Characterization of apolipoprotein-mediated HDL generation induced by cAMP in a murine macrophage cell line.

Abstract

Murine macrophage RAW264 were investigated for their response to lipid-free apolipoproteins. Preincubation of the cells with 300 microM dibutyryl cyclic (dBc) AMP for 16 h induced specific binding of apolipoprotein (apo) A-I to the cells and apoA-I-mediated HDL formation with cellular lipids, neither of which was detected in the absence of dBcAMP. Dose-dependent changes of the apoA-I specific binding and the apoA-I-mediated cholesterol release were largely superimposable. ApoA-II also mediated lipid release after the treatment of the cells with dBcAMP and effectively displaced the apoA-I binding to the cells. In contrast, cellular cholesterol efflux to lipid microemulsion and to 2-(hydroxypropyl)-beta-cyclodextrin was uninfluenced by the dBcAMP treatment. To induce the cellular reactivity with apoA-I, the incubation with dBcAMP required at least 6 h. Actinomycin D, cycloheximide, puromycin, and brefeldin A suppressed both the induction of apoA-I-mediated lipid release and the apoA-I specific binding to the cells. Analysis of the expression level of ABC1 mRNA by using reverse transcription-polymerase chain reaction and oligonucleotide arrays revealed that ABC1 mRNA was already expressed in the dBcAMP-untreated cells, and the dBcAMP treatment for 16 h enhanced its expression 9-13-fold. We conclude that dBcAMP selectively induces apolipoprotein-mediated cellular lipid release and accordingly high-density lipoprotein generation by inducing specific binding of apolipoprotein, but does not influence diffusion-mediated lipid efflux. The cell-apolipoprotein interaction seems to depend on cellular protein biosynthesis and transport. A substantial increase in the level of ABC1 mRNA caused by the dBcAMP treatment indicates that ATP-binding cassette transporter 1, the protein product of ABC1, may directly be responsible for the interaction, but the question about the absence of the interaction with its baseline expression level remains.