15-Lipoxygenase promotes resolution of inflammation in lymphedema by controlling Treg cell function through IFN-ß.

Lymphedema (LD) is characterized by the accumulation of interstitial fluid, lipids and inflammatory cell infiltrate in the limb. Here, we find that LD tissues from women who developed LD after breast cancer exhibit an inflamed gene expression profile. Lipidomic analysis reveals decrease in specialized pro-resolving mediators (SPM) generated by the 15-lipoxygenase (15-LO) in LD. In mice, the loss of SPM is associated with an increase in apoptotic regulatory T (Treg) cell number. In addition, the selective depletion of 15-LO in the lymphatic endothelium induces an aggravation of LD that can be rescued by Treg cell adoptive transfer or ALOX15-expressing lentivector injections. Mechanistically, exogenous injections of the pro-resolving cytokine IFN-ß restores both 15-LO expression and Treg cell number in a mouse model of LD. These results provide evidence that lymphatic 15-LO may represent a therapeutic target for LD by serving as a mediator of Treg cell populations to resolve inflammation.

CXCL4L1-fibstatin cooperation inhibits tumor angiogenesis, lymphangiogenesis and metastasis.

Anti-angiogenic and anti-lymphangiogenic drugs slow tumor progression and dissemination. However, an important difficulty is that a tumor reacts and compensates to obtain the blood supply needed for tumor growth and lymphatic vessels to escape to distant loci. Therefore, there is a growing consensus on the requirement of multiple anti-(lymph)angiogenic molecules to stop cell invasion efficiently. Here we studied the cooperation between endogenous anti-angiogenic molecules, endostatin and fibstatin, and a chemokine, the Platelet Factor-4 variant 1, CXCL4L1. Anti-angiogenic factors were co-expressed by IRES-based bicistronic vectors and their cooperation was analyzed either by local delivery following transduction of pancreatic adenocarcinoma cells with lentivectors, or by distant delivery resulting from intramuscular administration in vivo of adeno-associated virus derived vectors followed by tumor subcutaneous injection. In this study, fibstatin and CXCL4L1 cooperate to inhibit endothelial cell proliferation, migration and tubulogenesis in vitro. No synergistic effect was found for fibstatin-endostatin combination. Importantly, we demonstrated for the first time that fibstatin and CXCL4L1 not only inhibit in vivo angiogenesis, but also lymphangiogenesis and tumor spread to the lymph nodes, whereas no beneficial effect was found on tumor growth inhibition using molecule combinations compared to molecules alone. These data reveal the synergy of CXCL4L1 and fibstatin in inhibition of tumor angiogenesis, lymphangiogenesis and metastasis and highlight the potential of IRES-based vectors to develop anti-metastasis combined gene therapies.

Fibroblast growth factor 2 internal ribosome entry site (IRES) activity ex vivo and in transgenic mice reveals a stringent tissue-specific regulation.

Fibroblast growth factor 2 (FGF-2) is a powerful mitogen involved in proliferation, differentiation, and survival of various cells including neurons. FGF-2 expression is translationally regulated; in particular, the FGF-2 mRNA contains an internal ribosome entry site (IRES) allowing cap-independent translation. Here, we have analyzed FGF-2 IRES tissue specificity ex vivo and in vivo by using a dual luciferase bicistronic vector. This IRES was active in most transiently transfected human and nonhuman cell types, with a higher activity in p53 -/- osteosarcoma and neuroblastoma cell lines. Transgenic mice were generated using bicistronic transgenes with FGF-2 IRES or encephalomyocarditis virus (EMCV) IRES. Measurements of luciferase activity revealed high FGF-2 IRES activity in 11-d-old embryos (E11) but not in the placenta; activity was high in the heart and brain of E16. FGF-2 IRES activity was low in most organs of the adult, but exceptionally high in the brain. Such spatiotemporal variations were not observed with the EMCV IRES. These data, demonstrating the strong tissue specificity of a mammalian IRES in vivo, suggest a pivotal role of translational IRES- dependent activation of FGF-2 expression during embryogenesis and in adult brain. FGF-2 IRES could constitute, thus, a powerful tool for gene transfer in the central nervous system.

Translation of CUG- but not AUG-initiated forms of human fibroblast growth factor 2 is activated in transformed and stressed cells.

Four isoforms of the human fibroblast growth factor 2 (FGF-2), with different intracellular localizations and distinct effects on cell phenotype, result from alternative initiations of translation at three CUG and one AUG start codons. We showed here by Western immunoblotting and immunoprecipitation that the CUG-initiated forms of FGF-2 were synthesized in transformed cells, whereas "normal" cells almost exclusively produced the AUG-initiated form. CUG-initiated FGF-2 was induced in primary skin fibroblasts in response to heat shock and oxidative stress. In transformed cells and in stressed fibroblasts, CUG expression was dependent on cis-elements within the 5' region of FGF-2 mRNA and was not correlated to mRNA level, indicating a translational regulation. UV cross-linking experiments revealed that CUG expression was linked to the binding of several cellular proteins to FGF-2 mRNA 5' region. Since translation of FGF-2 mRNA was previously shown to occur by internal ribosome entry, a nonclassical mechanism already described for picornaviruses, the cross-linking patterns of FGF-2 and picornavirus mRNAs were compared. Comigration of several proteins, including a p60, was observed. However, this p60 was shown to be different from the p57/PTB internal entry factor, suggesting a specificity towards FGF-2 mRNA. We report here a process of translational activation of the FGF-2 CUG-initiated forms in direct relation with trans-acting factors specific to transformed and stressed cells. These data favor a critical role of CUG-initiated FGF-2 in cell transformation and in the stress response.

Efficient gene transfer in skeletal muscle with AAV-derived bicistronic vector using the FGF-1 IRES.

IRESs (internal ribosome entry sites) are RNA elements behaving as translational enhancers in conditions of global translation blockade. IRESs are also useful in biotechnological applications as they allow expression of several genes from a single mRNA. Up to now, most IRES-containing vectors use the IRES from encephalomyocarditis virus (EMCV), highly active in transiently transfected cells but long and not flexible in its positioning relative to the gene of interest. In contrast, several IRESs identified in cellular mRNAs are short and flexible and may therefore be advantageous in gene transfer vectors such as those derived from the adeno-associated virus (AAV), where the size of the transgene expression cassette is limited. Here, we have tested bicistronic AAV-derived vectors expressing two luciferase genes separated by the EMCV- or fibroblast growth factor 1 (FGF-1) IRES. We demonstrate that the AAV vector with the FGF-1 IRES, when administrated into the mouse muscle, leads to efficient expression of both transgenes with a stable stoechiometry, for at least 120 days. Interestingly, the bicistronic mRNA containing the FGF-1 IRES leads to transgene expression 10 times superior to that observed with EMCV, in vivo. AAV vectors featuring the FGF-1 IRES may thus be advantageous for gene therapy approaches in skeletal muscle involving coexpression of genes of interest.

cis-acting elements involved in the alternative translation initiation process of human basic fibroblast growth factor mRNA.

Four forms of basic fibroblast growth factor (bFGF) are synthesized from the same mRNA, resulting from alternative initiations of translation at three CUG start codons and one AUG start codon. The CUG- and AUG-initiated forms have distinct intracellular localizations and can modify cell phenotypes differently, indicating that control of the alternative expression of the different forms of bFGF has an important impact on the cell. In this study, we investigated the roles of the mRNA 5' untranslated region and the alternatively translated region located between the CUG and AUG codons in the regulation of alternative translation of the different forms of bFGF. Deletions and site-directed mutagenesis were carried out in bFGF mRNA leader, and translation was studied in vitro and in vivo. The results enabled us to identify five cis-acting RNA elements (two in the 5' untranslated region and three in the alternatively translated region) involved in the regulation of either global or alternative initiation of translation. Each of these elements had a specific effect on the level of synthesis of the different forms of bFGF. Furthermore, we showed that the 5' untranslated region regulatory elements had different effects on bFGF translation, depending on the translation system used. These results suggest that bFGF translation is modulated by cis-acting elements corresponding to secondary or tertiary RNA structures, which could be the targets of cell-specific trans-acting factors.

c-myc Internal ribosome entry site activity is developmentally controlled and subjected to a strong translational repression in adult transgenic mice.

The expression of c-myc proto-oncogene, a key regulator of cell proliferation and apoptosis, is controlled at different transcriptional and posttranscriptional levels. In particular, the c-myc mRNA contains an internal ribosome entry site (IRES) able to promote translation initiation independently from the classical cap-dependent mechanism. We analyzed the variations of c-myc IRES activity ex vivo in different proliferating cell types, and in vivo in transgenic mice expressing a bicistronic dual luciferase construct. c-myc IRES efficiency was compared to that of encephalomyocarditis virus (EMCV) IRES under the same conditions. The c-myc IRES was active but with variable efficiency in all transiently transfected cell types; it was also active in the 11-day- old (E11) embryo and in some tissues of the E16 embryo. Strikingly, its activity was undetected or very low in all adult organs tested. In contrast, EMCV IRES was very active in most cell types ex vivo, as well as in embryonic and adult tissues. These data suggest a crucial role of IRES in the control of c-myc gene expression throughout development, either during embryogenesis where its activity might participate in cell proliferation or later on, where its silencing could contribute to the downregulation of c-myc expression, whose deregulation leads to tumor formation.

Alternative translation of human fibroblast growth factor 2 mRNA occurs by internal entry of ribosomes.

Alternative initiations of translation of the human fibroblast growth factor 2 (FGF-2) mRNA, at three CUG start codons and one AUG start codon, result in the synthesis of four isoforms of FGF-2. This process has important consequences on the fate of FGF-2: the CUG-initiated products are nuclear and their constitutive expression is able to induce cell immortalization, whereas the AUG-initiated product, mostly cytoplasmic, can generate cell transformation. Thus, the different isoforms probably have distinct targets in the cell. We show here that translation initiation of the FGF-2 mRNA breaks the rule of the cap-dependent ribosome scanning mechanism. First, translation of the FGF-2 mRNA was shown to be cap independent in vitro. This cap-independent translation required a sequence located between nucleotides (nt) 192 and 256 from the 5' end of the 318-nt-long 5' untranslated region. Second, expression of bicistronic vectors in COS-7 cells indicated that the FGF-2 mRNA is translated through a process of internal ribosome entry mediated by the mRNA leader sequence. By introducing additional AUG codons into the RNA leader sequence, we localized an internal ribosome entry site to between nt 154 and 318 of the 5' untranslated region, just upstream of the first CUG. The presence of an internal ribosome entry site in the FGF-2 mRNA suggests that the process of internal translation initiation, by controlling the expression of a growth factor, could have a crucial role in the control of cell proliferation and differentiation.

A new 34-kilodalton isoform of human fibroblast growth factor 2 is cap dependently synthesized by using a non-AUG start codon and behaves as a survival factor.

Four isoforms of human fibroblast growth factor 2 (FGF-2) result from alternative initiations of translation at three CUG start codons and one AUG start codon. Here we characterize a new 34-kDa FGF-2 isoform whose expression is initiated at a fifth initiation codon. This 34-kDa FGF-2 was identified in HeLa cells by using an N-terminal directed antibody. Its initiation codon was identified by site-directed mutagenesis as being a CUG codon located at 86 nucleotides (nt) from the FGF-2 mRNA 5' end. Both in vitro translation and COS-7 cell transfection using bicistronic RNAs demonstrated that the 34-kDa FGF-2 was exclusively expressed in a cap-dependent manner. This contrasted with the expression of the other FGF-2 isoforms of 18, 22, 22.5, and 24 kDa, which is controlled by an internal ribosome entry site (IRES). Strikingly, expression of the other FGF-2 isoforms became partly cap dependent in vitro in the presence of the 5,823-nt-long 3' untranslated region of FGF-2 mRNA. Thus, the FGF-2 mRNA can be translated both by cap-dependent and IRES-driven mechanisms, the balance between these two mechanisms modulating the ratio of the different FGF-2 isoforms. The function of the new FGF-2 was also investigated. We found that the 34-kDa FGF-2, in contrast to the other isoforms, permitted NIH 3T3 cell survival in low-serum conditions. A new arginine-rich nuclear localization sequence (NLS) in the N-terminal region of the 34-kDa FGF-2 was characterized and found to be similar to the NLS of human immunodeficiency virus type 1 Rev protein. These data suggest that the function of the 34-kDa FGF-2 is mediated by nuclear targets.

Two independent internal ribosome entry sites are involved in translation initiation of vascular endothelial growth factor mRNA.

The mRNA of vascular endothelial growth factor (VEGF), the major angiogenic growth factor, contains an unusually long (1,038 nucleotides) and structured 5' untranslated region (UTR). According to the classical translation initiation model of ribosome scanning, such a 5' UTR is expected to be a strong translation inhibitor. In vitro and bicistronic strategies were used to show that the VEGF mRNA translation was cap independent and occurred by an internal ribosome entry process. For the first time, we demonstrate that two independent internal ribosome entry sites (IRESs) are present in this 5' UTR. IRES A is located within the 300 nucleotides upstream from the AUG start codon. RNA secondary structure prediction and site-directed mutagenesis allowed the identification of a 49-nucleotide structural domain (D4) essential to IRES A activity. UV cross-linking experiments revealed that IRES A activity was correlated with binding of a 100-kDa protein to the D4 domain. IRES B is located in the first half of the 5' UTR. An element between nucleotides 379 and 483 is required for its activity. Immunoprecipitation experiments demonstrated that a main IRES B-bound protein was the polypyrimidine tract binding protein (PTB), a well-known regulator of picornavirus IRESs. However, we showed that binding of the PTB on IRES B does not seem to be correlated with its activity. Evidence is provided of an original cumulative effect of two IRESs, probably controlled by different factors, to promote an efficient initiation of translation at the same AUG codon.

The use of IRES-based bicistronic vectors allows the stable expression of recombinant G-protein coupled receptors such as NPY5 and histamine 4.

Stable expression of G protein coupled receptors in cell lines is a crucial tool for the characterization of the molecular pharmacology of receptors and the screening for new antagonists. However, in some instances, many difficulties have been encountered to obtain stable cell lines expressing functional receptors. Here, we addressed the question of vector optimization to establish cell lines expressing the human neuropeptide Y receptor 5 (NPY5-R) or histamine receptor 4 (HH4R). We have compared bicistronic vectors containing viral or cellular internal ribosome entry sites (IRES), co-expressing the receptor and the neomycine resistance gene from a single mRNA, to a bigenic vector containing two distinct promoters upstream each different genes. This study is the first one to validate the use of three cellular IRESs for long-term transgene expression. Our results demonstrate for both NPY5-R and HH4R that the bicistronic vectors with EMCV, VEGF, FGF1A or FGF2 IRES provide clones expressing functional receptors with yields between 25% and 100%. In contrast, the bigenic vector provided no functional clones, related to a low expression of NPY5R mRNA. The cell lines expressing active receptor were stable after more than 50 passages. These data indicate that IRES-based bicistronic vectors are particularly appropriate to establish cell clones expressing active G-coupled protein receptors with a high yield. In the case of NPY5, it was a new way to produce such a stable cell line. Furthermore, the characteristics-presented herein-of this receptor pharmacological property are perfectly in line with those reported in the literature.

Role of fibroblast growth factor-2 isoforms in the effect of estradiol on endothelial cell migration and proliferation.

Both 17beta-estradiol (E2) and fibroblast growth factor-2 (FGF2) stimulate angiogenesis and endothelial cell migration and proliferation. The first goal of this study was to explore the potential link between this hormone and this growth factor. E2-stimulated angiogenesis in SC Matrigel plugs in Fgf2+/+ mice, but not in Fgf2-/- mice. Cell cultures from subcutaneous Matrigel plugs demonstrated that E2 increased both migration and proliferation in endothelial cells from Fgf2+/+ mice, but not from in Fgf2-/- mice. Several isoforms of fibroblast growth factor-2 (FGF2) are expressed: the low molecular weight 18-kDa protein (FGF2lmw) is secreted and activates tyrosine kinase receptors (FGFRs), whereas the high molecular weight (21 and 22 kDa) isoforms (FGF2hmw) remains intranuclear, but their role is mainly unknown. The second goal of this study was to explore the respective roles of FGF2 isoforms in the effects of E2. We thus generated mice deficient only in the FGF2lmw (Fgf2lmw-/-). E2 stimulated in vivo angiogenesis and in vitro migration in endothelial cells from Fgf2lmw-/- as it did in Fgf2+/+ mice. E2 increased FGF2hmw protein abundance in endothelial cell cultures from Fgf2+/+ and Fgf2lmw-/- mice. As shown using siRNA transfection, these effects were FGFR independent but involved FGF2-Interacting Factor, an intracellular FGF2hmw partner. This is the first report for a physiological role for the intracellular FGF2hmw found to mediate the effect of E2 on endothelial cell migration via an intracrine action.

Cell transformation results in the loss of the density-dependent translational regulation of the expression of fibroblast growth factor 2 isoforms.

Alternative initiation of translation at three CUG and one AUG start codons leads to the synthesis of four isoforms of fibroblast growth factor 2 (FGF-2) that have distinct intracellular localizations and affect the cell phenotype differently. We show here that the expression of FGF-2 CUG-initiated isoforms decreases in a cell-density-dependent manner in normal human skin fibroblasts (HSFs) concomitantly with the FGF-2 mRNA level. In contrast, CUG-initiated FGF-2 expression is constitutive in SK-HEP-1 cells and in HSFs transformed with SV40 large T antigen. Cell transfection using a plasmid containing the FGF-2 mRNA leader fused to chloramphenicol acetyl transferase demonstrated that up-regulation of the CUG codons depends on cis-elements located in this leader. Furthermore, UV cross-linking experiments revealed a correlation between CUG codons utilization and the binding of several proteins to the mRNA leader. On the basis of the presence of an internal ribosome entry site (IRES) in the FGF-2 mRNA, we used bicistronic vectors to transfect normal and transformed cells. The density-dependent regulation in normal HSFs was cap-dependent, whereas the constitutive CUG-initiated FGF-2 expression in transformed cells occurred essentially by an IRES-dependent mechanism. Unexpectedly, the use of the AUG start codon occurred exclusively by internal entry, which suggests the presence of a second independent IRES in the FGF-2 mRNA that would be constitutive. A study of the eIF-4E levels and of the 4E-BP1 phosphorylation state at increasing cell densities showed a decrease of the eIF-4E level, concomitant with 4E-BP1 dephosphorylation in normal cells but not in transformed cells. These data point out a complex mechanism for the regulation of FGF-2 isoforms expression involving both the cap-dependent and the cap-independent initiation of translation and favor a positive role of CUG-initiated FGF-2 in cellular proliferation and transformation.

Relief of ornithine decarboxylase messenger RNA translational repression induced by alternative splicing of its 5' untranslated region.

The ornithine decarboxylase enzyme (ODC) is the key regulator of polyamine synthesis and is a member of the cellular proto-oncogene family. Its expression becomes constitutively activated by carcinogens, viruses, and oncogenes. ODC mRNA has a long 5' untranslated region that could be important in the regulation of enzyme levels by affecting translation. To test this hypothesis, we have determined the role of this region on the constitutive ODC hyperexpression measured in AR4-2J cells, an azaserine-induced, tumor-derived pancreatic acinar cell line. Construction of expression vectors in which ODC 5' leader sequence was placed flanking the chloramphenicol acetyltransferase reporter gene allowed us to identify three AR4-2J specific, different alternatively spliced ODC 5' leaders. The 5' ends of exons 2 and 3 were lengthened by 17 and 13 bases, respectively. Translation performed in a cell-free system as well as in COS7 transient transfection experiments demonstrated that AR4-2J isoforms induce a strong increase in the rate of translation. These results provide evidence that alternative splicing observed in tumoral cells, coupled with translation regulation, relieves the translation repression mediated by the long and structured 5' untranslated region of the ODC proto-oncogene.

Tumour suppressor p53 inhibits human fibroblast growth factor 2 expression by a post-transcriptional mechanism.

Fibroblast growth factor-2 (FGF-2) is a powerful mitogen and angiogenic factor whose expression is strongly regulated at the translational level. The constitutive upregulation of FGF-2 isoforms in transformed cells prompted us to investigate the post-transcriptional effects of a tumour suppressor, p53, on FGF-2 expression. We show here in human primary skin fibroblasts that the cell density-dependent variation of FGF-2 mRNA translatability was inversely correlated with endogenous p53 expression. Transient cell transfection revealed an inhibitory effect of wild-type p53 on the expression of chimeric FGF--CAT proteins. RNAse mapping experiments ruled out any effect of p53 on FGF--CAT mRNA accumulation, suggesting a translational inhibition. This inhibition was mediated by the FGF-2 mRNA leader, but not by vascular endothelial growth factor or platelet derived growth factor mRNA leaders. Neither p53-like protein p73, nor p21/waf had any inhibitory activity. Furthermore a set of hot spot mutants of p53 bearing mutations in the DNA binding domain had no post-transcriptional inhibitory effect. In contrast a p53 mutant of the transactivating domain was still able to block FGF--CAT expression, indicating that the post-transcriptional activity of p53 described here was independent of the trans-activation of target genes. Such data reveal a novel mechanism by which p53 efficiently blocks the expression of a major proliferating, anti-apoptotic and angiogenic gene.

p53 directs conformational change and translation initiation blockade of human fibroblast growth factor 2 mRNA.

Tumour suppressor p53 has been shown to inhibit fibroblast growth factor 2 expression post-transcriptionally in cultured cells. Here we have investigated the mechanism responsible for this post-transcriptional blockade. Deletion mutagenesis of the FGF-2 mRNA leader revealed the requirement of at least four RNA cis-acting elements to mediate the inhibitory effect of p53 in SK-Hep-1 transfected cells, suggesting the involvement of RNA secondary or tertiary structures. Recombinant wild-type, but not Ala(143) mutant p53, was able to specifically repress FGF-2 mRNA translation in rabbit reticulocyte lysate, in a dose dependent manner. Sucrose gradient experiments showed that p53 blocks translation initiation by preventing 80S ribosome formation on an mRNA bearing the FGF-2 mRNA leader sequence. Interaction of wild-type and mutant p53 with different RNAs showed no significant correlation between p53 RNA binding activity and its translational inhibiting effect. However, by checking the accessibility of the FGF-2 mRNA leader to complementary oligonucleotide probes, we showed that the binding to RNA of wild-type, but not mutant p53, induced RNA conformational changes that might be responsible for the translational blockade. This strongly suggests that p53 represses FGF-2 mRNA translation by a direct mechanism involving its nucleic acid unwinding-annealing activity.

Translation of the human c-myc P0 tricistronic mRNA involves two independent internal ribosome entry sites.

The human c-myc proto-oncogene is transcribed from four alternative promoters (P0, P1, P2, and P3) giving rise to mRNAs having 5' leader sequences of various length. The c-myc P0 mRNA contains three open reading frames (ORFs), the last one encoding c-Myc1 and c-Myc2 proteins generated by alternative translation initiated at CUG and AUG codons. The middle ORF (MYCHEX1) and the 5' ORF (ORF1) code for proteins 188 and 114 amino acids in length, respectively. We and others previously identified an internal ribosome entry site (IRES) in P0 and P2 c-myc mRNAs, promoting the cap-independent translation of c-Myc1 and c-Myc2. Here, we report the presence of a second IRES (named IRES1) promoting the cap-independent translation of MYCHEX1 in c-myc P0 mRNA. Using deletion analysis, we mapped an 80-nt region essential for IRES1 activity. c-myc P0 mRNA is thus the first eukaryotic polycistronic mRNA described for which translation initiation of two different open reading frames (MYCHEX1 and c-Myc1/c-Myc2) involves internal ribosome entry.

CUG initiation codon used for the synthesis of a cell surface antigen coded by the murine leukemia virus.

Murine leukemia virus (MuLV) codes for two precursors of the group-specific antigens, Pr65gag and Pr75gag, in vivo. While Pr65gag is the precursor to the virion structural proteins, Pr75gag undergoes glycosylation and is found on the surface of the infected cell as gp85gag, and it is thought to play a role in virus maturation and spread. Pr65gag synthesis starts at an AUG codon within a favourable initiation context (AAUAUGG at positions 618 to 624). The gp85gag start codon is upstream but its precise location is not known. To map the initiation codon of gp85gag, we used deletion and site-directed mutagenesis of the leader sequence of MuLV RNA and in vitro translation of the RNAs. Synthesis of the MuLV gp85gag protein appears to be initiated at a CUG codon located within a favourable context (ACCCUGG at positions 354 to 359 for Moloney-MuLV). The possible function of gp85gag was investigated by expressing Moloney-MuLV and Friend-MuLV proviral DNA and mutants deficient for gp85gag synthesis in mouse and rat cells. The results indicate that the gp85gag protein probably facilitates the spread of virus infection in tissue culture.

Pyrimidine tract binding protein and La autoantigen interact differently with the 5' untranslated regions of lentiviruses and oncoretrovirus mRNAs.

Retrovirus genomic mRNA exhibits a several hundred nucleotides-long untranslated region (5' UTR) which encloses many control elements required for retrovirus replication. In addition, this 5' UTR contains translation regulatory elements, such as internal ribosome entry sites (IRESes) that have been described in oncoretroviruses, as well as in lentiviruses. UV cross-linking experiments suggested that the pyrimidine tract binding protein (PTB), a cellular protein known to regulate the activity of several picornaviral IRESes, binds to human T-cell leukemia virus (HTLV)-I RNA but not to lentiviral human immunodeficiency virus (HIV)-1, HIV-2 or simian immunodeficiency virus RNAs. To calculate the affinity of such RNA-protein interactions, we developed a new method based on the BIAcore technology. The absence of affinity of PTB for lentiviral RNAs was confirmed, whereas its affinity for HTLV-I RNAs was 1000-fold lower than for picornaviral RNAs. The BIAcore technology also revealed a significant affinity of the La autoantigen, previously described for its involvement in translational control of viral mRNAs, for HIV-1 and HTLV-I RNAs. Addition of recombinant PTB to in vitro translation experiments weakly enhanced translation initiation in the presence of HTLV-I IRES, suggesting that such an IRES requires additional trans-acting factors.

Estrogens and atherosclerosis.

Numerous epidemiological as well as experimental studies have suggested that estradiol (E2) prevents atherosclerosis development. However two controlled prospective and randomized studies in women using hormone replacement therapy (HRT) did not confirm this beneficial effect. We then decided to use mouse models of atherosclerosis to define the possible mechanisms involved and the reasons for the discrepancy. We have shown that, although serum cholesterol decreases, this influence on lipid metabolism is negligible. Surprisingly, E2 induces an inflammatory-immune response towards a T helper cell (Th1) profile with increasing interferon-gamma production that could destabilize atheromatous plaques, and could account for the increase in the frequency of cardiovascular events in women undergoing HRT. At the level of the endothelium, E2 induces an increase in nitric oxide (NO) biodisponibility, but this phenomenon does not concern the development of fatty streaks. Nevertheless, the atheroprotective effect is apparently mediated at the level of the endothelium by a mechanism that has still to be characterized in molecular terms. These new acquisitions constitute a basis for new pharmacological developments allowing the prevention of deleterious effects and preserving the beneficial ones.