Amy Lindstrom ('97) Biology Dept., Davidson
Akira Miyazaki and T.Y. Chang Dept. of Biochemistry, Dartmouth Medical School
Early signs of atherosclerosis begin with the growth
of plaques in the inner wall of the vascular lumen. The study of plaque
formation can contribute significantly to understanding the pathology of
this disease (Ross, 1993). The enzyme acyl-coenzyme A: cholesterol acyltransferase
(ACAT) begins the series of processes that eventually lead to early plaque
development. This enzyme, which is present inside macrophage cells, breaks
down low density lipoproteins forming macrophage foam cells that in turn
promote the growth of plaques. The effects of chemically modified low density
lipoproteins (modified LDLs) and a synthetic glucocorticoid hormone, dexamethasone
(Dex) on ACAT protein levels were investigated in human THP-1 cells, a monocytic
cell line that differentiates to macrophage upon phorbol ester (PMA) treatment.
Western blot analysis using the anti-ACAT antibody DM10 as a probe was performed.
We found that acetylated LDL (AcLDL), oxidized LDL (OxLDL), and Dex may
activate ACAT activity by increasing ACAT protein expression.
Atherosclerosis contributes to a number of cardiovascular diseases by encouraging blood clot formation (called a thrombus) on the surface walls of vessels. This arterial disease "is responsible for 50 % of all mortality in the USA, Europe and Japan" (Ross, 1993). The severe consequences related to atherosclerosis has spurred an increased interest in trying to understand the progression of this disease.
Atherosclerosis begins with the formation of plaques in a space between the endothelial cells and the smooth muscle cells called the intima (Figure 1).
Figure 1: Diagram showing a cutaway view of the cell layers which comprise the artery. The different layers, lumenal wall and the subendothelial space (called the intima) have been identified.
Fatty substances, primarily cholesterol, contribute to the composition of plaques. The development of plaques in the intimal region roughens the characteristically smooth wall surface of the vascular lumen (Figure 2). The lumenal wall then becomes more susceptible to thrombus formation.
Figure 2: Cross sectional diagram of an artery demonstrating how plaque development disrupts the smooth endothelial lining of the vessel cavity. The cell layers and lumen have been identified.
The development of a thrombus is due in part to the clumping together of blood platelets, broken off cellular fragments from megakaryocytes. Because of their fragile nature, when platelets near a roughened area of the lumen, some tend to stick to this disturbance and break open. When breakage occurs, they release clotting factors which induce the formation of a thrombus. If the thrombus becomes dislodged (called an embolus) and floats freely through the lumen, it would eventually block off blood flow through one of the arterial branches. Thus atherosclerosis and the components which cause it increase one's risk of enduring a stroke, myocardial infarction, massive pulmonary embolism and many other cardiovascular diseases due to blocked arteries. However, clot formation does not always result in the development of atherosclerosis. The formation of clots can also denote a necessary response to an injured vessel wall by sealing the broken area and preventing excessive bleeding.
The ACAT Enzyme
The primary focus of this paper discusses the molecular basis by which early plaque formation may occur. The membrane protein found in the endoplasmic reticulum, ACAT, plays significant roles in lipoprotein assembly and secretion, in steroid hormone production, and in dietary cholesterol absorption. Previous studies focusing on the enzymatic properties of ACAT revealed that this enzyme contributes significantly to early plaque development (Chang et al., 1993). . In 1993, Chang's laboratory reported the cloning of the human ACAT cDNA by somatic cell, genetic and molecular approaches (Chang et al., 1993). Under pathological conditions, ACAT's reaction products induce the formation of foam cells whose characteristic feature is the accumulation of cholesterol esters as fatty lipid droplets in macrophages and smooth muscle cells. The accumulation of these fatty lipid droplets is the first sign of early atherosclerotic plaque development. The study of monocyte-derived macrophages (MP) can help better our understanding in this early plaque development. MPs are able to take in modified LDLs by means of the scavenger receptor (ScRc) which then causes the formation of foam cells (Figure 3).
Figure 3: Schematic view of monocyte differentiation
Monocytes migrate from the vascular lumen to the intima where they differentiate into macrophages. The scavenger receptors on the surface of MPs take up modified LDLs which get esterfied by ACAT, forming MP foam cells.
MPs' response to modified LDLs and Dex could aid
in a better understanding of atherosclerosis. The glucocorticoid Dex was
used because it can activate the ScRc which may have an effect on ACAT protein
levels (Hirsch et al., 1986). Therefore, we tested the effects of
AcLDL, OxLDL and Dex on the level of ACAT protein in MP using an established
monocyte cell line THP-1 which differentiates into macrophages upon PMA
treatment (Via et al.,1989). We discovered that AcLDL, OxLDL and
Dex enhanced the levels of ACAT protein.
THP-1 cells, an established cell line of human monocytic leukemia cells, were obtained from American Type Cell Culture and used as a model of human monocyte-macrophages. Cells were resuspended in RPMI 1640 medium that contained 10% fetal bovine serum and 10 ug/ml of gentamicin (medium A) and were grown at 37 oC in a CO 2 incubator. We seeded 3 million cells per 60 mm petri dish in 3 ml of medium A that contained an additional 50 ng/ml PMA. Twenty- four hours after seeding, 40 ug/ml OxLDL, 40 ug/ml AcLDL or 1 uM Dex were added to the dishes. The medium was changed every day and the sixth day after seeding, the cells were harvested. For harvesting, the cells were washed two times with 4 ml of phosphate buffered saline (PBS) and solubilized with 100 ul of 10% SDS.
Preparation of DM10 Antibody
The polyclonal antibodies raised against human ACAT (DM10) were prepared using the methods described by Chang et al. (1995). Briefly, a GST-ACAT fusion protein containing the first 131 amino acids from the N-terminus of the ACAT human cDNA was used. To create the GST-ACAT construct, the nucleotides that coded for amino acids 1-131 were subcloned into a GST-fusion vector to produce the in-frame fusion construct. The GST-ACAT fusion protein was expressed in bacteria and gel-purified for subsequent injection into rabbits. The ACAT specific antibodies were purified from rabbit antisera by using a GST-ACAT-coupled Sepharose 6B affinity column. The antibodies were stored at 4 oC under sterile conditions.
Western Blot Analysis
Followed the procedures as described by Cheng et al. (1994). The SDS-PAGE was run at 30 mA per gel for 5 hours. The protein transfer to nitrocellulose membrane was done at 350 mA for 5 hours. The membrane was incubated with an anti-rabbit IgG antibody that was conjugated with horse radish peroxidase. This secondary antibody was used in conjunction with enhanced chemi-luminescence for detecting the probe (Amersham).
Effects of Modified LDLs and Dex on ACAT Protein Levels
Figure 4 represents a western blot analysis using the DM10 anti-ACAT antibody as a probe. Lane 1 contains freshly harvested THP-1 cells which were grown in medium A alone. Lanes 2-5 consist of cells that were grown in medium A plus PMA treatment. In addition, 24 hours after seeding, the cells in lanes 3-5 were treated with AcLDL, Dex or OxLDL respectively. In lane 1, a slight hint of a band appears at 50 kDa. In lane 2, treated only with PMA, the intensity of the band is enhanced marginally when compared to lane 1. In lanes 3, 4, and 5 the 50 kDa band has increased several fold when compared to the control lanes (lanes 1 and 2).
Figure 4: Western Blot demonstrating
the effects of modified LDLs and Dex on ACAT protein levels
The blot was probed with the anti-ACAT DM10 antibodies. Cells were incubated in the presence (lanes 2-5) or absence (lane 1) of PMA and analyzed along with AcLDL, Dex or OxLDL (lanes 3, 4, 5 respectively). All ACAT protein bands migrated to the expected 50 kDa migration distance.
The results of the western blot analysis in figure 4 reveal that PMA treatment alone (lane 2) can increase the ACAT protein level marginally. Since PMA induces THP-1 cells to differentiate from MC to MP, it appears that this cellular process increases the ACAT protein level. The effects of modified LDLs and Dex are more striking. As previously mentioned, in lane 3 ( + AcLDL) the ACAT protein level increased when compared to the control lanes. These observations are similar to those attained by the lab at the Parke-Davis Pharmaceutical Company with northern analysis measuring ACAT mRNA levels (Wang et al. 1995). Their experimental protocol was very similar to the one we followed. Our cells were treated with 40 ug/ml of AcLDL 24 hours after seeding whereas the Parke-Davis lab added 50 ug/ml of AcLDL and waited until day 3 to do so. However, in both experiments, cells were harvested on day 5. Hence on the sixth day after seeding, the Parke-Davis lab observed that the total mRNA of ACAT was 2 times greater than that of the control line with PMA alone. With the western analysis, we got similar results at the protein level with those of Parke-Davis mRNA observations although getting a larger fold increase. However, in a paper by Wang et al. (1996), ACAT gene expression was measured by northern analysis with human MCs and a different observation was found. They observed that the mRNA levels of ACAT remained constant between control cells and those treated with AcLDL. This discrepancy between their results and ours may suggest that our THP- 1 cancer cells and Wang et al.'s wild-type cells react differently to this differentiation process. Further experiments must be done to clarify this phenomenon.
Lane 4 reveals that Dex also enhances ACAT protein levels in THP-1 cells as does OxLDL in lane 5. The synthetic glucocorticoid dexamethasone may regulate the activity of the scavenger receptor (ScRc) in MP (Hirsch et al., 1986). Therefore, activation of the ScRc could be linked to the activity of ACAT. Perhaps Dex increases the activity of the ScRc which in turn promotes the production of ACAT. Another western analysis should be done measuring the level of ScRc protein plus/minus Dex treatment. OxLDL is recognized by the ScRc and appears to upregulate ACAT protein levels also. Since both modified LDLs are recognized by the ScRc, and Dex alone affects the activity of the ScRc, this leads to a theory that an increase in the activity of the ScRc induces an increase in the amount of ACAT protein levels. Once the ScRc is activated, it may release some chemical agent that helps to initiate the transcription of the ACAT gene. Thus the increase in ACAT mRNA will result in a greater level of ACAT protein after translation.
However, because the ACAT mRNA levels only doubled compared to the drastic increase in ACAT protein levels when treated with modified LDLs and Dex, this may suggest that the mRNA levels remain relatively constant. Perhaps the ACAT mRNA is stored inside the cytoplasm until it is needed for translation. The activation of the ScRc may induce a series of signals that enable the cytoplasmic mRNA to be translated by the ribosomes. Furthermore, the ACAT mRNA could be translated multiple times until it is finally degraded. This suggests that only a small amount of ACAT mRNA would be needed to obtain a large fold increase in ACAT protein. The findings by Wang et al.(1995) and by us suggest that this may be the case.
It appears that both modified LDLs and Dex enhance ACAT protein levels, but whether they promote ACAT gene transcription and/or translation is not known. More experiments must be done with a focus on the ScRc in particularly. By testing the effects of modified LDLs and Dex on mutated forms of the ScRc, it may increase our understanding of the molecular processes occurring which seem to enhance ACAT protein levels.
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