© 2000 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Induction of Ubiquitin-Conjugating Enzyme by Aggregated Low Density Lipoprotein in Human Macrophages and Its Implications for Atherosclerosis
From the Division of Molecular Hemopoiesis (J.K., Y.F., M.N., M.M.), Center for Molecular Medicine, Jichi Medical School, Tochigi; Katsuta Research Laboratory (J.K.), Hitachi Koki Co, Ltd, Ibaraki; Clinical Laboratories (N.K., A.T., I.S.), Omiya Medical Center, Jichi Medical School, Saitama; the Department of Internal Medicine (A.T.), Makioka City Hospital, Yamanashi; and Omgen Inc (N.H.), Tokyo, Japan.
Correspondence to Yusuke Furukawa, MD, Division of Molecular Hemopoiesis, Center for Molecular Medicine, Jichi Medical School, 3311-1 Yakushiji, Minamikawachi-machi, Kawachi-gun, Tochigi 329-0498, Japan. E-mail furuyu{at}jichi.ac.jp
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Abstract |
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Abstract—Recently, we have found that aggregated low density lipoprotein (agLDL) inhibits apoptosis of lipid-bearing macrophages, thereby facilitating foam cell formation and atherosclerosis. To clarify the mechanisms by which agLDL inhibits apoptosis of macrophages, we isolated the genes specifically induced by agLDL by using a subtraction-based cloning strategy. One of the cloned genes, termed low density lipoprotein (LDL)-inducible gene (LIG), encodes a human homologue of bovine ubiquitin-conjugating enzyme E2–25K. Although LIG mRNA was ubiquitously expressed among human tissues, including hematopoietic cells, the abundance of transcripts was markedly increased by agLDL treatment in activated monocytes. LIG mRNA expression was not enhanced by nonatherogenic lipoproteins such as native LDL and high density lipoprotein, suggesting a role in atherosclerosis. Polyubiquitination of intracellular proteins was observed in monocytes cultured with agLDL, which coincided with upregulation of LIG. Furthermore, ubiquitin-dependent degradation of p53, an inducer of apoptosis, was accompanied by LIG induction in agLDL-treated monocytes. The antiapoptotic effect of agLDL was abrogated by a specific proteasome inhibitor, which also increased the half-life of p53 in monocytes. These results suggest that LIG contributes to foam cell formation by the suppression of apoptosis of lipid-bearing macrophages through ubiquitination and subsequent degradation of p53.
Key Words: foam cell • apoptosis • subtraction cloning • ubiquitin • p53
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Introduction |
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Formation of the atherosclerotic lesion is a complex process involving arterial endothelium, monocytes/macrophages, and smooth muscle cells, as well as a regulatory network of growth factors and cytokines.1 Injury of arterial endothelial cells, which is caused by exposure to various agents, including oxidized LDL, leads to an increase in adherence of circulating monocytes to the endothelium and the subsequent migration of these cells into the subendothelial space, where they uptake large amounts of cholesterol ester. These cholesterol-laden macrophages are known as foam cells, which constitute the major component of a fatty streak, the earliest atherosclerotic lesion.2 An understanding of the mechanisms of foam cell formation is essential for prevention and treatment of many disorders caused by atherosclerosis and, thus, is clinically very important. However, little is known about the molecular basis of transformation of monocytes into foam cells, despite recent extensive investigations.3 4
It is now believed that foam cells are generated by the endocytosis of modified LDLs, such as oxidized LDL but not native LDL, by monocytes/macrophages.5 6 Recently, we found that apoptosis of activated monocytes is completely inhibited by one of the modified lipoproteins, aggregated LDL (agLDL),7 which is known to generate foam cells in vitro resembling those in atherosclerotic plaques.8 The suppression of apoptosis may spare lipid-bearing macrophages from activation-induced cell death, which normally takes place in such situations9 and thus contributes to foam cell formation. Although we observed that some molecular events, including downregulation of apoptosis-promoting proteases (interleukin-1ß–converting enzyme and CPP32) and upregulation of antiapoptotic cytokines (interleukin-1ß), are associated with the inhibition of apoptosis by agLDL,7 no information is available regarding the direct and specific effector molecules mediating this phenomenon. In subsequent studies, therefore, we attempted to isolate the gene(s) specifically induced by agLDL in monocytes by using a subtraction-based cloning strategy.10 In the present study, we report that one of the isolated genes encodes a human homologue of E2 ubiquitin-conjugating enzyme.11 This molecule may act as a direct mediator of the suppression of apoptosis by agLDL through polyubiquitination and subsequent degradation of cellular proteins with apoptosis-inducing properties, such as p53, suggesting that it plays an important role in foam cell formation of cholesterol-laden macrophages.
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Methods |
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Reagents
All chemical reagents, including N-acetyl-leucil-leucil-norleucinal (LLnL), were purchased from Sigma Chemical Co unless otherwise stated. Ubiquitin aldehyde was obtained from Boston Biochemical. Lipoproteins were isolated from the serum of normolipidemic donors by sequential ultracentrifugation at densities between 1.006 and 1.065 g/mL in an ultracentrifuge (model CP100a, Hitachi Koki Co) and were dialyzed at 4°C against PBS (pH 7.5). agLDL was made by vortexing for 60 seconds in Eppendorf tubes as previously described.12
Cell Preparation and Culture
Human monocytes were isolated from the peripheral
blood of healthy volunteers by an R5E elutriation cell-separating
system (Hitachi Koki Co) as described elsewhere.13
Monocyte-enriched fractions (>90% purity) were resuspended at 3 to
6x106 cells/mL in DMEM (Life Technologies Inc)
and cultured in the presence of 0.2% autologous serum.7 T
lymphocytes and granulocytes were isolated and cultured as previously
described.14 Human monocytic leukemia cell line THP-1 was
maintained in RPMI 1640 medium supplemented with 10% FCS. For
isolation of RNA for the cDNA library, THP-1 cells were seeded at an
initial concentration of 5x105 cells/mL in RPMI
1640 medium containing 1% lipoprotein-deficient serum and 100 nmol/L
phorbol 12-myristate 13-acetate (PMA) and cultured in the
absence or presence of 0.5 mg/mL agLDL for 2 days.
Isolation of LIGs by cDNA Subtraction
Isolation of LDL-inducible genes (LIGs) was
carried out with the polymerase chain reaction (PCR)-select cDNA
subtraction kit (Clontech Laboratories Inc).15
Briefly, tester and driver cDNAs were synthesized from
poly(A)+ RNA from THP-1 cells cultured with PMA
and agLDL and from THP-1 cells cultured with PMA alone, respectively.
Tester cDNA was ligated with 2 different adapters at 5' ends, and each
ligation product was separately hybridized with driver cDNA at
68°C for 12 hours. Then, the 2 hybridization samples were mixed and
incubated overnight at 68°C after adding fresh denatured driver cDNA
to further enrich the sequences specifically present in tester
cDNAs. The differentially expressed tester sequences, which possess
different adapters on their ends, were selectively amplified by nested
PCR with the use of primers corresponding to the sequences of each
adapter. The PCR products were directly inserted into a pGEM-T
vector (Promega) and subjected to DNA sequencing.16 The
nucleotide sequences have been submitted to the
GenBank/EMBL/DDBJ data bank with accession numbers AB022435 and
AB022436.
Northern Blotting
Total cellular RNA was isolated by cesium chloride
centrifugation with the use of a S100AT5 fixed-angle
rotor in a CS150GX ultracentrifuge (Hitachi Koki, Co). Samples
(10 µg per lane) were electrophoresed in 1% agarose gels containing
6% formaldehyde, 20 mmol/L MOPS, 5 mmol/L sodium acetate,
and 1 mmol/L EDTA and blotted onto Hybond N+
synthetic nylon membranes (Amersham Corp). Hybridization was carried
out according to the standard procedure.17
Western Blotting
For detection of polyubiquitinated conjugates, cultured
monocytes were collected by centrifugation at 5000 rpm
for 5 minutes and immediately resuspended in 1x SDS-PAGE loading
buffer (7% SDS, 330 mmol/L dithiothreitol, and 33% glycerol)
containing 5 µmol/L ubiquitin aldehyde and 4 mol/L urea to
stabilize ubiquitinated proteins.18 After they were boiled
for 5 minutes, the samples were directly applied to 10%
SDS-polyacrylamide gels (0.1x6.5x8.5 cm) and transferred onto
Immobilon-P polyvinylidene difluoride membranes (Millipore
Corp). After they were blocked, the membranes were incubated for 1 hour
with anti-ubiquitin monoclonal antibody (MBL Co) at a final
concentration of 5 µg/mL. The membranes were developed with the
enhanced chemiluminescence system (Amersham Corp) after incubation with
horseradish peroxidase–conjugated anti-mouse IgG antibody diluted
1:1000 for 1 hour.
To detect ubiquitination of p53, monocytes were cultured with PMA and agLDL in the presence of 50 µmol/L LLnL for the indicated periods and lysed in EBC buffer (25 mmol/L Tris-HCl, pH 8.0, 120 mmol/L NaCl, 0.5% nonidet P-40, 100 mmol/L sodium fluoride, and 200 µmol/L sodium orthovanadate) containing protease inhibitors and ubiquitin aldehyde.19 20 An equal amount (150 µg) of the samples was subjected to immunoprecipitation with anti-p53 monoclonal antibody (Transduction Laboratories). Immune complexes were separated on 10% SDS-polyacrylamide gels and blotted with anti-ubiquitin antibody as described above.
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Results |
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Isolation of LIG and Its Characterization
After 2 rounds of subtractive hybridization and nested PCR as described in Methods, we obtained 3 clones, S26–15-(2), S27–15-(1), and S27–15-(4), which were selectively expressed in agLDL-treated THP-1 cells but not in the cells cultured with PMA alone. Among them, S27–15-(1) (99 bp) and S27–15-(4) (101 bp) were found to be identical, whereas S26–15-(2) (98 bp) bore no homology to the other 2 clones. We named the former LIG and proceeded to determine its characterization. By searching the Expressed Sequence Tag (EST) database, we identified that the 3' half of S27–15-(1) and S27–15-(4) matched previously described cDNA sequences encoding bovine E2–25K ubiquitin-conjugating enzyme11 and human huntingtin interacting protein-2 (HIP-2), which was identified by virtue of its interaction with huntingtin, a molecule responsible for Huntington’s disease21 (Figure 1
, underlined). Then, we screened a cDNA library from THP-1 cells with
S27–15-(4) as a probe and obtained 2 clones encoding full-length
LIG cDNA. One clone consists of 973 nucleotides,
which encode an open reading frame corresponding to a protein of 200
amino acids; another clone is an 820-bp spliced variant, which lacks a
part of the coding region between nucleotides 64 and 216
(Figure 1). The full-length LIG protein shows 100% amino acid
identity to both human HIP-2 and bovine E2–25K ubiquitin-conjugating
enzyme. The active site cysteine is conserved at residues 92 and 41 of
the complete form and spliced variant, respectively (Figure 1, italicized), suggesting that LIG proteins act as ubiquitin-conjugating
enzymes.
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LIG/HIP-2 mRNA Expression in Normal
Human Tissues and Hematopoietic Cells
We examined the expression of LIG/HIP-2 mRNA in various
normal human tissues by using MTN blot II (Clontech). As shown in
Figure 2, 3 distinct transcripts of 5.5,
2.4, and 1.2 kb were detectable in all tissues examined. The 1.2-kb
transcript showed the strongest intensity, and the other 2 bands were
relatively weak. Among 8 tissues examined, the testis displayed the
highest expression, followed by peripheral blood
leukocytes. The abundance of LIG transcripts suggests that
LIG has some functions in peripheral blood leukocytes.
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To investigate the function of LIG in the hematopoietic system, we
examined LIG/HIP-2 mRNA expression in primary hematopoietic
cells and the effect of various stimulants on it. As shown in Figure 3
, LIG/HIP-2 transcripts were
readily detectable in T lymphocytes, monocytes, granulocytes, and bone
marrow mononuclear cells before stimulation. Because total cellular RNA
was used in this experiment, the intensity of LIG/HIP-2
messages was generally weaker than that of the
poly(A)+ blot in Figure 2, and 5.5-kb
transcript was masked by cross hybridization with 28S rRNA.
LIG/HIP-2 mRNA expression was not enhanced by any stimulants
in T lymphocytes and granulocytes (Figure 3 and data not shown).
However, the amount of 1.2-kb transcript was slightly upregulated when
monocytes were cultured with PMA, and it was markedly increased by
agLDL treatment, consistent with the method of isolation of
this gene.
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Kinetics of LIG/HIP-2 mRNA Induction
in Monocytes
Next, we analyzed kinetics of the induction of
LIG/HIP-2 mRNA in monocytes. When peripheral
blood monocytes were cultured with PMA alone for up to 48 hours, only a
marginal increase in LIG/HIP-2 transcripts was observed.
However, LIG/HIP-2 mRNA was significantly upregulated by
coculture with agLDL after 12 hours and reached a maximal level at 48
hours (Figure 4A). It is of note that the
1.2-kb transcript was selectively upregulated by agLDL, although its
significance is unclear. Then, we determined the optimal dose of agLDL
for LIG/HIP-2 mRNA induction. As shown in Figure 4B, LIG/HIP-2 transcripts were maximally expressed at a
concentration of 0.5 mg/mL. These results clearly indicate that
transcription of the LIG gene is really under the control of
agLDL. In addition, nonatherogenic lipoproteins, such as native LDL and
HDL, did not enhance LIG/HIP-2 mRNA expression in
PMA-activated monocytes (data not shown), suggesting that LIG
induction is specific for agLDL and, thus, closely related to
atherogenesis.
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Ubiquitin-Dependent Degradation of p53 in agLDL-Treated Monocytes
and Its Role in Suppression of Apoptosis
Finally, we investigated the mechanisms by which LIG induction
affects foam cell formation and atherosclerosis. Given
that LIG/HIP-2 mRNA encodes ubiquitin-conjugating enzyme, we
first investigated whether ubiquitination of intracellular proteins was
induced in agLDL-treated monocytes as a consequence of LIG induction.
As shown in Figure 5A, polyubiquitinated
conjugates as well as several medium-sized ubiquitin-protein conjugates
became detectable in agLDL-treated monocytes after 24 hours and reached
a maximal level at 48 hours, which coincided with upregulation of
LIG. Polyubiquitination was under the detection limits in
monocytes treated with PMA alone, consistent with the low
levels of LIG expression in these cells.
Then, we sought to determine the targets of polyubiquitination in
agLDL-treated monocytes. In keeping with our previous
observation,7 target molecules should possess
apoptosis-inducing properties, and accelerated degradation of
these proteins by the ubiquitin pathway results in suppression of
apoptosis in monocytes. Bearing in mind a recent report that
apoptosis caused by oxidized LDL, another inducer of foam
cells, is dependent on p53,22 we tested whether p53 was
polyubiquitinated and degraded in agLDL-treated monocytes. In the
presence of agLDL, p53 was downregulated in activated monocytes
and became almost undetectable after 48 hours [Figure 6B, LLnL(-)]. As shown in Figure 6C, DNA fragmentation, a hallmark of
apoptosis,23 was not observed in these cells,
whereas apoptosis was induced in monocytes treated with PMA
alone (data not shown, see Reference 77 ). Ubiquitination of p53 was
demonstrated when ubiquitin-dependent proteolysis was blocked by a
specific proteasome inhibitor, LLnL [Figure 6A, LLnL(+)]. In this condition, degradation of p53 was inhibited (Figure 6B), and the antiapoptotic effect of agLDL was
abrogated, as indicated by the appearance of a DNA ladder (Figure 6C). These results show that agLDL-induced suppression of
apoptosis is closely associated with ubiquitin-dependent
degradation of p53.
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Discussion |
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To clarify the molecular basis of foam cell formation of cholesterol-laden macrophages, we attempted to isolate the gene(s) specifically induced by agLDL in monocytes by using the subtraction-based technique: cDNAs from THP-1 cells cultured with PMA and agLDL were subtracted from cDNAs from THP-1 cells cultured with PMA alone, and then residual cDNAs, which were preferentially expressed in agLDL-treated cells, were amplified by PCR. One of the cloned genes, termed LIG, was found to encode a human homologue of E2 ubiquitin-conjugating enzyme.11 The predicted amino acid sequence of LIG protein perfectly matches that of HIP-2, which was recently isolated by virtue of its interaction with a protein responsible for neurodegenerative Huntington’s disease (huntingtin) by using the yeast 2-hybrid system.21 We also identified an alternatively spliced form of LIG, which has not been detected in HIP-2 and bovine E2 ubiquitin-conjugating enzyme. Although it is possible that the alternative splicing variant is specific for hematopoietic cells, further characterization is required to determine its significance. HIP-2 protein was shown to be resolved as 3 different molecular sizes (45, 28, and 25 kDa) on SDS-polyacrylamide gels.21 Most peripheral tissues express the 25-kDa form of HIP-2, whereas the 28-kDa protein is exclusively abundant in brain.21 Because the full-length HIP-2 transcript was demonstrated to encode the major 25-kDa protein, the alternative splicing form is unlikely to be functional.
Using poly(A)+ RNA from various normal human tissues, we detected LIG transcripts of 3 different sizes, 5.5, 2.4, and 1.2 kb, in all tissues as previously demonstrated for HIP-2.21 The strong signal in peripheral blood leukocytes suggests that LIG functions in the hematopoietic system. Although LIG was ubiquitously expressed among hematopoietic cells, its expression was markedly enhanced by agLDL in activated monocytes. Therefore, LIG/HIP-2 may possess certain tissue-specific or cell type–specific functions that are accomplished through ubiquitination of target proteins in each tissue or cell. In the case of monocytes, the specific function of LIG/HIP-2 may be related to foam cell formation and atherosclerosis, in view of the fact that LIG was selectively induced by agLDL but not nonatherogenic lipoproteins (native LDL and HDL).
Ubiquitination of intracellular proteins was observed in agLDL-treated monocytes, which coincided with upregulation of LIG. It is well known that protein ubiquitination is involved in a broad spectrum of cellular events such as stress response,24 cell differentiation,18 25 and programmed cell death.26 Several lines of evidence indicate that ubiquitination plays an important role in apoptosis in both positive and negative ways. For instance, Soldatenkov and Dritschilo27 reported that induction of apoptosis in Ewing’s sarcoma cells by ionizing radiation was accompanied by accumulation of intracellular ubiquitinated proteins. In contrast, Monney et al28 demonstrated that caspase-independent apoptosis was induced when ubiquitination was inhibited by blocking the activity of E1, the ubiquitin-activating enzyme. Similarly, 3 groups described rapid induction of apoptosis by the suppression of ubiquitin-mediated proteolysis with specific proteasome inhibitors.29 30 31 The latter results indicate that ubiquitination acts in favor of protecting cells from apoptosis, consistent with the putative function of HIP-2 and LIG in neuron- and lipid-bearing macrophages, respectively.
Substrates of ubiquitination include the products of tumor suppressor genes,32 oncoproteins,33 transcription factors,34 cell cycle regulators,35 and cell surface receptors.36 Ubiquitination of these molecules requires the cooperative action of 3 classes of proteins: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3).24 Although it is known that endogenous huntingtin is ubiquitinated,21 its biological significance has yet to be clarified. However, a recent report by Saudou et al37 suggests that ubiquitination acts to protect neuronal cells from apoptotic cell death; mutant huntingtin, as observed in patients with Huntington’s disease, induces neurodegeneration by an apoptotic mechanism, and ubiquitination of mutant huntingtin ameliorates this process. It is possible that HIP-2 is involved in huntingtin ubiquitination as E2 enzyme, although there is no direct evidence. A similar scenario may be applicable to LIG; ie, LIG suppresses apoptosis of lipid-bearing macrophages through ubiquitination of certain cellular proteins.
Monocytes undergo apoptosis when they are fully activated.9 It has been shown that the activation-induced death of monocytes is modulated by various factors, such as lipopolysaccharide, interleukin-1, and macrophage-colony stimulating factor.13 Our previous finding added agLDL to the list of such modulators; agLDL can inhibit apoptosis of lipid-bearing macrophages, thereby facilitating foam cell formation.7 It is reasonable to speculate that LIG contributes to this process through ubiquitin-mediated degradation of cellular proteins with apoptosis-inducing properties. In the present study, we defined p53 as a possible target of LIG, although many other proteins are ubiquitinated in agLDL-treated monocytes, as shown by the presence of polyubiquitinated conjugates and several medium-sized ubiquitin-protein conjugates on immunoblotting. p53 is a principal mediator of apoptosis in many situations, including irradiation-induced or anticancer drug–induced cell death. Recently, Kinscherf et al22 reported that p53 is also involved in apoptosis of macrophages caused by oxidized LDL. They demonstrated that p53 was detectable in apoptotic macrophages of human atherosclerotic plaques, where foam cells frequently undergo apoptosis in the later stage of atherosclerosis.
It is established that the abundance of p53 is tightly regulated by ubiquitin-mediated proteolysis. Evidence supporting this notion first came from the observation that human papilloma virus inactivates p53 for efficient replication by producing E6 protein, which mediates ubiquitination of p53 in concert with a cellular protein, E6-AP.32 Two species of E2, the ubiquitin-conjugating enzymes, have been described so far as a partner of the E6/E6-AP ubiquitin-p53 ligase (E3) complex.38 39 In the present study, we found that p53 is downregulated in agLDL-treated macrophages; this downregulation coincided with suppression of apoptosis. When ubiquitin-dependent proteolysis was blocked by a specific proteasome inhibitor, LLnL, the antiapoptotic effect of agLDL was abrogated along with stabilization of ubiquitinated p53. These results indicate that agLDL-induced suppression of apoptosis is closely associated with ubiquitin-dependent degradation of p53. Although plausible, it is unclear whether LIG is a direct mediator of this process. To confirm that LIG is a third member of the E2 enzymes for p53, we are currently investigating whether p53 is ubiquitinated by purified LIG protein in vitro and whether p53 degradation is inhibited by controlled downregulation of LIG activity in agLDL-treated cells. Furthermore, it is interesting to examine the changes in other components of the ubiquitin proteolytic system in agLDL-treated macrophages, because dramatic global increases in ubiquitin conjugate levels are seen in conjunction with upregulation of multiple components of the ubiquitin pathway in other systems, such as developmentally programmed death in Manduca sexta.26 40
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Acknowledgments |
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This study was supported in part by a grant-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan.
Received April 22, 1999; accepted August 30, 1999.
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