EFFECT OF REDUCED LOW-DENSITY-LIPOPROTEIN RECEPTOR LEVEL ON HEPG2 CELL CHOLESTEROL-METABOLISM

Low-density lipoproteins (LDL) are taken up by both LDL receptor (LDLr )-dependent and -independent pathways. In order to determine the impor tance of these pathways in the activity of the various enzymes that ar e important in maintaining the cellular cholesterol level in hepatic c ells, we created HepG2 cells expressing lower levels of LDLr. Thus Hep G2 cells were transfected with a constitutive expression vector (pRc/C MV) containing a fragment of LDLr cDNA inserted in an antisense manner . Stable transformants were obtained that showed significant reduction s of 42, 72 and 85% of LDLr protein levels compared with the control, as demonstrated by immunoblotting and confirmed by the LDL binding ass ay. The best inactivation was achieved with the construct containing t he first 0.7 kb of LDLr cDNA. Incubating the different HepG2 cell subt ypes with LDL showed similar association of apolipoprotein B (ape B) o r cholesteryl esters from LDL with the cells, indicating that the LDLr deficiency did not significantly affect LDL uptake by the cell. Howev er, apoB degradation was reduced significantly by 71-82% in the most L DLr-deficient HepG2 cells. We also found that 3-hydroxy-3-methylglutar yl-CoA reductase (HMGCoA red) activity is significantly increased by 3 2-35% in HepG2 cells expressing very low levels of LDLr that also demo nstrate a significant decrease of 20% in acyl-CoA: cholesterol acyltra nsferase (ACAT) activity. However, these effects are moderate compared with those observed when cells were incubated in lipoprotein-depleted medium. where a >900% increase in HMGCoA red activity and a loss of 6 0% of ACAT activity was observed. Thus, in HepG2 cells, different leve ls of LDLr affect LDL-apoB degradation, but have very little effect on LDL association, HMGCoA red and ACAT activities, revealing that LDLr is more important in the clearance of LDL-apoB than in HepG2 cell chol esterol homoeostasis, a role that should be attributable to both LDLr- dependent and -independent pathways.