Identification of cAMP analogue inducible genes in RAW264 macrophages

Abstract

RNA was isolated from RAW264 cells treated with or without 8-Br-cAMP and the differential display and subtractive hybridization methods were performed. One hundred and twenty-five differentially displayed bands were identified. Upon Northern blot analysis, only three of these bands were confirmed as cAMP inducible mRNAs, named cI-1, cI-2, and cI-3 (for cAMP inducible genes 1–3). The cI-3 probe was identical to a previously known gene, gly96. Using the novel cI-1 and cI-2 partial cDNAs as probes, a mouse macrophage cDNA library was screened and the two full length genes were cloned, sequenced, and characterized as encoding large hydrophobic proteins. One hundred and fifteen partial cDNA clones from a subtractive hybridization library were also screened by Northern blot and 64 were found to be cAMP inducible. Of these, 45 represented 31 known unique genes in the GenBank nr database (cI-4–34), and 19 clones representing 15 unique sequences were not in the nr database (cI-35–49). One of the previously known genes was ABC1, the Tangier disease gene, which was identified from four independent partial cDNAs. ABC1 was upregulated in RAW cells by cAMP, concurrent with the cAMP induction of lipid efflux to apolipoprotein A1.

Introduction

The presence of monocyte-macrophage derived foam cells in the arterial intima is the earliest visible stage of atherosclerosis [1]. Since cholesterol loading of macrophages does not lead to scavenger receptor downregulation, these cells continue to take up modified lipoproteins as long as the substrate is present. Thus, cholesterol efflux is an important pathway to unload these cells and either inhibit the progression or lead to the regression of atherosclerosis. Efflux of cholesterol from macrophages in vitro occurs by at least two distinct pathways. The first, as characterized by Rothblat and others, occurs as cholesterol diffuses from a membrane with a high cholesterol to phospholipid ratio to an acceptor particle, like high density lipoprotein, with a lower cholesterol to phospholipid ratio [2]. This pathway is thought to be mediated by the scavenger receptor SR-BI protein [3]. The second pathway, as characterized by Yokoyama and others, occurs as cholesterol and phospholipids are transferred from cells to lipid free apolipoproteins (apo), or synthetic peptide analogues [4], [5]. One apparent protein mediator of this pathway is the ABC1 gene, recently identified by several labs as the Tangier disease gene [6], [7], [8], [9]. Fibroblasts from Tangier disease subjects are deficient in lipid efflux to apoA1 [10], [11]. We previously reported that cAMP analogues induce apoE and apoA1 binding and uptake and promote cholesterol efflux from the RAW264 macrophage cell line to these apo acceptors [12]. This pathway is associated with coated pit endocytosis of apoA1 and its resecretion as a nascent lipoprotein [13]. Thus a study was performed to identify cAMP inducible genes that might be playing a role in this pathway.

RNA differential display and subtractive hybridization were used to identify cAMP inducible genes. Upon Northern blot confirmation, differential display yielded only three cAMP inducible cDNAs, while subtractive hybridization was much more efficient yielding 64 inducible cDNAs. Both screens identified known genes and genes that were not present in the GenBank nr database. We obtained the full length cDNA clones for two of the unknown genes and characterize their mRNA and protein sequence, as well as the time course of their induction by cAMP. ABC1, the Tangier disease gene, was identified by four independent partial cDNAs as a cAMP inducible gene in RAW264 cells. Antisense transfections have shown that ABC1 plays a role in lipid efflux to apoA1 [6], [9]. The current catalog of cAMP induced genes may be helpful in elucidating other genes that play a role in the cAMP mediated efflux of lipids to apoA1 in RAW264 cells.