Papers by Pedro Fernandez-salguero
Journal of Cell Science, Feb 22, 2004
The aryl hydrocarbon receptor (AhR) is a transcriptional regulator of genes involved in xenobioti... more The aryl hydrocarbon receptor (AhR) is a transcriptional regulator of genes involved in xenobiotic metabolism. Increasingly clear is also the role of the AhR in the control of cell growth and proliferation. By analyzing differential patterns of gene expression between wild-type (AhR+/+) and null (AhR-/-) mouse embryo fibroblasts (MEF), we have identified latent transforming growth factor-β binding protein 1 (LTBP-1) as a negatively AhR-regulated gene in the absence of xenobiotics. Ltbp-1 mRNA and protein expression were markedly increased in AhR-/-MEF. Furthermore, secreted LTBP-1 was elevated in the culture medium and the extracellular matrix of AhR-null MEF. Actinomycin D inhibited Ltbp-1 mRNA overexpression, suggesting regulation at the transcriptional level. AhR activation by dioxin (TCDD) downregulated Ltbp-1, again suggesting an AhR-regulated mechanism. Treatment of AhR+/+ MEF with transforming growth factor-β (TGF-β) downregulated AhR and, simultaneously, increased Ltbp-1, further supporting the role of this receptor in LTBP-1 expression. AhR-/-conditioned medium had higher levels of active and total TGF-β activity, suggesting a role for LTBP-1 in maintaining extracellular TGF-β concentrations. TGF-β did not appear to directly regulate Ltbp-1 given that addition of TGFβ neutralizing antibody or TGFβ protein to AhR-/-MEF had no effect on Ltbp-1 expression. AhR-/-MEF had lower levels of matrix metalloproteinase 2 (MMP-2) activity, which could not be attributable to MMP-2 mRNA downregulation or MMPinhibitors Timp-1 and Timp-2 overexpression. These data identify LTBP-1 as one of the few AhR-regulated genes not involved in xenobiotic metabolism and also support the implication of the AhR in controlling TGFβ activity and cell proliferation.
British Journal of Cancer, Feb 1, 1998
Individuals with a deficiency in the enzyme dihydropyrimidine dehydrogenase (DPD) may experience ... more Individuals with a deficiency in the enzyme dihydropyrimidine dehydrogenase (DPD) may experience severe life-threatening toxicity when treated with 5-fluorouracil (5-FU). As routine measurement of enzyme activity is not practical in many clinical centres, we have investigated the use of DNA mutation analysis to identify cancer patients with low enzyme levels. We have identified two new mutations at codons 534 and 543 in the DPD cDNA of a patient with low enzyme activity and screened the DNA from 75 colorectal cancer patients for these mutations and the previously reported splice site mutation (Vreken et al, 1996; Wei et al, 1996). In all cases, DPD enzyme activity was also measured. The splice site mutation was detected in a patient (1 out of 72) with low enzyme activity whereas mutations at codons 534 (2 out of 75) and 543 (11 out of 23) were not associated with low enzyme activity. These studies highlight the need to combine DPD genotype and phenotype analysis to identify mutations that result in reduced enzyme activity.
Neurotoxicology, Jun 1, 2010
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxicant that alters normal brain developm... more 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a potent toxicant that alters normal brain development and produces cognitive disability and motor dysfunction. However, after decades of intense study, the molecular mechanisms of TCDD-induced neurotoxicity, the signaling pathways involved and its molecular targets in neurons still remain unknown. TCDD acts as an exogenous ligand of the aryl hydrocarbon receptor (AhR) that becomes a key signaling molecule in the regulation of the toxic and carcinogenic properties of TCDD. We have used NGF-differentiated pheochromocytoma (dPC12) cells to determine the type of cell death that takes place by TCDD toxicity. TCDD induced cell death in dPC12 cultures with an EC(50) of 218+/-24 nM, similar to that obtained in undifferentiated PC12 cells, 171+/-31 nM. Nuclear fragmentation was observed after TCDD incubation in parallel to an increase in caspase-3 activity. Staurosporine, which readily induced apoptosis in dPC12 cells, showed a similar increase in caspase-3 activity and the characteristic pattern of nuclear fragmentation. Flow cytometry measurements showed that dPC12 cells in the presence of TCDD were positive for annexin V labeling but negative for propidium iodide staining. In addition, TCDD increased the area of the peak corresponding to hypodiploid (apoptotic) DNA content. All together these results support the hypothesis that TCDD toxicity in dPC12 cells takes place mainly through an apoptotic process.
Trends in Pharmacological Sciences, Oct 1, 1995
Journal of Cell Science, Nov 15, 1998
Journal of Molecular Biology, Oct 1, 2003
The dioxin receptor (AhR), in addition to its role in xenobiotic-induced carcinogenesis, appears ... more The dioxin receptor (AhR), in addition to its role in xenobiotic-induced carcinogenesis, appears to participate in cell proliferation, differentiation and organ homeostasis. Understanding potential mechanisms of activation of this receptor in the absence of exogenous ligands is therefore important to study its contribution to endogenous cellular functions. Using mouse embryo primary fibroblasts, we have previously shown that proteasome inhibition increased AhR transcriptional activity in the absence of xenobiotics. We suggested that proteasome inhibitiondependent AhR activation could involve an increase in the expression of the partner protein dioxin receptor nuclear translocator (ARNT). Since ARNT over-expression induced nuclear translocation of the AhR, and ARNT-deficient cells were unable to translocate this receptor to the nucleus upon proteasome inhibition, we have analyzed the effect of proteasome inhibition on the expression of regulatory proteins controlling ARNT levels. Treatment with the proteasome inhibitor MG132 increased endogenous Sp1 phosphorylation and its DNA-binding activity to the ARNT promoter. Sp1 phosphorylation and binding to the ARNT promoter, ARNT over-expression and AhR nuclear translocation were inhibited by GF109203X, a protein kinase C-specific inhibitor. In addition, MG132 stimulated protein kinase C activity in MEF cells with a pattern similar to that observed for ARNT expression. These data suggest that cellular control of protein kinase C activity, through Sp1 and ARNT, could regulate AhR transcriptional activity in the absence of xenobiotics.
Stem cell reports, Sep 1, 2021
Summary Mammalian embryogenesis is a complex process controlled by transcription factors that reg... more Summary Mammalian embryogenesis is a complex process controlled by transcription factors that regulate the balance between pluripotency and differentiation. Transcription factor aryl hydrocarbon receptor (AhR) regulates OCT4/POU5F1 and NANOG, both essential controllers of pluripotency, stemness and early embryo development. Molecular mechanisms controlling OCT4/POU5F1 and NANOG during embryogenesis remain unidentified. We show that AhR regulates pluripotency factors and maintains the metabolic activity required for proper embryo differentiation. AhR-lacking embryos (AhR−/−) showed a pluripotent phenotype characterized by a delayed expression of trophectoderm differentiation markers. Accordingly, central pluripotency factors OCT4/POU5F1 and NANOG were overexpressed in AhR−/− embryos at initial developmental stages. An altered intracellular localization of these factors was observed in the absence of AhR and, importantly, Oct4 had an opposite expression pattern with respect to AhR from the two-cell stage to blastocyst, suggesting a negative regulation of OCT4/POU5F by AhR. We propose that AhR is a regulator of pluripotency and differentiation in early mouse embryogenesis.
International Journal of Molecular Sciences, Mar 14, 2023
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
LOS OBJETIVOS DEL PRESENTE TRABAJO SON LOS SIGUIENTES: EN PRIMER LUGAR LA CARACTERIZACION DE LA I... more LOS OBJETIVOS DEL PRESENTE TRABAJO SON LOS SIGUIENTES: EN PRIMER LUGAR LA CARACTERIZACION DE LA INTERACCION CON PESTICIDAS DEL CITOCROMO P450 MICROXOMAL OBTENIDO TRAS EL TRATAMIENTO DE ANIMALES CON DIFERENTES XENOBIOTICOS, EN SEGUNDO LUGAR LA INMOVILIZACION Y ESTABILIZACION DE ESTE SISTEMA ENZIMATICO A DISTINTOS SOPORTES (POLIACRILAMIDA Y BIORREACTORES DE FIBRA POROSA. LOS RESULTADOS OBTENIDOS MUESTRAN QUE: (I) EL HERBICIDA PARAQUAT ES UN SUSTRATO SUICIDA QUE DESNATURALIZA LA DOTACION NATIVA DE CITOCROMO P450 E INHIBE FUERTEMENTE LA ACTIVIDAD NADPH REDUCTASA, (II) LA TOXICIDAD NO ES PRODUCIDA POR ALTERACION DE LOS PARAMETROS QUIMICO-FISICOS DE LA BICAPA LIPIDICA; (III) LA INMOVILIZACION DE CITOCROMO P450 POR UNION A GELES DE ACRILAMIDA ES MENOS IDONEA QUE LA INMOVILIZACION EN BIORREACTORES DE FIBRA POROSA PRESENTANDO EN ESTE CASO LA ENZIMA UNA MAYOR ESTABILIDAD FRENTE AL TIEMPO, LA FUERZA IONICA DEL MEDIO Y LA TEMPERATURA, OPERANDO LA REACCION CATALITICA BAJO CONTROL CINETICO. DE ESTE MODO EL CITOCROMO P450 INMOVILIZADO EN BIORREACTORES PUEDE SER EMPLEADO EN LA ELIMINACION DE COMPUESTOS TOXICOS DEL SISTEMA CIRCULATORIO DE MAMIFEROS, DE MODO SIMILAR A COMO SE REALIZAN LOS PROCESOS DE HEMODIALISIS.
International Journal of Molecular Sciences, Nov 29, 2022
This article is an open access article distributed under the terms and conditions of the Creative... more This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY
Scientific Reports, Sep 14, 2022
The liver is among the few organs having the ability to self-regenerate in response to a severe d... more The liver is among the few organs having the ability to self-regenerate in response to a severe damage compromising its functionality. The Aryl hydrocarbon receptor (Ahr) is a transcription factor relevant for the detoxification of xenobiotics but also largely important for liver development and homeostasis. Hence, liver cell differentiation is developmentally modulated by Ahr through the controlled expression of pluripotency and stemness-inducing genes. Here, 2/3 partial hepatectomy (PH) was used as a clinically relevant approach to induce liver regeneration in Ahr-expressing (Ahr +/+) and Ahr-null (Ahr −/−) mice. Ahr expression and activity were early induced after 2/3 PH to be gradually downmodulated latter during regeneration. Ahr −/− mice triggered liver regeneration much faster than AhR +/+ animals, although both reached full regeneration at the latest times. At initial stages after PHx, earlier regenerating Ahr −/− livers had upregulation of cell proliferation markers and increased activation of signalling pathways related to stemness such as Hippo-YAP and Wnt/β-catenin, concomitantly with the induction of pro-inflammatory cytokines TNFa, IL6 and p65. These phenotypes, together with the improved metabolic adaptation of Ahr −/− mice after PHx and their induced sustained cell proliferation, could likely result from the expansion of undifferentiated stem cells residing in the liver expressing OCT4, SOX2, KLF4 and NANOG. We propose that Ahr needs to be induced early during regeneration to fine-tune liver regrowth to physiological values. Since Ahr deficiency did not result in liver overgrowth, its transient pharmacological inhibition could serve to improve liver regeneration in hepatectomized and transplanted patients and in those exposed to damaging liver toxins and carcinogens. Abbreviations Ahr Aryl hydrocarbon receptor PH 2/3 Partial hepatectomy Under homeostatic conditions, the adult liver has a differentiated, non-proliferative and polyploid phenotype with negligible cell renewal needed for its metabolic, detoxifying and energetic functions 1,2. Injuries and severe pathological conditions can damage the liver eventually reducing its total mass. However, the liver is among the few organs in the body with an efficient regenerative capacity to recover its size, architecture and function 3,4. Two-thirds partial hepatectomy (PHx) in rodents represents a convenient model to investigate the complexity of the signalling pathways critical to control liver regeneration 5. Other models of liver regeneration use chemical compounds such as carbon tetrachloride (CCl 4) to cause an acute injury associated to inflammation and tissue necrosis 6. After PHx, hepatocytes are released from their quiescent state and, together with other hepatic cells, enter cell cycle and start to divide. This proliferative process is in a large part driven by the activation of the immune system with the release of IL6 and TNFα by macrophages 7. Thus, regeneration induced by PHx is mainly produced by the proliferation of quiescent hepatocytes with a marginal implication of liver stem cells 4,6,8. The central network of genes controlling the balance between pluripotency and differentiation is formed by transcription factors OCT4, NANOG and SOX2 9. These transcription factors maintain an undifferentiated
Nucleic Acids Research, Feb 15, 2016
Cell differentiation is a central process in development and in cancer growth and dissemination. ... more Cell differentiation is a central process in development and in cancer growth and dissemination. OCT4 (POU5F1) and NANOG are essential for cell stemness and pluripotency; yet, the mechanisms that regulate their expression remain largely unknown. Repetitive elements account for almost half of the Human Genome; still, their role in gene regulation is poorly understood. Here, we show that the dioxin receptor (AHR) leads to differentiation of human carcinoma cells through the transcriptional upregulation of Alu retrotransposons, whose RNA transcripts can repress pluripotency genes. Despite the genome-wide presence of Alu elements, we provide evidences that those located at the NANOG and OCT4 promoters bind AHR, are transcribed by RNA polymerase-III and repress NANOG and OCT4 in differentiated cells. OCT4 and NANOG repression likely involves processing of Alu-derived transcripts through the miRNA machinery involving the Microprocessor and RISC. Consistently, stable AHR knockdown led to basal undifferentiation, impaired Alus transcription and blockade of OCT4 and NANOG repression. We suggest that transcripts produced from AHR-regulated Alu retrotransposons may control the expression of stemness genes OCT4 and NANOG during differentiation of carcinoma cells. The control of discrete Alu elements by specific transcription factors may have a dynamic role in genome regulation under physiological and diseased conditions.
Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduct... more Previous studies suggested that the aryl hydrocarbon receptor (AhR) contributes to mice reproduction and fertility. However, the mechanisms involved remain mostly unknown. Retrotransposon silencing by piRNAs is essential for germ cell maturation and, remarkably, AhR has been identified as a regulator of murine <i>B1-SINE</i> retrotransposons. Here, using littermate <i>AhR<sup>+/+</sup></i> and <i>AhR</i><sup>−/−</sup> mice, we report that AhR regulates the general course of spermatogenesis and oogenesis by a mechanism likely associated with piRNA-associated proteins, piRNAs and retrotransposons. piRNA-associated proteins MVH and Miwi are upregulated in leptotene to pachytene spermatocytes with a more precocious timing in <i>AhR</i><sup>−/−</sup> than in <i>AhR<sup>+/+</sup></i> testes. piRNAs and transcripts from <i>B1-SINE</i>, <i>LINE-1</i> and <i>IAP</i> retrotransposons increased at these meiotic stages in AhR-null testes. Moreover, <i>B1-SINE</i> transcripts colocalize with MVH and Miwi in leptonaema and pachynaema spermatocytes. Unexpectedly, <i>AhR</i><sup>−/−</sup> males have increased sperm counts, higher sperm functionality and enhanced fertility than <i>AhR<sup>+/+</sup></i> mice. Contrary, piRNA-associated proteins and <i>B1-SINE</i> and <i>IAP</i>-derived transcripts are reduced in adult <i>AhR</i><sup>−/−</sup> ovaries. Accordingly, AhR-null female mice had lower numbers of follicles when compared with <i>AhR<sup>+/+</sup></i> mice. Thus, AhR deficiency differentially affects testis and ovary development possibly by a process involving piRNA-associated proteins, piRNAs and transposable elements.
Epigenetics & Chromatin, Mar 14, 2020
Transcriptional repression of Nanog is an important hallmark of stem cell differentiation. Chroma... more Transcriptional repression of Nanog is an important hallmark of stem cell differentiation. Chromatin modifications have been linked to the epigenetic profile of the Nanog gene, but whether chromatin organization actually plays a causal role in Nanog regulation is still unclear. Here, we report that the formation of a chromatin loop in the Nanog locus is concomitant to its transcriptional downregulation during human NTERA-2 cell differentiation. We found that two Alu elements flanking the Nanog gene were bound by the aryl hydrocarbon receptor (AhR) and the insulator protein CTCF during cell differentiation. Such binding altered the profile of repressive histone modifications near Nanog likely leading to gene insulation through the formation of a chromatin loop between the two Alu elements. Using a dCAS9-guided proteomic screening, we found that interaction of the histone methyltransferase PRMT1 and the chromatin assembly factor CHAF1B with the Alu elements flanking Nanog was required for chromatin loop formation and Nanog repression. Therefore, our results uncover a chromatin-driven, retrotransposon-regulated mechanism for the control of Nanog expression during cell differentiation.
HAL (Le Centre pour la Communication Scientifique Directe), 1995
To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms i... more To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in rodents, we disrupted the ligand-binding domain of the ␣ isoform of mouse PPAR (mPPAR␣) by homologous recombination. Mice homozygous for the mutation lack expression of mPPAR␣ protein and yet are viable and fertile and exhibit no detectable gross phenotypic defects. Remarkably, these animals do not display the peroxisome proliferator pleiotropic response when challenged with the classical peroxisome proliferators, clofibrate and Wy-14,643. Following exposure to these chemicals, hepatomegaly, peroxisome proliferation, and transcriptional activation of target genes were not observed. These results clearly demonstrate that mPPAR␣ is the major isoform required for mediating the pleiotropic response resulting from the actions of peroxisome proliferators. mPPAR␣-deficient animals should prove useful to further investigate the role of this receptor in hepatocarcinogenesis, fatty acid metabolism, and cell cycle regulation.
Molecular and Cellular Biology, Jun 1, 1995
To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms i... more To gain insight into the function of peroxisome proliferator-activated receptor (PPAR) isoforms in rodents, we disrupted the ligand-binding domain of the ␣ isoform of mouse PPAR (mPPAR␣) by homologous recombination. Mice homozygous for the mutation lack expression of mPPAR␣ protein and yet are viable and fertile and exhibit no detectable gross phenotypic defects. Remarkably, these animals do not display the peroxisome proliferator pleiotropic response when challenged with the classical peroxisome proliferators, clofibrate and Wy-14,643. Following exposure to these chemicals, hepatomegaly, peroxisome proliferation, and transcriptional activation of target genes were not observed. These results clearly demonstrate that mPPAR␣ is the major isoform required for mediating the pleiotropic response resulting from the actions of peroxisome proliferators. mPPAR␣-deficient animals should prove useful to further investigate the role of this receptor in hepatocarcinogenesis, fatty acid metabolism, and cell cycle regulation.
Mammalian embryogenesis is a complex process controlled by transcription factors that dynamically... more Mammalian embryogenesis is a complex process controlled by transcription factors that dynamically regulate the balance between pluripotency and differentiation. Transcription factor AhR is known to regulate Oct4/Pou5f1 and Nanog, both essential genes in pluripotency, stemness and early embryo development. Yet, the molecular mechanisms controlling Oct4/Pou5f1 and Nanog during embryo development remain largely unidentified. Here, we show that AhR is required for proper embryo differentiation by regulating pluripotency factors and by maintaining adequate metabolic activity. AhR lacking embryos (AhR-/-) showed a more pluripotent phenotype characterized by a delayed expression of differentiation markers of the first and second cell divisions. Accordingly, central pluripotency factors OCT4/POU5F1, NANOG, and SOX2 were overexpressed in AhR-/-embryos at initial developmental stages. An altered intracellular localization of these factors was observed in absence of AhR and, importantly, OCT4 had an opposite expression pattern with respect to AhR from the 2-cell stage to blastocyst, suggesting a negative regulatory mechanism of OCT4/POU5F by AhR. Hippo signalling, rather than being repressed, was upregulated in very early AhR-/-embryos, possibly contributing to their undifferentiation at later stages. Consistently, AhR-null blastocysts overexpressed the early marker of inner cell mass (ICM) differentiation Sox17 whereas downregulated extraembryonic differentiation-driving genes Cdx2 and Gata3. Moreover, the persistent pluripotent phenotype of AhR-/-embryos was supported by an enhanced glycolytic metabolism and a reduction in mitochondrial activity. We propose that AhR is a regulator of pluripotency and differentiation in early mouse embryogenesis and that its deficiency may underline the reduced viability and increased resorptions of AhR-null mice.
Rat liver microsomes can be immobilized in hollow fiber bioreactors under mild physical chemical ... more Rat liver microsomes can be immobilized in hollow fiber bioreactors under mild physical chemical conditions. The immobilized system shows a cytochrome P450 activity of about 20% of that of isolated microsomes, and retain more than 90% of the initial activity upon incubation for 24 h at 25 °C, or 3–4 weeks at −20°C. Thus, these immobilized systems appears to be suitable for technological applications, such as removal of xenobiotics from biological samples or from the blood of mammals.
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Papers by Pedro Fernandez-salguero