From the Divisions of Hematology-Oncology, Departments of Pediatrics and Medicine, Cornell University Medical College, New York, New York 10021
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The thiol amino acid homocysteine (HC)
accumulates in homocystinuria and homocyst(e)inemia, and is associated
with a wide variety of clinical manifestations. To determine whether HC
influences the cell's program of gene expression, vascular endothelial
cells were treated with HC for 6-42 h and analyzed by differential
display. We found a 3-7-fold, time-dependent induction of
a 220-base pair fragment, which demonstrated complete sequence identity
with elongation factor-1 (EF-1
), a member of the multimeric
complex regulating mRNA translation. Fibroblasts from cystathionine
-synthase
/
individuals also showed up to 3.0-fold increased
levels of mRNA for EF-1
, -
, and -
when compared with
normal cells, and treatment of normal cells with the HC precursor,
methionine, induced a 1.5-2.0-fold increase in EF-1
, -
, and -
mRNA. This induction was completely inhibited by cycloheximide and
reflected a doubling in the rate of gene transcription in nuclear
run-on analyses. In HC-treated endothelial cells, pulse-chase studies
revealed a doubling in the rate of synthesis of the thiol-containing
protein, annexin II, but no change in synthesis of the cysteineless
protein, plasminogen activator inhibitor-1. Thus, HC induces expression
of a family of acute translational response genes through a protein
synthesis-dependent transcriptional mechanism. This process
may mediate accelerated synthesis of free thiol-containing proteins in
response to HC-induced oxidative stress.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Homocysteine (HC)1 is an
intermediate thiol amino acid, which accumulates intracellularly and in
plasma in homocystinuria and homocyst(e)inemia (1). HC is formed upon
demethylation of methionine, and participates in the
transsulfuration pathway in which it condenses with serine to form
cystathionine. The most frequently encountered form of homocystinuria
results from deficiency of the pyridoxal-5'-phosphate (vitamin
B6)-dependent rate-limiting enzyme, cystathionine
-synthase (2, 3). In addition, the enzymes
5-methyltetrahydrofolate-homocysteine methyltransferase and
5,10-methylenetetrahydrofolate reductase participate in the
remethylation of HC, regenerating methionine in the presence of
5-methyltetrahydrofolate. Genetic or acquired deficiencies of these
enzymes are also causes of homocyst(e)inemia (4). Since HC is not a
dietary constituent, the sole source of HC in human tissues is
methionine. Elevations in plasma homocyst(e)ine have been associated
with a variety of clinical syndromes including thromboembolic vascular
disease, dislocation of the ocular lens, osteoporosis, neural tube
defects, and mental retardation (1, 5-7). The mechanisms for these
diverse effects are not understood.
Recent studies suggest that imbalances in the redox state of a cell may profoundly influence its functional activity. Oxidatively modified proteins, as may form in the presence of HC (8), undergo modified rates of cellular processing (9-11), follow alternative transport pathways (12, 13), and manifest functional abnormalities (6). In addition, several genes including reducing agent and tunicamycin-responsive protein (RTP), the stress protein GRP78/BiP, activating transcription factor 4 (ATF-4), and a methylenetetrahydrofolate dehydrogenase/cyclohydrolase have been found to be induced in endothelial cells exposed to high dose HC (14). In vascular smooth muscle cells, the cyclin A gene appears to be transcriptionally activated following exposure to HC (15, 16).
Elongation factor-1 (EF-1) is a multimeric protein that regulates the
efficiency and fidelity of mRNA translation in eukaryotic cells.
Expression of EF-1, the best studied of four subunits (
,
,
, and
), is regulated at both transcriptional and
post-transcriptional levels. In the present study, we show that HC,
either supplied exogenously to endothelial cells or produced
endogenously in cystathionine
-synthase-deficient fibroblasts,
up-regulates the EF-1 family of genes. This occurs through a
transcriptional mechanism that is protein
synthesis-dependent. Furthermore, EF-1 induction by HC is
associated with accelerated turnover of the thiol-containing protein,
annexin II, whereas synthesis of a cysteineless protein, plasminogen
activator inhibitor-1, is unchanged. These data suggest that the cell
may respond to deleterious effects of HC by induction of an acute
translational response by which damaged proteins may be efficiently
replenished.
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Materials--
DL-homocysteine,
L-cysteine, L-methionine, and cycloheximide
were purchased from Sigma. [-35S]dATP,
[
-32P]dCTP, and [
-32P]rUTP were
obtained from NEN Life Science Products. Plasmids containing cDNAs
encoding human EF-1
(81678) and EF-1
(78530) were from American
Type Culture Collection. A 28 S rRNA probe was kindly supplied by Dr.
Iris Gonzales, Department of Pathology, Hahneman University,
Philadelphia, PA. Polyclonal rabbit IgG directed against human EF-1
,
-
, -
, and -
subunits was generously provided by Dr. Wim
Möller (Leiden University, Leiden, The Netherlands). Affinity-purified rabbit anti-Dyctiostelium EF-1
was
kindly supplied by Dr. John Condeelis (Albert Einstein College of
Medicine, Bronx, NY). Polyclonal goat anti-human plasminogen activator
inhibitor-1 IgG (395G) was purchased from American Diagnostica.
Cell Culture and Treatment--
Human umbilical vein endothelial
cells (HUVEC) were harvested, propagated in M199, 0.3 mM
DL-methionine, 20% pooled human serum (17). Normal and
cystathionine -synthase
/
human fibroblasts (NIGMS Human Genetic
Mutant Cell Repository; GM 00751) were cultured in Earle's minimal
essential medium, 0.15 mM L-methionine, 20% fetal bovine serum. DL-Homocysteine,
L-cysteine, or L-methionine were added as 2- or
4-fold stock solutions.
Estimation of Free Thiols-- Fibroblasts or HUVEC from T75 flasks were washed three times with PBS, scraped into 5 ml of PBS, pelleted, and resuspended in 1 ml of PBS. Lysates were prepared by three cycles of freeze-thaw. Following centrifugation (15,000 × g, 10 min), supernatants were diluted 1:10 in 0.1 M Na2HPO4, pH 8.0, and treated immediately with 200 µM 5,5'-dithio-bis-(2-nitrobenzoic acid) (Ellman's reagent, 15 min, 21 °C). Absorbance at 412 nm was used to calculate free sulfhydryl content using a path length of 1 cm and molar extinction coefficient of 14,150.
Differential Display Analysis--
Total RNA from HUVEC treated
with or without DL-homocysteine was isolated by guanidinium
thiocyanate protein denaturation and phenol-chloroform extraction (18).
For differential display (19), RNA (0.2 µg) was reverse transcribed
in a buffer containing 50 mM Tris-HCl, 75 mM
KCl, 3 mM MgCl2, and 5 mM
dithiothreitol, pH 8.3, using degenerate primers T12MG,
T12MA, and T12MC, where M is A, G, or C. The
reaction mixture was heated to 65 °C (5 min), and then cooled
(37 °C, 10 min) prior to incubation with Moloney murine leukemia
virus reverse transcriptase (Life Technologies, Inc., 200 units,
37 °C, 60 min). The reaction was terminated by heating to 95 °C
(5 min), and then stored at 20 °C (18 h). Reverse transcribed
cDNA was amplified by polymerase chain reaction in the presence of
[
-35S]dATP using 10 arbitrary forward
primers (Genhunter Corp.; 5'-AGCCAGCGAA-3', 5'-GACCGCTTGT-3',
5'-AGGTGACCGT-3', 5'-GGTACTCCAC-3', 5'-GTTGCGATCC-3', 5'-GCAATCGATG-3',
5'-CCGAAGGAAT-3', 5'-GGATTGTGCG-3', 5'-CGTGGCAATA-3', 5'-TAGCAAGTGC-3')
and three oligo(dT) reverse primers, through 40 cycles consisting
of 94 °C (30 s), 40 °C (2 min), and 72 °C (30 s), and a final
incubation at 72 °C (5 min). Amplified cDNAs were resolved on a
6% polyacrylamide sequencing gel (1500 constant V, 3 h). Gels
were dried without fixation, and exposed to Kodak XAR film (
70 °C,
18 h). Differentially displayed bands were cut out, recovered by
ethanol precipitation in the presence of 3 M sodium acetate
(pH 5.2), and reamplified by polymerase chain reaction. Products were
isolated on 1% low melting point agarose and purified by ethanol
precipitation (20).
Northern Blot Analysis--
Total RNA from HUVEC or fibroblasts
(4-10 µg) was resolved on a 1.5% agarose denaturing formaldehyde
gel, and blotted to Zetaprobe (Bio-Rad) (21). Radiolabeled probes were
generated by random prime labeling and incubated with filters (18 h,
43 °C in 50% formamide or 48 °C in 25% formamide). Filters were
washed four times in increasingly stringent SSC solutions from 2 to
0.1×SSC at 21 °C, dried, and autoradiographed on Kodak XAR film
(70 °C). Signals were quantified by phosphorimaging and/or laser
densitometry.
DNA Sequencing-- Differentially displayed cDNAs were subcloned directionally into pBluescript KS+ (Stratagene) using EcoRI and XbaI restriction sites, and sequenced at the Rockefeller University DNA and Protein Sequencing Laboratory using T3 and T7 primers. Derived sequences were compared with GenBank and EMBL data bases.
Nuclear Run-on--
Nuclei were isolated by cell lysis in 0.5%
Nonidet P-40 from normal and cystathionine -synthase
/
fibroblasts, washed, and stored in liquid nitrogen in 50 mM
Tris-HCl, 40% glycerol (v/v), 5 mM MgCl2, 0.1 mM EDTA, pH 8.3 (22). Thawed nuclear suspensions (200 µl)
from either untreated cells, or cells treated with 0.3 mM
methionine or 0.3 mM cysteine (18 h), were incubated with
0.5 mM ATP, CTP, GTP, and [
-32P]UTP (800 Ci/mmol, 30 min, 30 °C). 32P-Labeled RNA was isolated by
phenol/chloroform/isoamyl alcohol (25/24/1; v/v/v; pH 5.2) extraction
and precipitation, and resuspended at equal cpm/ml in hybridization
buffer (10 mM TES, 0.2% SDS, 10 mM EDTA, 600 mM NaCl, pH 7.4). Denatured probes for EF-1
, -
, and
-
and 28 S RNA (8.3 µg) slot-blotted on nitrocellulose filters
were hybridized with labeled nuclear transcripts (65 °C, 36 h).
The filters were dried, autoradiographed with Kodak XAR film (24 h,
70 °C), and the signals quantified by phosphorimaging.
Western Blot and Enzyme-linked Immunosorbent Assay (ELISA)-- HUVEC or fibroblasts were washed three times with PBS (137 mM NaCl, 1.5 mM KH2PO4, 15 mM Na2HPO4·7H2O, 3 mM KCl, pH 7.4) and lysed by three cycles of freeze-thaw. Lysates were centrifuged at 15,000 × g, and the supernatants (5 µg/well in 50 µl of carbonate buffer: 15 mM Na2CO3, 35 mM NaHCO3, 3 mM NaN3, pH 9.6) used to coat wells of 96-well Nunc immunosorbent plates (18 h, 4 °C). The wells were blocked with bovine serum albumin (10 mg/ml in PBS/0.5% Tween 20, 2.5 h, 37 °C), washed and incubated with subunit-specific rabbit immune IgG (1:500 in PBS/0.5% Tween 20, 2.5 h, 37 °C), the specificities of which were verified by Western blot (23). The wells were washed three times with PBS, incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (1:500 in PBS/0.5% Tween 20, 2.5 h, 37 °C), washed again three times, and developed with p-nitrophenylphosphate in diethanolamine buffer (9.7% diethanolamine v/v, 3 mM NaN3, 0.01% MgCl2·6H2O, pH 9.8). Change in absorbance was evaluated over a 60-min time interval.
Metabolic Labeling and Immunoprecipitation-- HUVEC (80% confluent), untreated or treated with 5 mM DL-homocysteine (18 h), were washed three times with Hepes-buffered saline (HBS, 11 mM Hepes, 137 mM NaCl, 4 mM KCl, 11 mM glucose, pH 7.4) and incubated with methionine-free medium (4 h). Cells were then pulsed with 50 µCi of [35S]methionine per 75-cm2 flask (1 h), washed three times with HBS, and chased with complete medium with or without 5 mM homocysteine. Cells were treated with protease inhibitors and lysed by three cycles of freeze-thaw in HBS. Supernatants (500 × g, 10 min, 500 µl) were incubated with polyclonal anti-annexin II (24) or anti-PAI-1 (American Diagnostica 395G) for 18 h, treated with a 200-µl packed volume of Protein G Sepharose 4 Fast Flow beads (Amersham Pharmacia Biotech 17-0618-01) pre-equilibrated in 0.75 Tris, pH 8.8 (4 °C, 3 h), and washed three times in the same buffer (25). The beads were treated for 30 min (21 °C) with 100 µl of 5× PAGE sample buffer, and the samples electrophoresed on a 12% SDS-polyacrylamide gel (18 h). The gels were treated with EN3HANCE, dried, fluorographed, and analyzed by densitometric image analysis.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Identification of mRNAs Differentially Expressed by
Homocysteine--
Differential display analysis of mRNA from
untreated HUVEC, and HUVEC treated with 5 mM HC for 6, 18, and 42 h was carried out. Total intracellular thiol content of
HC-treated HUVEC peaked at 2.4 times base line within 4 h, and
remained at greater than twice base line from 6-18 h (Fig.
1). In HC-treated HUVEC, release of
neither 51Cr (26) nor lactate dehydrogenase differed from
that observed in untreated controls (8.1 ± 2.3 versus
9.1 ± 1.2 units/ml, respectively, S.E., n = 4),
indicating that cellular integrity was maintained. In two separate
experiments, approximately 10 discrete bands appeared to be up- or
down-regulated in the presence of HC, one of which (arrow)
showed a time-related increase in intensity (3.6-fold at 6 h,
4.8-fold at 18 h, and 6.9-fold at 42 h) (Fig.
2). Upon reamplification and Northern
blot analysis of mRNA from control and HC-treated HUVEC,
time-dependent expression could be confirmed only for this
band (Fig. 3C). This DNA
fragment was subcloned directionally into pBluescript KS+ using
EcoRI and XbaI restriction sites. Sequence
analysis revealed complete identity with bases 770-991 of the cDNA
for human elongation factor-1 (EF-1
), a protein involved in
regulation of mRNA translation (Table
I).
|
|
|
|
|
Regulation of EF-1 Transcripts by Endogenously Formed
Homocysteine--
To determine whether EF-1 and its partners,
EF-1
and -
, were up-regulated under conditions where homocysteine
is produced endogenously, normal human foreskin fibroblasts were
compared with homocystinuric fibroblasts which lack the enzyme
cystathionine
-synthase (CBS
/
). At rest, CBS
/
fibroblasts
contained approximately twice as much intracellular thiol as normal
fibroblasts (Fig. 1). When CBS +/+ fibroblasts were treated with 0.45 mM methionine, a homocysteine precursor, intracellular
thiols doubled within 4 h, and slowly returned to base line over
the next 14 h. CBS
/
fibroblasts, on the other hand, showed
roughly a doubling of intracellular thiol within 4 h, followed by
a continuous further increase over the ensuing 14 h. These
experiments verified that CBS
/
fibroblasts were unable to clear
methionine-induced intracellular thiols, whereas CBS +/+ cells did so
with relative efficiency.
|
|
|
Homocysteine Accelerates Turnover of a Thiol-containing Protein, Annexin II-- To assess the effect of HC on overall protein synthesis, incorporation of [35S]methionine into trichloroacetic acid-precipitable material was quantified in both control and HC-treated HUVEC. By this criterion, total protein synthesis was consistently reduced over 4-40 h to 63 ± 5% (S.E., n = 10) in the presence of HC. To determine the effect of EF-1 induction on turnover of a thiol-containing protein, pulse-chase metabolic labeling was conducted (Fig. 8A). Annexin II is a calcium-regulated phospholipid-binding protein that contains 4 cysteine residues, at least 2 of which exist in the reduced state (28). Following the initial [35S]methionine pulse, synthesis of annexin II proceeded 2.1 times as rapidly in HC-treated cells as in untreated control cells. By 16 h of chase, levels of immunoprecipitable annexin II in HC-treated cells averaged twice those observed in untreated control cells. The rate of disappearance of annexin II in HC-treated cells was 1.9 times greater than that of control cells. These data indicate that HC accelerates rates of annexin II synthesis and degradation. Because HC had no significant effect on annexin II mRNA levels at 16 h and 42 h (annexin II mRNA/GAPDH mRNA: 102% and 94% of untreated control, respectively), the observed increase in annexin II synthesis appears to reflect an enhancement in translational efficiency. In contrast, there was no significant difference in turnover of the non-thiol-containing, cysteineless protein, plasminogen activator inhibitor-1 (PAI-1) (Fig. 8B), as immunoprecipitable levels of PAI-1 differed by less than 15% throughout a 40-h time course. Thus, although turnover of a thiol-containing protein, annexin II, was accelerated in the presence of HC, metabolism of a non-thiol protein, PAI-1, was unaltered.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Protein translation plays a crucial role in processes governing
cell growth, proliferation, and differentiation (29-31). In most
eukaryotes, the two primary elongation factors, multimeric EF-1 and
monomeric EF-2, are primary sites of regulation of protein translation
(29). EF-1 consists of five subunits (2
), which promote GTP-driven delivery of aminoacyl tRNAs to the ribosome. The
EF-1
·GDP complex is converted to active EF-1
·GTP by the nucleotide exchange activities of EF-1
and EF-1
. The EF-1
moiety is known to enhance the nucleotide exchange activity of EF-1
, and may also serve to anchor the complex to membrane structures. Interestingly, EF-1
, which is homologous to EF-1
in the
C-terminal nucleotide exchange region, is unique among these factors in
that it contains a leucine zipper motif of unknown function.
EF-1 appears to play a central role in regulation of mRNA
translation, and alterations in levels of EF-1 subunit expression have
been reported in a variety of settings. Over-expression of EF-1 is
associated with increased translational fidelity in yeast (32), and
increased longevity in Drosophila (33). Loss of expression
of EF-1
, on the other hand, is accompanied by decreased rates of
protein synthesis and the onset of senescence in human fibroblasts
(34). Furthermore, EF-1
expression, possibly driven by the oncogene
v-fos (35), may lead to a malignant phenotype in human
pancreatic adenocarcinoma cells (36) and to increased susceptibility to
transformation in fibroblasts (37). EF-1
mRNA is overexpressed
in esophageal (38) and gastric (39) carcinomas.
Our data show increases varying from 1.5- to 4.5-fold in steady state
mRNA, mRNA transcription rate, and protein levels for EF-1
subunits in fibroblasts and endothelial cells treated with HC or
methionine for 6-42 h (Figs. 3, 4, 5, and 8). Although modest in some
cases, these changes are generally consistent in magnitude with EF-1
subunit responses to a number of previously reported stimuli. For
example, the EF-1 mRNA increase in response to p53 expression in
erythroleukemia cells was 2-5-fold (40). EF-1
mRNA increased
1.5-2.0-fold in response to ionizing radiation (41). At the protein
level, increases in the 3-5-fold range have been reported for both
EF-1
in maize endosperm stimulated by lysine (42), and for EF-1
in vascular smooth muscle cells treated with angiotensin II,
platelet-derived growth factor, or calf serum (43).
It is not clear why the effect of cysteine on transcription of EF-1 subunits differs so strikingly from the effect of HC. There is, however, ample precedent for the disparate effects of HC and cysteine on endothelial cell metabolism. For example, HC, but not cysteine, blocks carboxyl methylation of p21ras and inhibits cell proliferation (44). In addition, HC, but not cysteine, directly impairs the tissue plasminogen activator binding domain of its receptor, annexin II (26, 45), induces tissue factor expression (46), and reduces protein C activation (47). One possible explanation is that cysteine may be shunted toward synthesis of the antioxidant glutathione, a pathway that would prevent it from acting as an oxidant (48).
It is interesting that increased expression of both EF-1 and
mRNA upon exposure to HC or methionine can be inhibited by cycloheximide (Fig. 6). These data suggest that de novo
protein synthesis is required for induction by HC as appears to be the case for induction of EF-1
by ionizing radiation, another initiator of oxidative stress (41). Further, induction of two other proteins (cyclins A and D1) by HC is evident only after 12-24 h (15, 16),
suggesting that synthesis of an intermediate protein may be
required.
Oxidative stress induces a number of adaptive cellular responses. Free
cysteine-containing proteins are especially vulnerable to oxidation and
undergo rapid degradation (49). This is a likely explanation for the
increased rate of disappearance of annexin II, a free thiol protein
known to form a disulfide oxidation product in the presence of HC (45).
Although overall protein synthesis is reduced during a typical stress
response, synthesis of specific classes of adaptive proteins is known
to be increased (50). In addition to EF-1 subunits, other genes
up-regulated during the HC-induced stress response in endothelial cells
include glucose regulated chaperone protein 78 (GRP78/BiP), reducing
agent and tunicamycin-responsive protein (RTP), and activating
transcription factor 4 (ATF-4) (14). This altered program of
transcription may be orchestrated by factors such as HSF1, Sp-1, PEBP2,
HIF-1, AP-1, and NF-6B, which are activated upon exposure to reactive thiols (51-54). The promoter region of the EF-1 gene contains at
least eight Sp-1 sites as well as a single AP-1 site (55), suggesting
that it may be induced upon activation of these factors. To our
knowledge, the promoter regions for the EF-1
and -
genes have not
yet been characterized.
It is not yet clear what role increased levels of EF-1, -
, -
,
and -
may play in cells with elevated homocysteine. On the one hand,
homocysteine may stimulate an increase in synthesis of a select
population of polypeptides with which it forms mixed disulfides. Our
data suggest that the phospholipid-binding, free thiol-containing
fibrinolytic receptor annexin II is synthesized at an increased rate in
the presence of HC even though overall protein synthesis is decreased
by 40-50%. On the other hand, increased levels of EF-1 subunits might
also play a role in protein degradation, as homocysteinylation of
protein free thiols, as occurs at cysteine 9 of annexin II (45), may
mark a protein for degradation. Gonen et al. (56) have
recently suggested a role for EF-1
in the degradation of
N-acetylated proteins via the ubiquitin pathway.
In summary, our data demonstrate transcriptional induction of three EF-1 polypeptide subunits in response to pathophysiologic concentrations of homocysteine. This effect may contribute to the cell's adaptive response to oxidative stress. We postulate that elevated levels of EF-1 may serve to replenish homocysteinylated proteins destined for degradation. In addition, identification of the pathways of HC-mediated gene induction may illuminate mechanisms responsible for its diverse cellular effects and the may uncover adaptive cellular responses to HC-induced perturbation.
![]() |
ACKNOWLEDGEMENT |
---|
We acknowledge the expert technical assistance of Emil Lev.
![]() |
FOOTNOTES |
---|
* This work was supported by NIH grants HL 42493, HL 46403, and HL 58981.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Depts. of Pediatrics
and Medicine, Cornell University Medical College, 1300 York Ave., Box
45, New York, NY 10021. Tel.: 212-746-2034; Fax: 212-746-8809; E-mail:
khajjar{at}mail.med.cornell.edu.
1
The abbreviations used are: HC, homocysteine;
CBS, cystathionine -synthase; EF-1, translation elongation factor-1;
ELISA, enzyme-linked immunosorbent assay; HUVEC, human umbilical vein endothelial cells; PAI-1, plasminogen activator inhibitor-1; PBS, phosphate-buffered saline; TES,
N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|