Induction of Acute Translational Response Genes by Homocysteine
ELONGATION FACTORS-1alpha , -beta , AND -delta *

George Chacko, Qi Ling, and Katherine A. HajjarDagger

From the Divisions of Hematology-Oncology, Departments of Pediatrics and Medicine, Cornell University Medical College, New York, New York 10021

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

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-1delta (EF-1delta ), a member of the multimeric complex regulating mRNA translation. Fibroblasts from cystathionine beta -synthase -/- individuals also showed up to 3.0-fold increased levels of mRNA for EF-1alpha , -beta , and -delta 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-1alpha , -beta , and -delta 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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 beta -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-1alpha , the best studied of four subunits (alpha , beta , gamma , and delta ), 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 beta -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
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- DL-homocysteine, L-cysteine, L-methionine, and cycloheximide were purchased from Sigma. [alpha -35S]dATP, [alpha -32P]dCTP, and [alpha -32P]rUTP were obtained from NEN Life Science Products. Plasmids containing cDNAs encoding human EF-1alpha (81678) and EF-1beta (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-1alpha , -beta , -gamma , and -delta subunits was generously provided by Dr. Wim Möller (Leiden University, Leiden, The Netherlands). Affinity-purified rabbit anti-Dyctiostelium EF-1alpha 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 beta -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 [alpha -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 beta -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 [alpha -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-1alpha , -beta , and -delta 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
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Abstract
Introduction
Procedures
Results
Discussion
References

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-1delta (EF-1delta ), a protein involved in regulation of mRNA translation (Table I).


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Fig. 1.   Time course of free thiol accumulation in endothelial cells and fibroblasts. HUVEC treated with 5 mM HC (black-triangle), and CBS +/+ (bullet ), or CBS -/- fibroblasts (open circle ) treated with 0.45 mM methionine, were washed once, scraped into PBS, pelleted (500 × g, 10 min), resuspended in 1 ml of PBS, and lysed by three cycles of freeze-thaw in an ethanol-dry ice bath. Aliquots of the clarified cell lysates were analyzed for free thiol content using 5,5'-dithio-bis-(2-nitrobenzoic acid), and total protein using the bicinchoninic acid assay as described under "Experimental Procedures."


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Fig. 2.   Representative differential display of mRNA transcripts from HC-stimulated HUVEC. Confluent passage 2-4 HUVEC were treated with 5 mM DL-HC in regular medium for 0, 6, 18, and 42 h. Total RNA was harvested, reverse transcribed, and amplified by polymerase chain reaction as described under "Experimental Procedures." Transcripts were analyzed on a 6% polyacrylamide sequencing gel. Shown are products derived from degenerate primer T12MC (M = A, G, or C) and four arbitrary primers (A, 5'-AGCCAGCGAA-3'; B, 5'-GACCGCTTGT-3'; C, 5'-AGGTGACCGT-3'; D, 5'-GGTACTCCAC-3'). Arrowheads indicate bands selected for further analysis. The arrow indicates the band confirmed by Northern blot.


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Fig. 3.   Time course for the steady state EF-1 mRNA response in HC-treated HUVEC. A, EF-1alpha ; B, EF-1beta ; C, EF-1delta . Total RNA from HUVEC treated for various periods of time with 5 mM HC was probed in Northern blot analyses with cDNA fragments encoding the indicated EF-1 subunit. Signals were normalized for total RNA loaded per lane using a 28 S probe. Shown are means ± S.E., n = 3. (p < 0.01 by Student's two-tailed t test, except for * where value is not significantly different from control.)

                              
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Table I
EF-1delta , a transcript differentially displayed by homocysteine-treated HUVEC

EF-1delta is one member of a five-subunit complex that regulates the rate of mRNA translation. To determine whether mRNAs encoding related subunits EF-1alpha and EF-1beta were also up-regulated in the presence of HC, additional Northern hybridization analyses were carried out (Fig. 3, A and B). These studies revealed an increase in steady state mRNA for EF-1alpha , -beta , and -delta that was evident by 6-18 h, and maximal (2-4-fold) by 42 h. Thus, exposure of endothelial cells to HC appeared to coordinately up-regulate steady state mRNA for all three EF-1 subunits.

To determine the specificity and dose-response relationship of EF-1delta mRNA upon exposure to HC, HUVEC were treated with 5 µM to 5 mM HC or L-cysteine for 24 h (data not shown). Northern hybridization revealed a 1.4-3.0-fold dose-related increase in steady state mRNA levels in response to HC, but, interestingly, no significant response to L-cysteine in the same dosage range. These data indicated that the effect of HC was both specific and dose-dependent, occurring at concentrations of HC commonly seen in vascular disease (15-100 µM).

To determine whether up-regulation of EF-1 subunit mRNAs was reflected at the protein level, ELISAs were carried out (27) (Fig. 4). After 8 h, expression of EF-1alpha and -beta increased by 90-95%, while EF-1gamma and -delta rose by 60-65%. By 24 h, expression of all four subunits had increased by 2.5-3.5 times (p < 0.001). These data suggested that all components of the EF-1 complex are coordinately regulated, and increase significantly at the protein level in response to HC.


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Fig. 4.   Expression of EF-1 polypeptides in cultured endothelial cells and fibroblasts. A, HC-treated HUVEC. Cell lysates from HUVEC treated with 5 mM HC for 0, 8, or 24 h were analyzed by ELISA as described under "Experimental Procedures" (n = 3, mean ± S.E., p < 0.001 for each subunit at 8 and 24 h, except * where p < 0.02). B, CBS +/+ (normal) and CBS -/- (homocystinuric) fibroblasts. Cell lysates were prepared at 18 h as described for HUVEC. Data are normalized for total protein content and expressed as a ratio with control (n = 3, mean ± S.E., p < 0.001 for each subunit; Student's two-tailed t test).

Regulation of EF-1 Transcripts by Endogenously Formed Homocysteine-- To determine whether EF-1delta and its partners, EF-1alpha and -beta , were up-regulated under conditions where homocysteine is produced endogenously, normal human foreskin fibroblasts were compared with homocystinuric fibroblasts which lack the enzyme cystathionine beta -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.

As shown in Fig. 5, treatment of normal fibroblasts with 0.45 mM methionine led to a 1.3-1.7-fold increase in steady state mRNA levels for EF-1alpha , -beta , and -delta in Northern blot analyses (p < 0.001). Furthermore, normal fibroblasts treated with 1 mM HC showed a 1.5-2.5-fold increase in EF-1alpha , -beta and -delta mRNA (p < 0.001). In resting homocystinuric fibroblasts, steady state levels of EF-1alpha , -beta , and -delta mRNA were increased by 1.5-3.0-fold (p < 0.001). Supplemental methionine did not increase these levels further. These data indicated that steady state levels of EF-1alpha , -beta , and -delta mRNA are significantly up-regulated under conditions where intracellular homocysteine is increased. In addition, protein levels of EF-1 subunits in normal and homocystinuric fibroblasts were assessed by ELISA (Fig. 4B). Compared with CBS +/+ cells, CBS -/- fibroblasts showed a 2.5-4.5-fold increase in expression of EF-1 subunit protein (p < 0.001). To ascertain the mechanism by which EF-1 subunit mRNA was induced in response to methionine, time-course studies were undertaken (Fig. 6). In response to methionine, both EF-1alpha (Fig. 6A) and EF-1delta (Fig. 6B) mRNA steady state levels in CBS +/+ fibroblasts peaked within 4 h and remained elevated for up to 18 h. This response was inhibited in cells pretreated with cycloheximide (100 µM, 2 h). EF-1alpha mRNA levels also fell in response to cycloheximide, but recovered between 4 and 18 h. These data suggest that induction of both EF-1alpha and EF-1delta requires protein synthesis. Identical results were obtained when 5 mM HC was substituted for methionine, suggesting that methionine may act by conversion to HC.


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Fig. 5.   Effect of methionine and HC on EF-1 subunit mRNA levels in CBS +/+ and CBS -/- fibroblasts. Total mRNA was isolated from normal fibroblasts, either untreated or treated with 0.45 mM methionine or 1 mM HC (18 h), and CBS -/- fibroblasts, either untreated or treated with 0.45 mM methionine (18 h). RNA was probed in a Northern blot with cDNAs encoding EF-1alpha (A), -beta (B), and -delta (C). Signals were normalized using a 28 S ribosomal RNA probe. Shown are means ± S.E., n = 6. (p < 0.001 for each sample compared with untreated control, except delta  where p = 0.01; Student's two-tailed t test).


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Fig. 6.   Time course of EF-1 subunit mRNA induction in normal fibroblasts by methionine. Normal fibroblasts were treated with 0.45 mM methionine for 1, 2, 4, and 18 h. Steady state mRNA levels for EF-1alpha (A) or for EF-1delta (B) were estimated by Northern blot analysis of samples from cells either pretreated (+CHX) or not pretreated (-CHX) with cycloheximide (100 µM, 2 h). mRNA levels were normalized to 28 S RNA for each sample.

To determine whether induction of EF-1 subunits by methionine involved regulation at the transcriptional level, nuclear run-on experiments were conducted (Fig. 7). For all three genes, the rate of transcription in CBS +/+ cells increased 1.8-2.5-fold upon addition of 0.45 mM methionine (p < 0.001). Similarly, in CBS -/- cells, transcription was increased 1.8-2.5-fold compared with CBS +/+ cells (p < 0.001), and this rate was not augmented further upon addition of methionine. In contrast, addition of L-cysteine (0.3 mM) to the culture medium failed to significantly enhance the rate of transcription of EF-1alpha , -beta , or -delta in either CBS +/+ or CBS -/- cells. These data suggest that increased intracellular levels of methionine or homocysteine, but not cysteine, are specifically associated with increased transcription of EF-1alpha , -beta , and -delta mRNA.


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Fig. 7.   Effect of HC on EF-1alpha , -beta , and -delta rate of transcription: nuclear run-on analysis. Nuclei were isolated from normal (CBS +/+) or CBS -/- fibroblasts treated with or without 0.45 mM methionine as indicated. Labeled transcripts were prepared as described under "Experimental Procedures," and hybridized to denatured probes for EF-1alpha (A), EF-1beta (B), and EF-1delta (C) blotted to nitrocellulose. Signals were normalized to 28 S RNA. Shown are mean values ± S.E., n = 4, except Cys for which n = 1. The p values are < 0.001, 0.01, and 0.02 for *, **, and ***, respectively (Student's two-tailed t test).

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.


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Fig. 8.   Effect of HC on thiol protein synthesis and degradation: pulse-chase analysis. HUVEC, either untreated or pretreated with HC (5 mM, 18 h) were methionine-starved (4 h), pulsed with [35S]methionine (1 h), and chased in regular medium or medium containing 5 mM HC as described under "Experimental Procedures." At the indicated time intervals, cells were harvested, and the lysates immunoprecipitated with rabbit IgG directed against anti-annexin II (A) or goat IgG directed against plasminogen activator inhibitor-1 (B). Immunoprecipitates were resolved on SDS-polyacrylamide gels, fluorographed, and quantified by densitometry. Shown is one experiment representative of three.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

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 (alpha 2beta gamma delta ), which promote GTP-driven delivery of aminoacyl tRNAs to the ribosome. The EF-1alpha ·GDP complex is converted to active EF-1alpha ·GTP by the nucleotide exchange activities of EF-1beta and EF-1delta . The EF-1gamma moiety is known to enhance the nucleotide exchange activity of EF-1beta , and may also serve to anchor the complex to membrane structures. Interestingly, EF-1delta , which is homologous to EF-1beta 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-1alpha is associated with increased translational fidelity in yeast (32), and increased longevity in Drosophila (33). Loss of expression of EF-1alpha , on the other hand, is accompanied by decreased rates of protein synthesis and the onset of senescence in human fibroblasts (34). Furthermore, EF-1alpha 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-1gamma 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-1alpha mRNA increase in response to p53 expression in erythroleukemia cells was 2-5-fold (40). EF-1delta 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-1alpha in maize endosperm stimulated by lysine (42), and for EF-1beta 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-1alpha and delta  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-1delta 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-1alpha 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-1beta and -delta genes have not yet been characterized.

It is not yet clear what role increased levels of EF-1alpha , -beta , -gamma , and -delta 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-1alpha 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.

Dagger 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 beta -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
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Abstract
Introduction
Procedures
Results
Discussion
References

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