-
[show abstract]
[hide abstract]
ABSTRACT: Post-lactational involution has been reported to share common features with breast tumor development. A deep characterization of the signaling triggered after weaning would help to unveil the complex relationship between involution and breast cancer. NF-κB, a crucial factor in the involuting gland, might be an important regulatory node for signal amplification after weaning; however there is limited information about the identity of NF-κB-target genes and the molecular mechanisms leading to the selection of genes involved in a particular biological process. We identified 4532 target genes in mammary gland at 48h weaning, by genome-wide analysis of regions bound by RelA(p65)-NF-κB in vivo. It was found that among total RelA(p65)-NF-κB-enriched genes, only 268 bound the trans-activating complex p65/p300. Our results suggest that the latter represents a major complex preferentially involved in the modulation of the inflammatory response at 48 h of mammary gland involution. A genome-wide factor location analysis revealed that p65-binding had a heterogeneous distribution while the complex of p65 and its co-activator p300 were mainly bound to proximal promoters near transcription start sites. Moreover, our computational analysis predicts the existence of cooperating elements on RelA-NF-κB/p300-enriched genes that could explain preferential binding and modulation of gene expression during mammary gland involution.
Cellular Physiology and Biochemistry 01/2011; 28(5):833-46. · 2.86 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Id2 is a pleiotropic protein whose function depends on its expression levels. Id2-deficient cells show increased cell death. This study explored the molecular mechanisms for the modulation of Id2 expression elicited by GSH and oxidative stress in the liver of acetaminophen (APAP)-intoxicated rats. APAP-overdose induced GSH depletion, Id2 promoter hypoacetylation, RNApol-II released and, therefore, Id2 down-regulation. Id2 expression depends on c-Myc binding to its promoter. APAP-overdose decreased c-Myc content and binding to Id2 promoter. Reduction of c-Myc was not accompanied by decreased c-myc mRNA, suggesting a mechanism dependent on protein stability. Administration of N-acetyl-cysteine prior to APAP-overload prevented GSH depletion and c-Myc degradation. Consistently, c-Myc was recruited to Id2 promoter, histone-H3 was hyperacetylated, RNApol II was bound to Id2 coding region and Id2 repression prevented. The results suggest a novel transcriptional-dependent mechanism of Id2 regulation by GSH and oxidative stress induced by APAP-overdose through the indirect modulation of the proteasome pathway.
Free radical research 09/2010; 44(9):1044-53. · 2.22 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Proteomic studies in the mammary gland of control lactating and weaned rats have shown that there is an increased pattern of nitrated proteins during weaning when compared with controls. Here we report the novel finding that cathepsin D is nitrated during weaning. The expression and protein levels of this enzyme are increased after 8 h of litter removal and this up-regulation declines 5 days after weaning. However, there is a marked delay in cathepsin D activity since it does not increase until 2 days post-weaning and remains high thereafter. In order to find out whether nitration of cathepsin D regulates its activity, iNOS (inducible nitric oxide synthase)(-/-) mice were used. The expression and protein levels of this enzyme were similar to WT (wild-type) animals, but the proteolytic activity was significantly reduced during weaning in knockout compared to WT mice. in vitro treatment of recombinant human cathepsin D or lactating mammary gland homogenates with relatively low concentrations of peroxynitrite enhances the nitration as well as specific activity of this enzyme. Using MS, it has been shown that the residue Tyr168 was nitrated. All of these results show that protein nitration during weaning might be a signalling pathway involved in mammary gland remodelling.
Biochemical Journal 02/2009; 419(2):279-88. · 4.90 Impact Factor
-
Javier Pereda,
Javier Escobar,
Juan Sandoval,
José Luis Rodríguez,
Luis Sabater,
Federico V Pallardó, Luis Torres,
Luis Franco,
José Viña,
Gerardo López-Rodas,
Juan Sastre
[show abstract]
[hide abstract]
ABSTRACT: Glutathione depletion is a key factor in the development of acute pancreatitis. Our aim was to study the regulation of glutamate cysteine ligase, the rate-limiting enzyme in glutathione synthesis, in edematous or necrotizing pancreatitis in rats. Glutathione levels were kept low in necrotizing pancreatitis for several hours, with no increase in protein or mRNA levels of glutamate cysteine ligase subunits, despite binding of RNA polymerase II to their promoters and coding regions. The survival signal pathway mediated by ERK and c-MYC was activated, and c-MYC was recruited to the promoters. The failure in gene up-regulation seems to be due to a marked increase in cytosolic ribonuclease activity. In contrast, in edematous pancreatitis glutathione levels were depleted and rapidly restored, and protein and mRNA expression of glutamate cysteine ligase increased markedly due to enhanced transcription mediated by recruitment of c-MYC, NF-kappaB, and SP-1 to the promoters. No increase in cytosolic ribonuclease activity was found in this case. We propose a novel pathophysiological mechanism to differentiate necrotizing from edematous pancreatitis, which is the inefficient up-regulation of glutamate cysteine ligase caused by increased cytosolic ribonuclease activity in the severe form of the disease. This mechanism would abrogate a rapid recovery of glutathione levels.
Free Radical Biology and Medicine 05/2008; 44(8):1599-609. · 5.42 Impact Factor
-
Angel Rubio,
Elizabeth Guruceaga,
Mercedes Vázquez-Chantada,
Juan Sandoval,
L Alfonso Martínez-Cruz,
Victor Segura,
José L Sevilla,
Adam Podhorski,
Fernando J Corrales, Luis Torres,
Manuel Rodríguez,
Fabienne Aillet,
Usue Ariz,
Felix Martínez Arrieta,
Juan Caballería,
Antonio Martín-Duce,
Shelly C Lu,
M Luz Martínez-Chantar,
José M Mato
[show abstract]
[hide abstract]
ABSTRACT: We have integrated gene expression profiling of liver biopsies of NASH patients with liver samples of a mouse model of steatohepatitis (MAT1A-KO) to identify a gene-pathway associated with NASH.
Affymetrix U133 Plus 2.0 microarrays were used to evaluate nine patients with NASH, six patients with steatosis, and six control subjects; Affymetrix MOE430A microarrays were used to evaluate wild-type and MAT1A-KO mice at 15 days, 1, 3, 5 and 8 months after birth. Transcriptional profiles of patients with NASH and MAT1A-KO mice were compared with those of their proficient controls.
We identified a gene-pathway associated with NASH, that accurately distinguishes between patients with early-stage NASH and controls. Patients with steatosis have a gene expression pattern intermediate between that of NASH and controls. Promoter analysis revealed that 34 of the genes associated with NASH contained an Sp1 element. We found that Sp1 binding to these genes is increased in MAT1A-KO mice. Sp1 is also hyperphosphorylated in MAT1A-KO as well as in patients with NASH and steatosis.
A gene-pathway associated with NASH has been identified. We speculate that hyperphosphorylation of Sp1 may be involved in the genesis of steatosis and that other factors, such as oxidative stress, may trigger its progression to NASH.
Journal of Hepatology 05/2007; 46(4):708-18. · 9.26 Impact Factor
-
José L Rodríguez,
Abdelhalim Boukaba,
Juan Sandoval,
Elena I Georgieva,
M Ujue Latasa,
Elena R García-Trevijano,
Gaetano Serviddio,
Toshikazu Nakamura,
Matías A Avila,
Juan Sastre, Luis Torres,
José M Mato,
Gerardo López-Rodas
[show abstract]
[hide abstract]
ABSTRACT: Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine, the main methyl donor. Two MAT-encoding genes (MAT1A, MAT2A) are found in mammals. The latter is expressed in proliferating liver, dedifferentiation and cancer, whereas MAT1A is expressed in adult quiescent hepatocytes. Here, we report studies on the molecular mechanisms controlling the induction of MAT2A in regenerating rat liver and in proliferating hepatocytes. The MAT2A is up-regulated at two discrete moments during liver regeneration, as confirmed by RNApol-ChIP analysis. The first one coincides with hepatocyte priming (i.e. G0-G1 transition), while the second one takes place at the G1-S interface. Electrophoretic mobility shift assays showed that a putative E2F sequence present in MAT2A promoter binds this factor and ChIP assays confirmed that E2F1, E2F3 and E2F4, as well as the pocket protein p130, are bound to the promoter in quiescent liver. MAT2A activation is accompanied by changes in the binding of histone-modifying enzymes to the promoter. Interestingly, p130 is not displaced from MAT2A promoter during hepatocyte priming, but it is in the late expression of the gene at the G1-S transition. Finally, the transcription factor Sp1 seems to play a decisive role in MAT2A induction, as it binds the promoter when the gene is being actively transcribed. In summary, the present work shows that the molecular mechanism of MAT2A expression is different during G0-G1 or G1-S transition and this may be related to the distinct requirements of S-adenosylmethionine during liver regeneration.
The International Journal of Biochemistry & Cell Biology 02/2007; 39(4):842-50. · 4.63 Impact Factor
-
José L Rodríguez,
Juan Sandoval,
Gaetano Serviddio,
Juan Sastre,
María Morante,
Maria-Giulia Perrelli,
María L Martínez-Chantar,
José Viña,
Juan R Viña,
José M Mato,
Matías A Avila,
Luis Franco,
Gerardo López-Rodas, Luis Torres
[show abstract]
[hide abstract]
ABSTRACT: The Id (inhibitor of DNA binding or inhibitor of differentiation) helix-loop-helix proteins are involved in the regulation of cell growth, differentiation and cancer. The fact that the molecular mechanisms of liver regeneration are not completely understood prompted us to study the fate of Id2 in proliferating liver. Id2 increases in liver regeneration after partial hepatectomy, following the early induction of its gene. Co-immunoprecipitation shows that Id2 forms a complex with E2F4, p130 and mSin3A in quiescent liver and all these components are present at the c-myc promoter as shown using ChIP (chromatin immunoprecipitation). Activation of c-myc during hepatocyte priming (G0-G1 transition) correlates with the dissociation of Id2 and HDAC (histone deacetylase), albeit p130 remains bound at least until 6 h. Moreover, as the G0-G1 transition progresses, Id2 and HDAC again bind the c-myc promoter concomitantly with the repression of this gene. The time course of c-myc binding to the Id2 promoter, as determined by ChIP assays is compatible with a role of the oncoprotein as a transcriptional inducer of Id2 in liver regeneration. Immunohistochemical analysis shows that Id2 also increases in proliferating hepatocytes after bile duct ligation. In this case, the pattern of Id2 presence in the c-myc promoter parallels that found in regenerating liver. Our results may suggest a control role for Id2 in hepatocyte priming, through a p130 dissociation-independent regulation of c-myc.
Biochemical Journal 10/2006; 398(3):431-7. · 4.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The biological functions of vitamin E have been classically attributed to its property as a potent inhibitor of lipid peroxidation in cellular membranes. However, in 1991, Azzi's group first described that alpha-tocopherol inhibits smooth muscle cell proliferation in a protein kinase C (PKC)-dependent way, demonstrating a non-antioxidant cell signalling function for vitamin E. More recently, the capacity of alpha-tocopherol to modulate gene expression with the implication of different transcription factors, beyond its antioxidant properties, has also been established. This study was to determine the effect of vitamin E-deficiency on liver nuclear factor-kappa B (NF-kappaB) DNA-binding activity and the response of target antioxidant-defense genes and cell cycle modulators. Rats were fed either control diet or vitamin-E free diet until 60 or 90 days after birth. Vitamin E-deficiency enhanced liver DNA-binding activity of NF-kappaB [electrophoretic mobility-shift assay, (EMSA)] and up-regulated transcription of gamma-glutamylcysteine synthetase (gamma-GCSM; gamma-GCSC), cyclin D1 and cyclin E. We also showed down-regulation of p21(Waf1/Cip1) transcription. Western-blot analysis demonstrated that gamma-glutamylcysteine synthetase catalytic subunit (gamma-GCSC) and cyclin D1 showed a similar pattern to that found in the RT-PCR analysis. Moreover, chromatin immunoprecipitation (ChIP) assay demonstrated that NF-kappaB directly regulates transcription of gamma-GCS (both subunits) and cyclin D1 through the binding of NF-kappaB to the corresponding gene promoters, which was enhanced in vitamin E-deficiency. These findings show that vitamin E-deficiency induces significant molecular regulatory properties in liver cells with an altered expression of both antioxidant-defense genes and genes that control the cell cycle and demonstrate that liver NF-kappaB activation is involved in this response. Our results emphasize the importance of maintaining an adequate vitamin E consumption not only to prevent liver oxidative damage but also in modulating signal transduction.
Free Radical Research 11/2005; 39(10):1127-38. · 2.88 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: We describe a procedure, RNAPol-ChIP, to measure actual transcriptional rate. It consists of the detection, by chromatin immunoprecipitation (ChIP), of RNA polymerase II within the coding region of genes. To do this, the DNA immunoprecipitated with polymerase antibodies is analysed by PCR, using an amplicon well within the coding region of the desired genes to avoid interferences with polymerase paused at the promoter. To validate RNAPol-ChIP, we compare our results to those obtained by classical methods in several genes induced during either liver regeneration or acute pancreatitis. When short half-life mRNA genes are studied (e.g. c-fos and egr1), RNAPol-ChIP gives results similar to those of other procedures. However, in genes whose mRNA is more stable (e.g. the hemopexin, hpx, gene) RNAPol-ChIP informs on real-time transcription with results comparable to those of methods such as nuclear run-on or run-off, which require the isolation of highly purified nuclei. Moreover, RNAPol-ChIP advantageously compares with methods based on the analysis of steady-state mRNA (northern blot or RT-PCR). Additional advantages of RNAPol-ChIP, such as the possibility of combining it with classical ChIP analysis to study transcription-associated changes in chromatin are discussed.
Nucleic Acids Research 02/2004; 32(11):e88. · 8.03 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: In the lactating mammary gland, weaning produces mitochondrial cytochrome c release and nuclear DNA fragmentation, as determined by gel electrophoresis. This is followed by a significant decrease in lactation. Weaning for 2 h produces an early induction of the tumour suppressor/transcription factor p53, whereas the oncoprotein c-Jun and c-Jun N-terminal kinase are elevated after 24 h of weaning when compared with controls. The expression of p21(cip1) and p27(kip1), cyclin-dependent kinase inhibitors, was significantly higher in weaned rats when compared with control lactating rats. All the changes mentioned above also happen in the lactating mammary gland when propargylglycine, an inhibitor of the liver trans-sulphuration pathway, is administered. This effect is partially reversed by N -acetylcysteine administration. The administration of buthionine sulphoximine, an irreversible inhibitor of gamma-glutamylcysteine synthetase, to lactating rats produces a decrease in GSH levels and changes in protein concentrations and gene transcripts similar to those in rats with impaired trans-sulphuration pathway. These data suggest that the inter-tissue flux of GSH is an important mechanism of L-cysteine delivery to the lactating mammary gland and emphasize the importance of this physiological event in maintaining the gene expression required to sustain lactation.
Biochemical Journal 09/2003; 373(Pt 3):825-34. · 4.90 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Several clinical trials have revealed that individuals who were given beta-carotene and vitamin A did not have a reduced risk of cancer compared to those given placebo; rather, vitamin A could actually have caused an adverse effect in the lungs of smokers [Omenn, G.S., Goodman, G.E., Thornquist, M.D., Balmes, J., Cullen, M.R., Glass, A., Keogh, J.P., Meyskens, F.L., Valanis, B., Williams, J.H., Barnhart, S. & Hammar, S. N. Engl. J. Med (1996) 334, 1150-1155; Hennekens, C.H., Buring, J.E., Manson, J.E., Stampfer, M., Rosner, B., Cook, N.R., Belanger, C., LaMotte, F., Gaziano, J.M., Ridker, P.M., Willet, W. & Peto, R. (1996) N. Engl. J. Med. 334, 1145-1149]. Using differential display techniques, an initial survey using rats showed that liver RNA expression of c-H-Ras was decreased and p53 increased in rats with chronic vitamin A deficiency. These findings prompted us to evaluate the expression of c-Jun, p53 and p21WAF1/CIF1 (by RT-PCR) in liver and lung of rats. This study showed that c-Jun levels were lower and that p53 and p21WAF1/CIF1 levels were higher in chronic vitamin A deficiency. Vitamin A supplementation increased expression of c-Jun, while decreasing the expression of p53 and p21WAF1/CIF1. Western-blot analysis demonstrated that c-Jun and p53 showed a similar pattern to that found in the RT-PCR analyses. Binding of retinoic acid receptors (RAR) to the c-Jun promoter was decreased in chronic vitamin A deficiency when compared to control hepatocytes, but contrasting results were found with acute vitamin A supplementated cells. DNA fragmentation and cytochrome c release from mitochondria were analyzed and no changes were found. In lung, an increase in the expression of c-Jun produced a significant increase in cyclin D1 expression. These results may explain, at least in part, the conflicting results found in patients supplemented with vitamin A and illustrate that the changes are not restricted to lung. Furthermore, these results suggest that pharmacological vitamin A supplementation may increase the risk of adverse effects including the risk of oncogenesis.
European Journal of Biochemistry 04/2003; 270(7):1493-501. · 3.58 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Several clinical trials have revealed that individuals who were given β−carotene and vitamin A did not have a reduced risk of cancer compared to those given placebo; rather, vitamin A could actually have caused an adverse effect in the lungs of smokers [Omenn, G.S., Goodman, G.E., Thornquist, M.D., Balmes, J., Cullen, M.R., Glass, A., Keogh, J.P., Meyskens, F.L., Valanis, B., Williams, J.H., Barnhart, S. & Hammar, S. N. Engl. J. Med (1996) 334, 1150–1155; Hennekens, C.H., Buring, J.E., Manson, J.E., Stampfer, M., Rosner, B., Cook, N.R., Belanger, C., LaMotte, F., Gaziano, J.M., Ridker, P.M., Willet, W. & Peto, R. (1996) N. Engl. J. Med. 334, 1145–1149]. Using differential display techniques, an initial survey using rats showed that liver RNA expression of c-H-Ras was decreased and p53 increased in rats with chronic vitamin A deficiency. These findings prompted us to evaluate the expression of c-Jun, p53 and p21WAF1/CIF1 (by RT-PCR) in liver and lung of rats. This study showed that c-Jun levels were lower and that p53 and p21WAF1/CIF1 levels were higher in chronic vitamin A deficiency. Vitamin A supplementation increased expression of c-Jun, while decreasing the expression of p53 and p21WAF1/CIF1. Western-blot analysis demonstrated that c-Jun and p53 showed a similar pattern to that found in the RT-PCR analyses. Binding of retinoic acid receptors (RAR) to the c-Jun promoter was decreased in chronic vitamin A deficiency when compared to control hepatocytes, but contrasting results were found with acute vitamin A supplementated cells. DNA fragmentation and cytochrome c release from mitochondria were analyzed and no changes were found. In lung, an increase in the expression of c-Jun produced a significant increase in cyclin D1 expression. These results may explain, at least in part, the conflicting results found in patients supplemented with vitamin A and illustrate that the changes are not restricted to lung. Furthermore, these results suggest that pharmacological vitamin A supplementation may increase the risk of adverse effects including the risk of oncogenesis.
European Journal of Biochemistry. 03/2003; 270(7):1493 - 1501.
-
[show abstract]
[hide abstract]
ABSTRACT: Mitochondrial damage in rat liver induced by chronic vitamin A-deficiency was studied using three different groups of rats: (i) control rats, (ii) rats fed a vitamin A-free diet until 50 d after birth and (iii) vitamin A-deficient rats re-fed a control diet for 30 d. No statistical difference in body weight and food intake was found between control and vitamin A-deficient rats. Liver GSH concentration was similar in both groups. However, in vitamin A-deficient rats, the mitochondrial GSH/GSSG ratio was significantly lower and the levels of malondialdehyde (MDA) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo8dG) were higher when compared to control rats. These values were partially restored in re-fed rats. The mitochondrial membrane potential of vitamin A-deficient rats was significantly lower than in control rats and returned to normal levels in restored vitamin A rats. Two populations of mitochondria were found in vitamin A-deficient rats according to the composition of membrane lipids. One population showed a similar pattern to the control mitochondria and the second population had a higher membrane lipid content. This report emphasizes the protective role of vitamin A in liver mitochondria under physiological circumstances.
Free Radical Biology and Medicine 08/2000; · 5.42 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The phytopathogenic bacterium Pseudomonas syringae produces a fluorescent pigment when it is grown in iron-deficient media. This pigment forms a very stable Fe(III) complex that was purified in this form by using a novel procedure based on ultrafiltration and column chromatography. The Fe(III) complex has a molecular weight of 1,100 and contains 1 mol of Fe(III). The pigment is composed of an amino acid moiety with three threonines, three serines, one lysine, delta-N-hydroxyornithine, and a quinoline-type fluorescent chromophore. These features and its stability constant (in the range of 10) suggest that the fluorescent pigment of P. syringae is related to the siderophores produced by another Pseudomonas species.
Applied and Environmental Microbiology 08/1986; 52(1):157-60. · 3.83 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The chromatin structure of the promoter and proximal 5′ transcribed region of the Arabidopsis thaliana Adh gene has been studied in three experimental models: whole plants under aerobic conditions in which the gene is repressed, whole plants under flooding-induced anaerobiosis, in which the expression of the gene occurs in some plant tissues and yeast cells in which the Arabidopsis Adh DNA had been cloned but is not expressed at all. Experiments of indirect end labelling after DNase I and micrococcal nuclease digestion of nuclei allowed us to conclude that no positioned nucleosomes exist in plant nuclei on the proximal region of the promoter (up to −350), probably due to the presence of trans acting factors bound to promoter sequences. In the distal regions of the promoter a nucleosome is positioned covering a putative enhancer described by Dolferus et al. (Plant Physiol., 105 (1994) 1075–1087) in the repressed gene, but it is absent from active copies of the gene. Positioning of this nucleosome seems to be directed by sequence, as suggested by the theoretical analysis of the rotational determinants for positioning. Two-dimensional electrophoresis of Arabidopsis DNA indicates that an intrinsic curvature exists roughly between +200 and +350 and this motif directs nucleosome positioning at both sides.
Plant Science.
-
[show abstract]
[hide abstract]
ABSTRACT: We hypothesize that glutathione (GSH) fluctuations could have a prominent role in the modulation of c-myc expression through a mechanism affecting chromatin remodeling complexes. This could lead to an open chromatin structure accessible to transcription factors. We studied the in vivo effect of GSH depletion on these complexes bound to the c-myc promoter in the liver of l-buthionine-(S,R)-sulfoximine (BSO)-treated rats. Using chromatin immunoprecipitation we found that 3 h after BSO treatment the repressing complexes Id2 and Sin3A (part of a histone–deacetylase complex) were released from the c-myc promoter. STAT3 was phosphorylated and associated with its coactivator p300 with intrinsic acetyltransferase activity. Consequently, STAT3 was acetylated and bound to the c-myc promoter and histone H3 became hyperacetylated. At the same time, the RNApol II paused on the c-myc promoter was released, and the gene was overexpressed. After 6 h of BSO treatment, Id2/Sin3A returned to the c-myc promoter and the gene expression was down-regulated. Moreover, we observed a second peak of c-myc expression 48 h after BSO treatment, although at this time histone H3 was hypoacetylated and RNApol II paused, suggesting that this second peak was not subject to transcriptional control, but to posttranscriptional modulation. On the whole, our experiments suggest a novel mechanism for the effect of GSH on gene expression involving chromatin changes from a repressive to an open structure accessible to transcription factors such as STAT3.
Free Radical Biology and Medicine.
