. The human ACAT-1 cDNA was cloned by a somatic cell and molecular genetic approach. Chinese hamster ovary (CHO) cell mutants lacking ACAT activity (including clone AC29) were isolated (8); subsequent stable transfection experiments showed that human genomic DNAs complemented the ACAT deficiency in AC29 cells (9). A 1.2-kb human genomic DNA fragment was cloned from the stable transfectants. This fragment (designated as G2 DNA) led to the eventual cloning of a full-length human ACAT cDNA K1 (4011 bp in length). Expression of this cDNA, designated as ACAT-1, in AC29 cells complemented the ACAT deficiency of the mutant (10). Additional results showed that expressing this cDNA in insect cells, which do not contain endogenous ACAT-like activity, produced high levels of ACAT activity in vitro, confirming that this cDNA encodes the catalytic component of ACAT enzyme (11). The coding region of the ACAT gene has been mapped to chromosome 1q 25 (12). Protein sequence analysis revealed the ACAT-1 protein as a hydrophobic protein containing multiple transmembrane domains and sharing several peptide regions in common with other acyltransferases (10). Recombinant human ACAT-1 protein expressed in CHO cells has been purified to homogeneity; the homogeneous ACAT-1 protein remains catalytically active and uses cholesterol as a substrate in a highly cooperative manner (13). Homologues of human ACAT-1 cDNA have also been cloned from other species (reviewed in Ref. 1), including two yeast homologues (14,15). Disruption of the ACAT-1 gene in mice has been reported (16); the ACAT-1 gene-deficient mice exhibit marked reduction in cholesteryl ester levels in only selective tissues and not in all the tissues examined. These and other results led to the molecular cloning of ACAT-2 cDNA (17-19). The predicted amino acid sequence of ACAT-2 is homologous but distinct from that of ACAT-1. The physiological roles of ACAT-1 and ACAT-2 in various tissues of different species are currently under intense investigation by several laboratories. In humans, immunodepletion experiments suggest that the ACAT-1 protein plays major catalytic roles in hepatocytes, adrenal glands, macrophages, and kidneys, but not in the intestines (20).The 4.0-kb human ACAT-1 cDNA contains a single open reading frame of 1.65 kb. It also contains an unusually long 5Ј-untranslated region (5Ј-UTR; 1396 bp) and 965 bp of 3Ј-untranslated region. Using the coding region as probe, North-

Abstract-The acyl coenzyme A:cholesterol acyltransferase (ACAT) gene was first cloned in 1993 (Chang et al, J Biol Chem. 1993;268:20747-20755; designated ACAT-1). Using affinity-purified antibodies raised against the N-terminal portion of human ACAT-1 protein, we performed immunohistochemical localization studies and showed that the ACAT-1 protein was highly expressed in atherosclerotic lesions of the human aorta. We also performed cell-specific localization studies using double immunostaining and showed that ACAT-1 was predominantly expressed in macrophages but not in smooth muscle cells. We then used a cell culture system in vitro to monitor the ACAT-1 expression in differentiating monocytes-macrophages. The ACAT-1 protein content increased by up to 10-fold when monocytes spontaneously differentiated into macrophages. This increase occurred within the first 2 days of culturing the monocytes and reached a plateau level within 4 days of culturing, indicating that the increase in ACAT-1 protein content is an early event during the monocyte differentiation process. The ACAT-1 protein expressed in the differentiating monocytes-macrophages was shown to be active by enzyme assay in vitro. The high levels of ACAT-1 present in macrophages maintained in culture can explain the high ACAT-1 contents found in atherosclerotic lesions.

Interleukin-1 beta is believed to contribute to the pathophysiology of rheumatoid arthritis by activating collagenase gene expression. We have used a cell culture model of rabbit synovial fibroblasts to examine the molecular mechanisms of IL-1 beta-mediated collagenase gene expression. Stimulation of rabbit synovial fibroblasts with 10 ng/ml recombinant human IL-1 beta resulted in a 20-fold increase in collagenase mRNA by 12 h. Transient transfection studies using collagenase promoter-CAT constructs demonstrated that proximal sequences responded poorly to IL-1 beta, possibly due to insufficient activation of AP-1 by this cytokine. More distal sequences were required for IL-1 beta responsiveness, with a 4700 bp construct showing approximately 5-fold induction above control. To examine post-transcriptional mechanisms, transcript from a human collagenase cDNA was constitutively produced by the simian virus 40 early promoter. IL-1 beta stabilized the constitutively expressed human transcript. Furthermore, mutation of the ATTTA motifs in the 3' untranslated region of the human gene also stabilized the transcript. Finally, the rabbit collagenase 3' untranslated region destabilized a constitutively transcribed chloramphenicol acetyltransferase transcript. These data indicate that in addition to activating transcription, IL-1 beta increases collagenase transcript stability by reversing the destabilizing effects of sequences in the 3' untranslated region.

cytokine that exerts many pro-atherosclerotic effects in vivo, causes up-regulation of ACAT-1 mRNA in human blood monocyte-derived macrophages and macrophage-like cells but not in other cell types. To examine the molecular nature of this observation, we identified within the ACAT-1 P1 promoter a 159-base pair core region. This region contains 4 Sp1 elements and an IFN-␥ activated sequence (GAS) that overlaps with the second Sp1 element. In the monocytic cell line THP-1 cell, the combination of IFN-␥ and all-trans-retinoic acid (a known differentiation agent) enhances the ACAT-1 P1 promoter but not the P7 promoter. Additional experiments showed that all-trans-retinoic acid causes large induction of the transcription factor STAT1, while IFN-␥ causes activation of STAT1 such that it binds to the GAS/Sp1 site in the ACAT-1 P1 promoter. Our work provides a molecular mechanism to account for the effect of IFN-␥ in causing transcriptional activation of ACAT-1 in macrophage-like cells. ACAT1 is an intracellular enzyme responsible for catalyzing the intracellular formation of cholesteryl esters from cholesterol and long-chain fatty acyl-coenzyme A (1). In mammals, two ACAT genes have been identified (2-5). In adult human tissues, ACAT-1 is the major enzyme present in various tissues, including macrophages, liver (hepatocytes and Kupffer cells), and adrenal gland (6, 7). ACAT-1 is also present in the intestine; however, the major enzyme involved in the intestinal cholesterol absorption may be ACAT-2, which is mainly located in the apical region of the intestinal villi (7). The relative tissue distributions of ACAT-1 and ACAT-2 in mice and monkeys are not entirely consistent with those found in humans (8, 9) raising the possibility that the distribution of the two ACATs in various tissues may be species dependent. In macrophages and other cell types, a dynamic cholesterol-cholesteryl ester cycle exist; the formation of intracellular cholesteryl esters is catalyzed by ACAT-1, while the hydrolysis of cholesteryl esters is catalyzed by the enzyme neutral cholesteryl ester hydrolase (10, 11). The net accumulation of intracellular cholesteryl esters is affected at the substrate level, as well as at the levels of the enzymes ACAT and neutral cholesteryl ester hydrolase (12)(13)(14). The main mode of sterol-specific regulation of ACAT-1 has been identified at the post-translational level, involving allosteric regulation by its substrate cholesterol (1, 15). On the other hand, the cellular and molecular nature of non-sterolmediated ACAT-1 regulation remains largely unknown. Recently, using mouse macrophage-derived foam cells, Panousis and Zuckerman (12) reported that IFN-␥ increased the cellular cholesteryl ester content and reduced high density lipoproteinmediated cholesterol efflux; its cellular effects were attributed to its ability to increase ACAT-1 message (12) and to induce down-regulation of the Tangier Disease gene (the ABC1 transporter) (16). In the current work, we showed that IFN-␥ increased ACAT-1 message and protein ...

A rare form of human ACAT1 mRNA, containing the optional long 5 -untranslated region, is produced as a 4.3-kelonucleotide chimeric mRNA through a novel interchromosomal trans-splicing of two discontinuous RNAs transcribed from chromosomes 1 and 7 (Li, B. L., Li, X. L., Duan, Z. J., Lee, O., Lin, S., Ma, Z. M., Chang, C. C., Yang, X. Y., Park, J. P., Mohandas, T. K., Noll, W., Chan, L., and Chang, T. Y. (1999) J. Biol. Chem. 274, 11060 -11071). To investigate its function, we express the chimeric ACAT1 mRNA in Chinese hamster ovary cells and show that it can produce a larger ACAT1 protein, with an apparent molecular mass of 56 kDa on SDS-PAGE, in addition to the normal, 50-kDa ACAT1 protein, which is produced from the ACAT1 mRNAs without the optional long 5 -untranslated repeat. To produce the 56-kDa ACAT1, acat1 sequences located at both chromosomes 7 and 1 are required. The 56-kDa ACAT1 can be recognized by specific antibodies prepared against the predicted additional amino acid sequence located upstream of the N-terminal of the ACAT1 ORF . The translation initiation codon for the 56-kDa protein is GGC, which encodes for glycine, as deduced by mutation analysis and mass spectrometry. Similar to the 50-kDa protein, when expressed alone, the 56-kDa ACAT1 is located in the endoplasmic reticulum and is enzymatically active. The 56-kDa ACAT1 is present in native human cells, including human monocyte-derived macrophages. Our current results show that the function of the chimeric ACAT1 mRNA is to increase the ACAT enzyme diversity by producing a novel isoenzyme. To our knowledge, our result provides the first mammalian example that a trans-spliced mRNA produces a functional protein.Acyl-coenzyme A:cholesterol acyltransferase (ACAT) 1 is an intracellular enzyme that plays important roles in lipid metabolism. It catalyzes the formation of cholesteryl esters, using long-chain fatty acyl coenzyme A and cholesterol as the two substrates. In mammals, two ACAT genes have been identified (reviewed in Refs. 1-4). The first ACAT gene, acat1, was identified by isolating a human cDNA (ACAT cDNA K1) that functionally complements a Chinese hamster ovary cell mutant (clone AC29) lacking endogenous ACAT activity (5). The second ACAT gene, acat2, was identified by homology cloning, based on the nucleotide sequence of ACAT1 cDNA. The ACAT1 and ACAT2 proteins share extensive sequence homology at their C-terminal halves but not at their N-terminals. Both enzymes are integral membrane proteins. Human ACAT1 (hACAT1) contains seven transmembranes (6), whereas hACAT2 contains only two detectable transmembranes (7). A conserved histidine (His-460 in hACAT1 and His-432 in hACAT2), located within a long stretch of hydrophobic residues, may serve as an active site for ACAT catalysis (7,8). Human ACAT1 message and protein are present in many tissues and various cell types examined, including adrenal, kidney, hepatocytes, Kupffer cells, intestinal enterocytes, fibroblasts, macrophages, and neurons in the brain (5, 9 -12). In contrast, abundant ACA...

By using specific anti-ACAT-1 antibodies in immunodepletion studies, we previously found that ACAT-1, a 50-kDa protein, plays a major catalytic role in the adult human liver, adrenal glands, macrophages, and kidneys but not in the intestine. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in the intestine may be largely derived from a different ACAT protein. To test this hypothesis, we produced specific polyclonal anti-ACAT-2 antibodies that quantitatively immunodepleted human ACAT-2, a 46-kDa protein expressed in Chinese hamster ovary cells. In hepatocyte-like HepG2 cells, ACAT-1 comprises 85-90% of the total ACAT activity, with the remainder attributed to ACAT-2. In adult intestines, most of the ACAT activity can be immunodepleted by anti-ACAT-2. ACAT-1 and ACAT-2 do not form heterooligomeric complexes. In differentiating intestinal enterocyte-like Caco-2 cells, ACAT-2 protein content increases by 5-10-fold in 6 days, whereas ACAT-1 protein content remains relatively constant. In the small intestine, ACAT-2 is concentrated at the apices of the villi, whereas ACAT-1 is uniformly distributed along the villus-crypt axis. In the human liver, ACAT-1 is present in both fetal and adult hepatocytes. In contrast, ACAT-2 is evident in fetal but not adult hepatocytes. Our results collectively suggest that in humans, ACAT-2 performs significant catalytic roles in the fetal liver and in intestinal enterocytes. Acyl-coenzyme A:cholesterol acyltransferase (ACAT) 1 is an integral membrane protein located in the endoplasmic reticulum. It catalyzes the formation of cholesteryl esters from long

The first acyl-coenzyme A:cholesterol acyltransferase (ACAT) cDNA cloned and expressed in 1993 is designated as ACAT-1. In various human tissue homogenates, ACAT-1 protein is effectively solubilized with retention of enzymatic activity by the detergent CHAPS along with high salt. After using anti-ACAT-1 antibodies to quantitatively remove ACAT-1 protein from the solubilized enzyme, measuring the residual ACAT activity remaining in the immunodepleted supernatants allows us to assess the functional significance of ACAT-1 protein in various human tissues. The results showed that ACAT activity was immunodepleted 90% in liver (83% in hepatocytes), 98% in adrenal gland, 91% in macrophages, 80% in kidney, and 19% in intestines, suggesting that ACAT-1 protein plays a major catalytic role in all of the human tissue/cell homogenates examined except intestines. Intestinal ACAT activity is largely resistant to immunodepletion and is much more sensitive to inhibition by the ACAT inhibitor Dup 128 than liver ACAT activity.-Lee, O., C. C. Y. Chang, W. Lee, and T-Y. Chang. Immunodepletion experiments suggest that acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) protein plays a major catalytic role in adult human liver, adrenal gland, macrophages, and kidney, but not in intestines.

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