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Consequences of over-expression of rat Scavenger Receptor, SR-BI, in an adrenal cell model.

Reaven E, Nomoto A, Cortez Y, Azhar S - Nutr Metab (Lond) (2006)

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Geriatric Research, Education, and Clinical Center (GRECC), Department of Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, USA. eve.reaven@va.gov

ABSTRACT

Background: 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.

Methods: 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.

Results: 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 1 the formation of crater-like erosions of the surface with multiple double membraned channel structures lining the craters, and 2 dimerized formations of SR-BI lining the newly formed craters and associated double membraned channels.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus

Heavily immunostained surface crater-like formation with undulating double membranes in a cell co-transfected with SR-BI-V5 and SR-BI-cMyc constructs. Note: the numerous closely associated gold particles representing dimers of cMyc (large gold), V5 (small gold) and the combination of cMyc + V5 (see circles) with large+small gold.
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Figure 6: Heavily immunostained surface crater-like formation with undulating double membranes in a cell co-transfected with SR-BI-V5 and SR-BI-cMyc constructs. Note: the numerous closely associated gold particles representing dimers of cMyc (large gold), V5 (small gold) and the combination of cMyc + V5 (see circles) with large+small gold.

Mentions: In the high magnification micrograph of Fig. 6, we see another example of the cell surface double membraned craters described earlier in Fig. 4. In Fig. 6, however, the cell was co-transfected with SR-BI V5 (small gold) + SR-BI cMyc (large gold), and immunostained with monoclonal and polyclonal antibodies to the two tags as shown with different sized gold particles. The co-transfection in this case permits identifying SR-BI dimer formation morphologically, and provides the possibility of quantification of dimer and oligomer forms as shown in Table 3. Despite corrective measures, there is always concern about random gold clustering in solutions, and, in our effort to quantitate dimers, the possibility exists that closely associated gold particles may not necessarily represent true dimer formations. As shown in Fig. 6, it is possible to get around this issue by considering 3 possible types of closely associated gold particles (as defined in the Methods section): 2 closely associated large gold particles, 2 small gold particles, but also, closely associated large + small gold particles. In Fig. 6, and in other similar areas of SR-BI stained double membranes used to quantify dimer formation morphologically (Table 3), it is clear that the relative numbers of large + small gold combinations is substantial, and also similar in number to the other possible dimer combinations. This is a reality check: large/small gold dimer combinations indicate that antibodies for two entirely different proteins V5 and cMyc are not only present at the same cell site, but in close enough association to form numerous dimer formations when stained with their respective secondary antibodies tagged with different sized gold particles. Table 3 shows the results of quantifying SR-BI dimer combinations from cell surface related areas of SR-BI immunogold found in co-transfected cells, and indicates that when all the possible dimer formations are considered together, more than 40% of the gold found in these areas represents sites of SR-BI dimerization. SR-BI staining of regions with double membrane formations found inside transfected cells (as described in Fig. 5), also show substantial dimerization of SR-BI.


Consequences of over-expression of rat Scavenger Receptor, SR-BI, in an adrenal cell model.

Reaven E, Nomoto A, Cortez Y, Azhar S - Nutr Metab (Lond) (2006)

Heavily immunostained surface crater-like formation with undulating double membranes in a cell co-transfected with SR-BI-V5 and SR-BI-cMyc constructs. Note: the numerous closely associated gold particles representing dimers of cMyc (large gold), V5 (small gold) and the combination of cMyc + V5 (see circles) with large+small gold.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1764879&req=5

Figure 6: Heavily immunostained surface crater-like formation with undulating double membranes in a cell co-transfected with SR-BI-V5 and SR-BI-cMyc constructs. Note: the numerous closely associated gold particles representing dimers of cMyc (large gold), V5 (small gold) and the combination of cMyc + V5 (see circles) with large+small gold.
Mentions: In the high magnification micrograph of Fig. 6, we see another example of the cell surface double membraned craters described earlier in Fig. 4. In Fig. 6, however, the cell was co-transfected with SR-BI V5 (small gold) + SR-BI cMyc (large gold), and immunostained with monoclonal and polyclonal antibodies to the two tags as shown with different sized gold particles. The co-transfection in this case permits identifying SR-BI dimer formation morphologically, and provides the possibility of quantification of dimer and oligomer forms as shown in Table 3. Despite corrective measures, there is always concern about random gold clustering in solutions, and, in our effort to quantitate dimers, the possibility exists that closely associated gold particles may not necessarily represent true dimer formations. As shown in Fig. 6, it is possible to get around this issue by considering 3 possible types of closely associated gold particles (as defined in the Methods section): 2 closely associated large gold particles, 2 small gold particles, but also, closely associated large + small gold particles. In Fig. 6, and in other similar areas of SR-BI stained double membranes used to quantify dimer formation morphologically (Table 3), it is clear that the relative numbers of large + small gold combinations is substantial, and also similar in number to the other possible dimer combinations. This is a reality check: large/small gold dimer combinations indicate that antibodies for two entirely different proteins V5 and cMyc are not only present at the same cell site, but in close enough association to form numerous dimer formations when stained with their respective secondary antibodies tagged with different sized gold particles. Table 3 shows the results of quantifying SR-BI dimer combinations from cell surface related areas of SR-BI immunogold found in co-transfected cells, and indicates that when all the possible dimer formations are considered together, more than 40% of the gold found in these areas represents sites of SR-BI dimerization. SR-BI staining of regions with double membrane formations found inside transfected cells (as described in Fig. 5), also show substantial dimerization of SR-BI.

Bottom Line: 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.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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Geriatric Research, Education, and Clinical Center (GRECC), Department of Veterans Affairs Palo Alto Health Care System, Palo Alto, CA 94304, USA. eve.reaven@va.gov

ABSTRACT

Background: 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.

Methods: 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.

Results: 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 1 the formation of crater-like erosions of the surface with multiple double membraned channel structures lining the craters, and 2 dimerized formations of SR-BI lining the newly formed craters and associated double membraned channels.

Conclusion: 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.

No MeSH data available.


Related in: MedlinePlus