Evidence for sterol-independent regulation of low-density lipoprotein receptor activity in Hep-G2 cells.
The relationship between the serum factor(s)-mediated induction of low-density lipoprotein (LDL) receptor activity and changes in cellular cholesterol metabolism was examined in the human hepatoma cell line Hep-G2. Relative to incubation with serum-free media [Eagle's minimal essential medium (MEM) control], short-term (less than 8 h) incubation with medium containing 15% of either calf serum (MEM + serum) or the d greater than 1.25 fraction of calf serum (MEM + d greater than 1.25) produced a time- and concentration-dependent increase in the uptake of 125I-LDL. Immunoblotting with anti-(LDL receptor) antibodies demonstrated that this was correlated with a 2-fold increase in the amount of the mature 136,000 Da LDL receptor protein in detergent-solubilized Hep-G2 cell membranes. Incubation with MEM + serum, but not MEM + d greater than 1.25, increased the efflux of radiolabelled cholesterol from Hep-G2 cells. However, the induction of 125I-LDL uptake by MEM + d greater than 1.25 (2.3-fold) and MEM + serum (2.2-fold) was virtually identical. Addition of the d less than 1.063 lipoproteins of calf serum to MEM + d greater than 1.25 at their original or three times their serum concentration decreased the induction of 125I-LDL uptake by MEM + d greater than 1.25 by only 20-30%. Together, these results suggest that the stimulation of 125I-LDL uptake was not due to the presence of high-density lipoprotein, the absence of LDL or the stimulation of cholesterol efflux. MEM + serum stimulated 125I-LDL uptake in cells cholesterol-loaded by incubation with rat very-low-density lipoprotein with beta electrophoretic mobility (beta-VLDL). Compared to incubation with the MEM control, either MEM + serum or MEM + d greater than 1.25 produced time-dependent increases in the activity of 3-hydroxy-3-methylglutaryl-CoA reductase which also occurred in cholesterol-loaded cells. However, cholesterol biosynthesis, whether measured from 3H2O, [14C]acetate or [3H]mevalonic acid, was not increased. Incubation with MEM + serum or MEM + d greater than 1.25 did not affect [3H]oleate incorporation into cellular cholesteryl esters, hydrolysis of intracellular [3H]cholesteryl esters or the cellular mass of unesterified or esterified cholesterol. Incubation with MEM + serum or MEM + d greater than 1.25 produced a transient increase in the level of LDL receptor mRNA, reaching a maximum of 5-10-fold by 2 h and decreasing to near baseline levels by 4 h. Actinomycin D blocked the serum-factor-mediated induction of LDL receptor mRNA.(ABSTRACT TRUNCATED AT 400 WORDS)
Research Institute, Palo Alto Medical Foundation, Palo Alto, CA 94301.
J L
JL
Ellsworth
C
C
Chandrasekaran
A D
AD
Cooper
D000085:Acetates / Q000378:metabolism; D000818:Animals; D001769:Blood; D015152:Blotting, Northern; D015153:Blotting, Western; D002784:Cholesterol / Q000097:blood; D004247:DNA / Q000235:genetics; D015342:DNA Probes; D003609:Dactinomycin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006903:Hydroxymethylglutaryl CoA Reductases / Q000378:metabolism; D007700:Kinetics; D008077:Lipoproteins, LDL / Q000378:metabolism; D008114:Liver Neoplasms, Experimental / Q000378:metabolism*; D008297:Male; D008798:Mevalonic Acid / Q000378:metabolism; D019301:Oleic Acid; D009829:Oleic Acids / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis / Q000187:drug effects; D051381:Rats; D011919:Rats, Inbred Strains; D011973:Receptors, LDL / Q000378:metabolism*; D013261:Sterols / Q000378:metabolism*; D014407:Tumor Cells, Cultured
D000085:Acetates; D015342:DNA Probes; D008077:Lipoproteins, LDL; D009829:Oleic Acids; D012334:RNA, Neoplasm; D011973:Receptors, LDL; D013261:Sterols; D003609:Dactinomycin; D019301:Oleic Acid; D004247:DNA; D002784:Cholesterol; D006903:Hydroxymethylglutaryl CoA Reductases; D008798:Mevalonic Acid