Expression of Staphylococcus aureus clumping factor A in Lactococcus lactis subsp. cremoris using a new shuttle vector.

Staphylococcus aureus harbors redundant adhesins mediating tissue colonization and infection. To evaluate their intrinsic role outside of the staphylococcal background, a system was designed to express them in Lactococcus lactis subsp. cremoris 1363. This bacterium is devoid of virulence factors and has a known genetic background. A new Escherichia coli-L. lactis shuttle and expression vector was constructed for this purpose. First, the high-copy-number lactococcal plasmid pIL253 was equipped with the oriColE1 origin, generating pOri253 that could replicate in E. coli. Second, the lactococcal promoters P23 or P59 were inserted at one end of the pOri253 multicloning site. Gene expression was assessed by a luciferase reporter system. The plasmid carrying P23 (named pOri23) expressed luciferase constitutively at a level 10,000 times greater than did the P59-containing plasmid. Transcription was absent in E. coli. The staphylococcal clumping factor A (clfA) gene was cloned into pOri23 and used as a model system. Lactococci carrying pOri23-clfA produced an unaltered and functional 130-kDa ClfA protein attached to their cell walls. This was indicated both by the presence of the protein in Western blots of solubilized cell walls and by the ability of ClfA-positive lactococci to clump in the presence of plasma. ClfA-positive lactococci had clumping titers (titer of 4,112) similar to those of S. aureus Newman in soluble fibrinogen and bound equally well to solid-phase fibrinogen. These experiments provide a new way to study individual staphylococcal pathogenic factors and might complement both classical knockout mutagenesis and modern in vivo expression technology and signature tag mutagenesis.

Dictyostelium discoideum as an expression host for the circumsporozoite protein of Plasmodium falciparum.

We have used the cellular slime mold, Dictyostelium discoideum (Dd), to express the Plasmodium falciparum circumsporozoite protein (CS), a potential component of a subunit vaccine against malaria. This was accomplished via an expression vector based on the discoidin I-encoding gene promoter, in which we linked a sequence coding for a Dd leader peptide to the almost complete CS coding region (pEDII-CS). CS production at both the mRNA and protein levels is induced by starving cells in a simple phosphate buffer. Variation in pH or cell density does not seem to influence CS synthesis. CS-producing cells can be grown either on their normal substrate, bacteria, or on a semi-synthetic media, without affecting CS accumulation level. The CS produced in Dd seems similar to the natural parasite protein as judged by its size and epitope recognition by a panel of monoclonal antibodies. We constructed a second expression vector in which the CS is under the control of a Dd ras promoter. CS accumulation can then be induced by external addition of cAMP. Such a tightly regulated promoter may allow expression of proteins potentially toxic to the cell. Thus, Dd could be a useful eukaryotic system to produce recombinant proteins, in particular from human or animal parasites like P. falciparum.

Expression of human estrogen receptor mutants in Xenopus oocytes: correlation between transcriptional activity and ability to form protein-DNA complexes.

The human estrogen receptor (hER) is a trans-acting regulatory protein composed of a series of discrete functional domains. We have microinjected an hER expression vector (HEO) into Xenopus oocyte nuclei and demonstrate, using Western blot assay, that the hER is synthesized. When nuclear extracts from oocytes were prepared and incubated in the presence of a 2.7 kb DNA fragment comprising the 5' end of the vitellogenin gene B2, formation of estrogen-dependent complexes could be visualized by electron microscopy over the estrogen responsive element (ERE). Of crucial importance is the observation that the complex formation is inhibited by the estrogen antagonist tamoxifen, is restored by the addition of the hormone and does not take place with extracts from control oocytes injected with the expression vector lacking the sequences encoding the receptor. The presence of the biologically active hER is confirmed in co-injection experiments, in which HEO is co-introduced with a CAT reporter gene under the control of a vitellogenin promoter containing or lacking the ERE. CAT assays and primer extensions analyses reveal that both the receptor and the ERE are essential for estrogen induced stimulation of transcription. The same approach was used to analyze selective hER mutants. We find that the DNA binding domain (region C) is essential for protein--DNA complex formation at the ERE but is not sufficient by itself to activate transcription from the reporter gene. In addition to region C, both the hormone binding (region E) and amino terminal (region A/B) domains are needed for an efficient transcription activation.(ABSTRACT TRUNCATED AT 250 WORDS)

The transcriptional repressor REST determines the cell-specific expression of the human MAPK8IP1 gene encoding IB1 (JIP-1).

Islet-brain 1 (IB1) is the human and rat homologue of JIP-1, a scaffold protein interacting with the c-Jun amino-terminal kinase (JNK). IB1 expression is mostly restricted to the endocrine pancreas and to the central nervous system. Herein, we explored the transcriptional mechanism responsible for this preferential islet and neuronal expression of IB1. A 731-bp fragment of the 5' regulatory region of the human MAPK8IP1 gene was isolated from a human BAC library and cloned upstream of a luciferase reporter gene. This construct drove high transcriptional activity in both insulin-secreting and neuron-like cells but not in unrelated cell lines. Sequence analysis of this promoter region revealed the presence of a neuron-restrictive silencer element (NRSE) known to bind repressor zinc finger protein REST. This factor is not expressed in insulin-secreting and neuron-like cells. By mobility shift assay, we confirmed that REST binds to the NRSE present in the IB1 promoter. Once transiently transfected in beta-cell lines, the expression vector encoding REST repressed IB1 transcriptional activity. The introduction of a mutated NRSE in the 5' regulating region of the IB1 gene abolished the repression activity driven by REST in insulin-secreting beta cells and relieved the low transcriptional activity of IB1 observed in unrelated cells. Moreover, transfection in non-beta and nonneuronal cell lines of an expression vector encoding REST lacking its transcriptional repression domain relieved IB1 promoter activity. Last, the REST-mediated repression of IB1 could be abolished by trichostatin A, indicating that deacetylase activity is required to allow REST repression. Taken together, these data establish a critical role for REST in the control of the tissue-specific expression of the human IB1 gene.

A liver protein fraction regulating hormone-dependent in vitro transcription from the vitellogenin genes induces their expression in Xenopus oocytes.

Xenopus laevis oocytes were used to assay for trans-acting factors shown previously to be involved in the liver-specific regulation of the vitellogenin genes in vitro. To this end, crude liver nuclear extracts obtained from adult estrogen-induced Xenopus females were fractionated by heparin-Sepharose chromatography using successive elutions with 0.1, 0.35, 0.6, and 1.0 M KCl. When these four fractions were injected into oocytes, only the 0.6-M KCl protein fraction significantly stimulated mRNA synthesis from the endogenous B class vitellogenin genes. This same fraction induced estrogen-dependent in vitro transcription from the vitellogenin B1 promoter, suggesting that it contains at least a minimal set of basal transcription factors as well as two positive factors essential for vitellogenin in vitro transcription, i.e. the NF-I-like liver factor B and the estrogen receptor (ER). The presence of these two latter factors was determined by footprinting and gel retardation assays, respectively. In contrast, injection of an expression vector carrying the sequence encoding the ER was unable to activate transcription from the oocyte chromosomal vitellogenin genes. This suggests that the ER alone cannot overcome tissue-specific barriers and that one or several additional liver components participate in mediating tissue-specific expression of the vitellogenin genes. In this respect, we present evidence that the oocyte germinal vesicles contain an NF-I-like activity different from that found in hepatocytes of adult frogs. This observation might explain the lack of vitellogenin gene activation in oocytes injected with the ER cDNA only.

Valproate treatment of human cord blood CD4-positive effector T cells confers on them the molecular profile (microRNA signature and FOXP3 expression) of natural regulatory CD4-positive cells through inhibition of histone deacetylase.

Regulatory T cells (Tregs) play a key role in immune system homeostasis and tolerance to antigens, thereby preventing autoimmunity, and may be partly responsible for the lack of an appropriate immune response against tumor cells. Although not sufficient, a high expression of forkhead box P3 (FOXP3) is necessary for their suppressive function. Recent reports have shown that histones deacetylase inhibitors increased FOXP3 expression in T cells. We therefore decided to investigate in non-Tregs CD4-positive cells, the mechanisms by which an aspecific opening of the chromatin could lead to an increased FOXP3 expression. We focused on binding of potentially activating transcription factors to the promoter region of FOXP3 and on modifications in the five miRs constituting the Tregs signature. Valproate treatment induced binding of Ets-1 and Ets-2 to the FOXP3 promoter and acted positively on its expression, by increasing the acetylation of histone H4 lysines. Valproate treatment also induced the acquisition of the miRs Tregs signature. To elucidate whether the changes in the miRs expression could be due to the increased FOXP3 expression, we transduced these non-Tregs with a FOXP3 lentiviral expression vector, and found no changes in miRs expression. Therefore, the modification in their miRs expression profile is not due to an increased expression of FOXP3 but directly results from histones deacetylase inhibition. Rather, the increased FOXP3 expression results from the additive effects of Ets factors binding and the change in expression level of miR-21 and miR-31. We conclude that valproate treatment of human non-Tregs confers on them a molecular profile similar to that of their regulatory counterpart.

Expression of the inducible NO synthase in human monocytic U937 cells allows high output nitric oxide production.

Nitric oxide (NO) produced by inducible NO synthase (iNOS, NOS-2) is an important component of the macrophage-mediated immune defense toward numerous pathogens. Murine macrophages produce NO after cytokine activation, whereas, under similar conditions, human macrophages produce low levels or no NO at all. Although human macrophages can express iNOS mRNA and protein on activation, whether they possess the complete machinery necessary for NO synthesis remains controversial. To define the conditions necessary for human monocytes/macrophages to synthesize NO when expressing a functional iNOS, the human monocytic U937 cell line was engineered to synthesize this enzyme, following infection with a retroviral expression vector containing human hepatic iNOS (DFGiNOS). Northern blot and Western blot analysis confirmed the expression of iNOS in transfected U937 cells both at the RNA and protein levels. NOS enzymatic activity was demonstrated in cell lysates by the conversion of L-[3H]arginine into L-[3H]citrulline and the production of NO by intact cells was measured by nitrite and nitrate accumulation in culture supernatants. When expressing functional iNOS, U937 cells were capable of releasing high levels of NO. NO production was strictly dependent on supplementation of the culture medium with tetrahydrobiopterin (BH4) and was not modified by stimulation of the cells with different cytokines. These observations suggest that (1) human monocytic U937 cells contain all the cofactors necessary for NO synthesis, except BH4 and (2) the failure to detect NO in cytokine-stimulated untransfected U937 cells is not due to the presence of a NO-scavenging molecule within these cells nor to the destabilization of iNOS protein. DFGiNOS U937 cells represent a valuable human model to study the role of NO in immunity toward tumors and pathogens.

Characterization of the effects of IL-17F on CHO cell line generation

CHO is the most commonly used mammalian host for the generation of cell lines allowing for the production of high quality therapeutic proteins. The generation of such cell lines is a lengthy and resource-intensive process requiring extensive screening in order to isolate candidates with optimal characteristics, such as growth, stability and productivity. For this reason, the biotechnology industry invests much effort in attempts to optimize CHO expression systems in order to streamline and shorten the cell line selection process. Based on preliminary observations of a facilitated selection of CHO-GS cell lines expressing members of the IL-17 cytokine family, this study investigates the use of IL-17F as a novel enhancing factor for CHO cell line generation. Using two different CHO expression systems (exploiting GS and DHFR-based selection), we demonstrated that IL-17F expression caused a significant increase in the occurrence of colonies during the selection process. All colonies selected produced substantial amounts of IL-17F, suggesting that benefits were conferred, during selection, to those cells expressing the cytokine. Furthermore, transgene expression levels were significantly increased when the selection pressure was raised to a level that would not normally be permissive for colony selection (i.e. 100 |o.M MSX for the CHO-GS expression system or 1000 nM MTX for the CHO-DHFR system). Finally, IL-17F expression was also found to enhance the rate of appearance of clones during single cell subcloning in the absence of selection pressure. Overall, these benefits have the potential to allow a substantial reduction in the length of cell line generation while significantly increasing cell line productivity. Nevertheless, we found that the high IL-17F expression levels required to convey enhancing effects was a limitation when attempting to co-express IL-17F and a recombinant soluble protein of therapeutic interest from independent CMV promoters within the same expression vector. In order to understand and overcome this limitation, studies were designed to characterize the IL-17F enhancing effect at the molecular and cellular level. Regular supplementation of recombinant biologically-active IL-17F into the culture medium during cell line selection was not able to reproduce the enhancing effects of endogenous IL-17F expression. In addition, increased IL-17F expression correlated with increased CHO-GS selection transgene expression at the single cell level. This data suggested a possible effect of IL-17F on viral promoter activity or transgene mRNA stability. It also provided direct evidence that the cells expressing the highest amounts of IL-17F obtained the most benefit. Overall data obtained from these study implied that IL-17F may act through an intracellular mechanism, possibly exerted during secretion. We therefore initiated experiments designed to determine the specific compartment(s) within which IL-17F triggers its effect. This work has identified IL-17F as a potentially powerful tool to optimize the CHO cell line generation process. The characterization of this enhancing effect at the molecular level has given us several insights into overcoming the current limitations, thus paving the way for the development of a viable technology that can be exploited within the biotechnology industry. - La CHO est la cellule hôte de mammifere la plus couramment utilisée dans la création de lignée cellulaire produisant des protéines thérapeutiques de haute qualité. La génération de ces lignées cellulaires est un processus long et exigeant l'utilisation de techniques de sélection robustes afin d'isoler des candidats possédants les caractéristiques optimales de croissance, de productivité et de stabilité d'expression. Les industries biopharmaceutiques ont investi beaucoup d'efforts afin d'optimiser les systèmes d'expression CHO dans le but raccourcir la longueur du procédé de sélection de lignées cellulaires et aussi d'en augmenter l'efficacité. A partir d'observations préliminaires obtenues lors de la génération de lignées cellulaires CHO- GS exprimant une cytokine appartenant à la famille des IL-17, nous avons réalisé une étude portant sur l'utilisation de l'IL-17F humaine (IL-17F) comme nouveau facteur d'optimisation pour la génération de lignées cellulaires CHO. Nous avons démontré, en utilisant les deux systèmes de sélection et d'expression CHO couramment utilisés (le premier exploitant la GS et l'autre basée sur la DHFR), que l'expression de l'IL-17F permet une augmentation significative de la fréquence d'apparition de colonies durant le processus de sélection de lignées cellulaires. Les différentes colonies sélectionnées expriment des quantités substantielles d'IL-17F, suggérant un effet bénéfique lors de la sélection qui serait exclusivement conféré aux cellules exprimant la cytokine. En outre, le niveau d'expression du transgene se trouve significativement augmenté lorsque la pression de sélection est portée à un niveau habituellement trop élevé pour permettre la sélection de colonies (soit 100 |JM MSX pour le système d'expression CHO-GS ou 1000 nM MTX pour le système CHO- DHFR). Enfin, l'expression d'IL-17F permet également d'améliorer la vitesse d'apparition de clones pendant une étape de sous-clonage en l'absence de pression de sélection. L'ensemble de ces effets bénéfiques permettent une réduction substantielle de la durée de génération de lignées cellulaires tout en augmentant considérablement la productivité des lignées obtenues. Néanmoins, nous avons constaté que la nécessité d'exprimer des niveaux élevés d'IL-17F afin obtenir l'ensemble de ses effets bénéfiques devient une contrainte lors de l'utilisation d'un vecteur d'expression composé de deux promoteurs CMV indépendants pour la co-expression de la cytokine et d'une protéine soluble présentant un intérêt thérapeutique. Afin de mieux comprendre et de surmonter cette limitation, plusieurs études ont été effectuées dans le but de mieux caractériser l'effet de IL-17F au niveau subcellulaire. L'apport régulier en IL-17F recombinante et biologiquement active dans le milieu de culture lors de la sélection de lignées cellulaires ne permet pas de reproduire les effets bénéfiques observés par l'expression endogène d'IL-17F. En outre, nous avons constaté que, lors de l'utilisation du système CHO- GS, l'augmentation d'expression de 1TL-17F est corrélée à un accroissement de l'expression du marqueur de sélection au niveau cellulaire. Ces résultats suggèrent un possible effet d'IL- 17F sur l'activité des promoteurs viraux et ainsi fournissent une preuve directe que les cellules exprimant de haut niveau d'IL-17F sont celles qui en profitent le plus. L'ensemble de ces observations mettrait en avant que l'effet d'IL-17F se ferait selon un mécanisme intracellulaire. Nous avons donc étudié le(s) compartiment(s) spécifique(s) dans lequel IL-17F pourrait exercer son effet. Ce travail a permis de définir IL-17F comme un puissant outil pour l'optimisation des procédés de génération de lignées cellulaires CHO. La caractérisation de cette amélioration de l'effet au niveau moléculaire nous a donné plusieurs indications sur la manière de dépasser les limitations actuelles, ouvrant ainsi la voie au développement d'une technologie viable qui peut être exploitée pars l'industrie biotechnologique.

Vasoactive intestinal peptide, pituitary adenylate cyclase-activating peptide, and noradrenaline induce the transcription factors CCAAT/enhancer binding protein (C/EBP)-beta and C/EBP delta in mouse cortical astrocytes: involvement in cAMP-regulated glycogen metabolism.

We have described previously a transcription-dependent induction of glycogen resynthesis by the vasoactive intestinal peptide (VIP) or noradrenaline (NA) in astrocytes, which is mediated by cAMP. Because it has been postulated that the cAMP-mediated regulation of energy balance in hepatocytes and adipocytes is channeled at least in part through the CCAAT/enhancer binding protein (C/EBP) family of transcription factors, we tested the hypothesis that C/EBP isoforms could be expressed in mouse cortical astrocytes and that their level of expression could be regulated by VIP, by the VIP-related neuropeptide pituitary adenylate cyclase-activating peptide (PACAP), or by NA. We report in this study that in these cells, C/EBP beta and C/EBP delta are induced by VIP, PACAP, or NA via the cAMP second-messenger pathway. Induction of C/EBP beta and -delta mRNA by VIP occurs in the presence of a protein synthesis inhibitor. Thus, c/ebp beta and c/ebp delta behave as cAMP-inducible immediate-early genes in astrocytes. Moreover, transfection of astrocytes with expression vectors selectively producing the transcriptionally active form of C/EBP beta, termed liver-enriched transcriptional activator protein, or C/EBP delta enhance the glycogen resynthesis elicited by NA, whereas an expression vector producing the transcriptionally inactive form of C/EBP beta, termed liver-enriched transcriptional inhibitory protein, reduces this resynthesis. These results support the idea that C/EBP beta and -delta regulate gene expression of energy metabolism-related enzymes in astrocytes.

Use of the chicken lysozyme 5' matrix attachment region to generate high producer CHO cell lines.

Scaffold or matrix attachment region (S/MAR) genetic elements have previously been proposed to insulate transgenes from repressive effects linked to their site of integration within the host cell genome. We have evaluated their use in various stable transfection settings to increase the production of recombinant proteins such as monoclonal antibodies from Chinese hamster ovary (CHO) cell lines. Using the green fluorescent protein coding sequence, we show that S/MAR elements mediate a dual effect on the population of transfected cells. First, S/MAR elements almost fully abolish the occurrence of cell clones that express little transgene that may result from transgene integration in an unfavorable chromosomal environment. Second, they increase the overall expression of the transgene over the whole range of expression levels, allowing the detection of cells with significantly higher levels of transgene expression. An optimal setting was identified as the addition of a S/MAR element both in cis (on the transgene expression vector) and in trans (co-transfected on a separate plasmid). When used to express immunoglobulins, the S/MAR element enabled cell clones with high and stable levels of expression to be isolated following the analysis of a few cell lines generated without transgene amplification procedures.

Marked increase in the secretion of a fully antigenic recombinant carcinoembryonic antigen obtained by deletion of its hydrophobic tail.

Carcinoembryonic antigen (CEA) is a well-known tumor marker, consisting of a single heavily glycosylated polypeptide chain (mol. wt 200 kD), bound to the cell surface by a phosphatidylinositol-glycan anchor. The hydrophobic domain, encoded by the 3' end of the open reading frame of the CEA gene is not present in the mature protein. This domain is assumed to play an important role in the targeting and attachment of CEA to the cell surface. To verify this hypothesis, a recombinant CEA cDNA lacking the 78 b.p. of the 3' region, encoding the 26 a.a. hydrophobic domain, was prepared in a Rc/CMV expression vector containing a neomycin resistance gene. The construct was transfected by the calcium phosphate technique into CEA-negative human and rat colon carcinoma cell lines. Geneticin-resistant transfectants were screened for the presence of CEA in the supernatant and positive clones were isolated. As determined by ELISA, up to 13 micrograms of recombinant CEA per 10(6) cells was secreted within 72 hr by the human transfected cells and about 1 microgram by the rat cells. For comparison, two human carcinoma cell lines, CO112 and LS174T, selected for high CEA expression, shed about 45 and 128 ng per 10(6) cells within 72 hr, respectively. Western blot analysis showed that the size of the recombinant CEA secreted by the transfected human cells is identical to that of reference CEA purified from human colon carcinomas metastases (about 200 kD). The recombinant CEA synthesized by the transfected rat carcinoma cells has a smaller size (about 144 kD, possibly due to incomplete glycosylation), as has already been observed for CEA produced by rat colon carcinoma cells transfected with full-length CEA cDNA. The 100-fold increase in secretion of rCEA encoded by truncated CEA cDNA transfected in human cells confirms the essential role of this domain in the targeting and anchoring of the glycoprotein. These results suggest a new approach for the in vitro production of large amounts of CEA needed in research laboratories and for immunoassay kits.

Pore-forming pyocin S5 utilizes the FptA ferripyochelin receptor to kill Pseudomonas aeruginosa.

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.

IB1, a JIP-1-related nuclear protein present in insulin-secreting cells.

JIP-1 is a cytoplasmic inhibitor of the c-Jun amino-terminal kinase activated pathway recently cloned from a mouse brain cDNA library. We report herein the expression cloning of a rat cDNA encoding a JIP-1-related nuclear protein from a pancreatic beta-cell cDNA library that we named IB1 for Islet-Brain 1. IB1 was isolated by its ability to bind to GTII, a cis-regulatory element of the GLUT2 promoter. The IB1 cDNA encodes a 714-amino acid protein, which differs from JIP-1 by the insertion of 47 amino acids in the carboxyl-terminal part of the protein. The remaining 667 amino acids are 97% identical to JIP-1. The 47-amino acid insertion contains a truncated phosphotyrosine interaction domain and a putative helix-loop-helix motif. Recombinant IB1 (amino acids 1-714 and 280-714) was shown to bind in vitro to GTII. Functionally IB1 transactivated the GLUT2 gene. IB1 was localized within the cytoplasm and the nucleus of insulin-secreting cells or COS-7 cells transfected with an expression vector encoding IB1. Using a heterologous GAL4 system, we localized an activation domain of IB1 within the first 280 amino acids of the protein. These data demonstrate that IB1 is a DNA-binding protein related to JIP-1, which is highly expressed in pancreatic beta-cells where it functions as a transactivator of the GLUT2 gene.

Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor.

The homeodomain protein PDX-1, referred as IPF-1/STF-1/IDX-1, is a transcriptional factor that plays a critical role in the control of several genes expressed in the pancreatic islet. PDX-1 gene expression has been previously shown to be reduced in cultured beta-cell lines chronically exposed to high glucose concentrations. As the glucose transporter type 2 (GLUT2) gene expression is selectively decreased in the beta-pancreatic cells of experimental models of diabetes, we postulated that the loss of GLUT2 gene expression in the pancreatic islets of diabetic animals may be due to the loss of PDX-1 transacting function on the GLUT2 gene. We, therefore, investigated the potential role of PDX-1 in the transcriptional control of GLUT2. We have identified a repeat of a TAAT motif (5'-TAATA-ATAACA-3') conserved in the sequence of the human and murine GLUT2 promoters. Recombinant PDX-1 binds to this GLUT2TAAT motif in electrophoretic mobility shift experiments. PDX-1 antiserum detects the formation of the complex of PDX-1 with the GLUT2TAAT motif in nuclear extracts from the pancreatic insulin-secreting cell line, beta TC3. The GLUT2TAAT motif was mutated in the murine GLUT2 promoter (-1308/+49 bp) linked to a luciferase reporter gene and transfected into beta TC3 cells. Compared with the transcriptional activity of the wild type promoter, that of the mutated promoter decreases by 41%. Multiple copies of the GLUT2TAAT motif were ligated 5' to a heterologous promoter and transfected into a PDX-1-expressing cell line (beta TC3) and into cell lines lacking the homeobox factor (InR1-G9 and JEG-3). The GLUT2TAAT motif mediates the activation of the heterologous promoter in the PDX-1-expressing cell line but not in InR1-G9 or JEG-3 cell lines. Furthermore, cotransfection in a PDX-1-deficient cell line with the expression vector encoding PDX-1 transactivates specifically the heterologous promoter containing the multimerized GLUT2TAAT motif. These data demonstrate that the murine GLUT2 promoter is controlled by the PDX-1 homeobox factor through the identified GLUT2TAAT motif.