Transcriptional and post-transcriptional 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-containing immune complexes (LDL-IC) by human monocyte-derived macrophages led to the transformation of these cells into foam cells and induced a paradoxical increase in receptor-mediated binding of 125I-labeled LDL due to an increase in the number of LDL receptors (LDL-R). The same metabolic changes are also observed in PMA-treated THP-1 cells after incubation for 2 h with 150 microg/ml of immune complexes containing either native, oxidized (ox), or malondialdehyde (mda) LDL. After stimulation, PMA-treated THP-1 cells showed not only a 40-fold increase in 125I-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 LDL-R number. In this study we have investigated, in PMA-treated THP-1 cells, the regulatory mechanism(s) responsible for the increased receptor-mediated binding of LDL induced by LDL-IC. By Northern blot and nuclear run-on analysis we have shown transcriptional activation 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 LDL-R mRNA stability 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 demonstrated, 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 whether the increase in transcriptional activity of the LDL-R gene was secondary to changes in the cholesterol regulatory pool we performed experiments in which the cell cholesterol content was modified by the addition of either 25-hydroxycholesterol and mevalonate or inhibitors of ACAT activity (SA-58035 and progesterone). These experiments showed that the enhanced LDL-R expression was not affected by the addition of any of the above compounds. In conclusion, LDL-IC induced both transcriptional and post-transcriptional activation of the LDL-R gene in PMA-treated THP-1 cells and this induction was independent of the free cholesterol content of these cells.
Huang
Y
Y
Department of Medicine, Medical University of South Carolina, Charleston 29425, USA.
Ghosh
M J
MJ
Lopes-Virella
M F
MF
D000936:Antigen-Antibody Complex / Q000737:chemistry / Q000276:immunology; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism; D002788:Cholesterol Esters / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008077:Lipoproteins, LDL / Q000032:analysis*; D009000:Monocytes / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D011973:Receptors, LDL / Q000235:genetics*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014158:Transcription, Genetic
D000936:Antigen-Antibody Complex; D002788:Cholesterol Esters; D008077:Lipoproteins, LDL; D011973:Receptors, LDL; D002784:Cholesterol; D013755:Tetradecanoylphorbol Acetate