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

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.

LDL and cAMP cooperate to regulate the functional expression of the LRP in rat ovarian granulosa cells.

Rat ovarian granulosa rely heavily on lipoprotein-derived cholesterol for steroidogenesis, which is principally supplied by the LDL receptor- and scavenger receptor class B type I (SR-BI)-mediated pathways. In this study, we characterized the hormonal and cholesterol regulation of another member of the LDL receptor superfamily, low density lipoprotein receptor-related protein (LRP), and its role in granulosa cell steroidogenesis. Coincubation of cultured granulosa cells with LDL and N6,O2'-dibutyryl adenosine 3',5'-cyclic monophosphate (Bt2cAMP) greatly increased the mRNA/protein levels of LRP. Bt2cAMP and Bt2cAMP plus human hLDL also enhanced SR-BI mRNA levels. However, there was no change in the expression of receptor-associated protein, a chaperone for LRP, or another lipoprotein receptor, LRP8/apoER2, in response to Bt2cAMP plus hLDL, whereas the mRNA expression of LDL receptor was reduced significantly. The induced LRP was fully functional, mediating increased uptake of its ligand, alpha2-macroglobulin. The level of binding of another LRP ligand, chylomicron remnants, did not increase, although the extent of remnant degradation that could be attributed to the LRP doubled in cells with increased levels of LRP. The addition of lipoprotein-type LRP ligands such as chylomicron remnants and VLDL to the incubation medium significantly increased the progestin production under both basal and stimulated conditions. In summary, our studies demonstrate a role for LRP in lipoprotein-supported ovarian granulosa cell steroidogenesis.

Transforming growth factor-beta receptors localize to caveolae and regulate endothelial nitric oxide synthase in normal human endothelial cells.

Caveolae (sphingolipid- and cholesterol-rich, 100 nm flask-shaped invaginations of the cell membrane) serve as a nexus of cell signalling. In the present study caveolin-rich lipid raft domains were extracted from HUVEC (human umbilical-vein endothelial cells) using both density gradient and immunoprecipitation techniques, and demonstrated localization of the TGF-beta (transforming growth factor-beta) receptors TbetaRI and TbetaRII to the Cav-1 (caveolin-1)-enriched raft fractions of these normal, human endothelial cells. Immunoprecipitation demonstrated an association between TbetaRI and TbetaRII, as well as an association of the TbetaRs receptors with Cav-1 and eNOS (endothelial nitric oxide synthase), suggesting a mutual co-localization to caveolae; after treatment of HUVEC with 5 ng/ml TGF-beta1 for 15 min, however, co-precipitation of eNOS with TbetaRI, TbetaRII and Cav-1 was diminished. The loss of immunoprecipitable eNOS from Cav-1-enriched fractions was accompanied by a decrease both in phosphorylation of eNOS and in enzymatic activity (conversion of arginine into citrulline). No change in the localization of eNOS to morphologically distinct caveolae could be detected by electron microscopy after treatment of HUVEC with TGF-beta1 for 20 min. The results of these investigations provide evidence that TbetaRI interacts with eNOS in the caveolae of normal, human endothelial cells and has a regulatory function on basal eNOS enzymatic activity.

Dimerization of the scavenger receptor class B type I: formation, function, and localization in diverse cells and tissues.

This study has examined the dimeric/oligomeric forms of scavenger receptor class B type I (SR-BI) and its alternatively spliced form, SR-BII, in a diverse group of cells and tissues: i.e., normal and hormonally altered tissues of mice and rats as well as tissues of transgenic animals and genetically altered steroidogenic and nonsteroidogenic cells overexpressing the SR-B proteins. Using both biochemical and morphological techniques, we have seen that these dimeric and higher order oligomeric forms of SR-BI expression are strongly associated with both functional and morphological expression of the selective HDL cholesteryl ester uptake pathway. Rats and mice show some species differences in expression of SR-BII dimeric forms; this difference does not extend to the use of SR-B cDNA types for transfection purposes. In a separate study, cotransfection of HEK293 cells with cMyc and V5 epitope-tagged SR-BI permitted coprecipitation and quantitative coimmunocytochemical measurements at the electron microscope level, suggesting that much of the newly expressed SR-BI protein in stimulated cells dimerizes and that the SR-BI dimers are localized to the cell surface and specifically to microvillar or double membraned intracellular channels. These combined data suggest that SR-BI self-association represents an integral step in the selective cholesteryl ester uptake process.

Cholesterol uptake in adrenal and gonadal tissues: the SR-BI and 'selective' pathway connection.

A constant supply of cholesterol is needed as a substrate for steroid hormone synthesis in steroidogenic tissues. Although there are three potential sources, which could contribute to the 'cholesterol pool', needed for steroidogenesis (i.e., de novo synthesis, hydrolysis of stored cholesteryl esters and exogenous lipoproteins), current evidence suggests that plasma lipoproteins are the major source of cholesterol for steroid production in adrenal gland, ovary and, under certain conditions, testicular Leydig cells. In many species, steroid producing cells and tissues obtain this lipoprotein-cholesterol by a unique pathway in which circulating lipoproteins bind to the surface of the steroidogenic cells and contribute their cholesteryl esters to the cells by a 'selective' process. This is a process in which cholesterol is selectively absorbed while the lipoprotein remains at the cell surface. The discovery of a specific receptor for this process (scavenger receptor class B, type I, known as SR-BI) has revolutionized our knowledge about the selective uptake pathway. The present review summarizes the functional importance of the selective pathway as a bulk cholesterol delivery system for steroidogenesis, and attempts to detail the expression, regulation and characteristics of SR-BI as it is deployed in steroidogenic systems as a means of achieving cholesterol balance.

Scavenger receptor class BI and selective cholesteryl ester uptake: partners in the regulation of steroidogenesis.

The steroidogenic tissues have a special requirement for cholesterol, which is used as a substrate for steroid hormone biosynthesis. In many species this cholesterol is obtained from plasma lipoproteins by a unique pathway in which circulating lipoproteins bind to the surface of the steroidogenic cells and contribute their cholesteryl esters to the cells by a 'selective' process in which the whole lipoprotein particle does not enter the cell. This review describes the lipoprotein selective cholesteryl ester uptake process and its specific partnership with the HDL receptor, scavenger receptor class BI (SR-BI). It describes the characteristics of the selective pathway, and the molecular properties, localization, regulation, anchoring sites and potential mechanisms of action of SR-BI in facilitating cholesteryl ester uptake by steroidogenic cells.

Hormonal regulation of adrenal microvillar channel formation.

This study examined the in vivo relationship between expression of the HDL receptor scavenger receptor class B (SR-BI) and corresponding structural changes in the rat adrenocortical cell microvillar compartment. Using hormonal stimulation and withdrawal protocols, we were able to manipulate adrenal SR-BI levels and carry out qualitative and quantitative measurements correlating SR-BI expression with microvillar mass and microvillar channel formation. Young male rats were used as controls or treated with adrenocorticotropin hormone (ACTH) (24 h), 17alpha-ethinyl estradiol (17alpha-E2) (5 days), or dexamethasone (DEX) (24 h). Quantitative Western blot analysis and immunocytochemistry indicated that ACTH and 17alpha-E2 treatment greatly increased SR-BI expression in the adrenal (especially in the microvillar compartment of adrenocortical cells), whereas DEX treatment led to a decrease of SR-BI by all measurements. At the same time, striking ultrastructural changes occurred in the adrenocortical cell microvillar compartment: e.g., microvillar area and microvillar channel formation and complexity dramatically increased (compared with control values) after ACTH or 17alpha-E2 treatment, whereas the same values declined after DEX treatment. These measurements illustrate the exceptional flexibility and responsiveness of the microvillar compartment to hormonal stimuli, and suggest that regulation of SR-BI expression and structural configuration of the surface of steroidogenic cells goes hand in hand.