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Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1650-1657

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1999;19:1650-1657.)
© 1999 American Heart Association, Inc.

Vascular Biology

Identification and Cloning of a New Gene (2A3-2), Homologous to Human Translational Elongation Factor, Upregulated in a Proliferating Rat Smooth Muscle Cell Line and in Carotid Hyperplasia

Kazem Zibara; Marie-Claude Bourdillon; Elza Chignier; Chantal Covacho; John L. McGregor

From INSERM Unit 331, Faculty of Medicine Laënnec, Lyon, France.

Correspondence to Dr K. Zibara, INSERM U331, Laënnec Medical School, 8 rue G. Paradin, 69372 Lyon cedex 08, France. E-mail zibara{at}laennec.univ-lyon1.fr


*    Abstract
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 *Abstract
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Abstract—Smooth muscle cells (SMCs), before migration and proliferation in the intima of the vessel wall, change from a normal contractile to a pathological proliferating phenotype. The molecular regulatory mechanisms implicated in such phenotypic changes remain poorly understood. In this study, using differential display, we have isolated for the first time a new gene (2A3-2) that is overexpressed in a rapidly proliferating, but not synthetic, rat SMC line. This was further confirmed by northern blot performed on the 2 cell types. Moreover, balloon catheter injury of rat carotids showed, by a virtual northern technique, an upregulation of this new gene in hyperplasia vessels. This new gene (2A3-2, 1.2 kb) was present in skeletal muscle, heart, aorta, lung, liver, kidney, and spleen. In addition, 5' rapid amplification of cDNA ends (5' RACE) allowed the cloning and sequencing of this 1.2-kb gene. Comparison of this newly identified gene sequence with data banks showed a strong homology to human and bovine mitochondrial translational elongation factor. The 2A3-2 gene, identified in this study, may play a vital role in the cascade of events implicated in switching SMC phenotype from a quiescent to a proliferate one.


Key Words: smooth muscle cell • differential display • virtual northern • rat carotid hyperplasia • cell culture • translational elongation factor


*    Introduction
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 *Introduction
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Arterial smooth muscle cells (SMCs) are a major component of atherosclerotic and restenotic plaques.1 Migration and proliferation of these cells is known to play a key role in lesion formation and atherogenesis.2 3 4 During the initial stages of these pathological processes, arterial SMCs migrate into the intima, change the ratio of myofilaments to the endoplasmic reticulum and Golgi apparatus,5 6 proliferate, and produce extracellular matrix components.1 7 8 Subsequently, SMCs are transformed into foam cells by accumulating lipids.9 10 11 In these pathologies, SMCs undergo a phenotypic modulation12 13 where they change from a contractile to a synthetic and then to a proliferating state. Harvested SMCs, during cell culture passages, progressively lose their contractile phenotype and exhibit characteristics identical to those observed in synthetic SMCs of diffuse intimal thickening.13 14 In long-term cultures, certain rat aortic SMCs were shown to generate a transformed phenotype15 16 with several similarities to highly proliferating cells.17 Little is known at this stage about the cascade of genes implicated in changes of SMC phenotype.18

Proliferation of SMCs in the intima of the vessel wall is thought to be either the result of an inflammatory process1 or that of a monoclonal growth.19 20 According to Ross,1 atherosclerotic lesions result from an excessive inflammatory fibroproliferative response to various forms of insult to the endothelium and SMCs of the artery. A large number of growth factors, cytokines, and vasoregulatory molecules participate in this process. After lesion formation, endothelial cells secrete chemotactic factors that would induce proliferation of SMCs and lipid deposition. LDLs that have been oxidized also participate in the formation of foam cells and atherosclerotic lesions. On the other hand, Benditt19 and Benditt et al20 hypothesized that a neoplastic process could arise during atherogenesis in response to a mutational or viral event.19,20 Indeed, certain genes have been shown to be implicated in the pathobiology of atherosclerosis and cancer.21 22 This hypothesis has been supported by experimental data demonstrating the appearance of SMCs in aortas of animals injected with carcinogens alone,23 in combination with methoxamine,24 or with oncogenic Marek's herpes virus.25 Moreover, DNA from human plaques was shown to completely transform transfected NIH 3T3 cells.26 Other factors, such as bacterial27 (Chlamydia pneumoniae) or viral28 agents (herpes virus and cytomegalovirus), have been detected and may be involved in the initiation and perpetuation of atherosclerotic lesions. In the present study, gene expression in normal rat synthetic SMCs at passage 9 (P9 cells) were compared with rapidly proliferating SMCs (V8 cell line) by differential display.29 A differentially expressed gene, present at high levels in rapidly proliferating cultured SMCs, was also shown to be upregulated in balloon catheter–injured, but not in normal, carotids.


*    Methods
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Surgical procedures and animal care conformed strictly to the Guidelines of the National Institute of Health and Medical Research (decree No. 87–848 of October 19, 1987). Sprague–Dawley rats (species, Rattus rattus, strain, OFA, Iffa Credo, France) used in this study were anesthetized with an intraperitoneal injection of pentobarbital (0.11 mL/100 mg of body weight).

Cell Culture
Primary SMCs were obtained from explants of medial thoracic aortas from 7- to 8-week-old male Sprague–Dawley (250 g) rat and cultured as previously described.14 30 31 32 Cell samples were preserved in liquid nitrogen at passages 2 to 10 and then every 10 passages. SMCs at passage 9 were shown to be in a synthetic state. A spontaneously highly proliferating rat SMC line17 (V8) has been used in this study. This cell line was established from aortic media of adult rat and passaged >200 times.17 Both cell types were grown in MEM medium supplemented with 10% newborn calf serum. The growth rate of P200 passaged cells was shown to be higher (x1.4) than that of P9 synthetic cells.17 To reach, at the same time, a similar cell density and confluence, P9 and P200 cells were seeded respectively at 14x103 and 10x103 cells/cm2 in 25-cm2 Falcon flasks. These cells, confluent within 5 to 7 days, were trypsinized and aliquots counted (60x103 to 80x103 cells/cm2). Before RNA extraction, cultures were growth-arrested by incubation in MEM medium supplemented with 0.1% newborn calf serum for 24 hours.

Total and Poly(A)+ RNA Preparation
After cell culturing, cells were washed with Hanks' medium (Sigma) and used for the RNA preparation. Total RNA was extracted by using the guanidinium thiocyanate33 method. For differential display analysis, genomic DNA contamination was removed by DNase I (MessageClean, GenHunter). For cDNA library construction and rapid amplification of 5' cDNA ends (5' RACE), poly(A)+ RNA was isolated from total RNA using oligo(dT)30 primers (Oligotex mRNA kit, Qiagen).

Differential Display Analysis
Differential display was performed as previously described29 (RNAimage, GenHunter).

Reverse Transcription (RT) Reaction
Total RNA (0.2 µg) from each sample was reverse transcribed with 100 U of Moloney murine leukemia virus reverse transcriptase in the presence of 250 µmol/L dNTPs and 2 µmol/L H-T11 M (M can be either dA, dG, or dC, and H is the HindIII restriction site). The 20-µL RT reaction was reverse-transcribed for 1 hour at 37°C, then the enzyme was denatured by heating at 75°C for 5 minutes.

PCR Amplification
Single-strand cDNA mixture (2 µL) thus obtained was used for 8 different PCR reactions, each containing a different arbitrary primer from the 5' end. The 18-µL PCR mix included 2 µmol/L of the H-T11 M primer (same as RT), 2 µmol/L of a specific arbitrary primer, 25 µmol/L of dNTPs with 0.25 µL of [{alpha}-33P]dATP (2000 Ci/mmol, Amersham), and 1 U of Taq DNA polymerase (Perkin-Elmer). Thermal cycling amplification parameters (40 cycles), using GeneAmp PCR System 9600 (Perkin-Elmer), were as follows: 94°C (15 s), 40°C (2 minutes), 72°C (30 s), and a final 5-minute extension step at 72°C.

Separation by Electrophoresis
Only 3.5 µL of the PCR products was separated on a 6% denaturing polyacrylamide gel in tris-borate–EDTA buffer after addition of 2 µL of loading dye (95% formamide, 10 mmol/L EDTA, pH 8.0, 0.09% xylene cyanole, and 0.09% bromophenol blue). The gels were run for 4 hours at 1400 V, dried without fixation for 2 hours at 80°C, exposed for 72 hours, and then visualized by autoradiography.

Band Recovery, Cloning, and Sequencing
(1) Differentially expressed bands (upregulated or downregulated) were recovered under sterile conditions by excising the gel slice from the dried gel by using a razor blade. Each gel slice was placed in 100 µL of sterile water, boiled for 15 minutes to solubilize the DNA, and then ethanol-precipitated. (2) PCR-reamplification is done with 4 µL of purified fragment, using the same primer pair and PCR parameters that gave rise to the band. (3) Reamplified DNA fragments were run on a 1.5% agarose gel. Bands that succeeded to be reamplified were cloned into PCR II vector (TA cloning kit, Invitrogen). (4) For DNA sequencing, minipreps of plasmid DNA were performed,34 followed by the dideoxy sequencing method35 (T7 Sequencing Kit, Pharmacia).

Homologies With the Data Banks
The sequences obtained were compared with known sequences by searching in the different databases (eg, GenBank, EMBL, EST, and STS), using the BLAST (basic local alignment search tool)36 and FASTA37 programs.

Probes and Northern Blot
Total RNA was extracted as above, denatured, separated by electrophoresis in formaldehyde–MOPS–agarose gel and then transferred to a nylon membrane (Hybond, Amersham). After capillary blotting performed overnight, the membrane was baked for 2 hours at 80°C. Probes for northern blots were prepared following the random priming method (High Prime), using the PCR-amplified inserts in the PCR II vector described above, and then purified by using G-Sephadex (Quick Spin Columns, Boehringer). Prehybridization and hybridization were performed according to standard protocols.38 Blots were exposed with intensifying screens against a Kodak film for 1 week at -70°C. Similar loading of RNA was assessed by using the actin probe.

Balloon Catheter Injury of the Rat Left Carotid Artery
Carotid arteries were obtained from 10- to 14-week-old male Sprague–Dawley rats (350 g). Neointima formation was induced as previously described.39 In brief, the left carotid was exposed under an operating microscope (OPMI 7, Carl Zeiss). After incision of the left external carotid artery, the balloon catheter (2F Fogarty, Baxter) was introduced through the primitive carotid artery. Injury of the left primitive carotid was produced by passing the inflated balloon catheter back and forth into the carotid for 3 times. The balloon was sufficiently inflated to generate slight resistance and the catheter was then removed, to ligate the external artery. Rats were kept under ad libitum conditions for 3 weeks after balloon injury, after which they were anesthetized by urethane (Sigma) for vessel extraction. Each carotid artery was cut into 2 pieces; the first was immediately snap-frozen in liquid nitrogen for RNA extraction and the second was fixed, to be used for histological and morphological controls. The right carotid, considered the normal control artery, was also extracted. This experimental model is known to induce a neointimal hyperplasia similar to that observed in humans after angioplasty.

SMART Technique and Virtual Northern Blot
Because of limited amounts of total RNA, gene expression analysis of rat carotids was performed by a tandem of SMART-PCR (Clontech) and virtual northern, as opposed to a standard northern blot. A full description of the SMART technology, and its applications, is given in the Clontech manual. In brief, single-strand cDNA is synthesized with 1 µg of total RNA from various samples (P9 and V8 cells, aorta, and right and treated left carotids). The RT is performed for 1 hour at 42°C, using 200 U/µL of Superscript II transcriptase (GibcoBRL), 10 µmol/L of the cDNA synthesis primer (CDS), 10 µmol/L of SMART oligo, 10 mmol/L dNTP, and 20 mmol/L DTT. When the 5' end of the mRNA is reached, the enzyme's terminal transferase activity adds a few nucleotides, primarily deoxycytidine, to the 3' end of the cDNA. RT then switches templates and continues replicating to the end of the oligo at the 5' mRNA end. The RT reaction was then heated at 72°C for 5 minutes. The resulting full-length single-strand cDNA contains a sequence complementary to the SMART oligo, which is then used as the template in PCR to generate double-stranded cDNA. Choosing the optimal number of PCR cycles ensures that the double-stranded cDNA will remain in the exponential phase of amplification. In our experiments, the optimal number of cycles was 16 because the plateau was reached after 17 cycles. Only single-strand cDNA having the SMART sequence at the 5' end and the oligo(dT) at the 3' end are exponentially amplified by PCR. The 100-µL PCR reaction contains 10 mmol/L of dNTP, 10 µmol/L of the PCR primer (complementary to the SMART oligo and CDS primer), 2 µL of the 50x KlenTaq polymerase, and 10 µL of the 10x KlenTaq PCR buffer. PCR conditions are as follows: 1 step at 95°C (1 minute), followed by 16 cycles of 95°C (15 s), 65°C (30 s), and 68°C (6 minutes). A virtual northern blot is obtained by first running 0.5 µg of SMART-PCR–amplified cDNA on an agarose/ethidium bromide gel. Subsequently, the electrophoresed material is transferred to a nylon membrane (Hybond N+, Amersham), which is then probed with 32P-labeled 2A3-2 fragment, as in a standard northern blot. Actin is also used to assess similar loading.

5' Rapid Amplification of cDNA Ends (5' RACE)
To obtain the upstream 5' region of the new gene, the 5' RACE technique was performed basically by applying the touchdown PCR principle40 and by using Marathon cDNA Amplification and Advantage KlenTaq Polymerase kits (Clontech).

In the first step, single-strand cDNA is synthesized with 1 µg of V8 poly(A)+ RNA, using 10 µmol/L of the cDNA synthesis primer and Moloney murine leukemia virus reverse transcriptase for 1 hour at 42°C. DNA synthesis was verified by the addition of dNTPs among which 1 was radiolabeled with [{alpha}-32P]dCTP (1 µCi/µL, NEN).

The second step is the synthesis of double-stranded DNA performed at 16°C for 3 hours in an enzyme mixture containing Escherichia coli DNA polymerase I, RNase H, and E coli DNA ligase. These enzymes allow the synthesis of double-stranded cDNA, RNA degradation, and the formation of blunt ends, respectively. A 1% agarose gel electrophoresis is performed to estimate the quantity and quality of the double-stranded cDNA synthesized. The gel is then dried and put in contact with a Kodak film at -70°C to visualize the DNA smear.

The third step allows us to obtain a library of double-stranded cDNA, from V8 cells, by ligating an adapter to both ends of the double-stranded cDNA, using a T4 DNA ligase at 16°C overnight.

In the last step, an aliquot of the library is subjected to PCR. The 50-µL PCR reaction contains 10 µmol/L of dNTP, 10 µmol/L of the adapter primer (complementary to the cDNA adapter), 5 µL of the 50x KlenTaq polymerase, and 10 µmol/L of gene specific primer (GSP) complementary to the 3' differentially expressed fragment (2A3-2 GSP: 5'-GGGTAAAGTTATTAAATATACAATGTAATAAACG-3'). The mixture was subjected to a PCR step at 94°C (1 minute) followed by 33 cycles of 94°C (30 s), 60°C (30 s), and 68°C (2 minutes 15 s), and a 5-minute extension step at 72°C. The amplified DNA fragments were cloned into the PCR II vector and purified by using Qiagen Plasmid Midi Kit (Qiagen). The insert DNA is then sequenced commercially (Genome Express).


*    Results
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 up arrowIntroduction
 up arrowMethods
 *Results
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 down arrowReferences
 
Identification and Cloning of the 2A3-2 Gene
On initial differential display gels, we observed 51 bands, using 8 arbitrary and 3 RT primers, that were differentially expressed between synthetic and rapidly proliferating cells (P9 and V8). Low molecular weight bands gave little sequence information and were often, as verified by PCR reamplification and northern-blots, found to be false positives. Of 36 high molecular weight bands retained, 22 bands were reamplified and cloned into PCR II plasmid. Sequences from different clones were then sent to databases for identity and homology search. Northern-blot was then used, for some of the newly identified cDNA fragments, to assay levels of expression in both cell types. One of the genes that was clearly differentially displayed was band 2A3-2 (Figure 1Down). This cDNA fragment showed an upregulation, using northern blots, in rapidly proliferating cells compared with synthetic cells (Figure 2ADown). Quantification of 2A3-2 signals, reported to the actin levels, showed a 4-fold increase in the P200 compared with the P9 cells. This increased expression was repeatedly observed (n=3) in different northern blots. The molecular weight of 2A3-2, as shown by northern, was determined to be 1.2 kb.



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  		Figure 1. A representative differential display (DD) analysis showing the 2A3-2 cDNA band. Total RNA was extracted from synthetic (P9) and proliferating (V8) SMCs, then subjected to DD. Migration of PCR products was performed on a denaturing 6% polyacrylamide gel, using arbitrary primer AP2 and a 3' primer (dT11A). Lanes 1 and 2 correspond to synthetic and proliferating cells, respectively.



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  		Figure 2. Northern blot analysis with the 2A3-2 cDNA band. A, The 2A3-2 gene is upregulated in proliferating (V8) but not synthetic cells (P9). Quantification of 2A3-2 signals, reported to the ß-actin levels, showed a 4-fold increase in the P200 compared with the P9 cells. This increased expression of the 2A3-2 gene in P200 compared with P9 was repeatedly observed (n=3) in different northern blots. The 2A3-2 gene has a molecular weight of 1.2 kb as shown by the northern blot. Lanes 1 and 2 correspond respectively to synthetic and rapidly proliferating cells. B, Rat multiple-tissue northern-blot analysis with the 2A3-2 cDNA band. The blot contained 16 µg of total RNA from various rat tissues and was probed with the 2A3-2 cDNA fragment isolated by differential display. Sizes of RNA markers are shown on the left (in kb). Transcripts of ~1200 bp could be observed in all rat tissues analyzed. A lower abundant transcript of ~2.4 kb is observed for skeletal muscle. Lane 1, skeletal muscle; lane 2, heart; lane 3, lung; lane 4, liver; lane 5, spleen; lane 6, kidney; and lane 7, aorta.

Tissue Distribution of 2A3-2 Gene
A rat multiple-tissue northern blot, probed by the 2A3-2 cDNA band, showed the 1.2-kb gene to be present in skeletal muscle, heart, aorta, lung, liver, kidney, and spleen tissues (Figure 2BUp). Skeletal muscle contains not only the normal 1.2-kb mRNA, but also a lower abundant transcript of ~2.4 kb. This transcript may arise from the use of an alternative polyadenylation site.41 The multiple northern blot shows that the 2A3-2 gene is not an artifact induced by cell culturing but is present in different tissues.

SMART and Virtual Northern of Carotids
Neointimal hyperplasia of rat carotids, induced by balloon catheter injury, was used to investigate the in vivo role of this new gene in SMCs. Rat left carotids were treated by a balloon catheter to initiate SMC proliferation and neointima formation. In contrast, right untreated carotids were used as controls (Figure 3Down). Because the amount of total RNA extracted from 1 carotid was <2 µg, we used a tandem of SMART-PCR and virtual northern blot to assay expression levels of 2A3-2. This tandem approach of technologies gives information similar to that obtained by a standard northern blot. The 2A3-2 gene was shown to be upregulated (4-fold increase) in the left balloon-treated carotid, in comparison with the control right carotid. The virtual northern experiment was repeated and confirmed on 5 different blots. Virtual northern blots further confirmed 2A3-2 upregulation in V8, but not in P9, cells as previously observed by standard northern (Figure 4ADown). It is noteworthy that the expression of 2A3-2 in P9 cells, under quiescent (48 and 72 hours) or proliferating conditions (0, 4, 8, and 24 hours), was not altered by the state of the proliferation of the cells (Figure 4CDown).



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  		Figure 3. Comparison of untreated to balloon catheter–treated carotid. A, Normal control right carotid artery (magnification, x60 before reduction). B, Balloon-injured left carotid artery with neointima formation. Intimal hyperplasia (IH) resulted from the proliferation of SMCs within the original intimal layer. This was observed 3 weeks after injury (magnification, x60 before reduction). a indicates adventitia; m, medial layer; L, lumen; IH, intimal hyperplasia. Arrowheads ({blacktriangleup}) represent internal elastica lamina, IEL.



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  		Figure 4. Northern blot experiments showing the upregulation of 2A3-2 under in vitro and in vivo conditions. A, A typical virtual northern blot showing upregulation of 2A3-2 in P200 cells (lane 2) compared with P9 cells (lane 1). The above data, on 2A3-2, confirms those obtained by standard northern blots. Moreover, 2A3-2 is upregulated in a carotid with hyperplasia (lane 5) compared with a healthy carotid (lane 4) and aorta (lane 3). The gene transcript size (1.2 kb) was found to be the same under in vitro and in vivo conditions. Actin serves as a control for loading. B, Quantification of 5 different virtual northern blot experiments that includes the above data. Upregulation differences, for 2A3-2, between P9 and P200 cells (lanes 1 and 2) and healthy or balloon catheter–treated carotids (lanes 4 and 5) are significant (<0.01). C, Northern blot showing 2A3-2 expression in P9 cells under quiescent and proliferating states. Quantification of 2A3-2 signals, reported to the 28S levels, showed that the expression of this gene was not altered by the state of the proliferation of the cells (1:1 ratio in all lanes). It seems therefore that the level of gene expression, between P9 and V8 cells, is linked to the phenotypic state of the cells and not to their growth rate. Lanes 1, 2, 3, 4, 5, and 6 correspond to RNA levels after 0, 4, 8, 24, 48, and 72 hours of stimulation by FCS, respectively. The ribosomal 28S served as a control for loading and for quantification.

5' RACE and Sequencing of the 2A3-2 Gene
The full length of this gene was obtained by 5' RACE, using a cDNA library constructed from rapidly proliferating V8 cells. The molecular weight of the 5' RACE product was 1.2 kb as previously indicated by northern blot (Figure 5Down). The PCR product was amplified, purified, cloned, and sequenced (Figure 6ADown). The full 2A3-2 nucleotide sequence was then sent to the European Molecular Biology Laboratory (EMBL) database, using Sequin software, to get an accession number (AJ006151).



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  		Figure 5. 5' RACE-PCR amplification. The cDNA library (obtained from the proliferating cells) was used along with a primer coming from the 3' end of 2A3-2 band. The other primer in the PCR comes from the adapter that is already ligated to the cDNA library. The touchdown PCR technique was used during the 5' RACE, which allowed us to obtain the 5' end of the gene. The molecular weight of the 5' RACE–amplified 2A3-2 gene is 1.2 kb. This confirmed northern results and indicated that we had cloned the full gene. {phi}X174 is given on the left as a molecular weight marker.



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  		Figure 6. Analysis of the different parts of the rat EF-Ts gene (2A3-2). A, The nucleotide sequence (1149 bp) with the poly(A) signal (AATAAA) underlined. The poly(A) tail is designated by (A)n, whereas the initiation and stop codons are shown in boldface (ATG is at +1). It is noteworthy that both human and bovine EF-Ts genes have an intron of 224 bp at position 99 and they have a 5' untranslated region (5'UTR) of very short length (18 bp). B, The EF-Ts (2A3-2) predicted protein sequence (324 amino acids) where the methionine (M) and stop (Z) codons are shown in boldface. The mature protein (*284 amino acids) starts from the underlined Ser residue at position 41. C, The different reading frames of the gene. Open reading frame 2 contains 972 bp where the ATG on position 8 is the initiation codon. D, Different parts of the gene with the initiation and termination codons. The 5' UTR is only 7 bp in length and the 3' UTR is 170 bp.

Characteristics of the 2A3-2 Gene
The open reading frame of the sequenced gene (1149 bp) was identified and shown to contain 972 bp running from the ATG start codon to a TAG stop codon (Figure 6Up). This gene had a very short 5' untranslated region (7 bp long). The initiation codon (designated +1) is preceded by a G residue at position -3 and followed by a T residue at position +4. Analysis of numerous translational start sites shows that the consensus sequence has a purine at position -3 and a G residue at position +4.42 Finally, the cDNA contained 170 bp in the 3' untranslated region with a typical poly(A) signal43 (AATAAA) that was determined 27 bp upstream of the poly(A) tail. When the full-length nucleotide sequence was analyzed by searching for homologies in the GenBank database, using FASTA, it was observed to have a 72% homology with human and bovine mitochondrial elongation factors,44 EF-Ts.

The presumed initiating ATG was assigned to the first methionine and the predicted protein sequence of 324 amino acid residues was termed the 2A3-2 protein (Figure 6BUp). The deduced 2A3-2 protein sequence showed a calculated molecular mass of 27 kDa. The sequence was run against the Swiss Prot database, which confirmed the GenBank results. The homologies with the bovine and human EF-Ts were of 85% in the mature protein and of 70% in the overall protein sequence. These important homologies suggest that we have cloned the rat homologous EF-Ts gene. A comparison of our sequence with human and bovine EF-Ts genes is shown in Figure 7Down. It is noteworthy that the E coli protein sequence has 29% homology with the bovine sequence. NH2-terminal analysis of the sequence indicates that the rat EF-Ts mature form begins with Ser-41 in the long open reading frame (Figures 6BUp and 7Down). The mitochondrial import signal is thus 40 amino acids long and it is 55 amino acids in the bovine form. The mitochondrial import signals are usually not conserved between different species; however, they usually lack acidic amino acids, are enriched in basic and hydroxylated residues, and can form an amphiphilic {alpha}-helix or ß-sheet. The transit peptide for rat EF-Ts lacks acidic residues and is moderately rich in basic and hydroxylated residues (30%). To summarize, the protein sequence is 324 amino acids (the human protein is 294 amino acids and the bovine one is 338 amino acids), whereas the mature form of the protein is 284 amino acids (283 amino acids for both the bovine and the human homologs). The rat N-terminal part of the protein has 29 amino acids more than the human one.



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  		Figure 7. Multiple sequence alignment with the corresponding bovine and human EF-Ts genes. Sequence comparison of EF-Ts proteins from pig, rat, and human. The protein sequence of bovine EF-Ts is shown in 1-letter code and only the differences are indicated for the 2 other sequences. The mature rat protein starts from the underlined Ser residue at position 41 and constitutes 284 amino acids, whereas both bovine and human homologous EF-Ts proteins are of 283 amino acids. Dots indicates a missing residue; dashes, a similar amino acid.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study, using differential display, we have isolated for the first time a new gene (2A3-2, 1.2 kb) that is overexpressed in rapidly proliferating, but not synthetic, rat SMC line. Moreover, balloon injury of rat carotids showed, by a virtual northern technique, an upregulation of this new gene in hyperplasia vessels. The following several lines of evidence support the above statement: (1) Differential display shows an upregulation of 2A3-2 in rapidly proliferating SMCs (V8) but not synthetic (P9) cells. (2) These results, obtained by differential display, were confirmed by northern blots. (3) Multiple-tissue northern showed the presence of this 1.2-kb gene in different rat tissues. (4) SMART technique in tandem with virtual northern blot show 2A3-2 upregulation in balloon-injured carotids compared with controls. (5) 5' RACE technique allowed us to clone and sequence the full 1.2-kb gene. This new gene shows, on database search, an important homology to human and bovine mitochondrial EF-Ts.44 Moreover, one should note that EF-Ts is a nuclear encoded gene in mammals. Finally, on a human multiple-tissue northern-blot, Xin et al44 found a 2.4-kb transcript, as we did, present only in skeletal muscles.

In performing differential display on synthetic and highly proliferating SMCs, a number of bands (51) were isolated. Such differentially expressed bands showed, on sequencing and database search, total, partial, or no homologies to known proteins (Zibara et al, unpublished data, 1998). The 2A3-2 cDNA band, after northern blot analysis, was selected for further study, as it was observed to be upregulated in a rapidly proliferating cell line (V8). This gene did not show, at the initial stage of the study, any significant homology to known genes. However, when the full-length nucleotide sequence was obtained, a 72% homology with the bovine and human mitochondrial EF-Ts is observed. Moreover, the protein is 85% homologous to bovine and human EF-Ts. Our hypothesis, concerning the role of 2A3-2 or EF-Ts, is that increased expression of this new gene by SMCs takes place after repeated arterial insult or injury and that it participates in vascular remodeling. 2A3-2 or EF-Ts expression is 4-fold higher in injured carotids compared with controls, 3 weeks after balloon injury. To further understand the role of EF-Ts, in the modulation of SMCs from a contractile to a synthetic phenotype, in situ hybridization must be performed on carotids left for different periods of time (7 to 40 days) after balloon injury. It is noteworthy that the expression of EF-Ts in synthetic cells (P9), under quiescent (48 and 72 hours) or proliferating conditions (0, 4, 8, and 24 hours), was not altered by the proliferation state of the cells. It appears therefore that the level of gene expression, between P9 and V8, is linked to the phenotypic state of the cells and not to their growth rate.

Modulation of SMC phenotypes is known to induce the upregulation of several genes such as c-myc,45 c-myb,46 and c-fos,47 p65 subunit of NF-{kappa}B,48 ras proteins,49 Osteopontin,50 mitogen-activated protein kinases,51 angiotensin II,52 and cdk2 kinase.53 Moreover, some new genes were recently found to be upregulated in activated proliferating SMCs, such as sgk54 (serum and glucocorticoid-regulated kinase), type VIII collagen,55 nucleophosmin56 (a nuclear phosphoprotein implicated in the regulation of cell growth and protein synthesis), interferon-inducible protein-10,57 and BART-158 (balloon angioplasty–responsive transcript in rat carotid arteries). It is noteworthy that certain genes, such as the type II transforming growth factor-ß receptor (RII), may play a central role in atherosclerosis and cancerous tumor cell proliferation21 22 and are common to both diseases. It is conceivable that a certain common mechanism may be present in both pathologies. Finally, a recent study by Nishio et al59 showed that glucose induced, in bovine aortic SMCs, a human elongation factor 2 homologous gene that is a member of the GTPase family and that is essential for polypeptide synthesis. The 2A3-2 gene (EF-Ts), also identified in this study in SMCs, is essential for protein synthesis. Indeed, EF-Ts is a translation factor implicated in the elongation step and synthesis of all cellular proteins. Furthermore, a member of the elongation factor family (EF-Tu), to which EF-Ts belongs, facilitates the binding of aminoacyl-tRNA to the ribosome during the elongation cycle of protein biosynthesis. The elongation factor EF-Ts acts as a catalyst in the displacement of GDP from the EF-Tu*GDP complex and allows the binding of GTP. This reaction allows the ternary complex EF-Tu*GTP*aminoacyl-tRNA to be formed. Carefully mapping the cascade of genes involved in SMC migration and proliferation, in atherosclerosis and restenosis, may ultimately allow a better understanding of the SMC phenotype modulation.

Vascular SMC proliferation contributes to the pathogenesis of atherosclerosis. In addition, SMC proliferation is a key event in neointimal formation after balloon angioplasty and restenosis. The molecular signals that mediate these processes have not yet been identified. This study has identified a new gene, 2A3-2 or EF-Ts, not previously reported to be involved in SMC functions. Further work is needed to delineate the role of this new gene in vascular lesions.


*    Acknowledgments
 
This work was supported by a European Union grant (Biomed 2 project, European Network on Atherosclerosis [ENA], subvention No. 2AC RA021 B), and an MESR grant (MESR/ACC-SV9 No. 1A008A).

Received November 25, 1998; accepted December 3, 1998.


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