-retinoic acid in undifferentiated THP-1 cells

We have previously shown that acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) protein content increases significantly during the human monocyte-macrophage differentiation process. To gain further insight, we used undifferentiated human monocytic THP-1 cells as a model system with which to examine whether ACAT-1 mRNA and protein content can be increased by treating cells with 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ] or with 9cis -retinoic acid (9cis -RA), two agents known to upregulate the expression of various genes during the monocyte-macrophage differentiation process. Immunoblot analysis with anti-human ACAT-1 antibodies revealed that ACAT-1 protein was increased by 2.6-fold, using 1,25-(OH) 2 D 3 at a physiological concentration (100 pM). ACAT-1 protein was also increased when using 9cis -RA, but only at relatively high concentrations (0.1–1 M). Northern blot analysis revealed that among the four ACAT-1 mRNA transcripts (2.8, 3.6, 4.2, and 7.0 kb) examined, only the 2.8and 3.6-kb transcripts were selectively increased. On the basis of enzyme assays in vitro, ACAT activity was increased 3.0-fold by using 100 nM 1,25-(OH) 2 D 3 , and 1.8-fold by using 1 M 9cis -RA. Together, our results suggest that 1,25-(OH) 3 participates in ACAT-1 gene expression during the monocyte-macrophage differentiation process. — Maung, K. K., A. Miyazaki, H. Nomiyama, C. C. Y. Chang, T-Y. Chang, and S. Horiuchi. Induction of acyl-coenzyme A:cholesterol acyltransferase-1 by 1,25-dihydroxyvitamin D 3 or 9cis -retinoic acid in undifferentiated THP-1 cells. J. Lipid Res. 2001. 42: 181–187. Supplementary key words ACAT • ACAT-1 • monocyte-macrophage differentiation Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acyl-coenzyme A (1). ACAT plays essential roles in various physiological processes including cellular cholesterol homeostasis, hepatic lipoprotein assembly, and dietary cholesterol absorption in intestine (1). Under pathological conditions, ACAT plays a crucial role in the accumulation of cholesterol esters in macrophage-derived foam cells (2). The first ACAT-1 cDNA was cloned in 1993 (3). Genomic organization of human ACAT-1 was then determined (4). The full-length ACAT-1 cDNAs in other mammalian species have subsequently been cloned (5–7). The characteristics of the ACAT-1 gene knockout mouse (8) strongly suggested the presence of another ACAT gene ( ACAT-2 ), which was eventually cloned as a homolog of ACAT-1 (9–11). ACAT-1 is ubiquitously expressed in various tissues and cells including adrenal glands, kidney (3, 5, 7), and macrophages (12), whereas ACAT-2 is expressed only in intestine and liver (9–11). Immunodepletion experiments using ACAT-1-specific antibodies (DM10) suggested that the major ACAT isozyme in adult human liver is ACAT-1 (13). To support this interpretation, immunohistochemical staining of adult human liver with DM10 indicated that human hepatocytes amply express ACAT-1 (12). We demonstrated by immunohistochemical methods that ACAT-1 is highly expressed in human atherosclerotic lesions, particularly in macrophage-derived foam cells (14). We (14) and others (15) also found that the ACAT-1 mRNAs and protein content increase significantly when bloodborne human monocytes spontaneously differentiate into macrophages. The cellular factors involved in causing increases in ACAT-1 protein and mRNAs during the monocytic differentiation process have not been identified. In an attempt to identify such factors, we used THP-1 cells as a model for human monocytes (16–19) and tested various agents for their possible effects on ACAT-1 expression. We found that 1,25-dihydroxyvitamin D 3 [1,25-(OH) 2 D 3 ] and Abbreviations: ACAT, acyl-coenzyme A:cholesterol acyltransferase; FCS, fetal calf serum; HODE, hydroxyoctadecadienoic acid; 25-OH D 3 , 25-hydroxyvitamin D 3 ; 1,25-(OH) 2 D 3 , 1,25-dihydroxyvitamin D 3 ; PC, phosphatidylcholine; 9cis -RA, 9cis -retinoic acid; PMA, phorbol 12myristate 13-acetate; RARE, retinoic acid response element; RXR, retinoid X receptor; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; VDR, vitamin D receptor; VDRE, vitamin D response element. 1 To whom correspondence should be addressed. e-mail: [email protected] at P E N N S T A T E U N IV E R S IT Y , on F ebuary 1, 2013 w w w .j.org D ow nladed fom