Purification of growth hormone-specific transcription factor GHF-1 containing homeobox.

Pituitary-specific expression of the growth hormone (GH) gene is governed by a transcription factor, GHF-1, that binds to two sites within its promoter. Recently, GHF-1 was shown to be a member of the homeobox family of DNA-binding proteins. An important question is whether GHF-1 controls the expression of other pituitary specific genes, such as prolactin (Prl), expressed in closely related cell types. To this end, GHF-1 was purified from extracts of GH- and Prl-expressing pituitary tumor cells and identified as a 33-kilodalton polypeptide. Although GHF-1 bound to and activated the GH promoter, it did not recognize the Prl promoter. However, at least one other factor in the same extracts, which was easily separated from GHF-1, bound to several sites within the Prl but not the GH promoter. Antibodies to GHF-1 did not react with the Prl binding activity. These results imply that the pituitary-specific expression of GH and Prl is governed by two distinct trans-acting factors.

Function of the homeodomain protein GHF1 in pituitary cell proliferation.

Mutations that cause pituitary dwarfism in the mouse reside in the gene encoding the transcription factor growth hormone factor 1 (GHF1 or pit1). These dwarf mice (dw and dwJ) are deficient in growth hormone (GH) and prolactin (PRL) synthesis and exhibit pituitary hypoplasia, suggesting a stem cell defect. With antisense oligonucleotide technology, a cell culture model of this genetic defect was developed. Specific inhibition of GHF1 synthesis by complementary oligonucleotides led to a marked decrease in GH and PRL expression and to a marked decrease in proliferation of somatotrophic cell lines. These results provide direct evidence that the homeodomain protein GHF1 is required not only for the establishment and maintenance of the differentiated phenotype but for cell proliferation as well.

Growth hormone gene regulation: a paradigm for cell-type-specific gene activation.

The growth hormone (GH) gene is specifically expressed within specialized cells of the anterior pituitary. The central role in GH gene activation is played by GHF-1, a homeodomain protein that is itself specifically expressed in the anterior pituitary.

The transcription factor PLA-1/SKN-1A is expressed in human placenta and regulates the placental lactogen-3 gene expression.

Here we report the selective expression of two POU transcription factor genes, PLA-1 and OCT-1, in human placenta and choriocarcinoma cell lines JAR, JEG-3 and BeWo. Pla-1 protein binds to a POU-consensus DNA sequence in the human placental lactogen-3 (PL-3) promoter and it is capable of trans-activating its transcription up to 18-fold. Other tissue-specific or ubiquitous POU transcription factors such as Pit-1/GHF-1 or Oct-1 showed none or low levels of trans-activation of the PL-3 promoter. In addition, we identified an unique and highly charged region in the N-terminal portion of Pla-1 protein required for full trans-activation of the PL-3 promoter.

Modification of membrane permeability by animal viruses.

Animal viruses modify membrane permeability during lytic infection. There is a co-entry of macromolecules and virion particules during virus penetration and a drastic change in transport and membrane permeability at the late stages of the lytic cycle. Both events are of importance to understand different molecular aspects of viral infection, as virus entry into the cell and the interference of virus infection with cellular metabolism. Other methods of cell permeabilization of potential relevance to understand the mechanism of viral damage of the membrane are also discussed.

Regulation of the pituitary-specific transcription factor GHF-1/Pit-1 messenger ribonucleic acid levels by growth hormone-secretagogues in rat anterior pituitary cells in monolayer culture.

Pituitary-specific expression of the GH gene is dependent on a pituitary-specific transcription factor GH factor-1 (GHF-1), a homeodomain protein also known as pituitary-specific transcription factor-1 (Pit-1). The aim of this study was to investigate the regulation of GHF-1 messenger RNA (mRNA) levels in primary monolayer cultures of rat anterior pituitary cells. Specifically, in addition to direct activators of second messenger signaling systems, we studied the effects of different hormones, all of which are known to be involved in the regulation of somatotroph cell function. We found that GH-releasing hormone (GHRH) increased GHF-1 mRNA levels in a time- and dose-dependent fashion. GHF-1 mRNA levels were increased 2.5-fold (P < 0.01) after incubation for 2 h with 10(-8) M GHRH. Longer incubations (6, 12, or 24 h) with GHRH failed to show a similar stimulatory effect. A significant increase in GHF-1 mRNA concentration (1.7-fold, P < 0.01) was observed after a 2-h treatment with physiological concentrations (10(-11) M) of GHRH. The action of GHRH seems to occur at the transcriptional level without the need of protein synthesis. Thus, treatment of cells with actinomycin D (5 micrograms/ml) completely abolished GHRH-induced increase in GHF-1 mRNA levels. Cycloheximide (23 micrograms/ml) alone increased GHF-1 mRNA levels (6-fold increase after treatment for 12 h, P < 0.01), as well as potentiating GHRH-induced increase in GHF-1 mRNA concentration (9-fold increase after treatment with GHRH plus cycloheximide for 12 h, P < 0.01). The effect of GHRH on GHF-1 mRNA levels could be mimicked by direct activators of second messenger signaling systems such as forskolin (10(-5) M) or the phorbol ester tumor promoter tetradecanoyl phorbol acetate (TPA) (10(-6) M). Other peptides such as pituitary adenylate cyclase activating polypeptide-38 (10(-7) M) but not GHRP-6 (10(-10) to 10(-5) M), were also able to increase GHF-1 mRNA levels. Treatment of the cells with somatostatin (10(-6) M) for either 2 or 48 h failed to modify basal or GHRH-induced GHF-1 mRNA levels. In contrast, pretreatment of the cells with insulin-like growth factor-1 (5 nM) inhibited basal GHF-1 mRNA concentration as well as completely blunting the subsequent response to cells exposed to GHRH for 2 h. These data demonstrate that GHRH, acting at the transcriptional level and through a mechanism not dependent on protein synthesis, plays a stimulatory role on GHF-1 mRNA levels.(ABSTRACT TRUNCATED AT 400 WORDS)

A novel pituitary transcription factor is produced by alternative splicing of the human GHF-1/PIT-1 gene.

An alternative splice acceptor site in intron 1 of the human GHF-1/PIT-1 gene was sequenced. The use of this splice site is responsible for a 78-bp in-frame insertion upstream from exon 2 and leads to the hGHF-2/PIT-2 cDNA detected in normal human pituitary.

Molecular cloning of gilthead seabream (Sparus aurata) pituitary transcription factor GHF-1/Pit-1.

We report here the complete nucleotide sequence of a cDNA clone encoding Sparus aurata GHF-1/Pit-1 isolated from an expression library prepared from gilthead seabream pituitary gland poly(A)+ RNA. The cDNA sequence (saGHF-1/Pit-1) encodes a protein of 371 amino acids (aa) containing a POU domain (aa 194-343) and a transactivation, STA domain (aa 1-128). Northern blot hybridization of pituitary RNA detected a single 3.0 kb band and a rat GHF-1/Pit-1 antiserum was found to immunoreact with pituitary protein species of 42 kDa by Western blot analysis. When compared with mammalian GHF-1/Pit-1 aa sequence, the POU and STA domains of saGHF-1/Pit-1 protein show 83% and 48% aa identity, respectively. In spite of the low homology of the transactivation domain, saGHF-1/Pit-1 is able to activate the transcription of the human growth hormone promoter.

The transcription factor GHF-1, but not the splice variant GHF-2, cooperates with thyroid hormone and retinoic acid receptors to stimulate rat growth hormone gene expression.

The rat growth hormone (GH) promoter was significantly activated in non-pituitary cells by the expression of unliganded trioodothyronine (T3) and retinoic acid (RA) receptors. Furthermore, a strong ligand-dependent activation was found in the presence of the pituitary-specific transcription factor GHF-1. When compared with GHF-1, the splice variant GHF-2 showed a decreased ability to bind the cognate site in the GH promoter. As a consequence, expression of GHF-2 had little stimulatory effect on the GH promoter and did not show cooperation with T3 or RA receptors even in the presence of ligands. Furthermore, over-expression of GHF-2 inhibited the response to T3 and RA in pituitary cells. These results show that alternative splicing of the GHF-1 gene gives rise to two isoforms that differ in their transactivating properties and in their ability to synergize with the nuclear thyroid hormone and retinoic acid receptors on GH gene expression.

Expression of the pituitary transcription factor GHF-1/PIT-1 in cell types of the adult porcine adenohypophysis.

We describe the expression of the transcription factor GHF-1/PIT-1 in adult porcine adenohypophysis by a nonradioactive in situ hybridization (ISH) method using a digoxigenin-labeled cDNA probe corresponding to the entire coding region of rat GHF-1. GHF-1 transcripts were found in 71.7% of adenohypophyseal cells. We also report the simultaneous detection of GHF-1 mRNA and pituitary hormones by combined ISH and immunocytochemistry (IC) in dispersed adenohypophyseal cells, detected with an alkaline phosphatase-NBT/BCIP technique and with an immunoperoxidase-3-amino-9-ethylcarbazole (AEC) method, respectively. The combination of the two techniques neither abolished nor diminished their sensitivity or specificity. GHF-1 is expressed in all five of the cell types in the adult porcine adenohypophysis, showing that this method is suitable for simultaneous detection of transcripts and proteins at the single-cell level.

Differential display RT-PCR analysis of human choriocarcinoma cell lines and normal term trophoblast cells: identification of new genes expressed in placenta.

In this study, we performed the differential display technique to identify genes specifically expressed in human choriocarcinoma cell lines (JEG-3, JAR and BeWo) and normal placental term cells. Few differences were found among the expression profiles of the three choriocarcinoma cell lines and most of the differentially expressed genes were detected in normal term placenta. A total of 36 cDNA fragments were isolated and analysed. Of these, 19 sequences corresponded to regions in the human genome coding for potential novel genes. We confirmed by RT-PCR, the placental mRNA expression of three selected new human genes, on chromosomes 16q12, 9q32 and 6q22. The other 17 sequences showed high similarity to known human genes (like PSG3, FN1, PAI-2). Interestingly, the functions of five known proteins (from genes IK, TRA-1, HERPUD1, UBA-2, and TRAP240) have not yet been well characterized in placenta tissue. In addition, new alternative spliced mRNAs were detected for IK, TRAP240 and PLAC3 genes. The differential expression of the PAI-2 gene among the choriocarcinoma cell lines was also confirmed. The genes identified in this analysis will be of interest for future studies regarding both a better understanding of the biology of the trophoblast cell and the formation of placental tumors.

Implantation of the human embryo: research lines and models. From the implantation research network 'Fruitful'.

Infertility is an increasing problem all over the world, and it has been estimated that 10-15% of couples in fertile age have fertility problems. Likewise induced unsafe abortion is a serious threat to women's health. Despite advances made in assisted reproduction techniques, little progress has been made in increasing the success rate during fertility treatment. This document describes a wide range of projects carried out to increase the understanding in the field of embryo implantation research. The 'Fruitful' research network was created to encourage collaborations within the consortium and to describe our different research potentials to granting agencies or private sponsors.

Action of 3-methylquercetin on poliovirus RNA replication.

3-Methylquercetin is a natural flavone that powerfully blocks poliovirus replication. This compound inhibits selectively poliovirus RNA synthesis both in infected cells and in cell-free systems. Poliovirus double-stranded RNA (replicative forms) is still made in the presence of this inhibitor, whereas the synthesis of single-stranded RNA and the formation of replicative intermediates are drastically blocked.

Adenovirus late protein synthesis is resistant to the inhibition of translation induced by poliovirus.

Inhibition of host protein synthesis after poliovirus infection has been suggested to be a consequence of the proteolytic degradation of a p220 polypeptide necessary to translate capped mRNAs. However, the synthesis of several adenovirus late proteins on capped mRNAs was resistant to poliovirus inhibition. Thus, the hexon protein was still made 8 h after poliovirus superinfection. The synthesis of other adenovirus proteins such as the fiber was much more sensitive to poliovirus-induced inhibition than the hexon, either in the absence or in the presence of guanidine. Detailed densitometric analyses clearly showed the differential behavior of several adenovirus late mRNAs to poliovirus shut-off of translation. This is striking in view of the fact that a common leader sequence in the 5' termini is present in the adenovirus late mRNAs. The use of 3-methyl quercetin, an inhibitor of poliovirus RNA synthesis (Castrillo, J. L., Vanden Berghe, D., and Carrasco, L. (1986) Virology 152, 219-227), showed that translation of several capped adenovirus mRNAs took place in poliovirus-infected cells after the synthesis of host proteins had ceased. The poliovirus mRNA and the adenovirus mRNA coding for the hexon protein are very efficient mRNAs and have a leader sequence of more than 740 and 250 nucleotides, respectively, with very rich secondary structures making it difficult to predict how the scanning model will operate on these two mRNAs.

The inhibition of nucleic acid synthesis in encephalomyocarditis virus-infected L929 cells: effects on nucleoside transport.

Picornavirus infection induces a profound inhibition of labelling of newly synthesized RNA in some cell lines. EMC virus blocks transcription in L929 cells, particularly at early times during infection. This inhibition is not dependent on virus gene expression, since it occurs with UV-inactivated virus and also in the presence of translation inhibitors. The inhibition can be largely accounted for by the blockade of [3H]nucleoside transport, as suggested by the transport kinetics and incorporation of labelled nucleoside from preloaded cells. The inhibition of transport and incorporation into TCA-precipitable material was observed with pyrimidine (uridine, thymidine and cytosine) and purine nucleosides (adenosine and guanosine), but the blockade by EMC virus was higher with the latter nucleosides. Preloading of cells with any of these nucleosides resulted in a decreased effect on nucleoside incorporation into nucleic acid after virus infection. These results suggest that the inhibition of incorporation of labelled nucleosides into nucleic acid in EMC virus-infected cells can be explained, at least in part, by the decreased pool size of the phosphorylated nucleosides. These effects are not specific for L cells, because they are also observed in other cell lines.

3-Methylquercetin is a potent and selective inhibitor of poliovirus RNA synthesis.

3-Methylquercetin (3MQ) is a natural compound isolated from Euphorbia grantii that selectively inhibits poliovirus replication, but has no effect on encephalomyocarditis virus. When the compound is present from the beginning of infection, the bulk of viral protein synthesis is prevented, but the shut-off of host protein synthesis still occurs. Addition of 3MQ 3 hr after infection has a slight effect on viral protein synthesis, suggesting that this compound blocks a step of viral replication different from translation. Indeed, poliovirus RNA synthesis is potently blocked by 3MQ, i.e., 50% inhibition at 2 micrograms/ml (6.3 X 10(-6) M). No effect on encephalomyocarditis, nor on cellular RNA synthesis is observed even at 20 micrograms/ml. The inhibitory effect of 3MQ is reversible, since cells treated with this compound from the beginning of infection start to synthesize viral RNA and proteins when the compound is removed. Strikingly, other natural compounds structurally related to 3-methylquercetin such as quercetin, naringenin, naringin, morin, catechin, kaempferol, myricetin, phloretin, phlorizdin, and rutin do not block poliovirus replication.

Effects of extracellular cations on translation in poliovirus-infected cells.

The effect of pH and different concentrations of added monovalent and divalent cations on translation in poliovirus-infected HeLa cells has been examined. A strong effect on protein synthesis was observed when the concentration of sodium ions was modified. If cells were placed in a hypotonic medium after virus adsorption, no shut-off of cellular translation took place, nor were viral proteins synthesized. An increase in the multiplicity of infection partially overcame this effect. Reversal of the shut-off of cellular translation and inhibition of viral protein synthesis was achieved when cells were placed in hypotonic medium 2 h after infection. Modification of divalent cations (calcium or magnesium) had little or no effect on the pattern of translation. On the other hand, acidic pH (below 6) inhibited both cellular and viral protein synthesis, whereas basic pH had no influence. During infection the synthesis of poliovirus proteins reached a maximum at about 4 to 6 h and then declined. This inhibition of viral translation was partially prevented if cells were placed in a medium containing a high concentration of potassium although the cytopathic effect was prevented. These results indicate that viral protein synthesis and the cytopathic effect were, to a large extent, influenced by the external monovalent ion concentration.

Cation content in poliovirus-infected HeLa cells.

Poliovirus-infected HeLa cells increased their permeability to monovalent ions from the third hour post-infection. At that time infected cells lost their content of potassium ions as measured by efflux of the potassium analogue 86Rb+. The rate of release of 86Rb+ from cells increased as infection proceeded, and was not sensitive to ouabain or quinidine, inhibitors of the Na+/K+ ATPase and Ca2+-induced K+ release system, respectively. The leakage of 86Rb+ was only slightly sensitive to furosemide, an inhibitor of the Na+/K+/Cl- contransport system, suggesting that the mechanism of release involved an increased passive permeability of the cell membrane. The calcium content or its efflux did not vary significantly in the infected cells. Neither were there alterations in the intracellular pH throughout infection.

Pit-1 and Pit-2 role in growth hormone gene regulation.

The role of the POU domain transcription factor Pit-1 (GHF-1) in differentiation and proliferation of the somatotrophic lineage is well known. In our study of differential splicing of the PIT1 gene we found a new protein, Pit-2, in which 26 amino acids are inserted into the transactivation domain. Pit-2 can activate the growth hormone promoter but has lost the ability to activate the prolactin promoter or PIT1 promoter itself.

The P2 and P3 regions of the poliovirus genome are preferentially translated at alkaline pH in infected HeLa cells.

HeLa cells infected with poliovirus exclusively synthesized proteins coded by the P2 and P3 regions of the viral genome when they were placed in an alkaline medium lacking sodium ions. The amount of viral protein synthesized was augmented by increasing the concentration of KCl. Also in the presence of KCl, the cells continued synthesizing this altered pattern of proteins for longer times. This effect occurred in the presence of guanidine, it was reversible, and the normal pattern of poliovirus proteins reappeared when control medium was added, even when guanidine was present. These findings suggest that truncated viral RNA is not made under these conditions. Pactamycin and sodium fluoride, two known inhibitors of the initiation of translation, blocked protein synthesis in cells placed in alkaline medium, indicating the possibility that initiation at internal sites of the viral mRNA may take place under these conditions. Finally, the proteins synthesized were analysed by two-dimensional gel electrophoresis. The proteins migrated as authentic viral proteins indicating that if internal initiation takes place, at least some of these initiation events occur in phase with the initiation codon present in the poliovirus genome.