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Division of Endocrinology and Diabetology (N.C., M.B., R.W.J., A.M.C.) and Department of Physiology (Se.A., N.D.), Faculty of Medicine, University Hospital, CH-1211 Geneva, Switzerland; and Geriatric Research, Education, and Clinical Center (Sa.A.), Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304
Address all correspondence and requests for reprints to: Prof. Alessandro M. Capponi, Division of Endocrinology and Diabetology, University Hospital, 24 rue Micheli-du-Crest, CH-1211 Geneva 14, Switzerland. E-mail: capponi{at}cmu.unige.ch
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Until a few years ago, a key missing element in the study of HDL
metabolism was a well defined HDL receptor. Recent studies have
identified a physiologically relevant membrane receptor for HDL that
mediates cholesterol uptake through a selective pathway
(6, 7, 8). This receptor has been termed scavenger receptor
class B type I (SR-BI). SR-BI is believed to bind to the 
helical
repeats of apolipoprotein A-I (ApoA-I) (7). It has been
suggested that this interaction leads to the formation of a nonaqueous
channel through which CE move down their concentration gradient to the
plasma membrane (7). Because SR-BI has been shown to bind
not only HDL but also LDL and modified LDL, it is likely that this
receptor may also affect LDL metabolism in vivo
(9, 10, 11, 12). Tissue distribution studies revealed that SR-BI
is expressed in the liver, where it contributes to the clearance of
plasma CE in reverse cholesterol transport (4), and in
steroidogenic tissues, where it is particularly abundant in the adrenal
gland (13). This expression is induced by trophic hormones
such as human CG (13), ACTH (14, 15), and LH
(13). Also, HDL have been shown to be significantly more
effective than LDL in supplying cholesterol for corticosterone
production in adrenal glands of the rat and mouse (16, 17). HDL has also been reported to be a source of cholesterol
for steroidogenesis in human ovarian cells (18). However,
the potential role of HDL in mineralocorticoid biosynthesis and its
regulation by hormones of the cardiovascular system, particularly in
the human adrenal gland, have not been addressed adequately.
The aim of the present studies was to examine the ability of bovine adrenal glomerulosa cells and human NCI H295R adrenocortical cells to selectively take up CE from HDL when challenged with angiotensin II (AngII). In addition, we reasoned that if SR-BI is a physiologically relevant receptor for HDL-derived CE uptake and provides cholesterol for steroidogenesis, its expression might be the target of the same stimuli that enhance cholesterol uptake and aldosterone production. We provide here evidence that this is indeed the case: bovine adrenal glomerulosa cells and human NCI H295R cells efficiently internalize HDL-derived CE for aldosterone biosynthesis, both in the resting state and under AngII challenge. We also show that SR-BI is expressed in both cell models and that the increase in HDL-derived CE uptake observed upon AngII stimulation is accompanied by an increase in SR-BI protein and mRNA levels, demonstrating a coordinated regulation of SR-BI expression and of the intracellular steps leading to adrenal aldosterone biosynthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Methods
Bovine adrenal zona glomerulosa cell preparation. Bovine
adrenal glands were obtained from a local slaughterhouse. Primary
cultures of glomerulosa cells were prepared by enzymatic dispersion
with dispase and purified on Percoll density gradients as described
elsewhere (20). Cells were kept in serum-free medium in
the absence or the presence of AngII (10 nM) for 24 h
before the experiments, which were performed on the 4th day of
culture.
Human adrenocortical carcinoma cell culture (NCI H295R). NCI H295R cells were kindly provided by Dr. W.E. Rainey (University of Texas Southwestern Medical Center, Dallas, TX) and maintained in a 1:1 mixture of DMEM and Ham’s F12 medium containing pyridoxine, L-glutamine, and 15 mM HEPES (Life Technologies, Inc., Basel, Switzerland). The culture medium was supplemented with insulin, transferrin, selenium (ITS+, Becton Dickinson and Co. Labware, Bedford, MA), and Ultroser (Ligacon, Tagelswangen, Switzerland) as well as with antibiotics as described elsewhere (21).
Human lipoprotein preparation. LDL (density = 1.006–1.063 g/ml) and apolipoprotein E-poor HDL (HDL3; density = 1.125–1.21 g/ml) were isolated from human plasma by sequential ultracentrifugation as described previously (22). We used exclusively HDL3 to avoid lipoprotein-derived CE uptake via the classic LDL-receptor pathway. Cholesteryl BODIPY-human HDL (rec-hHDL) were reconstituted as described previously (23). In brief, a mixture of egg phosphatidylcholine (2.3 µmol), sphingomyelin (0.6 µmol), fluorescent cholesteryl BODIPY FL C12 as CE (2.24 µmol), unesterified cholesterol (0.6 µmol), and triolein (0.34 µmol) was dissolved in chloroform and dried under N2, then resuspended in a 10 mM Tris-HCl buffer (pH 8) containing 150 mM NaCl, 0.25 mM EDTA, and sonicated at 52 C using a Labsonic L sonicator (B. Braun, Melsungen, Germany) equipped with a microtip at a power setting of 30 W. After sonication for 40 min, the temperature was decreased to 42 C and 5 mg of delipidated hHDL3 apolipoprotein in 2 ml of 2.5 M urea was added dropwise. Sonication was continued for an additional 10 min. The sonicated sample was centrifuged for 20 min at 4000 x g and dialyzed against PBS for 6 h at 4 C. Finally, the reconstituted cholesteryl BODIPY-human HDL3 particles were isolated sequentially by ultracentrifugation (16 h, 150,000 x g at 20 C, then 25 h, 150 x g at 20 C, d = 1.21 g/ml) and dialyzed against PBS for 24 h at 4 C. Immediately before use, the preparation was dialyzed against serum-free culture medium, and the protein concentration of rec-hHDL3 was determined using the Bio-Rad Laboratories, Inc. AG reagent (Glattbrugg, Switzerland) and BSA as a standard. BODIPY-CE are substrates for acid CE hydrolase (lysosomal) but are not hydrolyzed by the neutral CE hydrolase (cytosolic) (23). Consequently, intracellular fluorescence is considered to be caused by intact CE internalized through a nonendocytic pathway (i.e. a selective pathway).
Fluorescence microscopy. For the study of the uptake and accumulation of rec-hHDL-derived CE, glomerulosa cells were grown on round (25 mm diameter) glass coverslips (0.35 x 106 cells/coverslip) and pretreated with or without AngII (10 nM) for 24 h before incubation with rec-hHDL (30 µg/ml) for periods varying from 5 min to 4 h. After incubation, each coverslip was washed four to five times in Krebs buffer and subsequently immersed in a thermostatic chamber (Harvard Apparatus, Holliston, MA). Cells were immediately imaged on an Axiovert S100TV microscope equipped for epifluorescence microscopy using a 100x (or 40x) oil-immersion objective (numerical aperture, 1.3) (Carl Zeiss, Feldbach, Switzerland), 488 ± 10 nm excitation (DeltaRam, Photon Technology International, Inc., Monmouth Junction, NJ), a 505DRLP dichroic mirror and a 535RDF40 emission filter (Omega Optical, Brattleboro, VT). Fluorescence emission from the rec-hHDL was captured using a cooled back-illuminated 16-bit charge-coupled device frame transfer camera (Princeton Instruments, Roper Scientific, Trenton, NJ). All of the equipment was controlled for image acquisition and analysis with Metamorph/Metafluor 4.1.2 software (Universal Imaging, West Chester, PA). The fluorescent images were processed with Metamorph in black and white or in pseudocolors.
Fluorescence quantification. Glomerulosa cells were grown on six-well plates (2 x 106 cells/well). After starvation, cells were pretreated with or without AngII (10 nM) for 24 h before incubation with rec-hHDL (30 µg/ml) for periods varying from 5 min to 4 h. After incubation, cells were washed five times in cold PBS containing 0.1% BSA, and then lipids were extracted with hexane-isopropanol (3:2, vol/vol) as described previously (24). Each sample was dried under N2 and then reconstituted with hexane/isopropanol before being transferred to a glass cuvette. Fluorescence was measured at excitation and emission wavelengths 503 and 540 nm, respectively, using a Jasco CAF-110 spectrofluorometer (Jasco Corporation, Tokyo, Japan).
Effect of anti-ApoA-I antibody on rec-hHDL uptake. To test the ability of the anti-ApoA-I antibody to prevent SR-BI-mediated CE uptake, rec-hHDL (30 µg/ml) were preincubated with a monoclonal anti-ApoA-I antibody (120 µg/ml; a generous gift from Dr. Jean-Michel Dayer, Division of Immunology and Allergology, University Hospital, Geneva, Switzerland) for 30 min at 4 C before being added to bovine glomerulosa cells for 60 min. Fluorescence microscopy and quantification of rec-hHDL uptake were then performed as described above.
Steroid measurement. For steroid production, adrenal glomerulosa cells were grown on 24-well plates (0.5 x 106 cells/well) and preincubated in the absence or the presence of 10 nM AngII for 24 h. Subsequently, cells were exposed or not to AngII and to increasing concentrations of hHDL3 (125 to 500 µg protein/ml) or hLDL (25 to 100 µg protein/ml) for 3 h in serum-free medium. Equivalent masses of CE available to cells in the supplied hHDL3 or hLDL were calculated on the basis of a 5:1 ratio. Pregnenolone was measured by RIA using a commercial antiserum. WIN 19758 (5 µM), an inhibitor of 3ß-hydroxysteroid dehydrogenase, was included in the incubation medium to prevent further metabolism of pregnenolone into progesterone. The aldosterone content of the incubation medium was measured by direct RIA using a commercially available kit (DSL Inc., Webster, TX). Pregnenolone and aldosterone production were normalized to milligram cellular proteins.
SDS-PAGE analysis and immunoblotting. SDS-PAGE was performed according to Laemmli (25). Aliquots from either bovine glomerulosa or NCI H295R cell lysates were resolved by SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Millipore Corp., Volketswil, Switzerland) that was incubated in blocking buffer (PBS, 0.2% Tween 20, 5% nonfat dry milk) for 2 h and then exposed either to rabbit polyclonal anti-SR-BI antibodies (1:1000 dilution) at 4 C for 2 h or to antihuman LDL receptor antibodies (1:250 dilution) at 4 C overnight. The membrane was thoroughly washed with PBS/Tween buffer (3 x 10 min) and then incubated for 1 h with horseradish peroxidase-labeled goat antirabbit IgG (CovalAb, Oullins, France). The polyvinylidene fluoride sheet was then washed (4 x 15 min), and the antigen-antibody complex was revealed by enhanced chemiluminescence using the Western blotting detection kit from Amersham Pharmacia Biotech and Kodak Biomax film (Rochester, NY).
RNA isolation and Northern blot analysis. Glomerulosa cell
total RNA was extracted using the RNAgents kit (Promega Corp., Zurich, Switzerland) according to the instructions of the
manufacturer. This system consistently yields 50–80 µg of total RNA
per 107 cells. For Northern blot analysis, 20–30
µg of RNA was size fractionated on a 1% formaldehyde agarose gel,
vacuum transferred onto a Hybond-N+ membrane, and
fixed by UV cross-linking. The integrity of the 18s and 28s RNA was
checked by ethidium bromide staining of the gel. Hybridization was
performed using rat SR-BI cDNA (1.6 kb) generated by RT-PCR of RNA
isolated from cAMP-treated rat granulosa cells (19). The
cDNA was labeled with [-32P]dCTP using the
Rediprime random primer labeling kit. Northern blots were prehybridized
in Rapid Hybridization Buffer at 65 C for 30 min. The
-32P-labeled probe (specific activity, 2
x 106 cpm/ng DNA) was then added, and the
incubation was continued for 2 h at 65 C. Blots were washed for 5
and 15 min successively at room temperature in 2x sodium
chloride/sodium citrate buffer (SSC), 0.1% SDS, then for 15 min
in 1x SSC, 0.1% SDS. The final wash was performed at 65 C for
15 min in 1x SSC, 0.1% SDS. RNA-cDNA hybrids were
visualized on Kodak Biomax film after a 12- to 24-h
exposure period. Blots were stripped and reprobed with mouse
glyceraldehyde-3-phosphate dehydrogenase cDNA (Ambion, Inc. Lugano, Switzerland) to assess RNA loading.
Analysis of data. Results are expressed as means ± SEM. The mean values were compared by ANOVA using Fisher’s test or two-way ANOVA when appropriate. P < 0.05 was considered as statistically significant. Quantitation of autoradiograms was performed using a Molecular Dynamics, Inc. (Sunnyvale, CA) Computing Densitometer and ImageQuant software.
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Results |
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, nonstimulated cells responded to hHDL3 with a
concentration-dependent increase in pregnenolone production, which
reached approximately 15-fold the basal values. Similarly, aldosterone
production was stimulated 16-fold at 500 µg/ml
hHDL3. This suggested that HDL can deliver
substantial amounts of cholesterol esters to glomerulosa cells to
support mineralocorticoid synthesis. Furthermore, AngII-pretreated
cells showed a concentration-dependent increase in pregnenolone and
aldosterone formation that was approximately 3- and 6-fold greater than
that observed in nonpretreated cells, respectively (Fig. 1A). We then
compared the steroidogenic response of glomerulosa cells supplied with
hHDL3 to that of cells exposed to hLDL. As shown
in Fig. 1B, no significant differences were observed in the
steroidogenic response to both lipoprotein species, either in the basal
state or after AngII pretreatment, a result suggesting that
hHDL3 and hLDL were equally effective.
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hHDL3 enhanced aldosterone production by 1.6-fold
in both control and AngII-stimulated cells (n = 6).
Uptake and intracellular accumulation of fluorescent CE in bovine
glomerulosa cells and human NCI H295R cells
We next examined the uptake and storage of
HDL3-derived CE within these cells, using
hHDL3 reconstituted with the nonhydrolyzable
fluorescent CE, rec-hHDL3. Figure 2 shows fluorescence images of
time-dependent CE uptake in intact bovine adrenal glomerulosa cells
that had been pretreated in the absence (panels b–d) or in the
presence (panels f–h) of AngII for 24 h. In the basal state and
in the absence of rec-HDL3 (Fig. 2, a and e),
glomerulosa cells display a faint fluorescence, presumably attributable
to intrinsic lipid droplets. In control cells, a narrow labeling of the
plasma membrane appeared within 30–60 min of exposure to
rec-HDL3. By 1–2 h of exposure to
rec-HDL3, control cells had accumulated massive
amounts of fluorescent CE within lipid droplets and increased membrane,
cytosolic, and perinuclear signals were observed. Importantly, at all
time points, the labeling in AngII-pretreated cells was more intense
compared with that in the corresponding control cells (panels f–h
vs. b–d). In Fig. 2i, AngII-prestimulated glomerulosa cells
are shown at a higher magnification after 4 h of exposure to
rec-HDL3. The numerous highly fluorescent spots
correspond in size, location, and number to lipid droplets, as
confirmed by a Nomarski image of the same cells (Fig. 2j).
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, panels k to r. These cells internalized
substantial amounts of hHDL3-derived CE in the
basal state in a time-dependent manner (Fig. 2, l–n), and this process
was markedly enhanced after a 24-h pretreatment with AngII (Fig. 2, p–r).
The major apolipoprotein component of HDL3 is
ApoA-I. Recent studies in ApoA-I knockout mice suggested that ApoA-I
may play a crucial role in the delivery of HDL cholesterol to
steroidogenic tissues (26). We preincubated
rec-HDL3 with a monoclonal antibody against
ApoA-I before adding it to glomerulosa cells for 60 min. Fluorescence
microscopy analysis revealed that HDL3-derived CE
uptake was markedly reduced in control cells in the presence of the
anti-ApoA-I antibody (Fig. 2, s and t). Similar results were obtained
in AngII-pretreated cells (Fig. 2, u and v), suggesting that a crucial
event in HDL3-derived CE uptake is mediated by
ApoA-I.
To further substantiate these qualitative results, we next quantified
the amounts of CE taken up by glomerulosa cells using fluorometry. As
shown in Fig. 3, a time-dependent
increase of CE uptake was observed in control cells. In agreement with
the fluorescence imaging data, AngII pretreatment significantly
increased CE uptake, reaching 175 ± 15% of controls after 2
h of exposure to rec-HDL (n = 3, P < 0.01) and
271 ± 8% after 4 h (n = 3; data not shown).
Qualitatively similar results were obtained in human NCI H295R cells:
AngII pretreatment enhanced CE uptake to 165 ± 8% of controls
(n = 3, P < 0.01) after a 4-h exposure to
rec-hHDL3. When rec-hHDL3
were first incubated with the anti-ApoA-I antibody, fluorometric
measurements revealed that CE uptake was reduced by 41 ± 3%
(n = 3, P < 0.05 vs. controls with no
antiserum) in both control and AngII-pretreated bovine glomerulosa
cells. In contrast, when we used a nonrelevant monoclonal antibody
directed against apolipoprotein E instead of the anti-ApoA-I antibody,
AngII-induced BODIPY-CE uptake was not significantly affected (86
± 6% of controls with no antibody, n = 3, NS).
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demonstrate the kinetics of expression
of SR-BI protein in bovine glomerulosa cells incubated in the absence
(basal) or in the presence of AngII. An immunoreactive band of
approximately 86 kDa, a molecular mass similar to that described for
murine SR-BI (6), was detected. In control cells, steady
state levels of SR-BI expression were maintained for at least 24
h. AngII treatment caused a time-dependent increase in SR-BI, reaching
203 ± 12% of control values (n = 4) after 24 h of
stimulation (Fig. 4B). This effect of AngII was concentration dependent
between 10-10 and 10-8 M
(Fig. 4C).
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). AngII increased SR-BI protein
expression by 230 ± 15% of control values after 24 h (Fig. 5A; n = 3, P < 0.001), whereas LDL receptor
expression was only modestly affected by AngII (122 ± 3% of
control values; n = 3, NS; Fig. 5B). In contrast, stimulation of
NCI H295R cells with dibutyryl cAMP for 24 h resulted in a
more pronounced induction of LDL receptor expression (180 ± 4%
of control values; n = 3, P < 0.001; Fig. 5B).
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shows that a transcript of 2.8 kb was detected. Densitometric analysis
indicates that AngII induced a 3-fold increase in SR-BI mRNA levels
compared with control values (n = 4; Fig. 6B). Cycloheximide did
not affect the AngII-elicited increase in SR-BI mRNA, suggesting that
transactivating factors are already present within the cell. However,
SR-BI mRNA induction by AngII was abolished by actinomycin D,
suggesting a transcriptional control of SR-BI expression by AngII. When
glomerulosa cells were incubated for 24 h in the presence of
hHDL3, a significant increase (152 ± 4%;
P < 0.05) in SR-BI mRNA was observed. Finally, after a
combined treatment with both AngII and hHDL3, the
transcriptional effect of AngII on SR-BI mRNA levels was maintained.
Very similar results were obtained in human NCI H295R cells. SR-BI mRNA
levels were increased to 188 ± 13% over controls after a 24-h
exposure to AngII (n = 3, P < 0.01). This
increase was not significantly altered by cycloheximide but was
completely prevented by actinomycin D. The addition of
HDL3 did not affect AngII-elicited induction of
SR-BI mRNA in human NCI H295R cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Three major conclusions can be drawn from the present studies. 1) Human NCI H295R cells and bovine adrenal glomerulosa cells do indeed "selectively" internalize and process HDL-derived CE as a significant source of precursor for steroid hormone biosynthesis. 2) AngII enhances HDL-derived CE uptake as well as SR-BI expression (protein and mRNA) in both cell types, confirming a tight link between the selective uptake, the HDL receptor SR-BI, and steroidogenesis. 3) In contrast to cAMP, which has been shown to exert a more pronounced induction on LDL receptors than on SR-BI protein in human adrenal cells (34), AngII behaves in the opposite manner, potently increasing SR-BI expression while barely affecting LDL receptors. This finding suggests the possibility that the cells may switch between HDL and LDL utilization depending on the type of agonist that challenges them.
The demonstration of efficient HDL-derived CE uptake supporting steroidogenesis in glomerulosa cells is based on functional and fluorescence data. Using HDL as CE donors, we show that a substantial increase in pregnenolone and aldosterone production occurs in bovine adrenal glomerulosa cells and human NCI H295R cells. Of importance here is the finding that utilization of HDL3-derived CE occurs even in the resting state. This result is in contrast to observations made in human and rat ovarian granulosa cells, in which selective CE uptake is dependent on gonadotropin stimulation (18, 23, 36). Furthermore, AngII and lipoproteins act synergistically in supporting steroidogenesis, as indicated by the fact that the combined addition of AngII and HDL3 is clearly more effective than either agent alone. We have previously demonstrated that AngII increases the mobilization of cholesterol to the inner mitochondrial membrane (37). In bovine adrenal fasciculata cells, ACTH-stimulated cortisol production was significantly greater in the presence of HDL compared with LDL (29). We report here that HDL and LDL are equally effective in supporting steroidogenesis in bovine glomerulosa cells, indicating that both the endocytic and selective pathways are functional in these cells. However, the contribution of SR-BI to LDL-derived CE selective uptake in bovine and human adrenal cells remains to be determined. Indeed, SR-BI has been shown to mediate the efficient uptake of LDL-derived CE via a selective uptake mechanism in Y1 adrenocortical cells and in SR-BI-transfected COS-7 cells (12) as well as in Chinese hamster ovary cells (10, 11). Furthermore, ovarian granulosa cells from LDL receptor knockout mice selectively internalize LDL, suggesting that SR-BI may also affect the metabolism of LDL in vivo (38). Most importantly, Leitersdorf et al. (39) have reported in earlier studies that AngII induces preferential uptake of the CE moiety over the protein moiety of LDL in bovine adrenal glomerulosa cells.
The BODIPY-CE fluorescence microscopy experiments allowed us to directly follow the fate of labeled CE within the cells. HDL3-derived CE uptake is a rapid process, visible within 5 min, in both bovine glomerulosa cells and human NCI H295R cells. In contrast to ovarian granulosa cells, in which the plasma membrane labeling has been reported to be minimal (40), the labeling of the cell membrane in both adrenal glomerulosa and human NCI H295R cells is pronounced and persistent, a finding consistent with the observation that the receptor concentrates in plasma membrane microdomains called caveolae (41, 42). Interestingly, most of the fluorescent CE accumulates within lipid droplets in areas essentially situated under the plasma membrane, where large numbers of mitochondria can be found (43, 44).
The fluorometric experiments allowed us to precisely quantitate total CE uptake while overcoming intrinsic difficulties of the microscopic approach, such as photobleaching of the probe (40). AngII pretreatment doubled HDL-derived CE uptake, both in glomerulosa cells and in human NCI H295R cells. These results are of the same order of magnitude as those obtained with [3H]CE-labeled HDL in human NCI H295R cells prestimulated with 8-bromo-cAMP (34) and in Leydig cells treated with human CG (35). In contrast, the selective uptake was found to be dramatically increased in rat ovarian granulosa cells stimulated with dibutyryl cAMP, reaching 10- to 15-fold the value measured in nonstimulated cells (23).
The specificity of the SR-BI-mediated CE uptake in adrenal cells was further demonstrated with an anti-ApoA-I antibody. Recent studies of CE selective uptake in steroidogenic tissues of ApoA-I knockout mice stress that ApoA-I plays a critical role in this process (26, 45). Using an antibody against ApoA-I, we demonstrate a significant reduction (41%) of HDL3-derived CE uptake in bovine glomerulosa cells stimulated or not with AngII. This finding is consistent with data showing that 40% of the ApoA-I bound to SR-BI can be chemically cross-linked to the receptor (7).
Our study demonstrates a tight association between the function of the selective pathway and SR-BI expression, both processes being increased to the same extent by AngII. SR-BI has been shown to be up-regulated by ACTH in the murine adrenal gland in vivo and in Y1BS1 adrenal cells in vitro, as well as by dibutyryl cAMP in ovarian granulosa cells and by 8-bromo-cAMP in human adrenal cells (14, 19, 32, 33, 34, 46). We report here, for the first time, that SR-BI is induced to a comparable level in bovine and human adrenocortical cells after challenge with AngII in a concentration-dependent manner. Importantly, although LDL-derived CE may be taken up by the cell through both the endocytic and selective pathways, the major impact of AngII stimulation is to increase preferentially the expression of SR-BI and not that of the LDL receptor and thereby to promote the selective uptake of CE. Our data indicate that LDL receptor expression is more sensitive to the cAMP signaling pathway than to the Ca2+-messenger system. This finding is in line with previous data showing that the level of induction of SR-BI expression by cAMP was consistently lower than that of the LDL receptor in human NCI H295R cells (34).
The increase in SR-BI protein content after a 24-h exposure to AngII was accompanied by a similar induction of SR-BI mRNA levels. This concomitant increase in SR-BI protein and mRNA has been reported in various steroidogenic cell types from different species after stimulation with activators of the cAMP pathway, suggesting a conservation between species of SR-BI regulation (13, 15, 28, 30, 34). The induction of SR-BI mRNA by AngII does not require de novo protein synthesis, similar to what has been observed in NCI H295R cells exposed to ACTH (34), indicating that the effects of AngII are independent of short-lived proteins. Moreover, actinomycin D abolished the increase in SR-BI mRNA triggered by AngII, suggesting a transcriptional control of the SR-BI gene by the hormone, as has been reported for ACTH in human adrenal cells (34). The transcription factor steroidogenic factor 1 (SF-1) has been reported to mediate the transcription of human and murine SR-BI genes by cAMP (47, 48). The potential role of SF-1 in AngII-induced transcription of the SR-BI gene will require further analysis.
An important question is whether the AngII-induced increase in SR-BI expression in both bovine glomerulosa cells and human NCI H295R cells is secondary to hormone-mediated changes in cellular cholesterol homeostasis or is the result of direct effects of AngII on SR-BI gene expression. Two sets of data support the latter hypothesis. First, in separate experiments, we have observed that AngII still increased SR-BI mRNA levels when glomerulosa cells were treated with aminogluthetimide to prevent cholesterol side chain cleavage and thereby cholesterol depletion (data not shown). This result speaks in favor of a direct effect of AngII on the SR-BI gene independent of cholesterol status. Consequently, an involvement of transcription factors such as sterol regulatory element binding proteins in AngII stimulation of SR-BI expression in bovine and human adrenocortical cells may be ruled out, although sterol regulatory elements have been described in the SR-BI promoter (49). It is worth mentioning that a sterol-independent regulatory element that binds the C/EBP transcription factor has been identified recently in the human LDL receptor promoter (50), in spite of the well documented regulation of this receptor by cellular cholesterol status and sterol regulatory element binding proteins (51, 52). Whether such a response element is also present in the SR-BI promoter is not known. Second, we found that the up-regulation of SR-BI by AngII is insensitive to HDL3 loading. This result is similar to that obtained in luteinized granulosa cells incubated with HDL (19) but is in contrast to what has been observed in other cell systems, in which LDL induce a down-regulation of the LDL receptor and other cholesterol-sensitive genes (53, 54, 55).
In conclusion, the present study shows that bovine and, more importantly, human adrenocortical cells take up massive amounts of HDL-derived CE as a substrate for steroid hormone production in both the basal state and under AngII stimulation. Although blood lipoprotein profiles in human and cow are in favor of LDL (56), and although several earlier reports have concluded that HDL are not effective cholesterol donors for human and bovine steroidogenic cells (17, 57, 58), our data clearly indicate that adrenocortical cells from these species efficiently take up and metabolize HDL-derived CE to support AngII-induced aldosterone biosynthesis. This finding is in keeping with in vivo studies of adrenal function in familial hypercholesterolemia and hypobeta-lipoproteinemia, which have suggested a potential role for HDL in human adrenocortical cholesterol metabolism (59, 60).
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Acknowledgments |
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Footnotes |
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1 These two authors contributed equally to this work. 
Abbreviations: AngII, Angiotensin II; ApoA-I, apolipoprotein A-I; BODIPY FL C12, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacene-3-dodecanoate; CE, cholesterol ester; h, human; HDL, high density lipoprotein; HDL3, apolipoprotein E-poor high density lipoproteins; LDL, low density lipoprotein; rec-hHDL, cholesteryl BODIPY-high density lipoprotein; SR-BI, scavenger receptor class B type I; SSC, sodium chloride/sodium citrate buffer.
Received February 8, 2001.
Accepted for publication June 11, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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M.-E. Roehrich, V. Mooser, V. Lenain, J. Herz, J. Nimpf, S. Azhar, M. Bideau, A. Capponi, P. Nicod, J.-A. Haefliger, et al. Insulin-secreting beta -Cell Dysfunction Induced by Human Lipoproteins J. Biol. Chem., May 9, 2003; 278(20): 18368 - 18375. [Abstract] [Full Text] [PDF]
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