Synthesis and application of a N-1' fluorescent biotinyl derivative inducing the specific carboxy-terminal dual labeling of a novel RhoB-selective scFv.

The fluorescent site-specific labeling of protein would provide a new, easy-to-use alternative to biochemical and immunochemical methods. We used an intein-mediated strategy for covalent labeling of the carboxy-terminal amino acid of a RhoB-selective scFv previously isolated from a phage display library (a human synthetic V(H) + V(L) scFv phage library). The scFv fused to the Mxe intein was produced in E. coli and purified and was then labeled with a newly synthesized fluorescent biotinyl cysteine derivative capable of inducing scFv-Mxe intein splicing. In this study, we investigated the splicing and labeling properties of various amino acids in the hinge domain between scFv and Mxe under thiol activation. In this dual labeling system, the fluorescein is used for antibody detection and biotin is used for purification, resulting in a high specific activity for fluorescence. We then checked that the purified biotinylated fluorescent scFv retained its selectivity for RhoB without modification of its affinity.

Zoledronic acid treatment impairs protein geranyl-geranylation for biological effects in prostatic cells.

BACKGROUND: Nitrogen-containing bisphosphonates (N-BPs) have been designed to inhibit osteoclast-mediated bone resorption. However, it is now accepted that part of their anti-tumor activities is related to interference with the mevalonate pathway. METHODS: We investigated the effects of zoledronic acid (ZOL), on cell proliferation and protein isoprenylation in two tumoral (LnCAP, PC-3,), and one normal established (PNT1-A) prostatic cell line. To assess if inhibition of geranyl-geranylation by ZOL impairs the biological activity of RhoA GTPase, we studied the LPA-induced formation of stress fibers. The inhibitory effect of ZOL on geranyl geranyl transferase I was checked biochemically. Activity of ZOL on cholesterol biosynthesis was determined by measuring the incorporation of 14C mevalonate in cholesterol. RESULTS: ZOL induced dose-dependent inhibition of proliferation of all the three cell lines although it appeared more efficient on the untransformed PNT1A. Whatever the cell line, 20 microM ZOL-induced inhibition was reversed by geranyl-geraniol (GGOH) but neither by farnesol nor mevalonate. After 48 hours treatment of cells with 20 microM ZOL, geranyl-geranylation of Rap1A was abolished whereas farnesylation of HDJ-2 was unaffected. Inhibition of Rap1A geranyl-geranylation by ZOL was rescued by GGOH and not by FOH. Indeed, as observed with treatment by a geranyl-geranyl transferase inhibitor, treatment of PNT1-A cells with 20 microM ZOL prevented the LPA-induced formation of stress fibers. We checked that in vitro ZOL did not inhibit geranyl-geranyl-transferase I. ZOL strongly inhibited cholesterol biosynthesis up to 24 hours but at 48 hours 90% of this biosynthesis was rescued. CONCLUSION: Although zoledronic acid is currently the most efficient bisphosphonate in metastatic prostate cancer management, its mechanism of action in prostatic cells remains unclear. We suggest in this work that although in first intention ZOL inhibits FPPsynthase its main biological actitivity is directed against protein Geranylgeranylation.

Geranylgeranyl transferase inhibition stimulates anti-melanoma immune response through MHC Class I and costimulatory molecule expression.

Defective antitumor immune responses are frequent consequences of defects in the expression of major histocompatibility complex (MHC) class I and costimulatory molecules. We demonstrated that statins, inhibitors of HMGCoA reductase, enhance mIFN-gamma induced expression of MHC class I antigens on murine B16F10 melanoma. GGTI-298, a geranylgeranyl transferase I inhibitor, but not FTI-277, a farnesyl transferase inhibitor, mimics this effect of statins. This effect is related to peptide transporter protein TAP1 up-regulation. Simultaneously, GGTI-298 induces the expression of CD80 and CD86 costimulatory molecules. C3 exoenzyme, which selectively inactivates Rho proteins, phenocopies the effects of GGTI-298, indicating a role for Rho proteins in these events. Furthermore, the treatment of B16F10 cells with GGTI-298 or C3 exoenzyme associated with mIFN-gamma induces in vivo tumor growth slowing down in immunocompetent but not in nu/nu syngeneic mice. Both in vivo injections and in vitro restimulation of splenocytes with GGTI-298- and mIFN-gamma-treated B16F10 cells induces an enhancement of specific CD8 T lymphocytes labeled by TRP-2/H-2K(b) tetramers. Finally, these effects are not limited to mouse models since they were also reproduced in two human melanoma cell lines. These observations indicate that protein geranylgeranylation as well as Rho protein are critical for costimulatory and IFN-gamma-dependent MHC class I molecule expression in melanoma.

Chemical-based translational induction of luciferase expression: an efficient tool for in vivo screening of protein farnesylation inhibitors.

We describe the development of a cell system for in vivo screening of inhibitors of the mevalonate pathway. To this aim, we have constructed a bicistronic mRNA, transcribed from a constitutive cytomegalovirus promoter, containing the Renilla reniformis luciferase RNA open reading frame sequence as first cistron and the Firefly luciferase RNA sequence as a second cistron. The intercistronic space is made of the R17 binding sequence of the bacteriophage R17 protein. A chimeric protein able to bind to a specific sequence in the hairpin and to induce internal ribosome entry in the RNA switches on translation of the second cistron. This chimeric protein is made up of the bacteriophage RNA binding domain (R17) fused to the ribosome recruitment core of the eIF-4G1 eukaryotic translation initiation factor and to the CAAX box of H-Ras addressing the protein to the plasma membrane where it is not efficient. Internal ribosome entry upstream of the Firefly cistron is therefore under the dependence of the mevalonate pathway inhibitors. Indeed, products that are able to inhibit protein farnesylation rescue the cytoplasmic location of the R17-eIF-4G-CAAX protein, which once more becomes a translation factor for the expression of the second cistron. To exemplify the system, the present work checks the ability of various antiestrogens to interfere with the mevalonate pathway. It seems that pure antiestrogen, able to selectively bind the estrogen receptor, is unable to switch on the second Firefly cistron although selective antiestrogen-binding-site ligands are able to do so.

TcRho1, a farnesylated Rho family homologue from Trypanosoma cruzi: cloning, trans-splicing, and prenylation studies.

Rho GTPases are members of the Ras superfamily and are involved in signal transduction pathways, including maintenance of cell morphology and motility, cell cycle progression, and transcription activation. We report the molecular identification in trypanosomatids (Trypanosoma cruzi) of the first member of the Rho family. The cloned Rho protein, TcRho1, shares approximately 40% homology with other members of the Rho family. Southern blot analysis revealed that TcRHO1 is a single copy gene per haploid genome, and Northern blot assays showed a transcript of 1200 nucleotides in length. Mapping the 5'-untranslated region of TcRHO1 transcripts revealed at least five different transcripts derived from differential trans-splicing. Three of the five transcripts contain the trans-splicing site within the coding region of the TcRHO1 gene. TcRho1 also contains the C-terminal sequence CQLF (CAAX motif), which is predicted to direct post-translation prenylation of the cysteine residue. A synthetic peptide containing this C-terminal motif, when tested against Q-Sepharose chromatography fractions from T. cruzi cytosol, was shown to be efficiently farnesylated, but not geranylgeranylated, despite the fact that the CAAX motif with X = Phe specifies geranylgeranylation by mammalian protein geranylgeranyltransferase I. Furthermore, immunoblot analyses of epimastigote protein with anti-S-farnesylcysteine methyl ester and anti-TcRho1 antisera strongly suggested that TcRho1 is farnesylated in vivo. The farnesylation of proteins such as Rho GTPases could be the basis for the selective cytotoxic action of protein farnesyltransferase inhibitors on trypanosomatids versus mammalian cells.

RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: evidenced in vivo by an anti-farnesyl cysteine antibody.

Protein isoprenylation is a lipid posttranslational modification required for the function of many proteins that share a carboxyl-terminal CAAX motif. The X residue determines which isoprenoid will be added to the cysteine. When X is a methionine or serine, the farnesyl-transferase transfers a farnesyl, and when X is a leucine or isoleucine, the geranygeranyl-transferase I, a geranylgeranyl group. But despite its CKVL motif, RhoB was reported to be both geranylgeranylated and farnesylated. Thus, the determinants of RhoB prenylation appear more complex than initially thought. To determine the role of RhoB CAAX motif, we designed RhoB mutants with modified CAAX sequence expressed in baculovirus-infected insect cells. We demonstrated that RhoB was prenylated as a function of the three terminal amino acids, i.e., RhoB bearing the CAIM motif of lamin B or CLLL motif of Rap1A was farnesylated or geranylgeranylated, respectively. Next, we produced a specific polyclonal antibody against farnesyl cysteine methyl ester allowing prenylation analysis avoiding the metabolic labeling restrictions. We confirmed that the unique modification of the RhoB CAAX box was sufficient to direct the RhoB distinct prenylation in mammalian cells and, inversely, that a RhoA-CKVL chimera could be alternatively prenylated. Moreover, the immunoprecipitation of endogenous RhoB from cells with the anti-farnesyl cysteine antibody suggested that wild-type RhoB is farnesylated in vivo. Taken together, our results demonstrated that the three last carboxyl amino acids are the main determinants for RhoB prenylation and described an anti-farnesyl cysteine antibody as a useful tool for understanding the cellular control of protein farnesylation.

Synthesis, binding and structure-affinity studies of new ligands for the microsomal anti-estrogen binding site (AEBS).

New compounds have been synthesized based on the structure of the anti-tumoral drug tamoxifen and its diphenylmethane derivative, N,N-diethyl-2-[(4-phenyl-methyl)-phenoxy]-ethanamine, HCl (DPPE). These new compounds have no affinity for the estrogen receptor (ER) and bind with various affinity to the anti-estrogen binding site (AEBS). Compounds 2, 10, 12, 13, 20a, 20b, 23a, 23b, 29 exhibited 1.1-69.5 higher affinity than DPPE, and compounds 23a and 23b have 1.2 and 3.5 higher affinity than tamoxifen. Three-dimensional structure analysis, performed using the intersection of the van der Waals volume occupied by tamoxifen in its crystallographic state and the van der Waals volume of these new compounds in their calculated minimal energy conformation, correlated well with their pKi for AEBS (r = 0.84, P<0.0001, n = 18). This is the first structure-affinity relationship (SAR) ever reported for AEBS ligands. Moreover in this study we have reported the synthesis of new compounds of higher affinity than the lead compounds and that are highly specific for AEBS. Since these compounds do not bind ER they will be helpful to study AEBS mediated cytotoxicity. Moreover our study shows that our strategy is a new useful guide to design high affinity and selective ligands for AEBS.

IL-6 promoter is modulated by the 24 kDa FGF-2 isoform fused to the hormone binding domain of the oestrogen receptor.

Fusion proteins consisting of the 24 kDa nuclear form of basic fibroblast growth factor (FGF-2), associated with the hormone binding domain of oestrogen receptor (HBD), convey oestrogen inducibility to FGF-2. When stable HBD-FGF-2 HeLa cell lines were transiently transfected with an interleukin 6 (IL-6) construct, the IL-6 promoter activity was downregulated by the addition of oestradiol. Moreover, in these cell lines, the function of the FGF-2 nuclear localisation sequence was abolished by its fusion to HBD, while addition of oestradiol restored the location of the chimera to the nucleus.

Structural similitudes between cytotoxic antiestrogen-binding site (AEBS) ligands and cytotoxic sigma receptor ligands. Evidence for a relationship between cytotoxicity and affinity for AEBS or sigma-2 receptor but not for sigma-1 receptor.

1-Benzyl-4-(N-2-pyrrolidinylethoxy)benzene (PBPE) is a cytotoxic derivative of the antitumoral drug tamoxifen. PBPE binds with high-affinity and specificity to the microsomal antiestrogen-binding site (AEBS). PBPE, as well as some other high-affinity AEBS ligands, shares structural features with high-affinity and selective sigma receptor ligands in the N-(arylethyl)-N-alkyl-2-(1-pyrrolidinyl)ethylamine class, such as BD1008, which are cytotoxic against tumoral cells. Based on these structural and pharmacological similitudes, we set out to examine whether AEBS and sigma receptors could be related binding sites. We showed that BD1008 had a high affinity for AEBS. However, prototypical sigma receptor ligands were very low-affinity competitors on AEBS. Surprisingly, AEBS ligands displayed a high affinity for sigma-1 and sigma-2 receptor subtypes, showing that AEBS and sigma receptor-binding sites were not mutually exchangeable. Moreover, phenytoin, which is an allosteric modulator of sigma-1 receptor, was a competitive inhibitor of [3H]tamoxifen on AEBS. These results suggest that the tamoxifen-binding site on AEBS and the sigma ligand-binding site on sigma receptors were not identical but related entities. We also showed here that the high-affinity and specific AEBS ligands also bound sigma receptors with high affinity. Moreover, the compounds that were capable of displacing tamoxifen from AEBS were cytotoxic against tumoral cells but not against the AEBS-deficient cell line Rtx-6. These results confirm that AEBS and sigma receptors might belong to the same family of proteins, and that the tamoxifen-binding site might be involved in the cytotoxicity of AEBS ligands and some classes of sigma compounds.

Inhibition of interleukin-6 promoter activity by the 24 kDa isoform of fibroblast growth factor-2 in HeLa cells.

The expression of the interleukin (IL-6) gene can be regulated by various activating or inhibitory stimuli. This modulation involves several regulatory binding sites on the IL-6 promoter, and appears to be in general cell-specific. We have previously described that the nuclear 24 kDa isoform of fibroblast growth factor-2 (FGF-2) is able to increase IL-6 gene expression in NIH-3T3 cells. The transduction pathway involved was shown to be distinct from the extracellular mode of action of the smallest 18 kDa FGF-2 isoform. In the present study, we show that 24 kDa FGF-2-encoding vectors transfected into HeLa cells inhibit various co-transfected constructs incorporating the promoter element of the IL-6 gene and either the luciferase or the chloramphenicol acetyltransferase units. This down-regulation occurs dose-dependently with the 24 kDa FGF-2, is IL-6-promoter-specific, and does not involve an autocrine loop of the growth factor, since exogenously added FGF-2 fails to modulate the IL-6 promoter activity. Furthermore, 24 kDa FGF-2 inhibits the activity of both the co-transfected deletion mutants IL-6(-224) and IL-6(-158), and the point-mutated IL-6 promoter constructs in which the activating protein-1, nuclear factor (NF)-IL-6 and NF-kappaB elements are disrupted. We identify a responsive region to 24 kDa FGF-2 between positions -158 and -109 on the IL-6 promoter, which notably contains a retinoblastoma control element.

Alternative initiation of translation accounts for a 67/45 kDa dimorphism of the human estrogen receptor ERalpha.

The estrogen receptor protein, in the nuclear receptor superfamily, carries two transactivator domains designated AF1 and AF2. The activity of AF2, localized in the carboxy-terminal region, is ligand-dependent, whereas AF1 (amino-terminal) seems to be activated via the MAPKkinase pathway. Uterine and mammary cells exhibiting large amounts of ERalpha were the first estrogen target organs demonstrated. The response intensity in these tissues is related to the affinity of the receptor and to the number of sites occupied by its ligand. Certain physiological and pharmacological phenomena of estrogen resistance associated with a truncated form of ERalpha (deleted in the AF1 domain) would seem however to challenge this assertion. The 45 kDa truncated form is unable to induce cell proliferation but can still increase the expression of certain genes. In this work we suggest that this 45 kDa ERalpha form may originate from differential regulation of translation of the mRNA encoding the ERalpha. In vitro translation studies and transient expression in COS-7 cells in vivo demonstrated a mechanism of translation regulation that produced from a given mRNA either the wild type ER 67 kDa form or the AF1 deleted ER 45 kDa isoform. Bicistronic vectors were used to demonstrate that the 45 kDa protein originates from translation initiation at AUG 174 induced by an internal ribosome entry.

Modifications of benzylphenoxy ethanamine antiestrogen molecules: influence affinity for antiestrogen binding site (AEBS) and cell cytotoxicity.

The antiestrogen binding site (AEBS) is a membranous protein complex that has been shown to be intimately linked with the antiproliferative and antiretroviral effects of certain antiestrogenic compounds such as tamoxifen (Tx). Various specific ligands of AEBS derived from benzylphenoxy ethanamine and a new benzoyl structure were synthesized either by modification of the aminoether side chain or by halogen substitution at the meta-, ortho-, and para position on the benzoyl group. Using the MCF-7 cellular strain and its RTx6 variant (a clone selected for its antigrowth resistance to tamoxifen), it was shown that under high drug concentrations the cytotoxicity of the ligands was directly correlated with their affinity for AEBS. In agreement with previous observations made on triphenylethylenic ligands, modification of the basic ethanamine side chain modulated the ligand affinities. Chloride in meta increased ligand efficacy, whereas chloride substitution in ortho and para decreased it. Effects on AEBS-positive MCF-7 cells were drug concentration- and time-dependent, whereas they were unspecific on the AEBS-negative RTx6 cell line. These cytotoxic effects were confirmed in the absence of estrogen receptor on human AEBS-positive uterine cervix cell carcinoma HeLa cells, but were non-specific on rat fibroblastic AEBS-negative (low concentration) NRK cells. The cytotoxicities of these ligands are related to their affinities for AEBS.

Overexpression of the FGF-2 24-kDa isoform up-regulates IL-6 transcription in NIH-3T3 cells.

We have isolated NIH-3T3 cell lines overexpressing the nuclear 24-kDa isoform of fibroblast growth factor (FGF)-2 and characterized its regulatory effect on the expression of interleukin-6 (IL-6) in these cells. The clone pRF5 expressing the highest level was able to grow in 1% serum medium to a high saturation density and acquired a radioresistance advantage. In pRF5 and another clone pRF1, IL-6 RNA levels were markedly increased. Studies with IL-6 promoter constructs revealed that IL-6 gene up-regulation occurred at the transcriptional level and did not involve the AP-1 binding site. Exogenously added 18-kDa isoform of FGF-2 (100 ng/ml) produced down-regulation of IL-6 involving an AP-1 binding site, thus suggesting a receptor-independent pathway for the intracellular 24-kDa isoform.

Microsomal epoxide hydrolase of rat liver is a subunit of theanti-oestrogen-binding site.

A tritiated photoaffinity labelling analogue of tamoxifen, [(2-azido-4-benzyl)-phenoxy]-N-ethylmorpholine (azido-MBPE), was used to identify the anti-oestrogen-binding site (AEBS) in rat liver tissue [Poirot, Chailleux, Fargin, Bayard and Faye (1990) J. Biol. Chem. 265, 17039-17043]. UV irradiation of rat liver microsomal proteins incubated with tritiated azido-MBPE led to the characterization of two photolabelled proteins of molecular masses 40 and 50 kDa. The amino acid sequences of proteolytic products from the 50 kDa protein were identical with those from rat microsomal epoxide hydrolase (mEH). Treatment of hepatocytes with anti-sense mRNA directed against mEH abolished AEBS in these cells. In addition we found that tamoxifen and N-morpholino-2-[4-(phenylmethyl)phenoxy]ethanamine, a selective ligand of AEBS, were potent inhibitors of the catalytic hydration of styrene oxide by mEH. However, functional overexpression of the human mEH did not significantly modify the binding capacity of [3H]tamoxifen. Taken together, these results suggest that the 50 kDa protein, mEH, is necessary but not sufficient to reconstitute AEBS.

Expression of estrogen receptors during growth of human pancreatic adenocarcinoma cells (Capan-1)-relationship with differentiation.

In steroid target tissues, the presence of the corresponding hormone receptors is indicative of hormone dependence. In an attempt to assess the possible role of steroid hormones in the mechanism of growth and/or differentiation of cancerous pancreatic duct cells, the expression of estrogen receptor (ERalpha) was evaluated in human cancerous pancreatic duct cells (Capan-1) maintained in culture. These cells were selected as they acquire progressively a high degree of differentiation during growth in culture. In the present study, we showed that Capan-1 cells during growth in steroid-free medium associate spontaneously, become polarized, and form duct-like structures, features that are indicative of a high degree of differentiation. Capan-1 cells were also found to express ERalpha and progesterone receptor (PR). Immunoenzymatic assay showed maximal expression of ERalpha (236 +/ 55 fmol/mg protein) on the first day of the exponential growth phase, followed by a marked fall in expression (76.3%). At the onset of the stationary phase (Day 5), ERalpha levels were below 10 fmol/mg protein, becoming undetectable by Day 7. A similar time course was observed for PR: 18 +/- 0.9 fmol/mg protein at the onset of the exponential growth phase and no expression during the stationary phase. Addition of estradiol to 1-d-old cultures resulted in a twofold increase in PR expression, suggesting an induction of PR expression by estrogen. Immunocytochemical analysis with anti-ERalpha-1D5 antibodies showed nuclear and cytoplasmic localization of ERalpha in Capan-1 cells in the first 24 h of culture followed by a progressive disappearance thereafter. We also showed that cellular multiplication was increased by estradiol and progesterone during the exponential growth phase, pointing to the involvement of steroid hormones in the proliferation of nonpolarized Capan-1 cells. These results indicate that the expression of ERalpha is linked to the state of differentiation of the cells and make Capan-1 cells a model of choice to study ER regulation in nontarget tissues.

17 beta-estradiol prevents fatty streak formation in apolipoprotein E-deficient mice.

The reality of the atheroprotective effect of estrogens is still a matter of debate, and its unknown mechanisms could involve favorable changes in blood lipids and lipoproteins and/or direct action at the level of the arterial wall. We used the recently developed animal model of atherosclerosis constituted by apolipoprotein E-deficient mice in an attempt to clarify these issues. Male and female animals, fed a low-fat chow diet, were treated with increasing doses of 17 beta-estradiol (E2) after castration and compared with testosterone treated and uncastrated (intact) animals. Total serum cholesterol, LDL-cholesterol, and HDL-cholesterol concentrations decreased under E2 treatment in each sex and were weakly correlated with lesion area. However, a highly significant correlation between lesion area and serum E2 levels also suggested a direct action of E2 on cells of the vascular wall. A dose-response curve analysis revealed that these activities were sex-dependent, with females being nearly twice as sensitive to E2 as males. It also revealed that the atheroprotective activity was recruited at higher E2 concentrations than those needed by other E2 target tissues such as uterus or functions such as apoA-1 and LDL production and/or clearance rates.

Ligands of the antiestrogen-binding site are able to inhibit virion production of human immunodeficiency virus 1-infected lymphocytes.

Since the discovery of human immunodeficiency retrovirus, the drug arsenal against retrovirus has rapidly increased. Concomitantly, new challenges in the therapy of acquired immune deficiency syndrome have arisen, including drug toxicities, drug resistance, and the development of various cancers as effective therapies prolong survival. Tamoxifen, a nonsteroidal antiestrogen with a low incidence of side effects, is widely used in cancer therapy; it is known to exert pleiotropic activities by binding essentially to the estrogen receptor and other unidentified proteins. In the present work, quantification of the p24 core protein of human immunodeficiency virus 1 produced by infected lymphocytes shows an inhibitory effect of tamoxifen on virion production. Moreover, we assume that this effect is not mediated by the estrogen receptor because antiestrogen ligands interacting with the antiestrogen-binding site exhibit efficacy related to their affinity for this site, although specific antiestrogens of the estrogen receptor are ineffective.

Estrogen synthesis, estrogen metabolism, and functional estrogen receptors in rat arterial smooth muscle cells in culture.

To investigate the mechanisms by which estrogen hormones influence the vascular system, the metabolism of these hormones and the functionality of estrogen receptors were characterized in rat aortic smooth muscle cells from secondary cultures, a widely studied model of vascular biology. Aromatase, estradiol-17 beta-hydroxysteroid dehydrogenase and 17-ketoreductase enzyme activities were demonstrated in these cells. The presence of functional estrogen receptor could also be demonstrated by estrogen-induced transactivating ability in transfection experiments using the luciferase gene reporter and an estrogen responsive element as transcriptional enhancer although the amplitude of the response was only in the range of 140 to 150%. Immunocytochemical analyses, using monoclonal antibodies that recognize epitopes in the A/B domain of the molecule, showed a predominant cytoplasmic localization of these estrogen receptors, even after estrogen addition to the culture medium. Western blot analysis using antibodies that recognize epitopes in the A/B or F domain gave a mol wt of 67,000. Analysis of the estrogen receptor messenger RNA showed that there was no deletion of the proto-signals for nuclear accumulation. The aromatase and dehydrogenase activity results, coupled with the estrogen receptor immunological, RNA analysis, and transfection data strongly support the contention that rat aortic smooth muscle cells are estrogen target cells. This in vitro model is convenient for studying the mechanisms of action of estrogen hormones that seem very peculiar in this cell population.

[Differential effect of estrogens in target tissues]. / Action différentielle des oestrogènes selon les tissus cibles.

Using ER 1D5 new antibody it is shown that the estrogen receptor has an extra-nuclear localisation in vascular cells. This work exhibits, moreover, that the polymorphism of the estrogen receptor is in part regulated at the translation step.

Oestrogen synthesis, oestrogen metabolism and functional oestrogen receptors in bovine aortic endothelial cells.

In order to investigate the mechanisms by which oestrogenic hormones influence the vascular system, we have studied their metabolism and the functioning of oestrogen receptors in bovine aortic endothelial cells from primo-secondary cultures, a widely studied model of vascular pathophysiology. We have demonstrated the enzymic activity of oestradiol-17 beta-hydroxysteroid dehydrogenase, 17-ketoreductase and aromatase in these cells. Immunocytochemical analyses, using two different monoclonal antibodies that recognize epitopes in the A/B domain of the oestrogen receptor, showed that this molecule has a predominantly cytoplasmic localization even after the addition of oestrogen to the culture medium. We showed that the hormone-receptor complexes were functional by demonstrating their transactivating ability in transfection experiments using the luciferase gene reporter and an oestrogen-responsive element transcriptional enhancer, although the amplitude of the response was in the range of only 140-150%: this was not a consequence of the presence of a specific limiting factor, but instead might be related to the peculiar subcellular localization of the oestrogen receptor.