Induction of Acute Translational Response Genes by Homocysteine

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-1δ (EF-1δ), a member of the multimeric complex regulating mRNA translation. Fibroblasts from cystathionine β-synthase −/− individuals also showed up to 3.0-fold increased levels of mRNA for EF-1α, -β, and -δ 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-1α, -β, and -δ 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.

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 220base 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 thiolcontaining 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 thiolcontaining proteins in response to HC-induced oxidative stress.
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-5Ј-phosphate (vitamin B6)-dependent rate-limiting enzyme, cystathionine ␤-synthase (2,3). In addition, the enzymes 5-methyltetrahydrofolate-homocysteine methyltransferase and 5,10-methyl-enetetrahydrofolate 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)(6)(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 synthesisdependent. 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.
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 ϫ g, 10 min), supernatants were diluted 1:10 in 0.1 M Na 2 HPO 4 , pH 8.0, and treated immediately with 200 M 5,5Ј-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.
Northern Blot Analysis-Total RNA from HUVEC or fibroblasts (4 -10 g) 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 0.1ϫSSC at 21°C, dried, and autoradiographed on Kodak XAR film (Ϫ70°C). Signals were quantified by phosphorimaging and/or laser densitometry.
DNA Sequencing-Differentially displayed cDNAs were subcloned directionally into pBluescript KSϩ (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.
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 Ci of [ 35 S]methionine per 75-cm 2 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 ϫ g, 10 min, 500 l) were incubated with polyclonal anti-annexin II (24) or anti-PAI-1 (American Diagnostica 395G) for 18 h, treated with a 200-l 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 l of 5ϫ PAGE sample buffer, and the samples electrophoresed on a 12% SDS-polyacrylamide gel (18 h). The gels were treated with EN 3 HANCE, dried, fluorographed, and analyzed by densitometric image analysis.

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 51 Cr (26) nor lactate dehydrogenase differed from that observed in untreated controls (8.1 Ϯ 2.3 versus 9.1 Ϯ 1.2 units/ml, respectively, S.E., n ϭ 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 KSϩ using EcoRI and XbaI restriction sites. Sequence analysis re-  (Table I).
EF-1␦ is one member of a five-subunit complex that regulates the rate of mRNA translation. To determine whether mRNAs encoding related subunits EF-1␣ and EF-1␤ 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-1␣, -␤, and -␦ 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-1␦ mRNA upon exposure to HC, HUVEC were treated with 5 M 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 M).
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-1␣ and -␤ increased by 90 -95%, while EF-1␥ and -␦ rose by 60 -65%. By 24 h, expression of all four subunits had increased by 2.5-3.5 times (p Ͻ 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.
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 Ϫ/Ϫ). At rest, CBS Ϫ/Ϫ fibroblasts contained approximately twice as much intracellular thiol as normal fibroblasts (Fig. 1). When CBS ϩ/ϩ fibroblasts were treated with 0. 45  that CBS Ϫ/Ϫ fibroblasts were unable to clear methionineinduced intracellular thiols, whereas CBS ϩ/ϩ 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-1␣, -␤, and -␦ in Northern blot analyses (p Ͻ 0.001). Furthermore, normal fibroblasts treated with 1 mM HC showed a 1.5-2.5-fold increase in EF-1␣, -␤ and -␦ mRNA (p Ͻ 0.001). In resting homocystinuric fibroblasts, steady state levels of EF-1␣, -␤, and -␦ mRNA were increased by 1.5-3.0-fold (p Ͻ 0.001). Supplemental methionine did not increase these levels further. These data indicated that steady state levels of EF-1␣, -␤, and -␦ 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 ϩ/ϩ cells, CBS Ϫ/Ϫ fibroblasts showed a 2.5-4.5-fold increase in expression of EF-1 subunit protein (p Ͻ 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-1␣ (Fig. 6A) and EF-1␦ (Fig. 6B) mRNA steady state levels in CBS ϩ/ϩ fibroblasts peaked within 4 h and remained elevated for up to 18 h. This response was inhibited in cells pretreated with cycloheximide (100 M, 2 h). EF-1␣ mRNA levels also fell in response to cycloheximide, but recovered between 4 and 18 h. These data suggest that induction of both EF-1␣ and EF-1␦ requires protein synthesis. Identical results were obtained when 5 mM HC was substituted for methionine, suggesting that methionine may act by conversion to HC.
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 ϩ/ϩ cells increased 1.8 -2.5-fold upon addition of 0.45 mM methionine (p Ͻ 0.001). Similarly, in CBS Ϫ/Ϫ cells, transcription was increased 1.8 -2.5-fold compared with CBS ϩ/ϩ cells (p Ͻ 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-1␣, -␤, or -␦ in either CBS ϩ/ϩ or CBS Ϫ/Ϫ cells. These data suggest that increased intracellular levels of methionine or homocysteine, but not cysteine, are specifically associated with   7. Effect of HC on EF-1␣, -␤, and -␦ rate of transcription: nuclear run-on analysis. Nuclei were isolated from normal (CBS ϩ/ϩ) or CBS Ϫ/Ϫ 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-1␣ (A), EF-1␤ (B), and EF-1␦ (C) blotted to nitrocellulose. Signals were normalized to 28 S RNA. Shown are mean values Ϯ S.E., n ϭ 4, except Cys for which n ϭ 1. The p values are Ͻ 0.001, 0.01, and 0.02 for *, **, and ***, respectively (Student's two-tailed t test). increased transcription of EF-1␣, -␤, and -␦ mRNA.
Homocysteine Accelerates Turnover of a Thiol-containing Protein, Annexin II-To assess the effect of HC on overall protein synthesis, incorporation of [ 35 S]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 Ϯ 5% (S.E., n ϭ 10) in the presence of HC. To determine the effect of EF-1 induction on turnover of a thiol-containing protein, pulsechase 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 [ 35 S]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␣⅐GDP complex is converted to active EF-1␣⅐GTP 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 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 p21 ras 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 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 [ 35 S]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. 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 upregulated 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)(52)(53)(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.