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J L Danan

Publications and source records attributed to J L Danan.

At least 19 recordsLinked to original sources
PubMed2005

Expression and localization of alpha-fetoprotein mRNA and protein in human early villous trophoblasts.

Alpha-fetoprotein (AFP) is a major plasma protein produced during human fetal life. It is a good marker for several possible disorders affecting gestation. We previously reported that afp gene expression, which takes place mainly in yolk sac and fetal liver, also occurs in normal human placenta, specifically in early pregnancy. The aim of the present study was to determine the precise location of AFP synthesis sites within the placental villi. In situ hybridization and immunohistochemical experiments were performed on sections obtained from placentas of first-trimester and full-term pregnancies. We found that the pattern of afp gene expression was restricted to specific villous trophoblastic areas in early placentas. Both afp transcripts and AFP protein were mainly located in discontinuous regions, at junctions between two villi and at budding sites. In contrast, no AFP expression was detected in the cytotrophoblastic extravillous proliferative zone or in other placental cell types. According to the earlier studies, no AFP synthesis was detected in placental villous tissue from full-term pregnancies, using in situ hybridization and immunohistochemistry.

Chorionic Villi↗
PubMed2000

Modulation of the far-upstream enhancer of the rat alpha-fetoprotein gene by members of the ROR alpha, Rev-erb alpha, and Rev-erb beta groups of monomeric orphan nuclear receptors.

Expression of the oncodevelopmental alpha-fetoprotein (AFP) gene is tightly regulated and occurs in the yolk sac, fetal liver and intestine, and cancerous liver cells. Transcription of the AFP gene is under the control of three enhancers that are very tissue specific. We have shown that the most upstream of these enhancers, located at -6 kb, works through the combined action of liver-enriched factors and nuclear receptors that bind to three regions of this DNA regulatory element. This study showed that orphan nuclear receptors of the ROR alpha, Re-verb alpha, and Rev-erb beta groups can bind as monomers with high affinity and specificity to an evolutionarily conserved AGGTCA motif in the functionally important region 1 of this AFP enhancer. Transient transfection experiments performed with human HepG2 hepatoma cells showed that overproduction of ROR alpha 4 stimulated the activity of the AFP enhancer in a dose-dependent manner, while that of Rev-erb alpha and Rev-erb beta had the opposite effect. These effects were highly specific and required the integrity of the AGGTCA motif. The action of these nuclear receptors also occurred in the context of the entire 7-kb regulatory region of the rat AFP gene. These results suggest that altering the amounts or activities of these orphan receptors in cells of hepatic or endodermal origin could modulate AFP gene expression in response to a variety of developmental or carcinogenic stimuli.

Animals↗
PubMed1998

Phorbol esters down-regulate alpha-fetoprotein gene expression without affecting growth in fetal hepatocytes in primary culture.

The effects of phorbol esters (phorbol-12,13-dibutyrate, PDB) on alpha-fetoprotein expression and cell growth were assayed by using fetal hepatocytes in primary culture. PDB acts synergistically with epidermal growth factor (EGF) to specifically decrease alpha-fetoprotein (AFP) mRNA levels, without affecting the expression of other genes of the same family, such as albumin and Vitamin D-binding protein (DBP). This effect is PDB-dose dependent, maximal effects being at 10 ng/ml. The implication of protein kinase C (PKC) in this effect seems clear since bisindolylmaleimide (BIS), a specific PKC inhibitor, completely blocks the PDB effect on AFP expression. Nuclear run-on experiments show that the decrease in AFP mRNA levels is mainly due to an inhibition in the transcription rate of the gene. Determination of PKC activities shows that fetal hepatocytes contain mainly Ca(2+)-independent isoenzymes, which patterns of activation was not modified by EGF plus PDB treatment with respect to PDB treatment. We have found that MAPK and JNK activities, c-jun and c-fos mRNA levels and AP-1 binding activity are notably increased when cells are incubated with both EGF and PDB, PDB does not stimulate growth of fetal hepatocytes, measured either as [3H]-thymidine incorporation into DNA or by cell cycle analysis using flow cytometry. All these results suggest that activation of PKC may affect liver gene expression rather than cell growth in fetal hepatocytes.

Albumins↗
PubMed1998

Rat yolk sac explants as a system for studying the regulation of endodermal genes: down-regulation of the alpha-fetoprotein gene by dexamethasone and phorbol ester.

The visceral yolk sac is a fetal membrane with essential placental functions. It is the major site of synthesis of alpha-fetoprotein (AFP), the most abundant plasma protein in the fetus. We developed a system of rat yolk sac explants in serum-free culture medium to study the regulation of endodermal gene expression in yolk sac. The explanted yolk sac tissues retained their double-sided morphology for up to 48 hours. The epithelial cells of both layers remained tightly joined on a basement membrane as seen by light and electron microscopy. This probably accounts for the continued expression of several endodermal cell-specific markers. The levels of mRNA encoding AFP, vitamin D-binding protein (DBP), hepatocyte nuclear factor 1alpha and beta transcription factors did not change during the 48-hour culture period. This reflects the stability of the differentiation state of the yolk sac endodermal cells. Dexamethasone and phorbol ester (TPA) specifically reduced the AFP mRNA level without affecting that of DBP. This suggests that these transduction pathways are functional in the yolk sac during this period of gestation and could be involved in the physiological down-regulation of AFP gene expression before birth. All these results show that this serum-free culture of rat yolk sac explants is a valuable system for further investigating the action of natural compounds and pharmacological drugs on endodermal gene expression during the embryonic and fetal periods.

Animals↗
PubMed1996

Chicken ovalbumin upstream promoter-transcription factor, hepatocyte nuclear factor 3, and CCAAT/enhancer binding protein control the far-upstream enhancer of the rat alpha-fetoprotein gene.

We have further characterized the most distal of the three alpha-fetoprotein (AFP) enhancers required for expression of the AFP gene in fetal hepatocytes and yolk sac endodermal cells. Almost total rat AFP enhancer 3 (E3) activity is driven by a 160-bp fragment at -6 kb containing three target regions for nuclear proteins that cooperate to stimulate transcription from the AFP and the thymidine kinase promoters in HepG2 hepatoma cells. Region 1, recently shown to be crucial for correct function of the enhancer in liver of transgenic mice, is recognized by two sets of transcription factors that bind to partly overlapping sites, 1a and 1b, in a noncooperative and nonexclusive manner. Site 1a contains a motif, AGGTCA, which is recognized by chicken ovalbumin upstream promoter transcription factors (COUP-TFs), but not by hepatocyte nuclear factor 4. Hepatocyte nuclear factor 3 (HNF3) and CCAAT/enhancer binding protein (C/EBP), which bind to regions 2 and 3, respectively, are likely responsible for the liver-specific E3 action. They play a key role by acting in synergy. The participation of nuclear receptors such as COUP-TFs, with C/EBP and HNF3, in the tight control of the distal AFP enhancer is a new, and perhaps key, step toward understanding the regulation and function of this enhancer, which may remain active throughout development.

Animals↗
PubMed1995

The c-jun proto-oncogene down-regulates the rat alpha-fetoprotein promoter in HepG2 hepatoma cells without binding to DNA.

The effects of a phorbol ester (TPA) and of members of the Jun and Fos oncoprotein family on the activity of the rat alpha-fetoprotein (AFP) promoter were checked by using transient expression experiments in HepG2 hepatoma cells. TPA blocked the activity of the rat AFP promoter in a dose-dependent manner. Overexpression of c-Jun specifically repressed the rat AFP promoter but not the albumin promoter. JunB and JunD were poorer inhibitors. c-Fos expression did not potentiate the negative effect of Jun. The Jun-induced repression does not require binding of c-Jun to the AFP promoter. DNase 1 footprinting experiments did not display any high affinity binding site for Jun on the AFP promoter. Integrity of the c-Jun DNA binding domain is not required for the c-Jun protein to block the AFP promoter. The N-terminal part of Jun, which contains the activating domain, is responsible for the repression as shown by using Jun-Gal4 chimera. Jun likely exerts its negative control on the AFP promoter via protein-protein interactions with a not yet identified trans-activating factor within the -134 to +6 region or with a component of the general machinery of transcription. Jun proteins can thus be key intermediates in regulatory cascades which result in the differential modulation of the AFP and albumin gene expression in the course of liver development and carcinogenesis.

Animals↗
PubMed1995

Hepatoma cell-specific expression of a retrovirally transferred gene is achieved by alpha-fetoprotein but not insulinlike growth factor II regulatory sequences.

To target gene expression to malignant hepatic cells, we have constructed recombinant retroviral vectors containing a reporter gene encoding nuclear beta-galactosidase (nls-LacZ) under transcriptional control of regulatory sequences from the rat alpha-fetoprotein (AFP) or human insulinlike growth factor II (IGFII) genes. The AFP and IGFII P3 promoters activate transcription during fetal development and are often reactivated in hepatocellular carcinoma (HCC). Infection of several cultured cell types with the retroviral vector containing the IGFII P3 sequence resulted in expression of the reporter gene in all cell lines tested, including those that do not produce IGFII. In contrast, selective expression was achieved by vectors containing the AFP transcriptional regulatory sequence. Nuclear beta-galactosidase activity was detectable in cells from lines that produce AFP, and not in cells that do not express the AFP gene. In most infected cell lines, retroviral RNA synthesis from the 5' LTR was inhibited, and deletion of the retroviral LTR enhancer did not change expression from either the IGFII P3-nls-LacZ or the AFP-nls-LacZ cassettes. After treatment of cells with 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor (EGF), the decrease in concentrations of endogenous AFP messenger RNA (mRNA) and nls-LacZ mRNA transcribed from the transferred AFP regulatory sequence were similar. In the context of an integrated provirus, the AFP transcriptional regulatory sequence is therefore subject to similar regulatory control as that of the endogenous gene. These data show that the AFP sequence, and not the IGFII P3 promoter we used, is suitable for targeting gene expression to malignant hepatic cells.

3T3 Cells↗
PubMed1995

[Several transcription factors participate in the functioning of the alpha-fetoprotein gene promoter].

The oncodevelopmentally regulated alpha-fetoprotein (AFP) gene offers a very good model system to better understand the molecular mechanisms which dictate the specificity of gene expression in liver and control its tight modulation in the course of development and carcinogenesis. Transcription factors of the CCAAT/enhance-binding protein (C/EBP), hepatocyte nuclear factor-1 (HNF-1), and nuclear factor-1 (NF-1) families can bind in vitro to the promoter of the rat AFP gene, which makes the expression of the AFP gene specific to the liver. We have evaluated the influence of some of these factors on the activity of the AFP promoter by transfection of HepG2 hepatoma cells with the appropriate expression vector plus a CAT plasmid under the control of the AFP promoter. A similar plasmid bearing the rat albumin promoter was used as a control. C/EBP alpha, and C/EBP beta acted as transactivators on the AFP promoter, while LIP, a truncated form of C/EBP beta, was a potent negative regulator of the promoter. Interestingly, HNF-1 beta was found to be more potent than HNF-1 alpha in activating the AFP promoter in the HepG2 cells. This effect was highly promoter and cell specific since it did not occur with the rat albumin promoter or in Chinese hamster ovary cells. HNF-1 beta, which is produced earlier than HNF-1 alpha during liver development, would thus have the greater influence on the AFP promoter in early development. Our results pointed to a key role that NF1 might play in the functioning of the AFP promoter. Indeed, overexpression of NF1 induced a specific decrease in the activity of the AFP promoter. Competition between NF1 and HNF-1 for binding to their overlapping binding sites on the AFP promoter would be critical for modulating its activity.

Animals↗
PubMed1994

Members of the CAAT/enhancer-binding protein, hepatocyte nuclear factor-1 and nuclear factor-1 families can differentially modulate the activities of the rat alpha-fetoprotein promoter and enhancer.

The promoter of the rat alpha-fetoprotein (AFP) gene, which makes the expression of the developmentally regulated AFP gene specific to the liver, is a putative target for transcription factors of the CAAT/enhancer-binding protein (C/EBP), hepatocyte nuclear factor-1 (HNF-1) and nuclear factor-1 (NF-1) families. We have evaluated the influence of these factors on the activity of the AFP promoter by transfection of HepG2 hepatoma cells with the appropriate expression vector plus a CAT plasmid under the control of the AFP promoter. A similar plasmid bearing the rat albumin promoter was used as a control. C/EBP alpha, C/EBP beta and D-binding protein (DBP) acted as trans-activators on the AFP promoter, whereas liver inhibitory protein (LIP), a truncated form of C/EBP beta, was a potent negative regulator of the promoter. C/EBP alpha also bound to and stimulated the activity of the AFP enhancer at -2.5 kb. Interestingly, HNF-1 beta was found to be more potent than HNF-1 alpha in activating the AFP promoter. This effect was specific, as it did not occur with the rat albumin promoter. HNF-1 beta, which is produced earlier than HNF-1 alpha during liver development, would thus have the greater influence on the AFP promoter in early development. Both HNF-1s allowed expression of the AFP promoter in cells of nonhepatic origin. Overexpression of NF-1 induced a specific decrease in the activity of the AFP promoter. This strongly suggests that competition between NF-1 and HNF-1 for binding to their overlapping binding sites on the AFP promoter is critical for modulating its activity. Thus changing combinations of these trans-acting factors may tightly modulate the AFP promoter activity in the course of liver development and carcinogenesis.

Animals↗
PubMed1990

Regulation of the rat alpha-fetoprotein gene expression in liver. Both the promoter region and an enhancer element are liver-specific and negatively modulated by dexamethasone.

Cis-acting elements involved in the control of rat alpha-fetoprotein gene expression in the liver and its modulation by glucocorticoid hormones were detected after transfection of chloramphenicol acetyltransferase constructs and their transient expression into two hepatoma cell lines. The proximal promoter region (-324 to -15) was found to contain all the information necessary for tissue-specific expression. It is also involved in the negative gene modulation by glucocorticoids and includes an activating regulatory domain allowing efficient expression in the HepG2 cells. Three regions within 7 kilobase pairs of the 5' extragenic sequences are capable of stimulating the chloramphenicol acetyltransferase activity driven by the alpha-fetoprotein promoter sequence. One of these regions, at about -2.5 kilobase pairs, contains a short indivisible 170-base pair DNA element that fulfills all the criteria of a tissue-specific enhancer, i.e. orientation and position independence, as well as cell-specific stimulation of gene expression driven by a homologous or heterologous promoter. The enhancing properties of this element are totally abolished by glucocorticoids. DNase I footprinting experiments indicate that several rat liver nuclear proteins interact with this enhancer element.

Animals↗
PubMed1989

A common liver-specific factor binds to the rat albumin and alpha-foetoprotein promoters in vitro and acts as a positive trans-acting factor in vivo.

Rat liver nuclear extracts were tested for the presence of factors which might be common to the transcriptional regulation of both the albumin and alpha-foetoprotein genes. Gel shift assay showed the formation of three complexes (I, II and III) with the albumin probe. Two of them (I and III) could be displaced by the alpha-foetoprotein promoter. Analysis of nuclear extracts from liver, kidney, spleen and brain and competition experiments using several oligonucleotides covering regions from the albumin and alpha-foetoprotein promoters showed that complex III results from the binding of the ubiquitous nuclear factor 1, while complex II involves a CCAAT-box-binding protein also detected in brain and spleen extracts. Complex I is formed upon binding of a liver-specific factor to a proximal element of the rat albumin promoter. This factor also binds to a similar sequence in the alpha-foetoprotein promoter and is closely related to the hepatocyte nuclear factor 1, as shown by competition experiments using an oligonucleotide covering its target sequence on the beta-fibrinogen promoter. Transfection competition experiments indicated that, in vivo, this factor acts as a positive trans-acting element in the expression of both the rat albumin and alpha-foetoprotein genes.

Albumins↗
PubMed1989

The organization of repetitive sequences in the albumin and alpha-fetoprotein gene loci in the rat.

The distribution of middle repetitive sequences in the genic and extragenic regions of the rat albumin and alpha-fetoprotein genes was analyzed. Their presence was determined by probing Southern blots of restriction fragments of albumin and alpha-fetoprotein genomic subclones with 32P-labeled total rat DNA. Repetitive sequences were detected in both genes. They were classified as weak, moderate and intense hybridizing elements according to the intensity of hybridization. Weak repetitive sequences were characterized as dG.dT repeats by using 32P-labeled poly-(dG.dT)(dC.dA) oligomer probe. They occurred in 5' and 3' extragenic regions of the two genes and in introns 4 and 5 of the albumin gene. The moderate repetitive sequence present in intron 6 of the albumin gene was identified as the rat SINES element. 4D12. The intense repetitive sequence, localized in the 3' non-coding region of the albumin gene, corresponded to the terminal segment of a rat high repeat long interspersed DNA family, L1Rn. 4D12 and L1Rn sequences were also scattered throughout the alpha-fetoprotein locus as moderate and intense repetitive elements, respectively, but their distribution was different from that of the albumin genomic region. These results indicate that repetitive sequences invaded the two loci in a non-conservative manner.

Animals↗
PubMed1988

A liver-specific factor and nuclear factor I bind to the rat alpha-fetoprotein promoter.

Proteins putatively involved in the transcriptional control of rat (alpha-fetoprotein (AFP) gene expression in the liver were identified by analyzing the in vitro binding of proteins from nuclear extracts of fetal and adult rat livers to the cloned rat AFP promoter region (-197 to +48) using a combination of gel shift and DNase I footprinting assays. Three stable and specific high affinity complexes (I, II, and III) were detected by gel shift analysis of fetal rat liver extracts. Complex II was specific to extracts from fetal liver while the two others (I and III) were also formed with extracts from adult liver. Complex I was highly liver-specific since it was not detected with extracts of kidney, spleen, and brain. DNase I and gel shift competition experiments using a synthetic oligonucleotide indicated that it is formed upon binding of a liver-specific factor to region -65 to -46 of the rat AFP promoter. This region, perfectly conserved in the rat, mouse, and man, had been previously shown to be crucial for liver-specific expression of the mouse AFP gene. The binding of this liver-specific factor to this DNA region may thus represent a key step in the specificity of expression of AFP gene in liver. Gel shift and DNase I footprinting competition experiments showed that complex III was formed by binding of the widely distributed nuclear factor I to the rat AFP promoter in the region -125 to -100. It is potentially significant that, in extracts from fetal liver, nuclear factor is also involved with another binding factor in the formation of the stage-specific complex II.

Animals↗
PubMed1987

The rat alpha-fetoprotein and albumin genes. Transcriptional control and comparison of the sequence organization and promoter region.

Functional and structural approaches were used to characterize the transcription units of the rat alpha-feto-protein (AFP) and albumin genes. A cell-free nuclear transcription assay and several genomic clones were used to show that: 1) the rate of transcription of these genes is closely related to the levels of corresponding mRNAs in the yolk sac and during rat liver development, indicating that the expression of the albumin and AFP genes is mainly regulated at the transcriptional level in the rat, and 2) the in vivo 5' end boundaries of the rat AFP and albumin transcription domains were mapped near the respective first exons. Due to the presence of repeated sequences, the 3' end boundary of both genes could not be accurately defined in the same manner. 3) No transcription could be detected until 7 kilobases upstream from the cap site of these genes. In addition, the organization of the rat AFP gene was analyzed by restriction endonuclease mapping, S1 nuclease mapping, and nucleotide sequencing. Our results indicate that: 1) the rat AFP gene is 20 kilobase pairs long and is split into 15 exons by 14 intervening sequences; 2) the transcription initiation site of the rat AFP gene is heterogenous; 3) the 5'-flanking region upstream from the rat AFP gene exhibits 60-90% similarity with the mouse and human AFP genes while no major nucleotide identity is found with the rat albumin gene; 4) a 90-base pair sequence present as one copy upstream from the rat and mouse AFP genes is present as two copies in the human genome; 5) several inverted repeats are mapped in the 5'-flanking region indicating potential stem-loop structures. One highly conserved structure encompasses an enhancer-like core sequence and the sequence recognized by the TGGCA-binding protein.

Age Factors↗
PubMed1985

Regulation of rat yolk sac 25-hydroxy- and 1,25-dihydroxyvitamin D3 24-hydroxylase activities.

The yolk sac of the pregnant rat which functions as a true placenta is a target organ for vitamin D. This tissue can hydroxylate in position 24 both 25-hydroxy- and 1,25-dihydroxyvitamin D3 (25-OHD3 and 1,25-(OH)2D3). The present report describes an in vitro model for the study of 1,25-(OH)2D3 action on the further metabolism of 25-OH[3H]D3 and 1,25-(OH)2[3H]D3 by yolk sac. The tissue explants were preincubated with 1,25-(OH)2D3 for 18 h in a serum-free culture medium. Physiological concentrations of 1,25-(OH)2D3 were the most effective in stimulating (7.5-fold) the 1,25-(OH)2D3 24-hydroxylase, while the 25-OHD3 24-hydroxylase stimulation (4-fold) required a 1,25-(OH)2D3 concentration of 10(-7) M. The stimulating effect of 1,25-(OH)2D3 on the 1,25-(OH)2D3 24-hydroxylase was temperature-dependent, and, since its was inhibited by actinomycin D and cycloheximide, required de novo protein synthesis. 1,24,25-(OH)3D3, 25-OHD3, and 24,25-(OH)2D3 were 10- to 1000-fold less potent than 1,25-(OH)2D3 in inducing the 1,25-(OH)2D3 hydroxylase. Our results strongly suggest that 1,25-(OH)2D3 regulated the 1,25-(OH)2D3 24-hydroxylase by a receptor-mediated process. Furthermore, 1,25-(OH)2D3 at 10(-9) M induced within 4 h an increase of its own degradation and the formation of an as yet unidentified major 1,25-(OH)2[3H]D3 metabolite. We conclude that the yolk sac can participate in the regulation of 1,25-(OH)2D3 concentration in the fetoplacental unit.

24,25-Dihydroxyvitamin D 3↗
PubMed1985

Presence of immunoreactive vitamin D-binding protein in rat yolk sac endodermal cells.

The visceral yolk sac is, in the rat, an organ which possesses true placental functions. We recently showed that yolk sac is involved in the control of metabolism and action of vitamin D in the fetoplacental unit, since its endodermal cells contain a 24-hydroxylase for vitamin D metabolites and the 1,25-dihydroxyvitamin D receptor. In the present work, by using indirect immunoperoxidase staining, we demonstrate that an immunoreactive vitamin D-binding protein (DBP) is present in this yolk sac throughout embryonic and fetal development. It is mainly located at the apex of the endodermal cells. Immunoprecipitation studies of radioactive proteins synthesized in vitro by yolk sac explants showed that yolk sac DBP, in contrast to alpha-fetoprotein, is not synthesized in situ by yolk sac. This result, combined with the location of DBP at the apex of the endodermal cells which face the uterus, strongly suggests that yolk sac DBP is of maternal origin. The concomitant presence in the endodermal cells of this DBP, of the 1,25-dihydroxyvitamin D receptor, and of the system hydroxylating vitamin D metabolites in position 24, certainly has considerable physiological significance.

Animals↗