TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL REGULATION OF LDL RECEPTOR GENE-EXPRESSION IN PMA-TREATED THP-1 CELLS BY LDL-CONTAINING IMMUNE-COMPLEXES

We have previously shown that uptake of low density lipoprotein-contai ning immune complexes (LDL-IC) by human monocyte-derived macrophages l ed to the transformation of these cells into foam cells and induced a paradoxical increase in receptor-mediated binding of I-125-labeled LDL due to an increase in the number of LDL receptors (LDLR). The same me tabolic changes are also observed in PMA-treated THP-1 cells after inc ubation for 2 h with 150 mu g/ml of immune complexes containing either native, oxidized (ox), or malondialdehyde (mda) LDL. After stimulatio n, PMA-treated THP-1 cells showed not only a 40-fold increase in I-125 -labeled LDL binding but also a 40-fold increase in the immunoreactive LDL-R protein, confirming that the increase in LDL binding is due to an increase LDLR number. In this study ive have investigated, in PMA-t reated THP-1 cells, the regulatory mechanism(s) responsible for the in creased receptor-mediated binding of LDL induced by LDL-IC. By Norther n blot and nuclear run-on analysis we have shown transcriptional activ ation of the LDL-R gene with a 7-fold increase in the LDL-R mRNA level in LDL-IC stimulated cells. Due to the marked difference between the increase in LDL-R mRNA and LDL-R protein, we estimated LDLR mRNA stabi lity using a inhibitor chase method and have shown that LDL-IC did not alter the LDL-R mRNA stability in THP-1 cells. We have also demonstra ted, using cycloheximide as a inhibitor of protein synthesis, that the marked increase in LDL-R protein observed in LDL-IC-stimulated THP-1 cells resulted from de novo synthesis of LDL-R protein. To determine w hether the increase in transcriptional activity of the LDLR gene was s econdary to changes in the cholesterol regulatory pool we performed ex periments in which the cell cholesterol content was modified by the ad dition of either 25-hydroxycholesterol and mevalonate or inhibitors of ACAT activity (SA-58035 and progesterone). These experiments showed t hat the enhanced LDL-R expression was not affected by the addition of any of the above compounds. In conclusion, LDL-IC induced both transcr iptional and post-transcriptional activation of the LDL-R gene in PMA- treated THP-I cells and this induction was independent of the free cho lesterol content of these cells.