Consequences of over-expression of rat Scavenger Receptor, SR-BI, in an adrenal cell model
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The plasma membrane scavenger receptor, SR-BI, mediates the 'selective uptake' process by which cholesteryl esters (CE) from exogenously supplied HDL are taken up by target cells. Recent work suggests that dimer and higher order oligomeric forms of the SR-BI protein are important to this process. SR-BI has been shown to be particularly associated with microvilli and microvillar channels found at the cell surface of steroidogenic cells, and a study with the hormone stimulated adrenal gland has shown impressive changes in the size and complexity of the microvillar compartment as the mass of CE uptake (and accompanying steroidogenesis) fluctuates. In the present study, we examine a cell line in which we overexpress the SR-BI protein to determine if morphological, biochemical and functional events associated with SR-BI in a controlled cell system are similar to those observed in the intact mammalian adrenal which is responsive to systemic factors.
Y1-BS1 mouse adrenocortical cells were transiently transfected using rat SR-BI-pcDNA6-V5-His, rat SR-BI-pcDNA6-cMyc-His or control pcDNA6-V5-His vector construct using a CaPO4 precipitation technique. Twenty four hours after transfection, cells were treated with, or without, Bt2cAMP, and SR-BI expression, CE uptake, and steroidogenesis was measured. SR-BI dimerization and cell surface architectural changes were assessed using immunoelectron microscopic techniques.
Overexpression of the scavenger receptor protein, SR-BI, in Y1-BS1 cells results in major alterations in cell surface architecture designed to increase uptake of HDL supplied-CEs. Changes include  the formation of crater-like erosions of the surface with multiple double membraned channel structures lining the craters, and  dimerized formations of SR-BI lining the newly formed craters and associated double membraned channels.
These data show that overexpression of the scavenger receptor protein, SR-BI (accompanied by suitable hormone treatment and lipoproteins) in susceptible mammalian cells – is associated with increased cholesterol uptake and SR-BI dimerization within a much enlarged and architecturally complex microvillar compartment. These changes duplicate the structural, biochemical and functional changes related to the uptake of HDL CEs normally signaled by the action of ACTH on intact adrenal tissue.
KeywordsCholesteryl Ester Size Gold Particle Small Gold Particle Fasciculata Cell Zona Fasciculata Cell
very low-density lipoprotein
scavenger receptor class B, type I
cholesteryl oleolyl ether
The selective uptake of cholesteryl esters (CE) from lipoprotein particles such as HDL is a process by which the HDL core-CE is taken into cells without parallel uptake and degradation of the HDL particle itself [1, 2]. It represents a major route for the delivery of CEs to steroid producing tissues of rodents and humans [3, 4]. Scavenger receptor, class B, type I (SR-BI), a member of the CD36 family of proteins , has been identified as an HDL receptor that mediates the uptake of HDL-CEs via this process [3, 4, 5, 6, 7], and immunochemical analyses indicate that it is expressed most abundantly in steroidogenic cells and liver [8, 9, 10, 11, 12, 13]. Our published data provide evidence that the physical state of the SR-BI protein (i.e., monomer, vs dimeric and higher order oligomeric forms of SR-BI), and architectural changes in the cell surface induced by the expression of SR-BI, play major roles in the functional efficiency of the selective pathway [14, 15].
Tissues from the rat adrenal gland cortex illustrate these findings particularly well . The microvillar surface of rat adrenal zona fasciculata cells show unusual flexibility and responsiveness to hormonal simulation. In control cells, the entire surface is covered by limp and disorganized appearing microvilli; occasionally the microvilli are upright, occasionally they lie sideways, and every so often the outer external plasma membrane of one microvillus associates with an adjacent microvillus or other plasma membrane surface forming a double membraned channel-like structure . It is such channels where circulating lipoproteins (HDL and LDL) have been shown to be trapped in vivo [16, 17], and where even small VLDL can often be found . These formations are highly sensitive to hormonal control in the rat adrenal. In ACTH or 17α-E2 treatment of rats there is huge increase in the number of adrenal fasciculata cell microvilli and microvillar channels and substantial architectural changes in the entire microvillar compartment of these cells . There is, as well, a large increment in SR-BI content (adjusted for the obvious increase in microvillar volume) associated with this compartment, and there are corresponding increases in selective HDL-CE uptake. In stark contrast, adrenal tissue from animals in which dexamethasone has been used to down regulate steroidogenesis shows a rapid decline in all these features; as compared to cells from control animals, cortical cell microvilli from dexamethasone treated animals are much reduced in number and complexity, few microvillar channels can be found, SR-BI is virtually gone from the compartment, and selective cholesterol uptake of HDL-CE is barely measurable .
In subsequent studies we have shown that these various SR-BI-related changes in the adrenal have a direct relationship to SR-BI dimer formation; i.e., the level of SR-BI dimerization (i.e. dimers + oligomers of a higher order) appears invariably associated with the level of selective HDL-CE uptake, SR-BI expression and changes in microvillar compartment architecture in the adrenals of treated animals . A similar relationship between SR-BI dimerization and selective cholesterol uptake has been shown also in cells from other steroidogenic tissues such as the ovary and testis, as well as in a variety of cell lines .
In the current study, we set out to learn if the SR-BI related changes observed in the adrenal gland could be reproduced in isolated cells grown in vitro; i.e., does overexpression of SR-BI in such cells lead directly to substantial cell surface membrane changes? We chose as a cell model a mouse adrenal tumor cell line (Y1-BS1 cells), which has certain desirable characteristics: i.e., cells with modest amounts of endogenous SR-BI, yet, like intact steroidogenic tissues, these cells have other essential cellular tools permitting hormone-stimulated steroid hormone (i.e., 20α-dihydroprogesterone) production.
Iodine-125I radionucleotide (carrier free, ~629 GBq/mg; ~17 Ci/mg) was purchased from PerkinElmer NEN® Radionucleotides (Boston, MA). [1α, 2α (N)-3H] cholesteryl oleolyl ether (1.78 TBq/mmol; 48.0 Ci/mmol) was obtained from GE Health Care/Amersham Arlington Heights, IL. 20α-Dihydroprogesterone, Bt2cAMP, leupetin, PMSF, aprotinin and pepstatin A, goat anti-rabbit IgG-horse-radish peroxidase (HRP) and rabbit-anti mouse IgG-HRP were purchased from Sigma Chemical Co. (St. Louis, MO). The LumiGLO Chemiluminescent Substrate System used in Western blotting was obtained from KPL (Gaithersburg, MD). Goat-anti-mouse IgG coupled to 10 nm colloidal gold and goat anti-rabbit IgG coupled to 15 nm colloidal gold were supplied by Ted Pella, Inc., (Reading, CA). All other reagents used were of analytical grade. Apo E-free high-density hHDL3 were isolated as described previously . For uptake and internalization studies, hHDL3 preparations were conjugated with residualizing labels, i.e., 125I-labeled dilactitol tyramine ([125I]DLT) and [3H]cholesteryl oleolyl ether ([3H]COE) 
Cell culture and transient transfection
Y1-BS1, a sub-clone of Y1 mouse adrenocortical cells with detectable levels of SR-BI  were obtained from Dr. David Williams (SUNY at Stony Brook, NY) in 1998. The cells are normally cultured in Ham's F10 medium supplemented with 15% equine serum, 2.5% fetal bovine serum and 1% penicillin/streptomycin. For transient expression experiments, Y1-BS1 cells were transfected with rat SR-BI-pcDNA6-V5-His, rat SR-BI-pcDNA6-c-Myc-His or control pcDNA6-V5-His vector construct using the CaPO4 precipitation technique . Transfection efficiency was determined with β-galactosidase plasmid DNA to be about 10–15% in Y1-BS1 cells. For some studies, 24 h after transfection cells were treated with ± Bt2cAMP (2.5 mM) for 24 h. All transfected cells were used for studies 48 h after transfection.
Cultures of Y1-BS1 cells were transfected with rSR-BI-V5-pcDNA6.1 (or vector control) constructs for 48 h. Twenty four hours after transfection, some cultures were treated with Bt2cAMP (2.5 mM) for an additional 24 h. Subsequently, cells were incubated for 3–5 h in the absence (basal) or presence of Bt2cAMP (2.5 mM) ± hHDL3 (500 μg protein/ml) as indicated. Following incubation, a suitable aliquot of the medium from each sample was collected, and steroids were extracted from the medium using methylene chloride and quantified by fluorescence in 65% sulfuric acid-35% ethanol using corticosterone as a standard .
Washed transfected and non-transfected cells were lysed in lysis buffer (125 mM Tris-HCl, pH 6.8, 2% SDS, 5% glycerol, 1% 2-mercaptoethanol, 100 mM PMSF, 10 μg/ml leupeptin, 20 μg/ml aprotinin, and 5 μg/ml pepstatin A). After incubation at 37°C for 15 min, each lysate was sonicated briefly to disrupt chromation (DNA) and then used for immunoblotting.
Aliquots of cell lysates were mixed with equal volumes of 2X Laemmli sample buffer [20 mM Tris-HCl, PH 6.8, 2% SDS (w/v), 10% sucrose (w/v), and 1% 2-mercaptoethanol] and subjected to 7% SDS-PAGE . For each sample, a constant amount of protein (10–40 μg) was loaded on the gel. Protein standards (myosin, 200 kDa; β-galactosidase, 116.3 kDa; phosphorylase b, 97.4 kDa; bovine serum albumin (BSA) 66.2 kDa; and ovalbumin, 45 kDa) were also loaded on the gel. After electrophoretic separation, the proteins were transferred to Immobilon® polyvinyldene difluoride (PVDF) membranes using standard techniques. The protein blots were incubated with either polyclonal rabbit anti-SR-BI or monoclonal anti-V5 for 2 h at room temperature probed with peroxidase-labeled anti-rabbit or anti-mouse IgG and visualized using the LumiGLO Chemiluminescent Substrate System. The resulting autographic chemiluminescence was visualized for different time points (1–10 min), and appropriate films were subjected to densitometric scanning.
Selective uptake of human HDL3-derived CEs
Cultures of Y1-BS1 cells were transfected with rSR-BI-V5-pcDNA6.1 (or vector control) constructs for 48 h. Twenty four hours after transfection, some cultures were treated with Bt2cAMP (2.5 mM) for an additional 24 h at 37°C. Cells were incubated with [125I] DLT- [3H] COE hHDL3 (100 μg/ml) for 5 h at 37°C. At the end of incubation, the cells were processed to determine 125I-radioactivity, or were extracted with organic solvents and the amounts of CEs and apoproteins internalized via the endocytic and selective pathways were computed [12, 21]; results are expressed as the net mass of CEs internalized.
a) Single immunostaining technique: Y1-BS1 cells transiently transfected with a SR-BI-V5 construct were incubated with Bt2cAMP (2.5 mM) for the final 24 h. The cells were harvested, fixed as previously described [14, 15], then stained en bloc for 10 min with 0.75% tannic acid prior to embedment in LRGold resin (Ted Pella). Ultrathin sections were immunostained using V5 mAb (40 μg protein/ml) in 1% BSA for 2 h at room temperature followed by secondary antibody staining with goat anti-mouse IgG conjugated to 10 nm gold.
b) Double immunostaining technique: Cells were fixed and processed as described above. Ultrathin sections were blocked with 3% BSA and then incubated with a mixture of V5-mAb (40 μg protein/ml) and cMyc-pAb (3 μg protein/ml) in 1% BSA for 2 h at room temperature. Note: to reduce non-specific staining, the polyclonal antibody (anti-cMyc) was pre-absorbed with 10% basal Y1-BS1 cell homogenate. The secondary antibody was a mixture of goat anti-mouse IgG conjugated to 10 nm gold (for V5 staining) and goat anti-rabbit IgG conjugated to 15 nm gold (for cMyc staining).
c) dimer quantitation: SR-BI dimers were determined morphologically by estimating the distance between two gold particles considering the lengths of two primary and secondary immunoglobulin molecules and corrected for possible molecule folding . The estimated 'dimer' length between 2 gold particles was calculated to be 2 times the width of a single cross sectional slice of plasma membrane (thus two times the historically accepted value of 100A), and judgment was made on high magnification photographs (36 K × 3) where plasma membrane width was clearly defined.
Biochemical/physiological features of Y1-BS1 cells
Steroid production by control (non-transfected) and SR-BI transfected Y1-BS1 mouse adrenocortical cells
20α-Dihydroprogesterone (ng/mg cell protein/5h ± SE)
Control (vector)-transfected Y1-BS1 Cells
• Bt2cAMP (2.5 mM)
4542 ± 990
• Bt2cAMP + hHDL3
11620 ± 2041
SR-BI transfected Y1-BS1 cells
• Bt2cAMP (2.5 mM)
5303 ± 1298
• Bt2cAMP + hHDL3
26340 ± 2059¶
Selective HDL-CE uptake by control Y1-BS1 cells and cells transiently overexpressing SR-BI.
Mass of selective HDL-CE uptake (ng CE/mg cell protein/5h ± SE)
Control (vector) transfected cells
607 ± 128
• Bt2cAMP (2.5 mM)
1802 ± 463a
SR-BI transfected cells
1623 ± 295b
• Bt2cAMP (2.5 mM)
3514 ± 48c,d
Due to the clear cut functional improvement in cholesterol uptake and steroidogenesis following the use of both HDL and cAMP in the SR-BI transfected cells, this treatment was utilized in all subsequent morphological studies utlilizing this cell line.
Morphological features of control Y1-BS1 cells
Y1-BS1 cells cultured in a medium supplemented with equine serum and fetal calf serum ± stimulation with Bt2cAMP are healthy looking cells with a busy cytoplasm and an active cell surface showing occasional patches of microvilli, caveoli, coated vesicles, etc. No unusual cytoplasmic organelles or regions with unusual filamentous activity are seen. Although aliquots of native (non-transfected) Y1-BS1 cells contain substantial amounts of SR-BI as measured by Western blotting (Fig. 1), individual cells immunostained for SR-BI at the electron microscope level show only light SR-BI staining. Treatment with B2cAMP in these cells tends to increase surface microvilli and increase SR-BI.
Morphological changes in Y1-BS1 cells transfected with SR-BI
Y1-BS1 cells are difficult to transfect and despite trials with multiple reagents, transfection with the calcium phosphate precipitation technique was found to be the most efficient. In all cases discussed, B2cAMP was used for 24 h prior to cell collection which seemed to produce the most dramatic results.
Surprisingly, in the SR-BI transfected Y1-BS1 cells observed in this study, Golgi and endoplasmic reticulum areas were stained only lightly, or not at all.
It is important to note that while all transfected Y1-BS1 cells showed a disrupted surface with a production of SR-BI labeled double membraned channels, not all transfected cells present the same level of change. It is not clear whether the difference between cells reflects the fact that some cells had more time (than other cells) to develop advanced architectural changes, whether we are able to view only a limited slice through any given transfected cell, or whether certain cells are simply more resistant to change than others.
Biochemical and morphological features of SR-BI dimer formation in SR-BI transfected Y1-BS1 cells
A previous biochemical study from this laboratory identified significant levels of SR-BI monomers in native Y1-BS1 cells, with an increased concentration of monomer SR-BI and some dimer SR-BI expression in cells treated with Bt2cAMP . Whereas, the SR-BI monomers are ~3 fold increased in the SR-BI transfected cells, SR-BI dimer/oligomer forms are found in substantially higher proportions relative to their monomer forms in the transfected cells, and are especially high in those cells treated with cAMP (data not shown) – the same category of cells which show increased steroidogenic capacity and selective cholesterol uptake function in table 2 above.
Immunogold Dimer Formation Associated with SR-BI-enriched Cell Surface Sites of SR-BI-V5 + SR-BI-cMyc Transfected Y1-BS1 Cells
Total gold in region measured
Large + small hetero dimer
Small + small homodimer
Large + large homodimer
Total* dimer gold
Dimer gold as percent of total gold
This study clearly shows that transfection of Y1-BS1 cells (a mouse adrenal cell line) with rat SR-BI using a calcium phosphate precipitation technique results in cells with a dramatically altered cell surface. One version of this disrupted surface can be described as an explosion of SR-BI stained vesicles -where the original plasma membrane (including caveoli, clathrin coated pits, typical microvillar structures) no longer exists, but appears to have become part of the vesicular mass. However, in most of the transfected cells one also sees the development of multiple and quite elaborate SR-BI stained double membraned channel structures at the cell surface, which in their width, their intertwining curving nature, and their general architecture resemble the complex double membraned structures seen associated with the microvillar compartment of hormone-stimulated adrenocortical or ovarian cells in tissues of the normal rat. It is clear that the morphological plasma membrane changes we see are always associated with SR-BI (as they are in intact sections of steroidogenic tissue), and in the case of the Y1-BS1 cell preparations, only cells showing accumulations of SR-BI, show any of the physical changes noted.
In addition to these cell surface related changes, SR-BI transfected cells may also show accumulations of SR-BI intracellularly, often associated with non-identifiable vacuoles or masses found deep within the cell. Along the edges of these masses, patches of SR-BI accumulation can occasionally be identified, and, no matter how big or small the SR-BI mass, it is always associated with double membraned structures – some circular, some long, but in every way identical morphologically to the double membraned channel structures associated with the cell surface. While it is possible that these intracellularly located structures are, in fact, connected to the cell surface (at a point not visible in our sections), it seems possible that these deep sites are where SR-BI has accumulated, but has not been appropriately transported to the cell surface. Perhaps, in such cells, the production of SR-BI is so large that transport and utilization of the protein at the surface does not have sufficient time (or sufficient delivery proteins or equipment) to occur. And, like in SF9 insect cells infected with SR-BII (Reaven & Azhar, unpublished observations), the stalled delivery system deposits the protein at some site, and with the help of available cell machinery, the double membraned channels meant for the surface are actually produced in situ.
How SR-BI is transported through the cell is not yet clear. The multiple glycosylation sites of SR-BI  strongly suggest that the nascent protein must pass through trans Golgi membranes, but remarkably little SR-BI is visible in, or around, the Golgi areas, despite the use of multiple antibody types (anti SR-BI against the C-terminal or extracellular domain (ECD) or V5 or cMYc tagged SR-BI). Indeed, the most heavily transfected cells do not seem to have easily identifiable Golgi areas at all, and one wonders if transport of the newly formed protein has exhausted the Golgi membranes-which then are distributed throughout the cell and perhaps form the basis for some of the intracellular sites of SR-BI which we do see. Likewise, the existing endoplasmic reticulum is not heavily labeled for SR-BI, though in occasional cells one can find patches of wavy ER-like membranes (some even with attached ribosomes) which appear to stain lightly for SR-BI. Perhaps passage of the nascent protein through these traditional membranes is too rapid a process to show up dramatically. Or, as is always possible, the antigenic sites of SR-BI may be masked at these early stages, and it is not until the protein later dimerizes in situ, or at the cell surface, that the protein becomes available for immunostaining.
What remains clear is that cellular sites where SR-BI is prominent (e.g. surface membrane + surface or intracellular craters) show a high degree of SR-BI dimerization. Approximately 40% of the gold particles found in such areas are in close contact with each other, and this remains true whether the immuno-gold represents staining of two SR-BI V5 molecules, two SR-BI cMyc molecules, or a combination of the two different tags (therefore a dimer composed of SR-BI-V5+SR-BI cMyc). Indeed, the double tagged dimer re-enforces the idea that SR-BI, whatever its identification tag, migrates to the same cell region, and is intimately involved in the construction of the double membraned channels which result.
Finally, to what extent do the attributes of an exquisitely complex cell surface of double membraned channels composed of highly dimerized molecules of SR-BI lead to heightened cell function? We suggest that cells with coils of double membraned channels containing dimerized SR-BI are capable of taking up vast numbers of HDL particles [13, 14] in any given preparation. Such cells with their substantially increased cell surface and heightened ability to attract and trap HDL will also deliver increased CE mass to the cell interior as substrate for increased steroid production.
These data indicate that overexpression of the scavenger receptor protein, SR-BI, in a receptive mouse adrenocortical tumor cell line (Y1-BS1) leads to a complex cell surface of double membraned channels endowed with highly dimerized molecules of SR-BI. As a result, this hormone-stimulated adrenal tumor cell overexpressing SR-BI, like cells of the ACTH stimulated rat adrenal, is capable of capturing increased numbers of HDL, internalizing increased amounts of cholesteryl esters and secreting increased levels of steroid hormone.
This work was supported by NIH Grants HL-33881 and DK-56339 and funds from the Office of Research and Development, Medical Research Service, Department of Veterans Affairs.
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