Effect of sterol carrier protein-2 gene ablation on HDL-mediated cholesterol efflux from cultured primary mouse hepatocytes.
Although HDL-mediated cholesterol transport to the liver is well studied, cholesterol efflux from hepatocytes back to HDL is less well understood. Real-time imaging of efflux of 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-amino)-23,24-bisnor-5-cholen-3beta-ol (NBD-cholesterol), which is poorly esterified, and [(3)H]cholesterol, which is extensively esterified, from cultured primary hepatocytes of wild-type and sterol carrier protein-2 (SCP-2) gene-ablated mice showed that 1) NBD-cholesterol efflux was affected by the type of lipoprotein acceptor, i.e., HDL3 over HDL2; 2) NBD-cholesterol efflux was rapid (detected in 1-2 min) and resolved into fast [half time (t((1/2))) = 2.4 min, 6% of total] and slow (t((1/2)) = 26.5 min, 94% of total) pools, consistent with protein- and vesicle-mediated cholesterol transfer, respectively; 3) SCP-2 gene ablation increased efflux of NBD-cholesterol, as well as [(3)H]cholesterol, albeit less so due to competition by esterification of [(3)H]cholesterol, but not NBD-cholesterol; and 4) SCP-2 gene ablation increased initial rate (2.3-fold) and size (9.7-fold) of rapid effluxing sterol, suggesting an increased contribution of molecular cholesterol transfer. In addition, colocalization, double-immunolabeling fluorescence resonance energy transfer, and electron microscopy, as well as cross-linking coimmunoprecipitation, indicated that SCP-2 directly interacted with the HDL receptor, scavenger receptor class B type 1 (SRB1), in hepatocytes. Other membrane proteins in cholesterol efflux [SRB1 and ATP-binding cassettes (ABC) A-1, ABCG-1, ABCG-5, and ABCG-8] and several soluble/vesicle-associated proteins facilitating intracellular cholesterol trafficking (StARDs, NPCs, ORPs) were not upregulated. However, loss of SCP-2 elicited twofold upregulation of liver fatty acid-binding protein (L-FABP), a protein with lower affinity for cholesterol but higher cytosolic concentration than SCP-2. Ablation of SCP-2 and L-FABP decreased HDL-mediated NBD-cholesterol efflux. These results indicate that SCP-2 expression plays a significant role in HDL-mediated cholesterol efflux by regulating the size of rapid vs. slow cholesterol efflux pools and/or eliciting concomitant upregulation of L-FABP in cultured primary hepatocytes.
Storey
Stephen M
SM
Departmens of Physiology and Pharmacology, Texas Veterinary Medical Center, Texas A & M University, College Station, TX 77843-4466, USA.
Atshaves
Barbara P
BP
McIntosh
Avery L
AL
Landrock
Kerstin K
KK
Martin
Gregory G
GG
Huang
Huan
H
Ross Payne
H
H
Johnson
Jeffery D
JD
Macfarlane
Ronald D
RD
Kier
Ann B
AB
Schroeder
Friedhelm
F
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives* / Q000378:metabolism; D018528:ATP-Binding Cassette Transporters / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D002784:Cholesterol / Q000031:analogs & derivatives* / Q000378:metabolism; D050556:Fatty Acid-Binding Proteins / Q000235:genetics / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D055786:Gene Knockout Techniques; D022781:Hepatocytes / Q000378:metabolism*; D047468:Immunoprecipitation; D007700:Kinetics; D053439:Lipoproteins, HDL2 / Q000378:metabolism; D053440:Lipoproteins, HDL3 / Q000378:metabolism*; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D010750:Phosphoproteins / Q000378:metabolism; D011485:Protein Binding; D051122:Scavenger Receptors, Class B / Q000378:metabolism; D022161:Transport Vesicles / Q000378:metabolism; D000098682:Steroidogenic Acute Regulatory Protein
C077527:7-nitrobenz-2-oxa-1,3-diazol-4-ylcholesterol; D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; C493320:Fabp1 protein, mouse; D050556:Fatty Acid-Binding Proteins; D053439:Lipoproteins, HDL2; D053440:Lipoproteins, HDL3; D010750:Phosphoproteins; C495125:Scarb1 protein, rat; D051122:Scavenger Receptors, Class B; D000098682:Steroidogenic Acute Regulatory Protein; C072087:sterol carrier proteins; D002784:Cholesterol; D009327:4-Chloro-7-nitrobenzofurazan
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