SCP-2/SCP-x gene ablation alters lipid raft domains in primary cultured mouse hepatocytes

Abstract

Although reverse cholesterol transport from peripheral cell types is mediated through plasma membrane microdomains termed lipid rafts, almost nothing is known regarding the existence, protein/lipid composition, or structure of these putative domains in liver hepatocytes, cells responsible for the net removal of cholesterol from the body. Lipid rafts purified from hepatocyte plasma membranes by a nondetergent affinity chromatography method were: i) present at 33 6 3% of total plasma membrane protein; ii) enriched in key proteins of the reverse cholesterol pathway [scavenger receptor class B type I (SR-B1), ABCA1, P-glycoprotein (P-gp), sterol carrier protein-2 (SCP-2)]; iii) devoid of caveolin-1; iv) enriched in cholesterol, sphingomyelin, GM1, and phospholipids low in polyunsaturated fatty acid and double bond index; and v) exhibited an intermediate liquid-ordered lipid phase with significant transbilayer fluidity gradient. Ablation of the gene encoding SCP-2 significantly altered lipid rafts to: i) increase the proportion of lipid rafts present, thereby increasing raft total content of ABCA1, P-gp, and SR-B1; ii) increase total phospholipids while decreasing GM1 in lipid rafts; iii) decrease the fluidity of lipid rafts, consistent with the increased intermediate liquid-ordered phase; and iv) abolish the lipid raft transbilayer fluidity gradient. Thus, despite the absence of caveolin-1 in liver hepatocytes, lipid rafts represented nearly one-third of the mouse hepatocyte plasma membrane proteins and displayed unique protein, lipid, and biophysical properties that were differentially regulated by SCP-2 expression. Although all cells synthesize cholesterol, only liver and steroidogenic tissues effect net cholesterol removal. Reverse cholesterol transport (RCT) from peripheral tissues to liver for oxidation and excretion is the major physiological pathway for the net cholesterol elimination from the body. Although increasing evidence indicates that plasma membrane microdomains, termed lipid rafts/caveolae, compartmentalize cell surface proteins that mediate HDLcholesterol uptake/efflux in peripheral cells, the existence and/or significance of such domains in hepatocyte plasma membranes is not completely clear, especially in view of the nearly negligible levels of caveolin-1 in liver. However, an overall scheme is beginning to evolve based largely on findings with peripheral cells (reviewed in Ref. 1).First, peripheral cells (fibroblasts, muscle, and endothelial cells) are rich in both lipid rafts and caveolae (a subset of lipid rafts rich in several components of the RCT pathway) but are relatively poor in intracellular cholesterol binding proteins such as sterol carrier protein-2 (SCP-2) that facilitate cholesterol retention rather than efflux (reviewed in Refs. 1, 2). Cholesterol effluxes from peripheral cells to HDL by a process facilitated by: i) the HDL receptor scavenger receptor class B type I (SR-B1) (3); ii) ABCA1, a protein that mediates cholesterol and phosphatidylcholine desorption from the pla...

Fluorescence Techniques Using Dehydroergosterol to Study Cholesterol Trafficking

“…Biochemical studies also support this concept and demonstrate that purified cholesterol-rich microdomains isolated from cultured cell (L-cell, MDCK, primary hepatocytes) represent nearly one-third of the plasma membrane, are rich in cholesterol as well as saturated/ monounsaturated fatty acylated phospholipids, and are comprised of physically distinct, liquidordered membrane phases intermediate between fluid liquid-crystalline and rigid gel phases (26,82,102,136,172,182,(215)(216)(217). Studies with purified microdomains from L-cell and MDCK plasma membranes showed that both exogenous (e.g.…”

“…When transformed cells (L-cells, MDCK cells) or primary mouse hepatocytes were cultured with DHE (micro-crystals or LUV) under conditions to maximize DHE incorporation and equilibration, biochemical fractionation by affinity chromatography of purified plasma membrane vesicles yielded sterol-rich lipid rafts/caveolae, caveolae, and lipid rafts, respectively (26,27,70,136). DHE was codistributed similarly as cholesterol into sterol-rich and -poor domains (26,27,70,82,102,135,136,172,182,196). Lipid raft/caveolae associated receptors such as the oxytocin receptor are sensitive to the structure of the sterol with which it interacts and both cholesterol and DHE, but not other sterols, are effective in reconstituting activity of this highly sterol-sensitive plasma membrane protein in cholesterol-depleted lipid rafts/caveolae (89)(90)(91).…”

“…micro-crystals, lipoproteins, LUV, or cyclodextrin complexes) may affect the distribution of fluorescent sterol to lipid rafts/caveolae or non-rafts (see Section 6.3 and respectively (26,27,70,136). DHE was codistributed similarly as cholesterol into sterol-rich and -poor domains (26,27,70,82,102,135,136,172,182,196). Lipid raft/caveolae associated receptors such as the oxytocin receptor are sensitive to the structure of the sterol with which it interacts and both cholesterol and DHE, but not other sterols, are effective in reconstituting activity of this highly sterol-sensitive plasma membrane protein in cholesterol-depleted lipid rafts/caveolae (89-91).…”

Structure and Function of the Sterol Carrier Protein-2 N-Terminal Presequence

“…Effect of SUV lipid composition on the α-helical content of proSCP-2 and SCP-2 N-terminal peptides upon membrane interaction. Circular dichroism spectra were obtained as described in Experimental Procedures: (A and B) peptide [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] Pro, (C and D) peptide Pro, (E and F) peptide Colocalization of Cy5-SCP-2 and Cy5-proSCP-2 with a plasma membrane marker in L cells. L cells were incubated in the presence of Cy5-SCP-2 (A-D) or Cy5-proSCP-2 (E-H) and the plasma membrane marker, AlexaFluor 488-cholera toxin B (AF488-CTB), and examined by laser scanning confocal microscopy (LSCM) as described in Experimental Procedures.…”

Overexpression of sterol carrier protein-2 differentially alters hepatic cholesterol accumulation in cholesterol-fed mice

“…Both in vitro and imaging studies have detected SCP-2 in plasma membrane cholesterol-rich microdomains in both caveolin-1-expressing (L-cell fibroblasts) and caveolin-1-deficient (hepatocytes) cells (14,15). In addition, SCP-2 directly interacts with plasma membrane cholesterol-rich microdomain proteins (e.g., caveolin-1) involved in cholesterol uptake/trafficking (16,17).…”