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A Aranda

Publications and source records attributed to A Aranda.

At least 55 records · Page 3Linked to original sources
PubMed1997

Influence of Ras and retinoic acid on nerve growth factor induction of transin gene expression in PC12 cells.

Nerve growth factor (NGF)- and ras-induced neuronal differentiation of PC12 cells is accompanied by expression of transin, a secreted metalloproteinase. Retinoic acid (RA) is known to exert important effects on neural cell proliferation and differentiation. In this study we have analysed different PC12 sublines which express either activated Ras or dominant negative p21N17 Ras, to evaluate the influence of retinoic acid (RA) on the response of the transin gene to NGF and Ras. There was a good correlation between neurite extension and induction of transin mRNA levels in the different subclones. NGF did not induce transin mRNA in cells which do not differentiate in response to this neurotrophin. In addition, incubation with RA did not detectably increase basal transin mRNA levels, but caused a significant increase in the transin response to NGF or Ras in cells in which these factors induce a neuronal morphology. Sequences contained within 750 base pairs of the 5' flanking region of the transin gene confer responsiveness to NGF and Ras, but do not mediate the stimulatory effect of RA. In addition, expression of oncogenic Raf increases transin promoter activity in PC12 cells, but a dominant-negative Raf mutant was unable to block NGF-induced transin activity suggesting the existence of a bifurcation downstream of ras in the signaling mechanism leading to transin expression by NGF.

Animals↗
PubMed1997

Growth factor ligands of tyrosine kinase receptors activate the Rous sarcoma virus promoter by a Ras- and Raf-dependent mechanism.

In PC12 cells, NGF and other ligands of tyrosine kinase receptors increased the activity of the Rous sarcoma virus (RSV) promoter. IGF-1 stimulated the RSV promoter in SH-SY5Y neuroblastoma cells. This promoter was also activated by oncogenic Ras and Raf in different cell types, and growth factor induction was inhibited by expression of dominant negative forms of ras and raf, showing that its effect is mediated by a Ras- and Raf-dependent mechanism. Therefore, the RSV promoter should not be used in the determination of transfection efficiency in the studies on regulation of gene expression by ligands of tyrosine kinase receptors, ras and raf.

Animals↗
PubMed1997

Analysis of the structure of a natural alternating d(TA)n sequence in yeast chromatin.

We address here the question of the in vivo structure of a natural alternating d(TA)n sequence found at the 3' region of the Saccharomyces cerevisiae FBP1 gene. This sequence consists of 13 TA pairs interrupted by a TT dinucleotide in the middle of the tract. Previous experiments with cruciform-specific nucleases S1 and Endonuclease VII demonstrated the presence in vitro of a cruciform in this region. We also showed this region to be part of a nuclease hypersensitive site flanked by nucleosomes in yeast chromatin. Here we demonstrate, by means of S1 in vivo footprinting, that in yeast plasmids also adopts in vivo a non B-DNA structure where is not a cruciform. A theoretical analysis of this region that it contains a site susceptible to superhelical stress duplex destabilization. The locations and conditions under which alternative structures form in the wild-type sequence and in deletion mutants agree with these theoretical predictions, suggesting that some kind of denaturation is the alternative structure adopted by the sequence in vivo. This suggests that negative superhelical stress sufficient for local denaturation exists in nucleosomal DNA. We also demonstrate by micrococcal nuclease digestions that the deletion of the alternating d(TA)n sequence modifies the chromatin hypersensitive site but does not affect nucleosome positioning.

Base Sequence↗
PubMed1997

Retinoic acid induces secretion of transforming growth factors by PC12 pheochromocytoma cells.

Conditioned medium from PC12 cells incubated with retinoic acid (RA) increases [3H]thymidine incorporation in normal rat kidney (NRK) fibroblasts and 3D9 epithelial cells. The medium also causes anchorage-independent growth of NRK cells, which is strongly potentiated either in the presence of EGF or after activation of latent forms of transforming growth factors (TGFs) by acidification. These results suggest that RA regulates the release of more than one growth factor by PC12 cells. Conditioned media from control or NGF-treated PC12 cells causes growth of NRK cells in soft agar only after acidification. An increase in expression of the TGF-beta1 gene is coincident with NGF-induced neuronal differentiation of PC12 cells. In addition, RA also causes a dose- and time-dependent increase in content of TGF-beta1 transcripts. This increase is, at least in part, secondary to transcriptional activation. Sequences responsible for the effect of RA and NGF are located in the 5'-flanking region of the TGF-beta1 gene. The TFG-beta1 gene has two promoters and in transient transfection assays RA and NGF significantly enhance the activity of constructs containing the second promoter. High-affinity TGF-beta1 receptors were undetectable in PC12 cells both before and after NGF or RA treatment. RA and NGF decrease PC12 cell proliferation and a neutralizing anti-TGF-beta1 antibody does not reverse this inhibition. In summary, an increase in expression and secretion of TGF-beta1 accompanies RA and NGF-induced PC12 cell growth arrest, but TGF-beta1 does not play an autocrine role in this inhibition.

Adrenal Gland Neoplasms↗
PubMed1997

Histone acetylation influences thyroid hormone and retinoic acid-mediated gene expression.

Thyroid hormone (T3) and retinoic acid (RA) receptors regulate transcription of the rat growth hormone (GH) gene through binding to a common hormone response element (HRE) in the promoter. We have investigated the effect of histone acetylation on hormone-dependent expression of the rat GH gene. We examined the effect of butyrate, which induces histone hyperacetylation, and trichostatin A (TSA), a highly specific inhibitor of histone deacetylases. GH-mRNA levels were significantly increased in pituitary GH4C1 cells incubated with T3 and RA, and this response was further stimulated in the presence of 1 mM butyrate. The effect of butyrate was mimicked by TSA. Butyrate and TSA also enhanced the activity of recombinant constructs containing the GH promoter directing chloramphenicol acetyl transferase (CAT) reporter gene expression. CAT activity increased by 4- to 8-fold after incubation with 1 nM T3 and 1 microM RA, and this response was stimulated 2- to 4-fold further in the presence of 0.25 mM butyrate. This concentration of butyrate did not influence basal expression of CAT. TSA produced a dose-dependent increase of CAT activity in the absence of ligands, and between 5 and 200 nM potentiated the effect of T3 and RA. These compounds also increased the hormonal response of constructs in which the HRE was linked to heterologous [mouse mammary tumor virus (MMTV) and thymidine kinase (TK)] promoters. With butyrate >1 mM, basal activity of the GH promoter increased by more than 10-fold and the effect of T3 and RA was no longer observed. Overexpression of T3 receptors was able to counteract the stimulation of basal CAT levels caused by butyrate. Thus, in the absence of ligand, the T3 receptor acts as a constitutive repressor of gene expression. Upon binding of the hormone, the T3 receptor is converted into an activator. Our findings suggest that histone acetylation, which alters chromatin structure, may play an important role in hormone-mediated transcriptional regulation.

Acetylation↗
PubMed1997

Differential regulation of pituitary-specific gene expression by insulin-like growth factor 1 in rat pituitary GH4C1 and GH3 cells.

We have compared the influence of insulin-like growth factor 1 (IGF-1) on pituitary gene expression in the rat cell lines GH4C1 and GH3. Incubation with IGF-1 increased PRL messenger RNA (mRNA) levels in GH4C1 cells by 4- to 5-fold but decreased the levels of PRL transcripts in GH3 cells. In addition, the levels of GH-mRNA that were not affected by IGF-1 in GH4C1 cells were significantly inhibited by the growth factor in GH3 cells. IGF-1 also decreased PRL and GH-mRNA response to T3, retinoic acid, and Fk in GH3 cells. Stability of PRL or GH transcripts was not altered by IGF-1 in GH3 cells, suggesting that the inhibitory effect is exerted at a transcriptional level. The pituitary-specific transcription factor GHF-1/Pit-1 activates both the GH and PRL promoters. As analyzed by Western blot, IGF-1 did not alter GHF-1/Pit-1 protein levels in GH4C1 cells but reduced the levels of the transcription factor in GH3 cells. This decrease is secondary to a reduction of GHF-1/Pit-1 transcripts in IGF-1-treated GH3 cells. Thus, a different effect of IGF-1 on the expression of GHF-1/Pit-1 in GH3 and GH4C1 cells is likely involved in the different regulation of GH and PRL gene in both cell types. IGF-1 increases the activity of the PRL promoter in transient transfection assays in GH4C1 cells by a Ras-dependent mechanism. Expression of oncogenic Ras(Val12) mimics the effect of IGF-1, and the dominant negative Ras(Asn17) blocks IGF-1-mediated stimulation of the PRL promoter in GH4C1 cells. Although IGF-1 did not stimulate the PRL promoter in GH3 cells, Ras(Val12) strongly activated the promoter in these cells. Hence, the machinery to activate Ras-dependent signaling is intact in GH3 cells. Moreover, IGF-1 stimulates the mitogen-activated protein kinase in GH3 cells, showing that the components linking the IGF-1 receptor to Ras are also active. These results suggest that, in addition to the Ras/mitogen-activated protein kinase pathway, IGF-1 could activate a different pathway and that the combination of both is required to elicit PRL gene expression by the growth factor. This second pathway may be defective in GH3 cells that respond to Ras but not to IGF-1.

Animals↗
PubMed1996

Ras- and Raf-mediated regulation of transforming growth factor beta 1 gene expression by ligands of tyrosine kinase receptors in PC12 cells.

Different ligands of tyrosine kinase receptors have neurotrophic or mitogenic effects in PC12 cells. NFG and FGF, which cause morphological differentiation, as well as EGF, that induces cell growth, produce a significant increase of TGF-beta1 transcripts in PC12 cells. Sequences responsible for the transcriptional effects of the growth factors are located in the 5'-flanking region of the TGF-beta1 gene. The TGF-beta1 gene has two promoters and the growth factors significantly enhance the activity of constructs containing either the first or the second promoter. A functional p21ras is required for the regulation of TGF-beta1 by ligands of tyrosine kinase receptors since expression of oncogenic ras in PC12 cells also increases TGF-beta1 transcripts, and a dominant inhibitory ras mutant blocks activation of TGF-beta1 gene expression by NGF. Oncogenic raf stimulates the activity of both promoters and a dominant negative raf also significantly inhibits growth factor activation. As determined by Mv1Lu cell proliferation inhibition assay, PC12 cells release a significant amount of TGF-beta1 in a latent form and incubation with growth factors or expression of oncogenic ras further increase TCF-beta1 production. These results suggest that during proliferation or growth factor-induced differentiation of sympathetic neurons there is an increase in TGF-beta1 that could be an important mediator of neural cells function.

Animals↗
PubMed1996

Comparison of the effects of retinoic acid and nerve growth factor on PC12 cell proliferation, differentiation, and gene expression.

Retinoic acid (RA) produced a dose-dependent inhibition of PC12 cell growth and the appearance of cell clusters without neurite extension. RA-induced cell clumping was similar to that caused by dexamethasone (Dx). Nerve growth factor (NGF) induced neurite extension, and the combination of RA plus NGF produced a maximal decrease in cell proliferation with a mixed morphology in which part of the cell population had neurites and part formed clumps. Transcriptional effects of RA were demonstrated by the increase in the activity of reporter constructs that contain an RA response element. RA also regulated expression of endogenous genes in PC12 cells. The retinoid produced a two- to threefold increase in level of p75LNGFR mRNA (the low-affinity NGF receptor), without altering expression of the trk protoon-cogene (the high-affinity NGF receptor carrying tyrosine kinase activity). RA also caused a transient increase in level of tyrosine hydroxylase (TH) mRNA (twofold after 16 h), which returned to basal levels and then decreased relative to basal levels at 48 h. The effect of NGF on the expression of these genes was identical to that produced by RA. However, incubation with Dx did not induce p75LNGFR mRNA and produced a strong and sustained increase of TH mRNA level (three- to fivefold after 48 h). These results show that, despite the common morphological changes produced by RA and glucocorticoids in PC12 cells, the biochemical changes caused by RA are similar to those produced by NGF. Therefore, RA could initiate a biochemical program of neuronal differentiation in PC12 cells, although a fully differentiated phenotype with neurite extension is not obtained.

Adrenal Medulla↗
PubMed1996

Vitamin D interferes with transactivation of the growth hormone gene by thyroid hormone and retinoic acid.

The thyroid hormone, retinoic acid (RA), and vitamin D regulate gene expression by binding to similar receptors which act as ligand-inducible transcription factors. Incubation of pituitary GH4C1 cells with nanomolar concentrations of vitamin D markedly reduces the response of the rat growth hormone mRNA to thyroid hormone triiodothyronine (T3) and RA. The stimulation of growth hormone gene expression by both ligands is mediated by a common hormone response element (TREGH) present in the 5'-flanking region of the gene, and the inhibition caused by vitamin D is due to transcriptional interference of the vitamin D receptor on this DNA element. No inhibition of the basal promoter activity by the vitamin was observed. The response to T3 and RA of a heterologous promoter containing this element, the palindromic T3- and RA-responsive sequence TREPAL, or a direct repeat of the same motif is also inhibited by vitamin D. In contrast, vitamin D strongly induces the activity of constructs containing a vitamin D response element, and neither T3 nor RA reduces vitamin D-mediated transactivation. Transfection with an expression vector for the retinoid X receptor alpha (RXR alpha) increases transactivation by T3 and RA but does not abolish the inhibition caused by the vitamin. Gel retardation experiments show that the vitamin D receptor (VDR) as a heterodimer with RXR weakly binds to the T3- and RA-responsive elements. Additionally, VDR displaces binding of T3 and RA receptors in a dose-dependent manner. Our data suggest the formation of TR-VDR and RAR-VDR heterodimers with RXR. The fact that the same response element mediates opposite effects of at least four different nuclear receptors provides a greater complexity and flexibility of the transcriptional responses to their ligands.

Animals↗
PubMed1996

Comparison of bronchoscopic diagnostic techniques with histological findings in brain dead organ donors without suspected pneumonia.

BACKGROUND: The techniques for recognising pneumonia in mechanically ventilated patients were evaluated as no "gold standard" is available to establish the diagnosis in these patients. METHODS: A prospective study was performed on nine brain dead organ donors not suspected of having pneumonia to assess the specificity of bacteriological results from different samples by comparing them with the histological findings from an open pulmonary biopsy specimen taken immediately after death through a mini-thoracotomy. RESULTS: Seven of the nine organ donors without clinical evidence of pulmonary infection and not on antibiotic therapy showed histological features of bronchopneumonia. There was no association between the histological findings and quantitative cultures of the lung biopsy specimen. CONCLUSIONS: Histological evidence of pneumonia was common in this group of ventilated patients who had no clinical signs of the disease.

Adult↗
PubMed1995

Nerve growth factor regulates transforming growth factor-beta 1 gene expression by both transcriptional and posttranscriptional mechanisms in PC12 cells.

PC12 cells express two transforming growth factor (TGF)-beta 1 transcripts, 1.7 and 2.5 kb in size. Other TGF-beta s are expressed at much lower levels. Incubation with nerve growth factor (NGF) produced a time- and dose-dependent increase in content of TGF-beta 1 transcripts. The level of the smaller mRNA increased little, whereas that of the 2.5-kb transcript increased more, with the latter becoming the predominant TGF-beta 1 message. NGF differentially regulated the stability of both mRNAs. The half-life of the 2.5-kb transcript was not altered by NGF; however, the half-life of the 1.7-kb mRNA was approximately 6 h and increased to > 30 h on incubation with NGF. In addition, induction of the 2.5-kb TGF-beta 1 mRNA by NGF required de novo protein synthesis, whereas induction of the 1.7-kb TGF-beta 1 mRNA was independent of protein synthesis. The NGF effect was independent of protein kinase C activation, which also preferentially induced the larger transcript. PC12 cells release a significant amount of TGF-beta 1, and incubation with NGF further increases TGF-beta 1 production. This factor is released in a latent form. These results indicate that an increase in expression and secretion of TGF-beta 1 accompanies neuronal differentiation in PC12 cells. Regulation of TGF-beta 1 gene expression by NGF is complex, involving both increased transcription of the TGF-beta 1 gene and stabilization of the smaller TGF-beta 1 transcript.

Animals↗
PubMed1995

Negative regulation of expression of the pituitary-specific transcription factor GHF-1/Pit-1 by thyroid hormones through interference with promoter enhancer elements.

Expression of the growth hormone gene is due to the presence of the pituitary-specific transcription factor GHF-1/Pit-1. The action of the thyroid hormone T3 is mediated by nuclear receptors that regulate transcription by interaction with DNA elements located near promoters of the regulated genes. In this study, we show that T3 inhibits expression of the GHF-1/Pit-1 gene in rat pituitary GH4C1 cells by a novel mechanism that involves transcriptional interference with other regulatory elements of the promoter. Sequences between bp -90 and -200 of the rat GHF-1/Pit-1 gene which do not contain a hormone response element but contain two cyclic AMP-responsive elements mediate most of the repressive effect of T3. The hormone reduces basal levels of GHF-1/Pit-1 promoter activity and antagonizes its response to cyclic AMP and the tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate). A similar repression is found with a heterologous promoter that contains four copies of the cyclic AMP-responsive element motif. This regulation provides a novel example of the cross-talk between the thyroid hormone receptor and the signal transduction pathways used by different hormones and growth factors. Additionally, T3 interferes with in vitro binding of GHF-1/Pit-1 to a positive autoregulatory element located at bp -45 to -63 and has a detectable inhibitory effect on the activity of a promoter construct which extends to bp -90 of 5'-flanking DNA. The regulation of the transcription factor provides a novel example of negative transcriptional regulation by thyroid hormones.

Animals↗
PubMed1995

Retinoic acid induces expression of the transcription factor GHF-1/Pit-1 in pituitary prolactin- and growth hormone-producing cell lines.

PRL and GH gene expression depend on the presence of the pituitary-specific transcription factor GHF-1/Pit-1. We have examined the effects of retinoic acid (RA) on the expression of the GHF-1/Pit-1 gene. RA induced a time- and dose-dependent increase in GHF-1/Pit-1 messenger RNA in the PRL-producing cell line 235-1. A maximal effect (a 2- to 3-fold increase) was obtained after 12-24 h of incubation with 1 microM RA. The level of the transcription factor determined by both Western blotting and gel retardation analysis with a GHF-1/Pit-1-binding site was increased in RA-treated cells compared to that in control cells. Sequences located between -400 and -90 bp mediated a 2- to 3-fold activation of the GHF-1/Pit-1 promoter by RA. The retinoid also increased the response to cAMP and phorbol esters that is mediated by two cAMP-responsive elements (CREs) located in the same promoter fragment. Both CREs are required for RA induction, as deletion of either CRE abolished the response to the retinoid RA also induced GHF-1/Pit-1 gene expression in GH4C1 cells, which produce both PRL and GH. T3 did not affect expression of the GHF-1/Pit-1 gene in 235-1 cells, but decreased basal GHF-1/Pit-1 messenger RNA and promoter activity in GH4C1 cells and blocked the stimulatory effect of RA.

Base Sequence↗
PubMed1994

Determination of the promoter elements that mediate repression of c-fos gene transcription by thyroid hormone and retinoic acid receptors.

Basal and stimulated activity of the c-fos promoter is reduced by triiodothyronine (T3) and retinoic acid (RA) in GH1 cells. We examined the influence of these ligands on the activity of reporter constructs containing the AP-1 site, the serum response element (SRE) and the cyclic AMP responsive element (CRE) of the c-fos promoter under control of an heterologous promoter. T3 and RA decreased the response of AP-1 and SRE sequences to phorbol esters, forskolin or serum but they did not reduce basal or forskolin-stimulated activity mediated by the CRE. Therefore, repression of c-fos gene expression by T3 and RA receptors appears to be exerted through transcriptional interference with the SRE and the AP-1 binding site of the promoter.

Base Sequence↗