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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Department of Biochemical Genetics, Medical
Research Institute, and the Department of Biomaterials Science, Faculty
of Dentistry, Tokyo Medical and Dental University; and the Department
of Viral Oncology, Cancer Institute, Japanese Foundation for Cancer
Research, Tokyo, Japan.
Activating transcription factor (ATF) 3 is a member of ATF/cyclic
adenosine monophosphate (cAMP)-responsive element binding protein (ATF/CREB) family of transcription factors and functions as a
stress-inducible transcriptional repressor. To understand the
stress-induced gene regulation by homocysteine, we investigated activation of the ATF3 gene in human endothelial cells. Homocysteine caused a rapid induction of ATF3 at the transcriptional level. This
induction was preceded by a rapid and sustained activation of c-Jun
NH2-terminal kinase/stress-activated protein kinase
(JNK/SAPK), and dominant negative mitogen-activated protein kinase
kinase 4 and 7 abolished these effects. The effect of
homocysteine appeared to be specific, because cysteine or homocystine
had no appreciable effect, but it was mimicked by dithiothreitol and
Elevated blood levels of homocysteine are
associated with an increased risk of vascular diseases, such as
arterial and venous thrombosis and arteriosclerosis.1-5
For example, patients with severe homocysteinemia resulting from an
inherited disorder of methonine metabolism exhibit significant vascular
diseases in childhood.2 Homocysteine has been reported to
influence several aspects of metabolism in cellular components of
vascular wall. It perturbs endothelial cell functions by enhancing
expression of tissue factor6 and factor V,7 or
by reducing production of activated protein C,8
thrombomodulin activity,9 von Willebrand factor,10 anti-thrombin III binding to anticoagulant
heparan sulfate on cell,11 and binding sites for tissue
plasminogen activator.12 It also increases the affinity of
apolipoprotein(a) for fibrin.13 In smooth muscle cells,
homocysteine has a positive effect on cyclin A gene transcription, thus
stimulating smooth muscle cell proliferation, which is a hallmark of
arteriosclerosis.14,15
It has been argued that homocysteine causes these multifold effects
through the reactivity of its sulfhydryl group. For example, homocysteine affects folding of proteins by reducing disulfide bonds,
thus impairing their function or preventing their export from
endoplasmic reticulum (ER).16-18 In addition, with
catalytic help from the copper ion, homocysteine produces superoxide
and hydrogen peroxide, which injure cells by oxidative
stress.4,5 These processes involve activation of signal
transduction pathway(s) as well as a cascade of gene expression. An
understanding of these processes at the molecular level is required to
elucidate the mechanism of cell injury by homocysteine. Recently,
dramatically altered gene expression has been reported in endothelial
cells exposed to homoysteine.16-18 Among up-regulated
genes are glucose responsive protein (GRP) 78/immunoglobulin binding
protein (Bip), nicotinamide adenine dinucleotide
(NAD)-dependent methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase,
activating transcription factor (ATF) 4, reducing agents and
tunicamycin-responsive protein, Herp, and a few novel genes. GRP78/Bip
is an ER-resident chaperon that is induced by agents or conditions
known to elicit ER stress.16-18 Thus, it is suggested that
homocysteine alters the cellular redox state and causes ER stress.
However, we have limited knowledge of the cellular responses to
homocysteine in terms of signal transduction and gene expression that
are not directly linked to the expression of resident ER protein.
Members of the ATF/cyclic adenosine monophosphate (cAMP)
responsive element binding (CREB) family of transcription factors recognize a consensus DNA sequence, TGACGTCA; have structurally similar
basic region/leucine zipper domains; and interact selectively with each
other to form heterodimers through their leucine zipper region.19-22 Members of the ATF1/CREB subfamily contain
similar phosphorylation sites and stimulate transcription in response to cAMP or calcium influx.23-25 In contrast, members of
the ATF2/CRE-binding protein 1 (CRE-BP1) subfamily share
similarity in the first 100 N-terminal and the last 13 C-terminal
residues but do not have A-kinase consensus sites. ATF3, a member of
the ATF2/CRE-BP1 subfamily, is thus unresponsive to elevated cAMP, but
is induced by stress stimuli such as carbon tetrachloride,
ischemia/reperfusion, and convulsion.20,26,27 It is also
highly induced in regenerating-liver or adenovirus-E1-transformed
cells.28,29 Although ATF3 functions as a transcriptional
repressor,26 its biological significance has not been well defined.
In this study, we investigated the ATF3 gene induction in endothelial
cells exposed to homocysteine and identified the cis-element of the
gene responsible for the induction. We also showed that homocysteine
activated the c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) signaling pathway through its reductive stress. The significance of the stress-induced ATF3 gene expression and
its involvement in the homocysteine toxicity are discussed.
Reagents and plasmids
Cell culture, stimulation by homocysteine, and RNA
preparation
Northern blot Total RNA (10 µg) was denatured and separated on a 1% agarose gel in 10 mmol/L sodium phosphate buffer, pH 7.0. The RNA was then transferred onto a Hybond-N+ nylon-membrane (Amersham Life Science, Buckinghamshire, UK) and baked at 80°C for 2 hours. The membrane was prehybridized in Rapid-hyb solution (Amersham Life Science) at 42°C for 2 hours and then hybridized with radiolabeled cDNA probe at 42°C for 2 hours. After washing twice in 0.1 × SSC/0.1% sodium dodecyl sulfate (SDS) at 65°C for 20 minutes, the membrane was exposed and analyzed by Bas 2500 Bio-image analyzer (Fujifilm Co, Tokyo, Japan). DNA sequences for full-length human ATF3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), 540 base pairs (bp) and 1.2 kilobases, respectively, were radiolabeled with [ -32P] deoxycytidine triphosphate (6000 Ci/mmol, Amersham) with the use of a random primer-labeling kit
(Takara, Otsu, Japan) and were used as probes.
Nuclear run-on assay HUVECs (1 × 107cells) were treated with 3 mmol/L homocysteine for 1 and 2 hours. Cells were collected and treated in 1 mL of NP-40 lysis buffer (10 mmol/L Tris-HCl, pH 7.4, 10 mmol/L NaCl, 3 mmol/L MgCl2, and 0.5% NP-40) at 4°C for 10 minutes, and their nuclei were pelleted at 3000 rpm for 5 minutes. The lysis procedure was repeated twice, and the nuclei were resuspended in 100 µL of storage buffer (50 mmol/L Tris-HCl, pH 8.3, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, and 40% glycerol) and frozen in liquid nitrogen. Elongation of nascent RNA chains was initiated by mixing the above nuclear suspension with 100 µL of reaction buffer (10 mmol/L Tris-HCl, pH 8.0, 5 mmol/L MgCl2, 300 mmol/L KCl, 0.5 mmol/L each of adenosine triphosphate (ATP), cytidine 5'-triphosphate, guanosine 5'-triphosphate, and 100 µCi of-32P] uridine triphosphate (3000 Ci/mmol,
Amersham) and incubating at 30°C for 30 minutes. RNA synthesis was
terminated by incubation with 5 µg/mL RNase-free DNase I (Boehringer
Mannheim Biochemicals, Mannheim, Germany) at 30°C for 10 minutes. The mixture was then digested with proteinase K (200 µg/mL)
in 10 mmol/L Tris-HCl, pH 7.4, 5 mmol/L EDTA, and 1% SDS at 50°C for
1 hour and extracted twice with phenol-chloroform after adding 30 µg
transfer RNA as a carrier. Radiolabeled RNA was further purified by
ethanol precipitation and dissolved in 100 µL of 50% formamide,
5 × SSC, 5 × Denhardt's solution, and 0.1% SDS.
Approximately 20 µg of human ATF3 and GAPDH cDNA were immobilized on
a Hybond-N membrane with the use of a slot-blot apparatus. After
denaturation, baking, and prehybridization of the membrane with
Rapid-hyb solution, it was hybridized with radiolabeled RNA at 42°C
overnight. The membranes were washed twice in 0.1 × SSC/0.1% SDS
at 65°C for 20 minutes and analyzed by Bas 2500 Bio-image analyzer.
Preparation of whole-cell extracts Whole-cell extracts from the homocysteine-treated HUVECs were obtained as follows. Briefly, cells (1.5 × 106 cells) were washed in phosphate-buffered saline (PBS), resuspended in 50 µL of lysis buffer (50 mmol/L Hepes-KOH, pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, 1.5 mmol/L MgCl2, 1 mmol/L egtazic acid, 0.1 mmol/L phenylmethanesulfonyl fluoride (PMSF), 10 µg/mL each leupeptin and aprotinin, 200 µmol/L sodium vanadate, 100 mmol/L NaF, and 10% glycerol), and incubated on ice for 10 minutes. The cells were centrifuged at 10 000 rpm for 10 minutes, and the supernatant was taken as whole-cell extract. The amounts of protein were quantitated by Lowry method.30Western blot We separated 20 µg of whole-cell extracts from the homocysteine-treated HUVECs on a 10% SDS-polyacrylamide gel and transferred it onto a nitrocellulose membrane. After blocking with PBS containing 5% skimmed milk, the membrane was incubated with the indicated primary antibody in the same buffer. After washing, the membrane was further incubated with alkaline phosphatase-conjugated anti-IgG antibody. The reacted band was visualized by chemiluminescence as in the protocol from Tropix Inc (Bedford, MA).Immunostaining of cells HUVECs grown and treated as described in the figure legends were fixed by immersion in cold acetone/methanol (1:1) for 10 minutes and then rinsed with 70% ethanol, 50% ethanol, and PBS. After blocking in PBS containing 2% bovine serum albumin, 0.2% Tween 20, and 6.7% glycerol at room temperature for 1 hour, the cells were incubated with antibody for 1 hour, washed with PBS, and sequentially incubated with fluorescence-labeled secondary antibody. For staining of nuclei, cells were treated with 4, 6-diamidino-2-phenylindole dihydrochloride (DAPI, Nacalai Tesque, Kyoto, Japan).Construction of the reporter gene plasmids Human ATF3 gene promoter-luciferase reporter plasmid pLuc-1850 was constructed by insertion of the promoter sequence from the 1850
to +34 region of pATF3CAT into a basic vector (PGV-B2, Toyo Ink, Tokyo,
Japan) containing the firefly luciferase gene.31 Deletion mutants containing various lengths of the 5' flanking region
were obtained by insertion of sequences from 632,110, and 84 to
+34 into the basic vector to obtain pLUC-632, pLUC-110, and pLUC-84,
respectively. Then pLUC-1850m, which contains mutations of the
ATF/cAMP responsive element (CRE) site from 92 to 85 of the gene,
was prepared with the use of the plasmid pLUC-1850 as the matrix by an
overlap extension polymerase chain reaction (PCR)
protocol.32 Briefly, 2 separate PCR products, 1 for each half of the hybrid, were generated with an antisense
(5'-CCAGGCTGACCAAATGCTAT-3') or a sense (5'-ATAGCATTTGGTCAGCCTGG-3')
mutated primer and outside primers. Two products of 1740 bp and 140 bp
were mixed, and the second PCR was performed with the use of the 2 outside primers. The insert was sequenced to confirm the mutations from
the normal sequence of 5'-TTACGTCA-3' to 5'-TTTGGTCA-3'.
Transient expression of the human ATF3-luciferase gene HUVECs were grown in EGM-2 culture medium to 70% to 80% confluency in a 60-mm dish (3 × 105 cells) prior to transfection. Plasmid DNA (2 to 5 µg) was vortex-mixed with 20 µL of SuperFect (Qiagen Inc, Chatsworth, CA) in 150 µL of EGM-2; then 1 mL of EGM-2 with 2% FBS was added. The cells were exposed to this solution at 37°C for 2 hours. After replacement of the transfection solution with 2 mL of fresh medium and further incubation for 2 hours, the cells were treated with 3 mmol/L homocysteine for 4 hours. The medium was then changed to 2 mL of fresh EGM-2 without homocysteine, and the cells were further incubated at 37°C for another 20 hours. Cell extracts were obtained by adding 0.4 mL of lysis buffer and centrifugation at 12 000 rpm for 1 minute, and the supernatants were assayed for firefly and sea pansy luciferase activity by means of a dual luciferase reporter assay system (Promega). As an internal control of transfection and expression, pRL-CMV (Toyo Ink) containing the sea pansy luciferase gene was used.Preparation of nuclear extracts of HUVECs Nuclear extracts were prepared from the untreated or homocysteine-treated HUVECs (2 × 107 cells). Cells were washed in PBS, pelleted, and resuspended in an equal volume of lysis buffer (10 mmol/L Tris-HCl, pH8.0, 60 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 0.1 mmol/L PMSF, and 0.5% NP-40). After incubation on ice for 5 minutes, the lysates were centrifuged at 2500 rpm in a microfuge for 4 minutes. The pelleted nuclei were briefly washed with lysis buffer without NP-40 and resuspended in an equal volume of extraction buffer (20 mmol/L Tris-HCl, pH8.0, 420 mmol/L NaCl, 1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, and 25% glycerol); then 5 mol/L NaCl was added to a final concentration of 400 mmol/L. After further incubation on ice for 10 minutes, the nuclei were briefly vortexed and centrifuged at 14 000 rpm for 5 minutes. The supernatant was used as nuclear extract.Electrophoretic mobility shift assay Nuclear extracts (2 µg of protein) of the control and homocysteine-treated HUVECs were incubated in a final volume of 20 µL of binding buffer (10 mmol/L Hepes-KOH, pH 7.9, 60 mmol/L KCl, 0.5 mmol/L EDTA, 5 mmol/L MgCl2, 1 mmol/L DTT, 0.1 mmol/L PMSF, 5 mmol/L -mercaptoethanol) containing 1 µg of polydI-dC and 0.5 ng
of radiolabeled DNA probe at room temperature for 30 minutes. For
supershift assays, each antibody was added and incubated for another 30 minutes. DNA probe was obtained by annealing 0.1 µg each of
oligonucleotides for sense and antisense sequences of ATF3 promoter
from 99 to 69, and radiolabeled with either 25 µCi
 -32P] ATP (6000 Ci/mmol) and 10 U polynucleotide kinase
or 50 µCi -32P] deoxyadenosine triphosphate (6000 Ci/mmol) and 2 U Klenow fragment of DNA polymerase. Mutant
oligonucleotides used for the competition experiment contained
substitutions in the normal sequence of 5'-TTACGTCA-3' to yield
5'-TTTGGTCA-3'. For in vitro dephosphorylation, nuclear extract was
first pre-incubated in the binding buffer with 5 mmol/L glucose and 1 U
hexokinase in order to deplete endogenous ATP, and then incubated with
2.2 unit of calf intestine alkaline phosphatase for 30 minutes. Binding
mixture was applied onto a 4% or 5% polyacrylamide slab gel in
Tris-borate EDTA buffer. After electrophoresis, the gel was dried on a
3MM Whatmann paper and visualized by Fuji Bas 2500 image analyzer.
Immunoprecipitation Nuclear extracts (60 µg of protein) prepared from the control cells and cells treated with 3 mmol/L homocysteine for 4 hours were incubated with 1 µg anti-ATF2 antibody in 100 µL of immunoprecipitation buffer (20 mmol/L Hepes-KOH, pH 7.5, 100 mmol/L KCl, 6 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L DTT, 0.5% NP-40, 0.1 mmol/L PMSF, 10 µg/mL each of antipain and leupeptine) at 4°C for 1 hour with gentle rocking. After addition of 20 µL of 50% (vol/vol) protein G-Sepharose beads (Pharmacia Biotech, Little Chalfont, UK) and further incubation at 4°C for 1 hour, the immune complexes were precipitated and washed 3 times. Specific proteins in the immune complexes were dissociated with 20 µL of IP buffer containing 0.8% deoxycholate and 1.2% NP-40. After centrifugation, 3 µL of the supernatants were analyzed by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.Data analysis Statistical significance was determined by Student paired t tests, and P values were shown.
Induction of ATF3 gene expression by homocysteine As shown in Figure 1A, treatment of HUVECs with homocysteine caused a rapid induction of ATF3 gene expression. The mRNA level reached its maximum in 2 to 4 hours, then gradually decreased up to 8 hours. Western blot analysis revealed that the ATF3 protein was also induced 4 to 6 hours after exposure (Figure 1B). When the concentration of homocysteine was titrated, it was found that a relatively high dose of homocysteine (1 to 3 mmol/L) was required for the maximum induction, although significant induction was observed at lower concentrations (0.05 to 0.3 mmol/L) (Figure 1C). Since the induction of ATF3 mRNA was almost abolished by pretreatment of cells with actinomycin D (Figure 1C; 3+ActD), we next performed a nuclear run-on assay. Figure 1D shows that nuclei from the homocysteine-treated cells exhibited higher activity of the ATF3 gene transcription than those from the control cells. These data clearly indicated that ATF3 is one of the immediate early responsive genes activated by homocysteine and its induction is regulated mainly at a transcriptional level.
Homocysteine-induced ATF3 gene expression involved activation of JNK/SAPK To elucidate the signal transduction by homocysteine, the effects of mitogen-activated protein (MAP) kinase inhibitors were examined. Neither p38 kinase-specific inhibitor SB203580 nor extracellular signal-regulated kinase (ERK)-specific inhibitor PD98059, alone or in combination, had any inhibitory effect (data not shown). As shown in Figure 2A, Western blotting demonstrated that JNK/SAPK and p38 MAP kinase were phosphorylated after the homocysteine treatment. Intriguingly, the phosphorylation of JNK/SAPK was rapid and sustained longer; it occurred within 30 minutes and persisted for at least 8 hours. In contrast, the activation of p38 was observed only after prolonged exposure to homocysteine (4 to 8 hours). We next studied the homocysteine-responsive ATF3 gene transcription by means of a reporter assay. As shown in Figure 2B, the 1850-bp region of the 5' flanking sequence fused to the luciferase reporter gene exhibited approximately 2-fold induction by homocysteine. This induction was not affected by MAP-kinase inhibitors specific for p38 and ERK MAP kinase (data not shown). Upon coexpression of the dominant negative MKK4 and 7, which are upstream MKKs for JNK/SAPK, the homocysteine-responsive activation of the reporter gene was inhibited. In Figure 2C-D, the activation of ATF3 and JNK/SAPK by homocysteine was examined in cells transfected with dominant negative MKK4 and 7. Homocysteine clearly induced the ATF3 expression (Figure 2Cvi-x) and activated JNK/SAPK (Figure 2Dvi-x). Under this condition, dominant negative MKK4 and 7 inhibited the induction of ATF3 protein as well as the activation of JNK/SAPK. These data strongly suggested that the ATF3 gene induction by homocysteine was mediated at least in part by the activation of JNK/SAPK through cascade signals from MKK4 and 7. We next assayed the homocysteine effect using the pathophysiologic concentration of 0.05 to 0.3 mmol/L. The induction of ATF3 gene in the reporter assay (Figure 3A) and the phosphorylation of JNK/SAPK (Figure 3B) were observed, while statistically significant activation was observed at 0.3 mmol/L homocysteine, which is a serum concentration in severe cases of homocysteinemia.
Effects of other thiol-containing compounds and tunicamycin To investigate specificity of the homocysteine-induced activation of JNK/SAPK and ATF3, the effects of other thiol-containing reagents and tunicamycin were examined. As shown in Figure 4A, dithiothreitol and-mercaptoethanol exhibited significant effects, while cysteine had
only a marginal effect. By contrast, homocystine, the disulfide form of
homocysteine, had essentially no effect, indicating that the sulfhydryl
group was important for the effect. Tunicamycin, a glycosylation
inhibitor and potent inducer of ER stress, significantly activated the
JNK/SAPK and ATF3 expression. N-acetyl-L-cysteine, an active oxygen scavenger,
had no inhibitory effect on the homocysteine effect. These data
together indicated that the effect of homocysteine was mediated through
the reactivity of its thiol group, could be mimicked by ER stress, and
did not involve the generation of reactive oxygen species.
Effects of homocysteine in aortic endothelial cells We next examined the effect of homocysteine in HAECs. As shown in Figure 4B, homocysteine activated the JNK/SAPK and ATF3 expression in HAECs as well as in HUVECs. It should be noted that human endothelial cells are quiescent in vivo. Thus, the effects of homocysteine were examined in confluent cells. Almost similar effects were observed in both subconfluent and confluent cells. Therefore, the homocysteine effect appeared to be independent of the proliferative activity of the cells.Homocysteine-responsive element of ATF3 gene promoter To determine the homocysteine-responsive element(s) of the ATF3 gene promoter, we next analyzed plasmid constructs containing sequential deletions by reporter assay. Figure 5 shows that the deletion mutants from1850 to 110 were as active as the original construct in responding
to homocysteine. In contrast, further deletion to 84 nearly abolished
the activation by homocysteine. The region from 92 to 84 of the
ATF3 promoter contains an ATF/CRE sequence, TTACGTCA, that differs from
the consensus sequence TGACGTCA in only one base. We next introduced 2 mutations into the ATF/CRE site of pLuc-1850 and assayed its
inducibility by homocysteine. These mutations completely abolished the
stimulation by homocysteine. Thus, the ATF/CRE site represents one of
the major elements responsible for ATF3 gene induction by
homocysteine.
Electrophoretic mobility shift assays of nuclear extracts from homocysteine-treated HUVECs Figure 6A shows electrophoretic mobility shift assays (EMSAs) using the ATF/CRE element as DNA probe. Extracts from the control cells produced 2 major bands, I and II (lane 2), while extracts from the homocysteine-treated cells exhibited markedly increased intensity of bands I and II with an appearance of a new band, band III (lane 3). Band I was composed of 2 bands of close mobility, of which the upper band appeared only after homocysteine treatment. Bands I, II, and III were all competed out by an oligonucleotide with the wild-type sequence (lanes 4 and 5), but not by a mutated oligonucleotide (lanes 6 and 7), indicating that these 3 bands were all specific. As shown in Figure 6B, dephosphorylation of extracts from the homocysteine-treated cells markedly reduced the intensity of bands I and II and slightly reduced that of band III, suggesting that modification by phosphorylation of factors could, at least in part, account for the increased binding in the homocysteine-treated cells. Incubation of the control extracts with homocysteine or incubation of the homocysteine-treated extracts with-mercaptoethanol in vitro did not alter their EMSA patterns (data
not shown). Next, we examined the composition of DNA-protein complexes
by supershift assay. As shown in Figure 6C, anti-ATF2 antibody produced
a strong supershifted band with concomitant reduction of bands I and II
in both the unstimulated and stimulated cells (lanes 4 and 12). Band
III in the stimulated cells was also supershifted by anti-ATF2 antibody
(lane 12). Among another antibodies tested, anti-c-Jun antibody
produced a supershifted band in both types of cells (lanes 7 and 15),
and anti-ATF3 antibody supershifted only in the stimulated cells (lane
13). Control antibody did not produce apparent supershifted bands
(lanes 3 and 11), and no antibody tested recognized the ATF/CRE probe
in the assay (lanes 8 and 16). Anti-ATF4 antibody did not produce an
apparent supershifted band, although it has been reported that ATF4 is
induced by homocysteine.16 Taken together, these data
demonstrated that ATF2 and c-Jun are involved in recognizing the
ATF/CRE site of the gene promoter in the control cells, and these
factors are activated to increase the binding in the
homocysteine-treated cells. Our results further indicate that the ATF3
protein is recruited into the activated complex(es) in the
homocysteine-stimulated cells.
Quantitative analysis of transcription factors and their association in the homocysteine-treated HUVECs Figure 7A shows immunoblot analysis of the whole-cell extract of homocysteine-treated cells. The amounts of ATF2 and c-Jun increased moderately after treatment. As shown in Figure 1B, ATF3 protein was also induced. Thus, homocysteine up-regulated the level of transcription factors involved in recognition of the ATF/CRE site of the ATF3 gene. We next analyzed their interaction in vivo by immunoprecipitation. Figure 7B clearly demonstrates that ATF2 and c-Jun were associated with each other in both the control and stimulated cells, but the molar amounts of the 2 proteins were increased in the activated cells (lanes 2, 3, 5, and 6). In stimulated cells, ATF3 protein was recruited into the activated ATF2/c-Jun complex (lane 9), although it was present in substoichiometric amounts relative to ATF2 and c-Jun.
Effects of ATF and Jun family transcription factors on promoter activity of ATF3 gene Since our results indicated that ATF2, c-Jun, and ATF3 are involved in recognizing the ATF/CRE site of the ATF3 gene, we investigated the effects of overexpression of these factors, alone or in combination, by reporter assay. Figure 8 shows that both ATF2 and c-Jun, alone (lanes 3 and 4; P < .05, n = 3) or in combination (lane 7; P < .01, n = 3), activated the ATF3 gene. In contrast, ATF3 repressed both basal (lane 5; P < .05, n = 3) and ATF2/c-Jun-activated (lanes 8, 10 and 12; P < .05, n = 3) gene expression. These effects were observed when the reporter gene contained the wild-type sequence of the ATF/CRE site (solid columns), but not when the plasmid contained 2 base mutations in the element (open columns). These data indicated that ATF2 and c-Jun transactivated ATF3 promoter, but that ATF3 had a repressive effect. ATF4 stimulated ATF3 gene activity (lane 6), but had no significant effect on the activity in combination with c-Jun or ATF2 (lanes 9 and 11).
In this study, we demonstrated that homocysteine activated JNK/SAPK and induced the expression of ATF3 in vascular endothelial cells and that the ATF/CRE site of the ATF3 gene promoter is a major element responsible for the induction. It should be noted that an ATF/CRE site has also been shown to mediate
cyclin A gene activation by homocysteine in vascular smooth muscle
cells, although the element in the cyclin A gene is recognized by ATF1
and CREB.15,33,34 The induction of the cyclin A gene was
reported to be approximately 2-fold in a reporter assay,15
an extent of activation similar to that of the ATF3 gene in our assay.
Three families of leucine zipper proteins, CREB, ATF, and c-Jun, have
been shown to bind ATF/CRE sites as a homodimer or heterodimer, thus
allowing transcriptional cross-talk between different signaling
pathways.20,35-38 We have shown here that the enhanced
binding to the ATF/CRE site in the homocysteine-treated cells was due
to an increase of phosphorylation and molar amounts of ATF2 and c-Jun.
This is consistent with the fact that JNK/SAPK efficiently
phosphorylates these factors39,40 and stabilizes them
against breakdown by the proteasome system.41,42 Such an
activation pathway has been reported for E-selectin gene activation by
TNF- The fact that the activation of JNK/SAPK preceded the induction of ATF3 mRNA strongly suggested that the JNK/SAPK activation is of major functional relevance to the ATF3 induction by homocysteine. It was further shown that dominant negative MKK4 and 7 inhibited the homocysteine-induced ATF3 expression and JNK/SAPK activation (Figure 2B-D). Dominant negative JNK1 and 2 had less inhibitory effect, possibly owing to their more direct involvement in the activation of ATF2 and c-Jun (data not shown). Therefore, it is intriguing to examine the specific upstream signals from homocysteine to the activation of MKK4/7. Differential regulation of MKKs by signals has already been reported.45,46 It has been proposed that homocysteine added to serum, with the catalytic help of 4 to 5 µmol/L copper ion, generates reactive oxygen species, including hydrogen peroxide, that subsequently damage endothelial cells via an oxidative process.4,5 In our experiments, however, culture medium contained far lower concentration of copper ion (8 nmol/L or less), and neither active oxygen scavenger (Figure 4A) nor exogenously added catalase (data not shown) inhibited the homocysteine-induced effects. Thus, it is considered that most of the homocysteine effect in this study was not mediated by oxidative stress. Recent works from other laboratories showed that homocysteine failed to elicit an oxidative stress,16-18 but did increase expression of GRP78 and C/EBP-homologous protein (CHOP)/GADD153, both of which respond to agents or conditions that adversely affect the function of ER. In fact, homocysteine is reported to inhibit the cell-surface expression of thrombomodulin9 and processing and secretion of von Willebrand factor10 by preventing their exit from the ER. In the present study, the homocysteine-induced activation of JNK/SAPK and ATF3 expression could be mimicked by other thiol compounds and tunicamycin, a potent inducer of ER stress. It is, therefore, strongly suggested that the homocysteine effect was caused by reductive ER stress. More recently, ER stress has been shown to be coupled to activation of JNK/SAPK through an ER-residential protein kinase, IRE1.47 IRE1 is capable of activating expression of the ER chaperon GRP78/Bip, as well as non-ER-resident proteins such as the transcription factor CHOP/GADD153.48 Therefore, ATF3 might represent one of these non-ER proteins induced by ER stress. The possibility that homocysteine activates JNK/SAPK through the IRE1-dependent pathway is now under investigation. Homocysteine has antiproliferative effects in endothelial cells.14,18,49 It reduced DNA synthesis, and this effect was specifically observed at pathophysiological concentration of homocysteine by inhibiting p21ras methylation.49 It is not yet clear whether the JNK/SAPK activation and ATF3 expression by homocysteine are related to the antiproliferative effect. In this study, the homocysteine effect was similarly observed in both subconfluent and confluent cells (Figure 4B). However, it is possible that ER stress induced by homocysteine may directly or indirectly affect the cell proliferative activity, since ER stress has been reported to induce G1 arrest and programmed cell death.50,51 More importantly, homocysteine caused the rapid and sustained activation of JNK/SAPK, while p38 activation occurred after prolonged exposure (Figure 2A). Although the significance of p38 activation is not known, sustained activation of JNK/SAPK has recently been reported to be well correlated with the induced apoptotic cell death.52-54 Since we observed that homocysteine induced cell death in endothelial cells (C.Z. and S.K, unpublished data, 2000), it is speculated that JNK/SAPK activation and ATF3 induction might represent a novel pathway and gene expression that are involved in ER-stress-induced cell death. The significance of ATF3 in this process is now under vigorous investigation. Significant effects of homocysteine in this study occurred at far
higher concentration than the pathophysiologic range of 0.05 to 0.3 mmol/L, at which small but significant effects were observed
(Figures 1C and 3). We speculate that it is not the extracellular but
the intracellular level of homocysteine that activates JNK/SAPK and
ATF3 expression, and that cells are capable of metabolizing exogenous
homocysteine through such enzymatic activities as cystathionine Finally, the present study demonstrated for the first time that homocysteine activates JNK/SAPK by its reductive stress and subsequently induces the transcriptional repressor ATF3 in endothelial cells. Although the functional role of ATF3 in the homocysteine-induced endothelial cell dysfunction has not been established, ATF3 might have a role in the altered gene expression involved in endothelial cell growth and proliferation. More investigations, including identification of the upstream signal from homocysteine to the activation of JNK/SAPK and the downstream target genes regulated by ATF3, will be required to clarify the significance of ATF3 induction in vascular endothelial cell dysfunction associated with homocysteinemia.
We thank Dr Tsonwin Hai for the gift of pETATF3 and pATF3CAT; Dr Richard N. Kolesnick for dominant negative MKK4/SEK1; Dr Kinichiro Oda for expression plasmids for ATF2, ATF4, and c-Jun; and Drs Joan W. Conaway and Peter Hawkes for critical reading and editing of the manuscript.
Submitted January 24, 2000; accepted May 15, 2000.
Supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan, and from National Space Development Agency of Japan and Japan Space Forum (S.K).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Shigetaka Kitajima, Department of Biochemical Genetics, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo-ku, Tokyo, 113-8510, Japan; e-mail: kita.bgen{at}mri.tmd.ac.jp.
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Top
Abstract
Introduction
P),
anti-p38 (p38), or anti-phospho p38
(p38-
P < .01, n = 3.
(C,D) HUVECs were transfected with plasmids for the empty vector (i,
vi), dnMKK4 (ii, vii), dnMKK7 (iii, viii), or both (iv, ix) and then
stimulated with 3 mmol/L homocysteine. ATF3 expression and activation
of JNK/SAPK were detected by immunostaining with anti-ATF3 (panel C)
and anti-phospho JNK/SAPK (panel D) antibodies in the transfected
cells, which were detected by green fluorescent protein (GFP)
fluorescence (indicated by arrows in i-v). Cells were transfected with
the empty vector but not treated by homocysteine (v, x). In the lower
panels, ATF3-positive cells (panel C) or
JNK-
P < .001, n = 3.
B/ATF2-stimulated E-selectin gene promoter,