-3 -ol-15-onef |
27-Hydroxylationg |
22 |
41 |
19
|
| 6 |
C5-3 ,25-ol |
7 -Hydroxylation |
37 |
77 |
40
|
| 9 |
C5-3 ,27-ol |
7 -Hydroxylation |
33 |
79 |
46
|
| 7 |
C5-3 ,7 ,25-ol |
3-Oxidation/isomerization |
23 |
49 |
26
|
| 10 |
C5-3 ,7 ,27-ol |
3-Oxidation/isomerization |
30 |
60 |
30
|
|
C4-27-ol-3-onef |
27-Oxidation to
acid |
31 |
38 |
7
|
| 11 |
C4-7 ,27-ol-3-one |
27-Oxidation to
acid |
33 |
34 |
1
|
| 13 |
C5-3 ,27-ol-7-one |
27-Oxidation to
acid |
7 |
33 |
26
|
|
C4-25-ol-3-onef |
None |
33 |
37 |
4
|
| 8 |
C4-7 ,25-ol-3-one |
None |
26 |
31 |
5
|
| 16 |
CA4-7 -ol-3-one |
None |
30 |
47 |
17 |
|
|
a
For abbreviations and steroid names, see Table I.
|
|
b
Metabolism observed in normal human fibroblasts.
|
|
c
Difference in degree of suppression of HMG-CoA reductase in
normal and transformed fibroblasts induced by LDL or the oxysterol.
|
|
d
The activities of HMG-CoA reductase in normal and
transformed fibroblasts were 58 and 96 pmol/min/mg of protein,
respectively.
|
|
e
Results also shown in Fig. 3.
|
|
f
Oxysterol not normally detected in medium or cells
(fibroblasts) after incubation with FCS.
|
|
g
Not studied, but concluded from the results in Table VIII.
|
|
Since the production of 7 ,27-dihydroxy-4-cholesten-3-one and
27-hydroxy-7-oxocholesterol had started at the time when a significant
suppression (73%) of the activity of HMG-CoA reductase occurred (3-8
h after exposing normal fibroblasts to lipoproteins, Fig.
4), there was a possibility that the two events were
related. However, if a hindered conversion of LDL cholesterol and
autooxidation products to side-chain hydroxylated
3-oxo- 4 steroids and 27-hydroxy-7-oxocholesterol was
causing the defective response to the former sterols in the transformed
cells, then the same was expected to be seen in normal fibroblasts when
treated with inhibitors of the sterol metabolizing enzymes. Table
VIII shows the effects of treating normal human
fibroblasts with CsA and thereby preventing 27-hydroxylation
in the cells. Consistent with the behavior of transformed
fibroblasts, the suppressive effects on HMG-CoA reductase of LDL
cholesterol or oxysterols, which were 27-hydroxylated in untreated
cells, decreased or were abolished when CsA was present in the medium.
Table VIII also shows that 3 -hydroxy-5 -cholest-8(14)-en-15-one, a
potent suppressor of HMG-CoA reductase with an oxo group in the D-ring,
most likely had to be 27-hydroxylated before being active (25). As
expected, CsA did not interfere with the activity of 27-hydroxylated
sterols. Since the metabolism of 7 -hydroxy-4-cholesten-3-one was
limited to 27-hydroxylation in normal fibroblasts (see above), its
metabolite 7 ,27-dihydroxy-4-cholesten-3-one (or possibly the acid)
was likely to be a true suppressor of HMG-CoA reductase.
Fig. 4.
Time-response curves for the LDL-induced
production of 7 ,27-dihydroxy-4-cholesten-3-one ( ),
27-hydroxy-7-oxocholesterol ( ), and
7 ,25-dihydroxy-4-cholesten-3-one ( ), and suppression of HMG-CoA
reductase ( ) in normal human fibroblasts. The cells (protein
content: 1.7 mg; dish size: 143 cm2) were incubated with
medium (15 ml) containing 10% FCS and were harvested at the indicated
times. The concentration of cholesterol in FCS was 1.2 mM.
All cells were preincubated for 24 h in media containing 10% LDS (see
also Table III). For comparison, the production of
27-hydroxycholesterol ( ) is also shown.
Attempts to prevent 7 -hydroxylation in normal cells using
ketoconazole, a general cytochrome P-450 inhibitor (26), were
unsuccessful. This drug had a significant effect on the 27-hydroxylase
when added to the medium 30 min before the sterols, but the effects on
the 7 -hydroxylase were inconsistent. No further attempts were made
to find an inhibitor of the sterol 7 -hydroxylase.
Suppression of HMG-CoA Reductase by LDL and Oxysterols in Human
Malignant Cells
In order to determine if a defective metabolism
of and response to LDL are limited to virus-transformed fibroblasts or
are characteristic of malignant cells in general, the behavior of other
human tumor cell lines were also studied. Breast carcinoma, colonic
carcinoma, and malignant melanoma cell lines were selected because of
their high incidence in humans. Table IX shows that the
effects of LDL and selected oxysterols on HMG-CoA reductase in these
neoplastic cells were similar to those observed in the transformed
fibroblasts, although the corresponding normal cells were not available
for comparison. Thus, LDL and the oxysterols with a 3 -hydroxy group
had little or no effect on HMG-CoA reductase, whereas side-chain
hydroxylated steroids with a 3-oxo- 4 structure were
potent suppressors also in these cells.
Table IX.
Effects of LDL and oxysterols on HMG-CoA reductase in human malignant
cells
The effects of LDL (2% FCS; cholesterol concentration, 1.2
mM) and of selected oxysterols (0.12 µM in
media containing 10% LDS) on HMG-CoA reductase in breast and colonic
carcinoma cells and malignant melanoma cells were tested. The
activities of HMG-CoA reductase were determined after incubating the
cells for 24 h. All cells had been preincubated for 24 h in media
containing 10% LDS. For comparison, the corresponding values on
transformed human fibroblasts are also shown.
The effects of LDL (2% FCS; cholesterol concentration, 1.2
mM) and of selected oxysterols (0.12 µM in
media containing 10% LDS) on HMG-CoA reductase in breast and colonic
carcinoma cells and malignant melanoma cells were tested. The
activities of HMG-CoA reductase were determined after incubating the
cells for 24 h. All cells had been preincubated for 24 h in media
containing 10% LDS. For comparison, the corresponding values on
transformed human fibroblasts are also shown.
| Steroid structurea |
Activity of HMG-CoA
reductase
|
| Breast carcinoma cells |
Colonic carcinoma
cells |
Malignant melanoma cells |
Transformed
fibroblastsb |
Median
|
|
|
% controlc
|
| LDL cholesterol |
67 |
79 |
121 |
68 |
74
|
C5-3 -ol-7-one |
96 |
91 |
123 |
100 |
98
|
5 -C8(14)>-3 -ol-15-one |
66 |
80 |
101 |
41 |
73
|
C5-3 ,25-ol |
d |
94 |
d |
77 |
86
|
C5-3 ,27-ol |
75 |
86 |
75 |
79 |
77
|
C5-3 ,7 ,27-ol |
d |
74 |
73 |
60 |
73
|
| C4-25-ol-3-one |
43 |
55 |
d |
37 |
43
|
| C4-27-ol-3-one |
48 |
54 |
59 |
38 |
51 |
|
|
a
For abbreviations and steroid names, see Table I.
|
|
b
Corresponding values for normal fibroblasts were 18-38%.
|
|
c
The activities of HMG-CoA reductase in breast carcinoma
cells, colonic carcinoma cells, malignant melanoma cells, and
transformed fibroblasts were 59, 120, 137, and 96 pmol/min/mg of
protein, respectively.
|
|
d
Not studied.
|
|
Consistent with the results on transformed fibroblasts, incubations of
breast and colonic carcinoma cells (1.1 and 1.3 mg of cell protein,
respectively) with lipoproteins (10% FCS labeled with
[3H]cholesteryl oleate in 10 ml of medium) for 48 h
yielded essentially no 27-hydroxylated oxysterols or acids (<10
pmol/mg of cell protein). The total uptake of LDL was calculated to be
about 15%/mg of protein by breast cancer cells, but was much lower
(about 2%/mg of protein) by the colonic cancer cells. Thus, the low
production of side-chain hydroxylated oxysterols could at least partly
be due to a shortage of LDL receptors in the latter cells. When the
colonic cells (1.5 mg of protein) were incubated with unlabeled (1.2
nmol) and 3H-labeled 25-hydroxycholesterol for 48 h
(exactly as described for fibroblasts above), only small amounts
(<5%; <3%/mg of protein) were converted to
7 ,25-dihydroxy-4-cholesten-3-one. Since a large portion (about 40%)
of the sterol was not metabolized, this suggested that the activity of
the 7 -hydroxylase was low in these cells. Interestingly, and in
contrast to fibroblasts, a major portion of 25-hydroxycholesterol
(about 50%) was sulfated in the 3-position by the colonic cells. Thus,
in addition to deficiencies of the 27- and/or 7 -hydroxylating
enzymes in breast and colonic carcinoma cells, a shortage of LDL
receptors and an increased sulfation rate in the latter could
contribute to a low production of potent 27-hydroxylated
metabolites.
In contrast, the rates of metabolism of LDL cholesterol and oxysterols
were increased in malignant melanoma cells (1.0 mg of cell protein)
when incubated with lipoproteins (10% FCS) for 48 h. Yet the responses
to LDL and oxysterols were similar to those seen in the other tumor
cells (Table IX). However, like in liver cells (19, 27, 28),
27-hydroxylated metabolites were extensively converted to their
corresponding acids, the major ones being
7 -hydroxy-3-oxo-4-cholestenoic acid and
3 -hydroxy-7-oxo-5-cholestenoic acid (2.6 and 2.0 nmol/mg of cell
protein/48 h, respectively). Consequently, the accumulation of the
27-hydroxylated suppressors were low also in these cells, as reflected
by their concentrations in media. The amounts of
7 ,27-dihydroxy-4-cholesten-3-one and 27-hydroxy-7-oxo-cholesterol
were only about 0.2 and 0.06 nmol/mg of cell protein/48 h,
respectively. These studies show that common to all these tumor cell
lines were a defective response to LDL and a low intracellular
concentration (accumulation) of 7 ,27-dihydroxy-4-cholesten-3-one and
27-hydroxy-7-oxo-cholesterol, which was due to either a decreased
formation or an increased metabolism.
DISCUSSION
Cultured human fibroblasts have been widely used in studies of
regulatory mechanisms of cholesterol homeostasis (1, 2, 3, 4, 5, 6, 29, 30, 31, 32, 33).
Although oxysterols have documented suppressive effects on HMG-CoA
reductase (34, 35, 36, 37, 38), their formation in human fibroblasts has not been
studied until we recently showed that fibroblasts converted LDL
cholesterol to 27-hydroxycholesterol (6). We also showed that this
oxysterol is an important mediator between LDL and the suppression of
HMG-CoA reductase in the cells (6). The involvement of oxysterols in
the regulation of cholesterol homeostasis, their formation, and
biological activity in human fibroblasts have now been investigated
further.
In the absence of LDL, a production of oxysterols in normal fibroblasts
was not observed. In the presence of LDL, normal fibroblasts produced
several oxysterols and C27-acids in addition to
27-hydroxycholesterol. These oxygenated C27-steroids were
all formed from LDL cholesterol or autooxidation products of
cholesterol by a relatively extensive metabolism. Fig. 5
shows a simplified scheme of their metabolic pathways and their
conversion into potent HMG-CoA reductase suppressors in normal human
fibroblasts. Briefly, LDL is internalized by LDL receptors. Following
hydrolysis of cholesteryl esters, a portion of LDL cholesterol is
converted to 27-hydroxycholesterol, which is metabolized further to
7 ,27-dihydroxy-4-cholesten-3-one and the corresponding
C27-acid. However, the latter two sterols can also be
formed from the autooxidation product 7 -hydroxycholesterol (Fig. 5),
and their origin will therefore depend on the presence and cellular
uptake of LDL as well as of 7 -hydroxycholesterol. It is interesting
that the metabolic pathways of these sterols in fibroblasts show so
many similarities to those in human liver cells (19, 27, 28). This
suggests that many of the reactions of sterols seen in fibroblasts may
be occurring generally in human cells (see also below) although the
activities of the catalyzing enzymes may vary in different tissues.
The two other major autooxidation products, 7-oxocholesterol and
7 -hydroxycholesterol, were both 27-hydroxylated and then oxidized to
the corresponding C27-acids (Fig. 5). A large portion of
7 -hydroxycholesterol was also converted to 7-oxocholesterol and its
side-chain oxygenated metabolites. 25-Hydroxycholesterol is only a
minor autooxidation product of cholesterol, and it was found to be
metabolized analogous to that of 27-hydroxycholesterol, except that it
will not form an acid. Interestingly, 25-hydroxylation of sterols and
the formation of 7 ,25-dihydroxy-4-cholesten-3-one can occur in human
fibroblasts under certain conditions. This was observed when the cells
were incubated in excess of LDL or 7 -hydroxy-4-cholesten-3-one (see
above), when the incubation time was extended or when the fibroblasts
genetically lacked 27-hydroxylase activity (i.e. in
fibroblasts from patients with cerebrotendinous xanthomatosis). In the
latter cells, 7 ,25-dihydroxy-4-cholesten-3-one was by far the major
oxysterol formed (0.9-1.1 nmol/mg of cell protein) after incubation
with media containing 10% FCS for 48 h, and this could explain how
these cells can respond to LDL (see below) (6). Thus, 25-hydroxylation
of sterols may serve as an extra hydroxylating system in cells, being
active when the capacity of the 27-hydroxylase is exceeded by
substrate.
Exposure of lipoproteins to oxygen (air) prior to or during the
incubations will result in autooxidation of cholesterol, and the
amounts of the products can be considerable, especially when
LDL-particles have been isolated from serum. The concentrations of
autooxidation products are usually low in the circulation of man (39),
but their presence has to be considered when the biological effects of
LDL on cultured cells are being studied. Most of these sterols will
contaminate the LDL particles (40), and, thus, the biological effects
of LDL cholesterol are difficult to distinguish from those of the
autooxidation products. In order to limit the amounts of autooxidation
products present in our incubations, we have deliberately used intact
serum instead of purified LDL.
As expected, LDL cholesterol and the autooxidation products
7 -hydroxy, 7 -hydroxy-, and 25-hydroxycholesterol and
7-oxocholesterol all had strong suppressive effects on HMG-CoA
reductase in normal fibroblasts (29, 37). However, none of these was
particularly active in transformed fibroblasts. When the effects of
their metabolites were determined in the latter cells, only
7 ,27-dihydroxy-4-cholesten-3-one,
7 ,25-dihydroxy-4-cholesten-3-one, and 27-hydroxy-7-oxo-cholesterol
were potent suppressors of HMG-CoA reductase. The apparently normal
response to these metabolites suggested that the regulatory defect in
transformed cells was localized prior to and not beyond their
formation. In transformed fibroblasts, their formation was shown to be
hindered by reduced activities of enzymes catalyzing 27-hydroxylation,
7 -hydroxylation, and 3-oxidation of sterols. Additional support that
this could underlie the defective response to LDL (and oxysterols) in
transformed fibroblasts was that the regulatory defect could be induced
also in normal fibroblasts by obstructing 27-hydroxylation of sterols
with CsA (Table VIII).
Although the three most potent suppressors of HMG-CoA reductase had
different structures, there were apparent similarities between them as
illustrated in Fig. 6. Common to the sterols were an oxo
group with a conjugated double bond in the steroid nucleus and a distal
hydroxyl group in the side chain. Additional hydroxyl groups in the
steroid nucleus did not seem to affect their biological activity (15).
Interestingly, 3 -hydroxy-5 -cholest-8(14)-en-15-one (25) also
belongs to this group after 27-hydroxylation. This was in agreement
with the results obtained for this compound (see Tables VII, VIII, IX). These
sterols did not seem to be metabolized further in order to be active.
For example, saturation of the double bond resulted in an almost
complete loss of their activity (15), reduction of the oxo group to a
hydroxyl group also decreased the activity as seen when the activity of
27-hydroxy-7-oxo-cholesterol was compared with that of
7 ,27-dihydroxycholesterol. Further oxidation to an acid was
apparently not required, since the active 25-hydroxylated sterols
did not form acids. However, the corresponding steroids with a
27-carboxy group may also be active, when present in a nonionized form.
Furthermore, we did not find any evidence that additional oxygen groups
were required in the steroid nucleus or in the side chain for these
sterols to be active. Due to the flexibility of the side chain, its
hydroxyl group and the oxo group (with double bond) in the steroid
nucleus may not have to be situated at certain positions (Fig. 6).
Thus, in addition to 25- and 27-hydroxylated 3-oxo- 4
steroids, the corresponding 24-hydroxylated steroid was found to be a
highly potent suppressor of HMG-CoA reductase. These results suggest
that the oxysterols may bind to a common receptor protein, which could
be involved in the suppression of HMG-CoA reductase. To our knowledge,
none of the oxysterols shown in Fig. 6 has been used in the search for
such a receptor. The relatively extensive metabolism of sterols in
fibroblasts (Fig. 5) and the apparently few structural requirements for
being a suppressor of HMG-CoA reductase (Fig. 6) may explain how such a
wide range of different oxysterols can be biologically active in the
cells. On the other hand, not until an oxysterol receptor has been
found, can the true nature of its ligands be established with
certainty.
Fig. 6.
Structures of the three naturally occurring
cholesterol derivatives that were potent suppressors of HMG-CoA
reductase also in transformed fibroblasts. The sterols
7 ,27-dihydroxy-4-cholesten-3-one (I),
7 ,25-dihydroxy-4-cholesten-3-one (II), and
27-hydroxy-7-oxo-cholesterol (III) did not seem to require
further metabolism in order to suppress HMG-CoA reductase in human
fibroblasts. Common to these sterols is the presence of an oxo group
with a conjugated double bond in the steroid nucleus and a distal
hydroxyl group in the side chain. The sterols are drawn in such a way
that their apparent structural similarities are illustrated. For
comparison, 27-hydroxylated 3 -hydroxy-5 -cholest-8(14)-en-15-one
(IV) is also shown.
Out of the three suppressors formed,
7 ,27-dihydroxy-4-cholesten-3-one seems to be biologically most
important under normal conditions (see above). It was quantitatively
the major suppressor, it was rapidly formed and it was derived from LDL
cholesterol. Although suppressors may also be formed from autooxidation
products of cholesterol, the latter sterols were evidently not required
for fibroblasts to maintain their cholesterol homeostasis. For
simplicity we are now calling 7 ,27-dihydroxy-4-cholesten-3-one
``cytosterone'' in analogy with the names of potent steroid hormones
and since the sterol can be formed in several different human
tissues.
In a separate study, we have found that 27-hydroxylated
3-oxo- 4 steroids decrease the syntheses of HMG-CoA
reductase and LDL receptors in
fibroblasts.2 These effects were expected
of a suppressor derived from LDL (4). However, the formation of
7 ,27-dihydroxy-4-cholesten-3-one or the other suppressors did not
seem to be required for stimulating esterification of sterols in the
cells. In fact, this reaction may be triggered by
3 -hydroxy- 5 steroids with a hydroxyl group in the
side chain, since 25-hydroxycholesterol was more esterified in the
transformed than the normal fibroblasts (Table VI). This is in
agreement with the previous finding that mutant cells being resistant
to the actions of oxysterols are able to respond with esterification of
sterols (41). A binding protein for this group of oxysterols have been
characterized previously (42, 43, 44, 45), but we do not know if this protein
can be involved in the actions of acyl-CoA:cholesterol
acyltransferase.
The formation of 7 ,27-dihydroxy-4-cholesten-3-one from LDL
cholesterol was obstructed by low activities of sterol-metabolizing
enzymes in the transformed fibroblasts. The mechanisms underlying these
multiple enzyme deficiencies are not known. However, when normal
fibroblasts were incubated for 48 h with conditioned media (containing
10% LDS) from transformed fibroblasts and then were exposed to
lipoproteins for 24 h, the uptake of LDL increased by 40%, while the
formation of 27-oxygenated steroids decreased by 20%. This may suggest
that a substance (or substances) could have been released from the
transformed cells into the medium, which affected the regulatory
response to LDL also in normal cells. In relation to this, the
observation that interleukin 1 and tumor necrosis factor
stimulate the activity of the 7 -hydroxylating enzyme in rat ovaries
is interesting (46). According to our results and in contrast to the
speculations of the authors and others (47), this would be expected to
increase the suppression of HMG-CoA reductase by side-chain
hydroxylated oxysterols (and possibly by LDL) if rat cells behave in
the same way as human fibroblasts. Thus, our observation that LDL
cholesterol and autooxidation products seem to be metabolized prior to
being biologically active implies that cholesterol homeostasis in cells
can be regulated by factors that determine the activities of the
sterol-metabolizing enzymes.
Common to all the tumor cell lines studied was a low intracellular
accumulation of active HMG-CoA suppressors due to a decreased formation
or an increased metabolism. Although an altered sterol metabolism may
be a common cause of a defective feedback control in tumor cells, it
cannot be excluded that other defects may also exist in some cancer
cells. Nonetheless, a reduced response to LDL seems to be
characteristic of malignant cells (11), and it has been speculated that
this phenomenon may be essential for the growth of tumor cells by
increasing the cellular supply of cholesterol and intermediates in the
mevalonate pathway (11). This hypothesis can now be tested, since the
enzyme deficiencies in tumor cells can be by-passed and their
cholesterol homeostasis normalized by the described HMG-CoA reductase
suppressors. Preliminary results show that the growth of transformed
fibroblasts is stopped in a cell cycle specific way by these
suppressors.
In conclusion, the results of this study provide information about how
LDL and various oxysterols suppress HMG-CoA reductase in human cells,
about structural requirements of oxysterols for being suppressors of
HMG-CoA reductase, and about mechanisms underlying a defective
regulatory response to LDL in neoplastic cells. The results may
stimulate the interest in finding a receptor protein which can bind
this new group of oxysterols, in finding factors regulating cholesterol
homeostasis in cells by affecting the activities of sterol-metabolizing
enzymes and in elucidating the biological significance of malignant
cells having a defective response to LDL.
FOOTNOTES
*
This work was supported by Swedish Medical Research Council
Grant 03X-7890, Swedish Cancer Society Grant 2992-B94-05-XBC, and the
Karolinska Institute. The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
``advertisement'' in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
To whom correspondence should be addressed: Dept. of Clinical
Chemistry, Karolinska Hospital, S-171 76 Stockholm, Sweden.
1
The abbreviations used are: LDL, low density
lipoprotein; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; FCS, fetal
calf serum; LDS, lipoprotein-deficient serum; CsA, cyclosporin A; HPLC,
high performance liquid chromatography.
2
M. Axelson and S. Vitols, manuscript in
preparation.
Acknowledgments
We thank Kristina Garmark and Birgitta
Mörk for excellent technical assistance.
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