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M Beato

Publications and source records attributed to M Beato.

At least 37 records · Page 2Linked to original sources
PubMed1998

Transformation-dependent susceptibility of rat hepatic stellate cells to apoptosis induced by soluble Fas ligand.

Cytokine-driven activation of hepatic stellate cells (HSC) in tissue injury and inflammation is a key pathogenetic event in liver fibrogenesis leading to an expanded pool of matrix producing myofibroblasts (MFB) which represent the transformed counterpart of HSC. We hypothesize that expansion of the pool of MFB might also be accomplished by modulation of apoptosis, which plays an opposite and complementary role to mitosis in the cellular homeostasis. We characterized the susceptibility of HSC in primary culture and of MFB in secondary culture to apoptosis induced by the soluble Fas ligand (sFasL) and related the effects to the expression levels of Fas (APO-1/CD95) and some major proapoptotic and contra-apoptotic protooncogenes. MFB showed a dose-dependent apoptotic reaction upon exposure to sFasL as evidenced by a strong increase of nucleosomal DNA fragments, loss of cellular DNA, positive TUNEL reaction, and annexin staining. The effect was found only if protein synthesis (cycloheximide) or RNA synthesis (actinomycin D) were arrested. HSC maintained for various times in primary culture were completely resistant to sFasL in combination with cycloheximide, but in late primary cultures (day 7 onward) an increasing susceptibility to sFasL-mediated apoptosis was developed. By semiquantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis and alkaline phosphatase-anti-alkaline phosphatase staining Fas receptor was identified both in HSC and MFB at comparable expression levels. The expression of the contra-apoptotic protooncogenes bcl-2 and bcl-xl was found to be much stronger in early HSC than in late HSC and MFB as shown by ribonuclease protection assay. The expression of bcl-2 was additionally confirmed by semiquantitative RT-PCR and immunoblotting. Proapoptotic bax was found in comparable quantities at the RNA level in HSC and MFB but at the protein level MFB showed increased bax expression. It is concluded that transformation of HSC to MFB is paralleled by an increasing sensitivity to sFasL-mediated apoptosis, which might be related to a strong decrease of bcl-2 and bcl-xl expression, leading to a preponderance of proapoptotic gene expression in MFB. Modulation of apoptotic susceptibility of transforming HSC could be an important complementary pathway in the pathogenesis of fibrosis.

Animals↗
PubMed1998

Extracellular K+ induces locomotor-like patterns in the rat spinal cord in vitro: comparison with NMDA or 5-HT induced activity.

Bath-application of increasing concentrations of extracellular K+ elicited alternating motor patterns recorded from pairs of various lumbar ventral roots of the neonatal rat (0-2 days old) spinal cord in vitro. The threshold concentration of K+ for this effect was 7.9 +/- 0.8 mM (mean +/- SD). The suprathreshold concentration range useful to evoke persistent motor patterns (lasting >/=10 min) was very narrow ( approximately 1 mM) as further increments elicited only rhythmic activity lasting from 20 s to a few minutes. On average, the fastest period of rhythmic patterns was 1.1 +/- 0.3 s. Intracellular recording from lumbar motoneurons showed that raised extracellular K+ elicited membrane potential oscillations with superimposed repetitive firing. In the presence of N-methyl--aspartate (NMDA) or non-NMDA receptor blockers [R(-)-2-amino-phosphonovaleric acid or 6-cyano-7-nitroquinoxaline-2,3-dione, respectively] extracellular K+ increases could still induce motor patterns although the threshold concentration was raised. Serotonin (5-HT) also induced alternating motor patterns (threshold 15 +/- 7 microM) that were consistently slower than those induced by high K+ or NMDA. Ritanserin (1 microM) prevented the locomotor-like activity of 5-HT but not that of high K+ provided the concentration of the latter was further increased. Subthreshold concentrations of K+ became effective in the presence of subthreshold doses of 5-HT or NMDA, indicating mutual facilitation between these substances. The fastest pattern frequency was observed by raising K+ or by adding NMDA. In the presence of 5-HT, the pattern frequency was never as fast even if NMDA (or high K+) was coapplied. Furthermore, application of 5-HT significantly slowed down the K+- or NMDA-induced rhythm, an effect strongly potentiated in the presence of ritanserin. It is suggested that the operation of the spinal locomotor network was activated by rises in extracellular K+, which presumably led to a broad increase in neuronal excitability. Whenever the efficiency of excitatory synaptic transmission was diminished (for example by glutamate receptor antagonism), a larger concentration of K+ was required to evoke locomotor-like patterns. The complex effect (comprising stimulation and inhibition) of 5-HT on alternating pattern generation appeared to result from a dual action of this substance on the spinal locomotor network.

2-Amino-5-phosphonovalerate↗
PubMed1997

Transcription factor access to chromatin.

The question of how sequence-specific transcription factors access their cognate sites in nucleosomally organized DNA is discussed on the basis of genomic footprinting data and chromatin reconstitution experiments. A classification of factors into two categories is proposed: (i) initiator factors which are able to bind their target sequences within regular nucleosomes and initiate events leading to chromatin remodelling and transactivation; (ii) effector factors which are unable to bind regular nucleosomes and depend on initiator factors or on a pre-set nucleosomal structure for accessing their target sequences in chromatin. Studies with the MMTV promoter suggest that the extent and number of protein-DNA contacts determine whether a factor belongs to one or the other category. Initiator factors have only a few DNA contacts clustered on one side of the double helix, whereas effector factors have extensive contacts distributed throughout the whole circumference of the DNA helix. Thus, the nature of DNA recognition confers to sequence-specific factors their specific place in the sequential hierarchy of gene regulatory events.

Animals↗
PubMed1997

Binding of NF1 to the MMTV promoter in nucleosomes: influence of rotational phasing, translational positioning and histone H1.

To analyse the role of rotational orientation and translational positioning of nucleosomal DNA on transcription factor binding we have generated a series of mutant MMTV promoters containing insertions of various lengths between the hormone-responsive region and the binding site for NF1. These various MMTV promoter fragments were assembled in mononucleosomes and used for structural studies and binding experiments. We show that the insertions change the rotational phase and translational positioning of the NF1 site as predicted if the sequences upstream of the insertion site were the main determinants of nucleosome phasing. In band shift experiments with recombinant NF1 we cannot detect binding of the protein to NF1 sites included within the limits of a nucleosome, independent of their rotational orientation. Moving the NF1 site closer to the nucleosome border also did not permit NF1 binding. This behaviour probably reflects the way NF1 binds DNA, namely it almost completely surrounds the circumference of the double helix establishing a large number of contacts with the bases and the backbone. In contrast to the wild-type and short insertion mutants, NF1 bound readily to nucleosomes containing 30 or 50 bp insertions which placed the NF1 site at the nucleosome edge or within linker DNA. NF1 binding to the linker DNA was unaffected by incorporation of histone H1 into the nucleosome particle. These findings are discussed in relation to chromatin remodelling initiated by steroid hormones during induction of the MMTV promoter.

Animals↗
PubMed1997

Contribution of NMDA and non-NMDA glutamate receptors to locomotor pattern generation in the neonatal rat spinal cord.

The motor programme executed by the spinal cord to generate locomotion involves glutamate-mediated excitatory synaptic transmission. Using the neonatal rat spinal cord as an in vitro model in which the locomotor pattern was evoked by 5-hydroxytryptamine (5-HT), we investigated the role of N-methyl-D-aspartate (NMDA) and non-NMDA glutamate receptors in the generation of locomotor patterns recorded electrophysiologically from pairs of ventral roots. In a control solution, 5-HT (2.5-30 microM) elicited persistent alternating activity in left and right lumbar ventral roots. Increasing 5-HT concentration within this range resulted in increased cycle frequency (on average from 8 to 20 cycles min-1). In the presence of NMDA receptor antagonism, persistent alternating activity was still observed as long as 5-HT doses were increased (range 20-40 microM), even if locomotor pattern frequency was lower than in the control solution. In the presence of non-NMDA receptor antagonism, stable locomotor activity (with lower cycle frequency) was also elicited by 5-HT, albeit with doses larger than in the control solution (15-40 microM). When NMDA and non-NMDA receptors were simultaneously blocked, 5-HT (5-120 microM) always failed to elicit locomotor activity. These data show that the operation of one glutamate receptor class was sufficient to express locomotor activity. As locomotor activity developed at a lower frequency than in the control solution after pharmacological block of either NMDA or non-NMDA receptors, it is suggested that both receptor classes were involved in locomotor pattern generation.

2-Amino-5-phosphonovalerate↗
PubMed1997

Crosslinking of progesterone receptor to DNA using tuneable nanosecond, picosecond and femtosecond UV laser pulses.

UV laser crosslinking is a potentially powerful tool to investigate transient DNA-protein interactions and binding kinetics in intact cells. As the processes underlying UV laser crosslinking are not fully understood, we have performed a study of the influence of laser pulses with different physical parameters on crosslinking of the progesterone receptor to an oligonucleotide containing a hormone-responsive element. We also studied the influence of the various parameters on the amount of laser-irradiated DNA that can be correctly primer extended as an operational measurement of DNA integrity. A strong influence of pulse intensity and pulse length on the crosslink yield was found, likely due to a change in the 'two photon' processes responsible for crosslinking. The highest efficiency of protein crosslinking to DNA was achieved with femtosecond pulses and should be sufficient to enable use of this technique for in vivo studies.

Animals↗
PubMed1997

Progestins prevent apoptosis in a rat endometrial cell line and increase the ratio of bcl-XL to bcl-XS.

Endometrial cell proliferation and cell death are regulated by ovarian hormones. The fall of ovarian progesterone in late secretory phase, or the artificial withdrawal of ovarian hormones during early pregnancy, are followed by programmed cell death of uterine epithelial cells. Aspects of this cell-specific response have been reproduced in a newly established rat endometrial cell line which expresses functional progesterone receptor. At low concentrations of serum and in the absence of glucocorticoids, these cells were dependent on progestins for survival. Removal of progesterone or addition of the antiprogestins RU38486 or ZK98299 led to a substantial increase of apoptotic cells indicated by the accumulation of internucleosomally degraded DNA. The hormonal control of cell proliferation and cell death correlated with the overall quantity and distribution of the different bcl-X transcripts. Progesterone administration not only increased total bcl-X mRNA level but also shifted the quantitative ratio between the different mRNA isoforms in favor for the apoptosis inhibiting form, bcl-XL, compared with the apoptosis promoting form, bcl-XS. These effects were rapid and could not be prevented by inhibitors of protein synthesis. As the low level of bcl-2 and bax mRNA was not influenced by progesterone treatment, the observed changes in total amount of bcl-X transcripts and spliced isoforms could represent the mechanism by which progesterone controls cell death in epithelial cells of the endometrium.

Animals↗
PubMed1997

Nucleosome-mediated synergism between transcription factors on the mouse mammary tumor virus promoter.

In unstimulated mammalian cells and in Saccharomyces cerevisiae, the mouse mammary tumor virus (MMTV) promoter is silent and organized into positioned nucleosomes, one of which encompasses the binding sites for glucocorticoid receptor (GR) and nuclear factor I (NFI). Glucocorticoid induction in vivo involves a functional synergism between GR and NFI and simultaneous occupancy of the promoter sites for both proteins that cannot be reproduced on naked DNA. The role of chromatin in the process of induction was investigated by manipulating the nucleosome density in yeast strains carrying a regulated histone H4 gene. Following depletion of nucleosomes, independent transactivation by NFI or by GR, as well as binding of the individual proteins to the MMTV promoter, were enhanced, in agreement with a repressive function of nucleosomes. In contrast, NFI-dependent hormone induction of the promoter and the simultaneous binding of receptor and NFI were compromised by nucleosome depletion. This effect could be partly mediated by a cryptic binding site for the receptor that is functional only in the nucleosomal context. Thus, positioned nucleosomes do not only account for constitutive repression of the MMTV promoter, but also participate in induction by mediating cooperative binding and functional synergism between GR and NFI.

CCAAT-Enhancer-Binding Proteins↗
PubMed1997

Afferent inputs modulate the activity of a rhythmic burst generator in the rat disinhibited spinal cord in vitro.

Application of strychnine and bicuculline to the isolated spinal cord of the neonatal rat induces spontaneous bursting of regular rhythmicity (cycle period approximately 30 s). This phenomenon is important because it shows that a spinal network, made up by excitatory connections only, generates a very reliable rhythmic pattern. To find out how signals from the periphery or higher centres might influence the operation of the rhythmogenic network, the present experiments examined whether synaptic inputs from dorsal root (DR) or ventrolateral (VL) afferent fibers could modulate this spontaneous rhythmicity. This issue was addressed with intracellular recording from motoneurons or extracellular recording from ventral roots after eliciting bursting with strychnine plus bicuculline. Single electrical shocks (0.1 ms; intensity 1-4 times threshold) applied to one DR reset spontaneous bursting without altering its period or duration. Repetitive stimulations at periods ranging from 20 to 2 s entrained bursts on a 1:1 basis. Burst duration was shorter at lower stimulation periods whereas burst amplitude was unchanged. The lowest stimulation period compatible with burst entrainment depended on stimulus strength. At stimulation periods <2-s entrainment was always lost and spontaneous bursts unexpectedly returned even if electrical pulses still elicited ventral root reflexes. Such spontaneous bursts had similar properties as those recorded in the absence of electrical pulses. Analogous results were obtained with VL stimulations. It is concluded that the spinal rhythmogenic network was highly susceptible to external synaptic inputs, which paced burst generation whereas burst duration was adapted to interstimulus interval. A scheme is provided to explain the modulatory role of synaptic inputs as well as the escape of bursting from fast stimulus entrainment in terms of a rhythmogenic network functionally separated from reflex pathways activated by DR or VL tracts.

Afferent Pathways↗
PubMed1997

Point mutation in the ligand-binding domain of the progesterone receptor generates a transdominant negative phenotype.

A short conserved alpha-helix in the carboxyl-terminal activation function of the ligand-binding domain of steroid hormone receptors, called AF2, is important for ligand-dependent transactivation of inducible genes. We have generated two AF2 mutants of the B isoform of human progesterone receptor (PRB): a point mutant, PRBE911A, and a short deletion, PRB delta907-913R. The two mutants are expressed at levels comparable to the wild type receptor in transfected cells. The PRBE911A mutant showed similar hormone- and DNA- binding affinities as the wild type receptor, whereas the PRB delta907-913R mutant was defective in hormone and DNA binding. Both mutants were inactive when transiently transfected in CV-1 cells, which do not express endogenous PR. However, the point mutant, but not the deletion mutant, inhibited transactivation by cotransfected wild type PRB in a hormone-dependent fashion. The activity of endogenous PR in T47D cells or of endogenous glucocorticoid receptor in HeLa cells was also inhibited by the PRBE911A, but not by the deletion mutant. The point mutant was less active when introduced into an N-terminal truncated form of PR, where it gave rise to proteins that formed homodimers with poor affinity for DNA, but were able to form heterodimers with PRB. The negative dominant phenotype of the PRBE911A mutant likely originates from competition with wild type receptors for binding to DNA and will be useful for mechanistic studies of receptor function.

Amino Acid Sequence↗
PubMed1996

An inhibitor domain in Sp3 regulates its glutamine-rich activation domains.

Sp3 is a ubiquitously expressed human transcription factor closely related to Sp1 and Sp4. All three proteins contain a highly conserved DNA binding domain and two glutamine-rich regions, suggesting that they possess similar activation functions. In our previous experiments, however, Sp3 failed to activate transcription. Instead, it repressed Sp1-mediated transcriptional activation, suggesting that it is an inhibitory member of this family of regulatory factors. Here we show that Sp3 can also act as a positive regulator of transcription. The glutamine-rich domains on their own have a strong activation function and interact with the TATA box binding protein (TBP)-associated factor dTAFII110. However, in full-length Sp3 as well as in Gal4-Sp3 fusion proteins, both activation domains are silenced by an inhibitory domain located between the second glutamine-rich region and the DNA binding domain. The inhibitory domain completely suppressed transcriptional activation when fused to a heterologous glutamine-rich domain but only moderately suppressed transcription when linked to an acidic activation domain. Site-directed mutagenesis identified a stretch of highly charged amino acid residues essential for inhibitor function. Substitution of the amino acid triplet KEE by alanine residues within this region changed the almost transcriptionally inactive Sp3 into a strong activator. Our results suggest that the transcriptional activity of Sp3 might be regulated in vivo by relief of inhibition.

3T3 Cells↗
PubMed1996

Moderate increase in histone acetylation activates the mouse mammary tumor virus promoter and remodels its nucleosome structure.

The mouse mammary tumor virus (MMTV) promoter is regulated by steroid hormones through a hormone-responsive region that is organized in a positioned nucleosome. Hormone induction leads to a structural change of this nucleosome which makes its DNA more sensitive to cleavage by DNase I and enables simultaneous binding of all relevant transcription factors. In cells carrying either episomal or chromosomally integrated MMTV promoters, moderate acetylation of core histones, generated by treatment with low concentrations of the histone deacetylase inhibitors sodium butyrate or trichostatin A, enhances transcription from the MMTV promoter in the absence of hormone and potentiates transactivation by either glucocorticoids or progestins. At higher concentrations, histone deacetylase inhibitors reduce basal and hormone induced MMTV transcription. Inducing inhibitor concentrations lead to the same type of nucleosomal DNase I hypersensitivity as hormone treatment, suggesting that moderate acetylation of core histone activates the MMTV promoter by mechanisms involving chromatin remodeling similar to that generated by the inducing hormones.

Acetylation↗
PubMed1996

17beta-Estradiol induces cyclin D1 gene transcription, p36D1-p34cdk4 complex activation and p105Rb phosphorylation during mitogenic stimulation of G(1)-arrested human breast cancer cells.

MCF-7 human breast cancer cells express functional estrogen receptor and grow in response to estrogen stimulation. G(1)-synchronized MCF-7 cells, made quiescent by exposure to the HMG-CoA reductase inhibitor Simvastatin in estrogen-free medium, readily resume cell cycle progression upon stimulation with 17beta-estradiol (E(2)), even under conditions where polypeptide growth factor-triggered signal transduction pathways are inhibited by the continuous presence of Simvastatin in the culture medium. Under these conditions, cyclin D(1) gene transcription is transiently induced within the first 1-9 h of stimulation, as shown by the accumulation of cyclin D(1) mRNA and protein (p36(D(1))) in the cell and by enhanced expression of stably transfected D(1) promoter-luciferase hybrid genes. Estrogen-induced p36(D(1)) associates readily with p32(cdk2) and p34(cdk4), but not with p31(cdk5), which is however abundantly expressed in these cells. Only p36(D(1))-p34(cdk4) complexes are activated by E(2), as detected in cell extracts by immunoprecipitation with anti-D(1) antibodies followed by assessment of phosphotransferase activity toward the retinoblastoma (Rb) gene product and by analysis of p105(Rb) phosphorylation in vivo. An estrogen-responsive regulatory region has been mapped within the first 944 bp upstream of the transcriptional startsite of the human D(1) gene. Sequence analysis of this DNA region reveals that the cis-acting elements responsive to estrogen are likely to be different in this case from the canonical EREs.

Breast Neoplasms↗
PubMed1996

Chromatin structure of the MMTV promoter and its changes during hormonal induction.

1. The packaging of nuclear DNA in chromatin determines the conversion of the genetic information into a defined phenotype by influencing the availability of DNA sequences for interactions with regulatory proteins and transcription factors. 2. We have studied the influence of the first level of chromatin organization, the nucleosome, on the activity of the mouse mammary tumor virus (MMTV) promoter. The MMTV promoter is strongly transcribed in response to steroid hormones but is virtually silent in the absence of hormonal stimuli. Full hormonal induction requires binding of the hormone receptors to four hormone-responsive elements (HREs), as well as binding of nuclear factor I (NFI) and the octamer transcription factor 1 (OTF-1 or Oct-1) to sites located between the HREs and the TATA box. A full loading with transcription factors cannot be achieved on free DNA due to steric hindrance between hormone receptor and NFI and between NFI and OTF-1. 3. The low basal activity of the MMTV promoter is most likely due to its organization in a positioned nucleosome. In the intact cell, as well in reconstituted chromatin, the regulatory region of the MMTV promoter is wrapped around a histone octamer in a precise rotational orientation, which permits access of the hormone receptors to only two of the four HREs, while precluding binding of NFI and OTF-1 to their respective sites. Upon hormone induction, the nucleosome is remodeled and the path of its DNA altered in a way which makes the nucleosomal dyad axis more accessible to DNase I and enables occupancy of all relevant sites: the four HREs, as well as the binding sites for NFI and OTF-1. 4. These results suggest that the nucleosomal organization of the MMTV promoter not only is responsible for the low activity prior to hormone treatment, but also may be a prerequisite for full loading with transcription factors after hormone induction. We conclude that the DNA contains topological information which modulates the expression of the genetic program.

Animals↗
PubMed1996

Chromatin structure and the regulation of gene expression: remodeling at the MMTV promoter.

The role of the various levels of chromatin organization in the control of eukaryotic gene expression is discussed on the basis of recent advances in our understanding of chromatin structure in well-defined model systems. Particular attention is devoted to the precise structure and the possible functions of positioned nucleosomes and to the enzymatic mechanism of nucleosome remodeling. Some of the principles involved are illustrated with genomic footprinting results obtained with the mouse mammary tumor virus promoter, the nucleosomal organization of which is remodeled following transcriptional induction by steroid hormones. In this system a positioned nucleosome is responsible for transcriptional repression prior to hormone administration and participates in hormonal induction by facilitating the functional interaction among transcription factors on the promoter.

CCAAT-Enhancer-Binding Proteins↗
PubMed1996

Transcriptional regulation by steroid hormones.

Steroid hormones influence the transcription of a large number of genes by virtue of their interaction with intracellular receptors, which are modular proteins composed of a ligand binding domain, a DNA binding domain, and several transactivation functions distributed along the molecule. The DNA binding domain is organized around two zinc ions and allows the receptors to bind as homodimers to palindromic DNA sequences, the hormones responsive elements (HRE), is such a way that each homodimer contacts one half of the palindrome. Since the two halves are separated by three base pairs, the two homodimers contact the same face of the double helix. Before hormone binding, the receptors are part of a complex with multiple chaperones which maintain the receptor in its steroid binding conformation. Following hormone binding, the complex dissociates and the receptors bind to HREs in chromatin. Regulation of gene expression by hormones involves an interaction of the DNA-bound receptors with other sequence-specific transcription factors and with the general transcription factors, which is partly mediated by co-activators and co-repressors. The specific array of cis regulatory elements in a particular promoter/enhancer region, as well as the organization of the DNA sequences in nucleosomes, specifies the network of receptor interactions. Depending on the nature of these interactions, the final outcome can be induction or repression of transcription. The various levels at which these interactions are modulated are discussed using as an example the promoter of the Mouse Mammary Tumor Virus and its organization in chromatin.

Animals↗