Localization of human acyl-coenzyme A: cholesterol acyltransferase-1 (ACAT-1) in macrophages and in various tissues.
Journal: 2000/February - American Journal of Pathology
ISSN: 0002-9440
Abstract:
To investigate the distribution of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) in various human tissues, we examined tissues of autopsy cases immunohistochemically. ACAT-1 was demonstrated in macrophages, antigen-presenting cells, steroid hormone-producing cells, neurons, cardiomyocytes, smooth muscle cells, mesothelial cells, epithelial cells of the urinary tracts, thyroid follicles, renal tubules, pituitary, prostatic, and bronchial glands, alveolar and intestinal epithelial cells, pancreatic acinar cells, and hepatocytes. These findings showed that ACAT-1 is present in a variety of human tissues examined. The immunoreactivities are particularly prominent in the macrophages, steroid hormone-producing cells, followed by hepatocytes, and intestinal epithelia. In cultured human macrophages, immunoelectron microscopy revealed that ACAT-1 was located mainly in the tubular rough endoplasmic reticulum; immunoblot analysis showed that the ACAT-1 protein content did not change with or without cholesterol loading; however, on cholesterol loading, about 30 to 40% of the total immunoreactivity appeared in small-sized vesicles. These vesicles were also enriched in 78-kd glucose-regulated protein (GRP 78), a specific marker for the endoplasmic reticulum. Immunofluorescent microscopy demonstrated extensive colocalization of ACAT-1 and GRP 78 signals in both the tubular and vesicular endoplasmic reticulum before and after cholesterol loading. These results raise the possibility that foam cell formation may activate an endoplasmic reticulum vesiculation process, producing vesicles enriched in the ACAT-1 protein.
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Am J Pathol 156(1): 227-236

Localization of Human Acyl-Coenzyme A:Cholesterol Acyltransferase-1 (ACAT-1) in Macrophages and in Various Tissues

From the Second Department of Pathology*
and the Department of Biochemistry,
Kumamoto University School of Medicine, Kumamoto, Japan; and the Department of Biochemistry,
Dartmouth Medical School, Hanover, New Hampshire
Accepted 1999 Sep 4.

Abstract

To investigate the distribution of acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1) in various human tissues, we examined tissues of autopsy cases immunohistochemically. ACAT-1 was demonstrated in macrophages, antigen-presenting cells, steroid hormone-producing cells, neurons, cardiomyocytes, smooth muscle cells, mesothelial cells, epithelial cells of the urinary tracts, thyroid follicles, renal tubules, pituitary, prostatic, and bronchial glands, alveolar and intestinal epithelial cells, pancreatic acinar cells, and hepatocytes. These findings showed that ACAT-1 is present in a variety of human tissues examined. The immunoreactivities are particularly prominent in the macrophages, steroid hormone-producing cells, followed by hepatocytes, and intestinal epithelia. In cultured human macrophages, immunoelectron microscopy revealed that ACAT-1 was located mainly in the tubular rough endoplasmic reticulum; immunoblot analysis showed that the ACAT-1 protein content did not change with or without cholesterol loading; however, on cholesterol loading, about 30 to 40% of the total immunoreactivity appeared in small-sized vesicles. These vesicles were also enriched in 78-kd glucose-regulated protein (GRP 78), a specific marker for the endoplasmic reticulum. Immunofluorescent microscopy demonstrated extensive colocalization of ACAT-1 and GRP 78 signals in both the tubular and vesicular endoplasmic reticulum before and after cholesterol loading. These results raise the possibility that foam cell formation may activate an endoplasmic reticulum vesiculation process, producing vesicles enriched in the ACAT-1 protein.

Abstract

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) is a key enzyme involved in cellular cholesterol metabolism. It catalyzes the formation of cholesteryl esters from cholesterol and long-chain fatty acyl-coenzyme A. 1 ACAT activities are present in various tissues such as liver, intestines, adrenal glands, and aorta and are involved in intracellular cholesterol storage, lipoprotein assembly, steroid hormone production, and dietary cholesterol absorption. 2 Previous studies showed that ACAT is a membrane-bound enzyme; its activity is found only in the membrane fractions of intracellular organelles, especially in the rough endoplasmic reticulum. 3,4 More recently, molecular probes of ACAT have become available. In 1993, the first cDNA of ACAT, designated as ACAT-1, was cloned from a human THP-1 cell cDNA library by using a somatic cell and molecular genetic approach. 5 The sequence of human ACAT-1 cDNA led to the clonings of its homologues from various other species, including mice. 6 ACAT-1 gene knockout mice were produced. 7,8 Analyses of these mice showed that ACAT activities were significantly decreased in selected tissues examined but not in the livers, strongly suggesting that one or more additional ACAT genes, distinct from the ACAT-1 gene, probably exist in mice. More recently, a different ACAT cDNA, designated ACAT-2, was cloned. 9-11 The sequence of ACAT-2 is homologous but different from that of ACAT-1. Earlier, two different ACAT-like genes were found in the simple eukaryote Sachromycetes saravesea. 12,13 The exact roles of ACAT-1 and ACAT-2 in different species remain unknown. Their physiological functions are currently under intensive investigation in several laboratories.

Recently, specific polyclonal antibodies against the ACAT-1 protein from humans or mice have been produced. 14-16 In human cells and tissues, these antibodies recognize the ACAT-1 protein as a single 50-kd protein in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 14 Using an antibody against the human ACAT-1 protein, DM10, we performed immunoblot analysis and ACAT enzyme activity assay and found that human monocyte-derived macrophages expressed high levels of ACAT-1 protein and high ACAT enzyme activity during the early stage of monocyte/macrophage differentiation. 17 We also demonstrated that the ACAT-1 protein was amply present in macrophages, but not in smooth muscle cells, within the atherosclerotic lesions of human aorta. 17 Other than the atherosclerotic lesions, little is known about the distribution of ACAT-1 protein in normal human organs at the cellular level, particularly in those that play important roles in cholesterol homeostasis. At the single cell level, using the specific anti-ACAT-1 antibody (DM10) for immunofluorescent microscopy, Chang et al reported in human melanoma cells that ACAT-1 is mainly located in endoplasmic reticulum. 14 Using a similar method, Khelef et al reported in murine macrophages that a minor portion of ACAT-1 protein resides in membranes other than the endoplasmic reticulum 16 ; the non-rough endoplasmic reticulum localization of ACAT-1 may be the trans-Golgi network. 18 The distribution of ACAT-1 at the ultrastructural level in any cell type has not been reported yet.

In the present study, we examined by immunohistochemistry the distribution and localization of ACAT-1 in various normal human tissues. In addition, to investigate the intracellular localization of ACAT-1 at both the protein and cellular levels, we performed immunoblot analysis, immunoelectron microscopy, and immunofluorescent microscopy of cultured human macrophages before and after cholesterol loading by treating cells with acetylated low density lipoprotein (AcLDL).

+++, strongly positive; ++, positive; +, slightly positive; −, not detected.

Acknowledgments

We thank Ms. Makiko Tanaka and Mr. Osamu Nakamura, Second Department of Pathology, Kumamoto University School of Medicine, for skillful technical assistance.

Acknowledgments

Footnotes

Address reprint requests to Dr. Kiyoshi Takahashi, Second Department of Pathology, Kumamoto University School of Medicine, 2–2-1 Honjo, Kumamoto 860-0811, Japan. E-mail: .pj.ca.u-otomamuk.cidem.ujiak@imoan

Footnotes

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