Departments of Molecular Genetics (G.C., H.S., H.H.H.), Internal Medicine (L.Z., K.L.P., T.H., H.H.H.), and Pathology (J.A.R.) University of Texas Southwestern Medical Center at Dallas Dallas, Texas 75235
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ABSTRACT |
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More than 20 yr ago, HDL was found to be the major source of cholesterol for steroidogenesis in rodents (46). The dominant role of HDL in maintaining cholesterol ester stores in steroidogenic tissues is reflected by the marked lipid depletion seen in the adrenocortical cells of mice lacking apolipoprotein AI, the major apolipoprotein of HDL (7). The adrenal cortical cells of rats made hypolipidemic by treatment with either high-dose estrogen or 4-aminopyrazolopyrimidine are also lipid depleted (5, 8, 9).
Cholesterol delivery to steroidogenic tissues from HDL differs from the well characterized LDL receptor pathway (5, 6). The cholesterol uptake from HDL is selective; lipids are transported into the cell without the concomitant uptake and degradation of the apolipoproteins (10). In contrast, after LDL binds to its cell surface receptor, the entire particle is taken up by receptor-mediated endocytosis and delivered to lysosomes, where the apoproteins are degraded and the cholesterol esters are enzymatically hydrolyzed to release cholesterol (11).
The protein that mediates the selective uptake of lipids from HDL was identified 3 yr ago by Krieger and colleagues (12) and named scavenger receptor, class B, type I (SR-BI) (12). SR-BI is expressed at highest levels in those tissues and cell types most active in selective uptake in vivo: the liver, the zona fasciculata and zona reticularis of the adrenal glands, the theca cells and corpus luteum of the ovaries, and the Leydig cells of the testes (1216). Antibodies to the extracellular domain of SR-BI block HDL-cholesterol ester uptake and HDL-stimulated synthesis of steroids in cultured mouse adrenocortical cells (17). This finding is consistent with SR-BI playing a major role in supplying steroidogenic cells with cholesterol. Furthermore, SR-BI knockout mice have lipid-depleted adrenal glands and a 2- to 3-fold increase in plasma levels of HDL-cholesterol (18). Mice with a reduced amount of SR-BI showed decreased selective uptake of cholesterol esters (19).
Trophic hormones, acting by a cAMP-dependent protein kinase pathway (20), induce the expression of both the LDL receptor and SR-BI (9, 13, 21, 22). Trophic hormones fail to increase LDL receptor activity in adrenocortical cells when steroidogenesis is inhibited and the intracellular cholesterol content is maintained by the addition of exogenous lipoproteins (9). These observations are consistent with the model in which trophic hormones deplete intracellular cholesterol stores by stimulating steroidogenesis and thereby indirectly increase LDL receptor activity (23).
The mechanism by which trophic hormones up-regulate SR-BI expression is not known. SR-BI levels are elevated in the adrenal glands of multiple strains of genetically manipulated mice that are hypolipidemic, including some apoAI-/- mice (24) and mice in which the lecithin-cholesterol acyl transferase or the hepatic lipase genes have been inactivated (24, 25). These observations suggest that the levels of SR-BI, like the LDL receptor, may be regulated by the intracellular content of cholesterol.
cAMP acts synergistically with the nuclear hormone receptor steroidogenic factor 1 (SF-1) to activate the genes encoding multiple components of the steroidogenic pathway (26). All cytochrome P450 steroid hydroxylases and the steroidogenic acute regulatory protein (StAR), which mediates transport of cholesterol from the cytoplasm to the inner mitochondrial membrane, are regulated by SF-1 via SF-1-responsive promoter elements (27, 28). The human CLA-1/SR-BI gene also contains a consensus SF-1 binding motif in its promoter region (29). We previously showed that SF-1 binds to this site in a sequence-dependent manner and that this element is required for high-level expression of SR-BI promoter constructs in cultured adrenocortical cells (29).
In this paper, we explore the regulation of SR-BI mRNA expression in steroidogenic tissues of the mouse and examine the relative roles of trophic hormones (via cAMP and SF-1) and the intracellular concentration of cholesterol in regulating the levels of SR-BI in cultured mouse Yl adrenocortical cells.
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RESULTS |
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Adjacent sections from E10.5 and E11.5 embryos were hybridized with the SR-BI antisense probe. No staining was seen in the urogenital ridge on E10.5 in either male or female embryos (panels C and F); the only tissue that stains at this early time point is the premordial liver. On E11.5, a signal for SR-BI mRNA was present in the genital ridge of the male embryo (panel I), but only a marginal signal was apparent in the female embryo (panel L). Thus, SR-BI mRNA is first expressed in the genital ridge of the male embryo at E11.5, which is approximately 2 days after the first appearance of SF-1 mRNA and 1 day after the initial appearance of StAR (31) and the cholesterol side chain cleavage enzyme (SCC) (30). The level of SR-BI mRNA expression in the genital ridge at this stage was lower in female than male mice. In contrast to SR-BI, no sex-dependent differences in the expression levels of StAR or SCC mRNA were apparent at this time point (30, 31).
No SR-BI mRNA was detected in the hepatic primordia at day 9.5, which is the first day this structure can be identified (data not shown). In both the male and female embryos, low-level staining was apparent at E10.5 within the septum transversum of the hepatic/biliary primordia. Significant levels of SR-BI mRNA expression were detected in the liver primordia on E11.5 of the male and female embryos (panels I and L).
Sexually Dimorphic Tissue Expression of SR-BI Mirrors SF-I during
Fetal Development
In male mice, the testes become histologically distinct at
approximately E12.5 as they organize into round, cord-like
structures (the testicular cords), which contain both Sertoli cells and
primordial germ cells and ultimately develop into seminiferous
tubules. The Leydig cells, which reside between the testicular cords in
the interstitial region, synthesize testosterone. High levels of
expression of both SF-1 and SR-BI mRNAs are seen within the testes
during this time period (Fig. 2A). The
patterns of expression of SF-1 and SR-BI mRNAs within the testes were
overlapping but not identical (Fig. 2A
). SR-BI mRNA has a more
restricted and punctate pattern of distribution, with patches of
intense signal corresponding to the Leydig cells, thus resembling
patterns previously reported for SCC and StAR (30, 31). The signal for
SF-1 was more generalized, consistent with expression in both fetal
Sertoli and Leydig cells at this stage of development (30).
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The expression of SR-BI in the liver was similar in the male
and female embryos. SR-BI was expressed in the liver at E12.5 and E14.5
(panels C and F in Fig. 2) but the levels declined by E16.5. In
contrast, prior immunocytochemical studies failed to detect SR-BI in
the murine fetal liver through E17.5 (32). The apparent discrepancy
between the in situ hybridization and immunocytochemical
studies most likely reflects different sensitivities of the assays. It
also is possible that an alternatively spliced form of SR-BI, which is
not recognized by the antibody used for the immunocytochemical studies,
is expressed in the liver at these time points. The SR-BI gene is
alternatively spliced at its 3'-end (33). The antibody used for the
immunocytochemical studies only detects the major form of SR-BI
(referred to as SR-BI or SR-BI.1) (12), whereas our antisense probe
detects both SR-BI and SR-BII (also called SR-BI.2). More likely, the
absence of any immunodetectable hepatic SR-BI in murine embryos at
these time points reflects a level of expression that is too low or too
diffuse to detect by immunostaining.
From these studies, we conclude that SR-BI expression in the developing gonads is sexually dimorphic and generally correlates with the expression of SF-I. The pattern and timing of expression of SR-BI in the developing embryo closely resemble those described for the transcripts of two other key participants in the steroid biosynthetic pathway, SCC and StAR (30, 31).
SR-BI is Expressed at High Levels in the Fetal Adrenal Gland
SF-1 transcripts can be detected at E12.5 in the cells that
comprise the adrenal primordium (30). Within 24 h, high levels of
expression of both SF-1 and SR-BI transcripts were seen throughout the
adrenal gland (Fig. 3, panels AC).
These results correlate well with prior immunocytochemical studies that
revealed the first expression of immunodetectable SR-BI in the adrenal
at E14.5 (32). By E16.5 the chromaffin cell precursors have migrated
into the central portion of the gland to form the adrenal medulla. SF-1
and SR-BI are not expressed in the adrenal medulla (13), so the
staining pattern of the adrenal gland by E16.5 is doughnut shaped.
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The antibody used in these studies detects only the SR-BI/SR-BI.l
transcript, which is the predominant mRNA transcript in steroidogenic
cells (36). The other mRNA transcript, SR-BII (or SR-BI.2), differs in
sequence in the last 39 amino acids and thus is not detected by our
antibody (33). To determine whether (Bu)2 cAMP increases
the levels of both the SR-BI and SR-BII transcripts, we used an RNase
protection assay to assess the relative levels of the two transcripts
in the Y1-BS1 and MA-10 cells. When we used this assay to assess the
relative proportion of SR-BI and SR-BII in the testes, approximately
75% of the total SR-BI mRNA was SR-BII (data not shown), which is
similar to that previously reported (36). At all time points, more than
95% of the total mRNA (SR-BI plus SR-BII) in both Y1 cells and MA-10
cells was SR-BI (Fig. 5B). Moreover, a pronounced increase in the
levels of SR-BI mRNA was seen in both cell lines, with a time course
congruent with that seen for SR-BI protein (Fig. 5A
). Thus, there is no
evidence for differential regulation of the two mRNA transcripts in
response to (Bu)2 cAMP in the Y1 and MA-10 cells.
SR-BI Levels Are Regulated by Intracellular Cholesterol Levels
Independently of Trophic Hormones in YI-BSI Cells at Both a
Transcriptional and Posttranscriptional Level
Cholesterol substrate for adrenal steroidogenesis comes from three
sources: endogenous synthesis from acetyl CoA, hydrolysis of
intracellular cholesterol esters, and uptake from circulating
lipoproteins. ACTH dramatically up-regulates expression of SR-BI in the
adrenocortical cells of the mouse adrenal gland (18). Is the
up-regulation of SR-BI a direct effect of trophic hormones, or the
result of depletion of intracellular cholesterol due to the induction
of steroidogenesis?
To determine whether SR-BI levels are regulated by changes in the
intracellular concentration of cholesterol, we inhibited endogenous
cholesterol synthesis with compactin (10 µM), and
steroidogenesis with aminoglutethimide (5 µg/ml). To document that
steroid hormone production was effectively inhibited, we measured the
level of progesterone in the medium, which fell into the undetectable
range (data not shown). Aminoglutethimide treatment without the
addition of lipoproteins did not alter the level of SR-BI (data not
shown). Addition of 50 µg/ml LDL to the media of
aminoglutethimide-treated cells resulted in a 4-fold decrease in the
amount of immunodetectable SR-BI protein (Fig. 6A). No further decrease in the level of
SR-BI was seen when the concentration of LDL in the media was increased
to 100 µg/ml.
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To determine whether the decrease in SR-BI protein was due to a
reduction in the level of SR-BI mRNA, we used our RNase protection
assay to assess the effect of aminoglutethimide and LDL on the amount
of SR-BI and SR-BII mRNA. The levels of both SR-BI and SR-BII fell by
approximately 20% with the addition of LDL (Fig. 6B). Under these
conditions, the reduction in the level of SR-BI mRNA was less than that
of immunodetectable SR-BI (Fig. 6A
), suggesting posttranscriptional
regulation of SR-BI. This could be caused by a decrease in the
efficiency of mRNA translation or an increase in the degradation of
SR-BI protein. Alternatively, the epitope that binds the anti-SR-BI
antibody may be masked under these conditions.
To differentiate between these models, we assessed the half-life of
SR-BI by performing a pulse-chase experiment. After a 1-h pulse, the
level of immunoprecipitable SR-BI declined over the 12-h chase at the
same rate in the cells incubated in the presence of absence of
aminoglutethimide and LDL. The t1/2 in both is 7 h
(Fig. 7), which is slightly longer than
what was previously reported for recombinant SR-BI that was stably
expressed in Chinese hamster ovary cells (38). These results suggest
that LDL and aminoglutethimide treatment does not decrease SR-BI
protein by increasing the rate of degradation of SR-BI.
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Taken together, these experiments demonstrate that SR-BI is regulated by both cAMP analogs and the intracellular content of cholesterol; the effect of the cAMP analog appears to largely override the suppressive effect of the increase in intracellular cholesterol.
SR-BI Expression in the Adrenal Glands of Newborn
StAR-/- Knockout
(StAR-/-) Mice
StAR-/- mice are unable to efficiently
transport cholesterol from the cytoplasm across the mitochondria
membrane and thus accumulate cholesterol within their steroidogenic
cells (39). These mice have elevated circulating levels of ACTH due to
their adrenal insufficiency (39). In situ hybridization
studies were conducted to compare the levels of SR-BI transcripts in
newborn StAR-/- and wild-type mice (Fig. 9A). Although the adrenocortical
architecture is distorted by the abundant lipid deposits in the
StAR-/- adrenals (top panels), the
level of SR-BI transcripts is high (bottom panels). Thus,
the levels of SR-BI mRNA are high in the
StAR-/- adrenocortical cells despite their
increased cellular content of cholesterol, presumably due to the
elevated circulating levels of ACTH.
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DISCUSSION |
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The timing and cell-specific distribution of expression of SR-BI within the murine embryonic adrenal glands and gonads are similar to those of two other genes that encode proteins in the steroidogenic pathway, StAR and SCC (30, 31). StAR plays a key role in the acute response of steroidogenic cells to trophic hormones by mediating transport of cholesterol from cytoplasm to the inner mitochondrial membrane where SCC catalyzes the first reaction in steroidogenesis (40). The mRNA transcripts for StAR and SCC are first detected in murine embryos 12 days after the initial appearance of SF-1 transcripts in urogenital ridge (30, 31). Therefore, the SCC and StAR transcripts are both present in the early developing testes and adrenal glands, but not the ovaries, again resembling the expression pattern of SR-BI. The ontogeny of expression of SR-BI mRNA in the developing embryo, and its similarity to the pattern of expression of two other SF-1-regulated genes (StAR and SCC), strongly suggest that SR-BI is part of the coordinated response of steroidogenesis to trophic hormones and plays an important role in embryonic, as well as adult, steroidogenic tissues.
If SF-1 is responsible for activating transcription of SR-BI, then it
would be expected that SR-BI expression would not be present in the
steroidogenic tissues of mice that do not express SF-1. We could only
examine SR-Bl expression in SF-1 knockout mice before E12 because,
after this time point, gonads and adrenal primordium undergo
apoptosis (30, 35). As shown in Fig. 4, comparable levels of SR-BI
transcripts were detected in the developing liver of wild-type and SF-1
knockout embryos at E11.5, consistent with the fact that SF-1 is not
expressed in this organ and thus cannot regulate SR-BI. In contrast,
SR-BI was expressed in the genital ridge of the wild-type embryo, but
not SF-1 knockout mice. To the extent that the developing gonads are
still intact at this relatively early developmental stage, this finding
suggests that SF-1 plays important roles in SR-B1 expression in
vivo.
Although the expression patterns of SF-1 and SR-BI mRNA in the developing rodent are similar, they are not identical. In the testes, SF-1 is expressed in the fetal Sertoli cells as well as Leydig cells. SR-BI has an expression pattern in the testes that is similar to that of SCC and StAR (30, 31); all three transcripts are detected only in the steroidogenic cells of the interstitium. In other embryonic tissues where SF-1 mRNA is present, like the pituitary and ventromedial hypothalamus (30), we found no detectable SR-BI transcripts (data not shown). Conversely, SR-BI is expressed at significant levels in some tissues that do not express SF-1, such as the embryonic liver. As discussed in the results, the most likely explanation for the absence of any immunodetectable hepatic SR-BI in murine embryos is that the level of expression is either too low or too diffuse to be detected by immunostaining.
To further define the mechanisms that regulate SR-BI in steroidogenic tissues, we examined its expression in cAMP-stimulated cultured murine adrenocortical (Y1-BS1) and testicular carcinoma (MA-10) cells. SR-BI mRNA and protein levels were dramatically increased upon cAMP stimulation in both cell lines. The kinetics of SR-BI induction by cAMP analogs in these cell lines differed somewhat from that which was previously reported for StAR (31). The increase in SR-BI levels lags behind that of StAR by approximately 2 h. The time course of SR-BI induction in response to cAMP analogs in MA-10 cells is similar to that seen in rat granulosa cells (42). In the unstimulated state, these two cell lines are similar in that they synthesize no steroid hormones and express no SR-BI; after cAMP stimulation it takes approximately 6 h before any detectable increase in SR-BI protein is apparent.
In the absence of any trophic hormones, SR-BI levels appear to be regulated by the intracellular content of cholesterol. Our results are compatible with the demonstration that SR-BI expression remains high in desensitized, lipid-depleted, rat luteal cells, which cannot respond to trophic hormones (16); these lipid-depleted cells have high levels of immunodetectable SR-BI protein, as well as HMG CoA reductase and the LDL receptor. In our studies with Y1-BS1 cells, the levels of immunodetectable SR-BI fall into the nondetectable range when cells are cultured for 24 h in the presence of LDL (100 µg/ml). The reduction in SR-BI protein mass was greater than the fall in the level of SR-BI mRNA, which is consistent with a posttranscriptional mechanism. We found no evidence that the decrease in SR-BI in the lipoprotein-supplemented Y1-BS1 cells is due to an increased SR-BI degradation. Further studies will be required to define the posttranscriptional mechanism responsible for the observed dissociation between the levels of SR-BI mRNA and protein.
Even when Y1-BS1 cells are provided sufficient exogenous LDL to reduce HMG-CoA reductase to trace levels, administration of cAMP resulted in a considerable increase in SR-BI. These results suggest that trophic hormones up-regulate SR-BI expression directly rather than by depleting intracellular cholesterol stores. These data are consistent with the studies of Dexter et al. (2) who showed that ACTH stimulates the uptake of cholesterol from lipoproteins in the adrenal glands of hypophysectomized rat, even if steroidogenesis is completely inhibited. The results of our studies in cultured cells are similar to the findings of Gwynne et al. (4), who examined the effect of aminoglutethimide on the ACTH-stimulated uptake of radiolabeled cholesterol in rat adrenal slices. They showed that aminoglutethimide did not affect cholesterol uptake despite increasing the cellular cholesterol content by 5-fold. In granulosa cells, like the Y1-BSI cells, high levels of SR-BI were maintained even in the presence of high concentrations of lipoproteins (41).
Further evidence that trophic hormones override the effect of intracellular cholesterol concentrations on SR-BI expression is the finding that SR-BI levels are not decreased in the adrenal glands of StAR knockout mice (39). These mice are unable to efficiently transport cholesterol into the mitochondria of steroidogenic tissues and thus fail to synthesize sufficient steroid hormones to suppress pituitary ACTH secretion. As a consequence, the StAR-/- mice accumulate massive amounts of cholesterol in their adrenal glands and have elevated plasma levels of ACTH (39). Despite having cholesterol-laden adrenocortical cells, these mice have normal to elevated levels of SR-BI, which presumably are maintained by the high levels of circulating trophic hormones.
Taken together, the results of these studies are consistent with SR-BI being part of the repertoire of SF-1-responsive genes in steroidogenic tissues and the major pathway by which cholesterol is delivered for steroid hormone biosynthesis in the mouse. The physiological importance of this regulation may be to ensure that SR-BI will be up-regulated during times of stress, even if the adrenal gland is replete with cholesterol. This regulatory mechanism presumably ensures that the organism always has a sufficient supply of cholesterol available for maximal steroidogenesis in times of stress. This formulation is consistent with the extensive and careful studies of Reaven et al. (42), who showed that the selective uptake pathway is optimally designed for the dramatic increase in cholesterol delivery that is required upon stimulation.
Finally, it is important to note that multiple lines of mouse Y1 adrenocortical cells have been developed (20). These cell lines differ in their responsiveness to trophic hormones and are likely to differ in their expression levels of SR-BI. In some of the YI adrenal cell lines, exogenous HDL fails to reduce HMG-CoA reductase activity or incorporation of HDL-cholesterol into steroids (23). It is likely that these cell lines have a dysfunctional SR-BI receptor pathway.
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MATERIALS AND METHODS |
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Animals
Timed pregnant NIH Swiss mice were purchased from Harlan
Laboratories (Indianapolis, IN). Noon of the day on which the
copulatory plug was detected was designated 0.5 day of the gestation
(E0.5). Pregnant mice were killed by cervical dislocation at the time
intervals indicated, and the embryos were dissected, fixed in 4%
paraformaldehyde, and embedded in paraffin. Serial sagittal sections of
4 µm thickness were prepared using a microtome (44). To determine the
sex of each embryo, a small aliquot of tissue was taken from the yolk
sac and placed in 500 µl of 50 mM KCl, 1.5 mM
MgCl2, 10 mM Tris HCl, pH 8.5, 0.01% (wt/vol)
gelatin, 0.45% (vol/vol) NP40, 0.45% (vol/vol)Tween 20, and 100
µg/ml proteinase K (Sigma Chemical Co.) at 55 C
overnight. PCR was used to amplify a fragment from the mouse SRY gene
(45). The amplification reaction included 1 µl of the yolk sac lysate
as template and two oligonucleotides (5'-TCATGAGACTGCCAACCACAG-3' and
5'-CATGACCACCACCACCACCAA-3'). The PCR products were size fractionated
on a 1% (wt/vol) agarose gel, the gel was stained with ethidium
bromide, and the bands were visualized using UV light.
In Situ Hybridization
In situ hybridization was performed as previously
described (44). A 2.4-kb fragment containing the mouse SR-BI cDNA
(kindly provided by Dr. Monty Krieger, Massachusetts Institute of
Technology, Cambridge, MA) was subcloned into pcDNA3
(Invitrogen, Carlsbad, CA). The plasmid was linearized
using HindIII or XhoI to generate labeled
antisense and sense RNA probes, respectively, employing the In
Vitro Transcription System (Promega Corp., Madison,
WI). The probes were partially hydrolyzed by incubating them with 200
mM Na2CO3, pH 10.2, at 60 C for 25
min. Serial sections were deparaffinized and then allowed to hybridize
with the probes (1 x 10 (6)cpm/ml) using Hybridization Cocktail
(Amresco, Solon, OH) at 55 C overnight. The sections were washed and
the slides were dipped in NTB2 liquid emulsion (Eastman Kodak Co., Rochester,NY) diluted 1:1 in H20. The slides
were incubated at 4 C for 23 days and placed in Dektol developing
solution (Eastman Kodak Co.) and counterstained with
hematoxylin. Photographs of the slides were taken using a Eclipse
E1000M microscope (Nikon, Melville, NY) linked to a video
system (Media Cybernetics, Silver Spring, MD). Green and red
fluorescent filters was used under dark field illumination for the
sections incubated with the SF-1 and SR-BI probe, respectively.
Immunoblot Analysis of SR-BI, LDL Receptor, and HMG-CoA
Reductase
The Y1-BS1 cells were maintained in medium A (1:1 mixture of
DMEM and Hams F-12 medium, plus 100 U/ml penicillin and 100 µg/ml
streptomycin sulfate) with 15% horse and 2% FCS. On day 4 the medium
was switched to medium A plus 10% NLPPS. After 24 h, the medium
was supplemented with 1 mM (Bu)2cAMP. The same
protocol was used for MA-10 cells except that the cells were maintained
in Waymouths MB 752/1 medium plus 15% horse serum. Cultured cells were
washed twice with ice-cold PBS before being collected in 2 ml of PBS.
The cells were isolated by centrifugation at 1300 x g
for 5 min and resuspended in lysis buffer [1% (vol/vol) Triton, 50
mM Tris, 2 mM CaCl2, 80
mM NaCl, pH 8.2] containing protease inhibitors (0.5
mM phenylmethylsulfonylfluoride, 10 µg/ml
leupeptin, 5 µg/ml pepstatin A, and 2 µg/ml aprotinin). After a
15-min incubation on ice, the samples were centrifuged at 16,000
x g for 10 min. The protein concentration of the lysates
was determined using a BCA Protein Assay kit (Pierce Chemical Co., Rockford, IL). Cell lysates for immunoblots to detect
HMG-CoA reductase were prepared as described previously (37).
Approximately 50 µg of each cell lysate were reduced by the addition
of ß-mercaptoethanol to 1.5% and then size fractionated on a 6.5%
SDS-polyacrylamide gel. The proteins were transferred to Hybond C Extra
Transfer membrane (Amersham Pharmacia Biotech), and
immunoblot analysis was performed using a rabbit antibovine LDL
receptor antiserum (1:1000) (46), a rabbit antipeptide polyclonal
antibody directed against the last 14 amino acids of mouse SR-BI (10
µg/ml), and IgG-A9, a monoclonal antibody to HMG-CoA reductase (5
µg/ml) (37). Immunoblot analyses were performed using the Enhanced
Chemiluminescence Western Blotting Detection Kit (Amersham Pharmacia Biotech) according to the manufacturers
instructions, and then the filters were exposed to Reflection NEF film
(DuPont NEN, Wilmington, DE). The images were
scanned into a Power 7500/100 MacIntosh computer, and the relative
intensities of the bands were quantified using NIH Image 1.61
(http://rsb.info.nih.gov/nih-image/download.html).
Immunoprecipitation
On day 0, Y1-BS1 cells were plated at 500,000 cells per well in
a 6-well dish and grown for 2 days in medium A plus 15% horse serum
and 2% FCS. On day 3 the medium was changed to medium A with 10%
NLPPS. After 24 h the cells were incubated in methionine- and
cysteine-free DMEM medium (ICN Biochemicals, Inc.,
Costa Mesa, CA) for 30 min and then pulsed with Trans-label
methionine-cysteine (200 µCi/ml)(ICN Biochemicals, Inc.)
for 1 h. The cells were then chased in medium A plus 10% NLPPS
plus 2 mM cold methionine with or without 50 µg/ml of
LDL, compactin (10 µM), and 5 µg/ml of
aminoglutethimide. Cells were lysed at the indicated time points and
SR-BI was immunoprecipitated exactly as described (38) except that the
Protein A Sepharose was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
RNase Protection Assay
A 307-bp PCR fragment containing the sequence that encodes amino
acids 397499 of the mouse SR-BI cDNA was amplified from total murine
hepatic RNA using RT-PCR (Stratagene, La Jolla, CA) and
two oppositely oriented oligonucleotides with the following sequences:
5'-GGGCAAACAGGGAAGATCGAGCCA-3' and 5'-ACCGTGCCCTTGGCAGCTGGTGAC-3'. The
PCR product was subcloned into pGEMT Easy vector (Promega Corp.) and the insert was sequenced. The plasmid was linearized
using NcoI, and an in vitro transcription
reaction was carried out in the presence of [-32P]-CTP
and SP6 polymerase (Promega Corp.) for 1 h at 37 C
using the Riboprobe in vitro Transcription System
(Promega Corp.). The reaction product was incubated in 1 U
of RQ DNase (Promega Corp.) for 15 min at 37 C to digest
the DNA template. The reaction mixture was then diluted with RNase-free
water to a final volume of 50 ml, extracted once with 50 ml
phenol/chloroform (1:1), and then purified using a G50 spin column (5
Prime
3 Prime, Inc., Boulder, CO). A HybSpeed kit from Ambion, Inc. (Austin, TX) was used for the RNase protection assay. A
total of 1 x 105 cpm were mixed with 10 µg of total
cellular RNA that was isolated from the cultured cells using RNA STAT
(Tel-Test, Friendswood, TX). The RNase protection
assay was performed as recommended by the manufacturer. The protected
fragments were resolved on a 6% denaturing polyacrylamide gel, dried,
and exposed to Reflection NEM film for the indicated times. The bands
were quantified using a phosphoimager (Fuji Photo Film Co., Ltd., Stamford, CT).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grants HL-20948 (H.H.H.), DK-54028 (K.L.P.), and the Perot Family Fund.
Received for publication February 1, 1999. Revision received May 17, 1999. Accepted for publication June 3, 1999.
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REFERENCES |
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