Y. Huang et al., TRANSCRIPTIONAL AND POSTTRANSCRIPTIONAL REGULATION OF LDL RECEPTOR GENE-EXPRESSION IN PMA-TREATED THP-1 CELLS BY LDL-CONTAINING IMMUNE-COMPLEXES, Journal of lipid research, 38(1), 1997, pp. 110-120
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.