RESUMEN
The aryl hydrocarbon receptor (AHR) is a markedly established regulator of a plethora of cellular and molecular processes. Its initial role in the detoxification of xenobiotic compounds has been partially overshadowed by its involvement in homeostatic and organ physiology processes. In fact, the discovery of its ability to bind specific target regulatory sequences has allowed for the understanding of how AHR modulates such processes. Thereby, AHR presents functions in transcriptional regulation, chromatin architecture modifications and participation in different key signaling pathways. Interestingly, such fields of influence end up affecting organ and tissue homeostasis, including regenerative response both to endogenous and exogenous stimuli. Therefore, from classical spheres such as canonical transcriptional regulation in embryonic development, cell migration, differentiation or tumor progression to modern approaches in epigenetics, senescence, immune system or microbiome, this review covers all aspects derived from the balance between regulation/deregulation of AHR and its physio-pathological consequences.
Asunto(s)
Receptores de Hidrocarburo de Aril , Transducción de Señal , Receptores de Hidrocarburo de Aril/metabolismo , Homeostasis , Xenobióticos , Regulación de la Expresión GénicaRESUMEN
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/ß-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.
Asunto(s)
Regeneración Hepática , Receptores de Hidrocarburo de Aril , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Hepatectomía , Hígado/metabolismo , Hígado/cirugía , Ratones , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Aging impairs organismal homeostasis leading to multiple pathologies. Yet, the mechanisms and molecular intermediates involved are largely unknown. Here, we report that aged aryl hydrocarbon receptor-null mice (AhR-/-) had exacerbated cellular senescence and more liver progenitor cells. Senescence-associated markers ß-galactosidase (SA-ß-Gal), p16Ink4a and p21Cip1 and genes encoding senescence-associated secretory phenotype (SASP) factors TNF and IL1 were overexpressed in aged AhR-/- livers. Chromatin immunoprecipitation showed that AhR binding to those gene promoters repressed their expression, thus adjusting physiological levels in AhR+/+ livers. MCP-2, MMP12 and FGF secreted by senescent cells were overproduced in aged AhR-null livers. Supporting the relationship between senescence and stemness, liver progenitor cells were overrepresented in AhR-/- mice, probably contributing to increased hepatocarcinoma burden. These AhR roles are not liver-specific since adult and embryonic AhR-null fibroblasts underwent senescence in culture, overexpressing SA-ß-Gal, p16Ink4a and p21Cip1. Notably, depletion of senescent cells with the senolytic agent navitoclax restored expression of senescent markers in AhR-/- fibroblasts, whereas senescence induction by palbociclib induced an AhR-null-like phenotype in AhR+/+ fibroblasts. AhR levels were downregulated by senescence in mouse lungs but restored upon depletion of p16Ink4a-expressing senescent cells. Thus, AhR restricts age-induced senescence associated to a differentiated phenotype eventually inducing resistance to liver tumorigenesis.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina , Receptores de Hidrocarburo de Aril , Envejecimiento/metabolismo , Animales , Senescencia Celular/fisiología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Fibroblastos/metabolismo , Hígado/metabolismo , Ratones , Receptores de Hidrocarburo de Aril/genéticaRESUMEN
Transcription factor aryl hydrocarbon receptor (AHR) has emerged as one of the main regulators involved both in different homeostatic cell functions and tumor progression. Being a member of the family of basic-helix-loop-helix (bHLH) transcriptional regulators, this intracellular receptor has become a key member in differentiation, pluripotency, chromatin dynamics and cell reprogramming processes, with plenty of new targets identified in the last decade. Besides this role in tissue homeostasis, one enthralling feature of AHR is its capacity of acting as an oncogene or tumor suppressor depending on the specific organ, tissue and cell type. Together with its well-known modulation of cell adhesion and migration in a cell-type specific manner in epithelial-mesenchymal transition (EMT), this duality has also contributed to the arise of its clinical interest, highlighting a new potential as therapeutic tool, diagnosis and prognosis marker. Therefore, a deregulation of AHR-controlled pathways may have a causal role in contributing to physiological and homeostatic failures, tumor progression and dissemination. With that firmly in mind, this review will address the remarkable capability of AHR to exert a different function influenced by the phenotype of the target cell and its potential consequences.
RESUMEN
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.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Desarrollo Embrionario/genética , Receptores de Hidrocarburo de Aril/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Embrión de Mamíferos , Metabolismo Energético , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Genotipo , Glucólisis , Vía de Señalización Hippo , Ratones , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Estrés Oxidativo , Transporte de Proteínas , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
Non-small cell lung adenocarcinoma (NSCLC) bearing K-RasG12D mutations is one of the most prevalent types of lung cancer worldwide. Aryl hydrocarbon receptor (AHR) expression varies in human lung tumors and has been associated with either increased or reduced lung metastasis. In the mouse, Ahr also adjusts lung regeneration upon injury by limiting the expansion of resident stem cells. Here, we show that the loss of Ahr enhances K-RasG12D-driven NSCLC in mice through the amplification of stem cell subpopulations. Consistent with this, we show that K-RasG12D;Ahr-/- lungs contain larger numbers of cells expressing markers for both progenitor Clara (SCGB1A1 and CC10) and alveolar type-II (SFTPC) cells when compared to K-RasG12D;Ahr+/+-driven tumors. They also have elevated numbers of cells positive for pluripotent stem cells markers such as SOX2, ALDH1, EPCAM, LGR5 and PORCN. Typical pluripotency genes Nanog, Sox2 and c-Myc were also upregulated in K-RasG12D;Ahr-/- lung tumors as found by RNAseq analysis. In line with this, purified K-RasG12D/+;Ahr-/- lung cells generate larger numbers of organoids in culture that can subsequently differentiate into bronchioalveolar structures enriched in both pluripotency and stemness genes. Collectively, these data indicate that Ahr antagonizes K-RasG12D-driven NSCLC by restricting the number of cancer-initiating stem cells. They also suggest that Ahr expression might represent a good prognostic marker to determine the progression of K-RasG12D-positive NSCLC patients.
RESUMEN
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.
Asunto(s)
Elementos Alu , Ensamble y Desensamble de Cromatina , Proteína Homeótica Nanog/genética , Receptores de Hidrocarburo de Aril/metabolismo , Factor de Unión a CCCTC/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Humanos , Proteína Homeótica Nanog/metabolismo , Unión Proteica , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteínas Represoras/metabolismoRESUMEN
Aryl hydrocarbon receptor (AhR) deficiency alters tissue homeostasis. However, how AhR regulates organ maturation and differentiation remains mostly unknown. Liver differentiation entails a polyploidization process fundamental for cell growth, metabolism, and stress responses. Here, we report that AhR regulates polyploidization during the preweaning-to-adult mouse liver maturation. Preweaning AhR-null (AhR-/-) livers had smaller hepatocytes, hypercellularity, altered cell cycle regulation, and enhanced proliferation. Those phenotypes persisted in adult AhR-/- mice and correlated with compromised polyploidy, predominance of diploid hepatocytes, and enlarged centrosomes. Phosphatidylinositol-3-phosphate kinase (PI3K), extracellular signal-regulated kinase (ERK), and Wnt/ß-catenin signaling remained upregulated from preweaning to adult AhR-null liver, likely increasing mammalian target of rapamycin (mTOR) activation. Metabolomics revealed the deregulation of mitochondrial oxidative phosphorylation intermediates succinate and fumarate in AhR-/- liver. Consistently, PI3K, ERK, and Wnt/ß-catenin inhibition partially rescued polyploidy in AhR-/- mice. Thus, AhR may integrate survival, proliferation, and metabolism for liver polyploidization. Since tumor cells tend to be polyploid, AhR modulation could have therapeutic value in the liver.
RESUMEN
The aryl hydrocarbon receptor (AhR) is well-known for its major contributions to the cellular responses against environmental toxins and carcinogens. Notably, AhR has also emerged as a key transcription factor controlling many physiological processes including cell proliferation and apoptosis, differentiation, adhesion and migration, pluripotency and stemness. These novel functions have broadened our understanding of the signalling pathways and molecular intermediates interacting with AhR under both homeostatic and pathological conditions. Recent discoveries link AhR with the function of essential organs such as liver, skin and gonads, and with complex organismal structures including the immune and cardiovascular systems. The identification of potential endogenous ligands able to regulate AhR activity, opens the possibility of designing ad hoc molecules with pharmacological and/or therapeutic value to treat human diseases in which AhR may have a causal role. Integration of experimental data from in vitro and in vivo studies with "omic" analyses of human patients affected with cancer, immune diseases, inflammation or neurological disorders will likely contribute to validate the clinical relevance of AhR and the possible benefits of modulating its activity by pharmacologically-driven strategies. In this review, we will highlight signalling pathways involved in human diseases that could be targetable by AhR modulators and discuss the feasibility of using such molecules in therapy. The pros and cons of AhR-aimed approaches will be also mentioned.
Asunto(s)
Receptores de Hidrocarburo de Aril/metabolismo , Animales , Epigénesis Genética , Variación Genética , Humanos , Neoplasias/genética , Receptores de Hidrocarburo de Aril/genética , Transducción de SeñalRESUMEN
Recent experimental evidences from cellular systems and from mammalian and non-mammalian animal models highlight novel functions for the aryl hydrocarbon/dioxin receptor (AhR) in maintaining cell differentiation and tissue homeostasis. Notably, AhR depletion stimulates an undifferentiated and pluripotent phenotype likely associated to a mesenchymal transition in epithelial cells and to increased primary tumorigenesis and metastasis in melanoma. In this work, we have used a lung model of epithelial regeneration to investigate whether AhR regulates proper tissue repair by adjusting the expansion of undifferentiated stem-like cells. AhR-null mice developed a faster and more efficient repair of the lung bronchiolar epithelium upon naphthalene injury that required increased cell proliferation and the earlier activation of stem-like Clara, Basal and neuroepithelial cells precursors. Increased basal content in multipotent Sca1+/CD31-/CD4- cells and in cells expressing pluripotency factors NANOG and OCT4 could also improve re-epithelialization in AhR-null lungs. The reduced response of AhR-deficient lungs to Sonic Hedgehog (Shh) repression shortly after injury may also help their improved bronchiolar epithelium repair. These results support a role for AhR in the regenerative response against toxins, and open the possibility of modulating its activation level to favor recovery from lesions caused by environmental contaminants.
Asunto(s)
Pulmón/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Animales , Diferenciación Celular , Células Cultivadas , Pulmón/efectos de los fármacos , Pulmón/fisiología , Ratones , Naftalenos/toxicidad , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Hidrocarburo de Aril/genéticaRESUMEN
The aryl hydrocarbon receptor (AhR) has roles in cell proliferation, differentiation and organ homeostasis, including the liver. AhR depletion induces undifferentiation and pluripotency in normal and transformed cells. Here, AhR-null mice (AhR-/-) were used to explore whether AhR controls liver regeneration and carcinogenesis by restricting the expansion of stem-like cells and the expression of pluripotency genes. Short-term CCl4 liver damage was earlier and more efficiently repaired in AhR-/- than in AhR+/+ mice. Stem-like CK14 + and TBX3 + and pluripotency-expressing OCT4 + and NANOG + cells expanded sooner in AhR-/- than in AhR+/+ regenerating livers. Stem-like side population cells (SP) isolated from AhR-/- livers had increased ß-catenin (ß-Cat) signaling with overexpression of Axin2, Dkk1 and Cyclin D1. Interestingly, ß-Cat, Axin2 and Dkk1 also increased during regeneration but more notably in AhR-null livers. Liver carcinogenesis induced by diethylnitrosamine (DEN) produced large carcinomas in all AhR-/- mice but mostly premalignant adenomas in less than half of AhR+/+ mice. AhR-null tumoral tissue, but not their surrounding non-tumoral parenchyma, had nuclear ß-Cat and Axin2 overexpression. OCT4 and NANOG were nevertheless similarly expressed in AhR+/+ and AhR-/- lesions. We suggest that AhR may serve to adjust liver repair and to block tumorigenesis by modulating stem-like cells and ß-Cat signaling.
Asunto(s)
Transformación Celular Neoplásica/genética , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Neoplasias Hepáticas/etiología , Neoplasias Hepáticas/prevención & control , Regeneración Hepática/genética , Receptores de Hidrocarburo de Aril/genética , Animales , Biomarcadores , Transformación Celular Neoplásica/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/complicaciones , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Dietilnitrosamina/efectos adversos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Inmunofenotipificación , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Noqueados , Receptores de Hidrocarburo de Aril/metabolismoRESUMEN
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 Piwi-interacting RNAs (piRNAs) is essential for germ cell maturation and, remarkably, AhR has been identified as a regulator of murine B1-SINE retrotransposons. Here, using littermate AhR+/+ and AhR-/- mice, we report that AhR regulates the general course of spermatogenesis and oogenesis by a mechanism likely to be 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 AhR-/- than in AhR+/+ testes. piRNAs and transcripts from B1-SINE, LINE-1 and IAP retrotransposons increased at these meiotic stages in AhR-null testes. Moreover, B1-SINE transcripts colocalize with MVH and Miwi in leptonema and pachynema spermatocytes. Unexpectedly, AhR-/- males have increased sperm counts, higher sperm functionality and enhanced fertility than AhR+/+ mice. In contrast, piRNA-associated proteins and B1-SINE and IAP-derived transcripts are reduced in adult AhR-/- ovaries. Accordingly, AhR-null female mice have lower numbers of follicles when compared with AhR+/+ mice. Thus, AhR deficiency differentially affects testis and ovary development possibly by a process involving piRNA-associated proteins, piRNAs and transposable elements.
Asunto(s)
Proteínas Argonautas/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , ARN Helicasas DEAD-box/genética , Ovario/metabolismo , Receptores de Hidrocarburo de Aril/genética , Retroelementos/genética , Testículo/metabolismo , Animales , Proteínas Argonautas/metabolismo , ARN Helicasas DEAD-box/metabolismo , Femenino , Fertilidad , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Masculino , Meiosis , Ratones , ARN Interferente Pequeño/metabolismo , Regulación hacia ArribaRESUMEN
2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is a xenobiotic agent with high persistency that induces neurotoxic effects altering neurodevelopment and behavior. The molecular mechanisms and the signaling pathways involved in TCDD-mediated neurotoxicity, together with the search of its molecular targets in neurons are under intense study. We have previously shown that high nanomolar concentrations of TCDD for incubation times of minutes induce apoptosis in SHSY5Y human neuroblastoma cells by the disruption of calcium homeostasis, affecting membrane structural integrity. In this work, we have analyzed the effect of low nanomolar concentrations of TCDD for incubation times of hours to define the role of aryl hydrocarbon receptor which can be activated at those concentrations. TCDD induces toxicity in SHSY5Y human neuroblastoma cells under these experimental conditions with an EC50 value of approximately 3nM at 24h of incubation time. Transient transfection of a hairpin RNA for AhR protects against TCDD neurotoxicity, suggesting that AhR is mediating the dioxin effect. Altogether, these results support the hypothesis that TCDD toxicity in SHSY5Y neuroblastoma cells depends on dioxin concentration and time of incubation, with a main role of aryl hydrocarbon receptor at low nanomolar TCDD concentrations.
Asunto(s)
Neuronas/efectos de los fármacos , Neurotoxinas/farmacología , Dibenzodioxinas Policloradas/farmacología , Receptores de Hidrocarburo de Aril/metabolismo , Antioxidantes/farmacología , Apoptosis/efectos de los fármacos , Caspasa 3/metabolismo , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Neuroblastoma/patología , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores de Hidrocarburo de Aril/genética , Resveratrol , Estilbenos/farmacología , Factores de Tiempo , TransfecciónRESUMEN
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.
Asunto(s)
Elementos Alu , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Diferenciación Celular/genética , Regulación Neoplásica de la Expresión Génica , Receptores de Hidrocarburo de Aril/fisiología , Teratocarcinoma/genética , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Carcinoma/patología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Núcleo Celular/metabolismo , Humanos , Ratones , MicroARNs/metabolismo , Proteína Homeótica Nanog/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Regiones Promotoras Genéticas , ARN Polimerasa III/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Teratocarcinoma/enzimología , Teratocarcinoma/metabolismo , Teratocarcinoma/patología , Teratoma/genética , Teratoma/metabolismo , Transcripción Genética , Tretinoina/farmacologíaRESUMEN
BACKGROUND: The dioxin (AhR) receptor can have oncogenic or tumor suppressor activities depending on the phenotype of the target cell. We have shown that AhR knockdown promotes melanoma primary tumorigenesis and lung metastasis in the mouse and that human metastatic melanomas had reduced AhR levels with respect to benign nevi. METHODS: Mouse melanoma B16F10 cells were engineered by retroviral transduction to stably downregulate AhR expression, Aldh1a1 expression or both. They were characterized for Aldh1a1 activity, stem cell markers and migration and invasion in vitro. Their tumorigenicity in vivo was analyzed using xenografts and lung metastasis assays as well as in vivo imaging. RESULTS: Depletion of aldehyde dehydrogenase 1a1 (Aldh1a1) impairs the pro-tumorigenic and pro-metastatic advantage of melanoma cells lacking AhR expression (sh-AhR). Thus, Aldh1a1 knockdown in sh-AhR cells (sh-AhR + sh-Aldh1a1) diminished their migration and invasion potentials and blocked tumor growth and metastasis to the lungs in immunocompetent AhR+/+ recipient mice. However, Aldh1a1 downmodulation in AhR-expressing B16F10 cells did not significantly affect tumor growth in vivo. Aldh1a1 knockdown reduced the high levels of CD133(+)/CD29(+)/CD44(+) cells, melanosphere size and the expression of the pluripotency marker Sox2 in sh-AhR cells. Interestingly, Sox2 increased Aldh1a1 expression in sh-AhR but not in sh-AhR + sh-Aldh1a1 cells, suggesting that Aldh1a1 and Sox2 may be co-regulated in melanoma cells. In vivo imaging revealed that mice inoculated with AhR + Aldh1a1 knockdown cells had reduced tumor burden and enhanced survival than those receiving Aldh1a1-expressing sh-AhR cells. CONCLUSIONS: Aldh1a1 overactivation in an AhR-deficient background enhances melanoma progression. Since AhR may antagonize the protumoral effects of Aldh1a1, the AhR(low)-Aldh1a1(high) phenotype could be indicative of bad outcome in melanoma.
Asunto(s)
Aldehído Deshidrogenasa/metabolismo , Transformación Celular Neoplásica/metabolismo , Melanoma/metabolismo , Melanoma/patología , Receptores de Hidrocarburo de Aril/metabolismo , Aldehído Deshidrogenasa/genética , Animales , Línea Celular Tumoral , Movimiento Celular/genética , Modelos Animales de Enfermedad , Expresión Génica , Técnicas de Silenciamiento del Gen , Genes Reporteros , Humanos , Neoplasias Pulmonares/secundario , Melanoma/genética , Melanoma Experimental , Ratones , Imagen Molecular , Metástasis de la Neoplasia , Células Madre Neoplásicas/metabolismo , Receptores de Hidrocarburo de Aril/genéticaRESUMEN
The persistent xenobiotic agent 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces neurotoxic effects that alters neurodevelopment and behavior both during development and adulthood. There are many ongoing efforts to determine the molecular mechanisms of TCDD-mediated neurotoxicity, the signaling pathways involved and its molecular targets in neurons. In this work, we have used SHSY5Y human neuroblastoma cells to characterize the TCDD-induced toxicity. TCDD produces a loss of viability linked to an increased caspase-3 activity, PARP-1 fragmentation, DNA laddering, nuclear fragmentation and hypodiploid (apoptotic) DNA content, in a similar way than staurosporine, a prototypical molecule of apoptosis induction. In addition, TCDD produces a decrease of mitochondrial membrane potential and an increase of intracellular calcium concentration (P < 0.05). Finally, based on the high lipophilic properties of the dioxin, we test the TCDD effect on the membrane integrity using sarcoplasmic reticulum vesicles as a model. TCDD produces calcium efflux through the membrane and an anisotropy decrease (P < 0.05) that reflects an increase in membrane fluidity. Altogether these results support the hypothesis that TCDD toxicity in SHSY5Y neuroblastoma cells provokes the disruption of calcium homeostasis, probably affecting membrane structural integrity, leading to an apoptotic process.
Asunto(s)
Apoptosis/efectos de los fármacos , Calcio/metabolismo , Homeostasis/efectos de los fármacos , Espacio Intracelular/metabolismo , Neuronas/metabolismo , Neuronas/patología , Dibenzodioxinas Policloradas/toxicidad , Anisotropía , Línea Celular Tumoral , ADN/metabolismo , Fragmentación del ADN/efectos de los fármacos , Diploidia , Humanos , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Espacio Intracelular/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Neuroblastoma/patología , Neuronas/efectos de los fármacos , Retículo Sarcoplasmático/efectos de los fármacos , Retículo Sarcoplasmático/metabolismoRESUMEN
2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a prototypical environmental contaminant with neurotoxic properties that alters neurodevelopment and behavior. TCDD is a ligand of the aryl hydrocarbon receptor (AhR), which is a key signaling molecule to fully understand the toxic and carcinogenic properties of dioxin. Much effort is underway to unravel the molecular mechanisms and the signaling pathways involved in TCDD-induced neurotoxicity, and to define its molecular targets in neurons. We have used cerebellar granule cells (CGC) from wild-type (AhR+/+) and AhR-null (AhR-/-) mice to characterize the cell death that takes place in neurons after TCDD toxicity. TCDD induced cell death in CGC cultures from wild-type mice with an EC(50) of 127±21 nM. On the contrary, when CGC neurons from AhR-null mice were treated with TCDD no significant cell death was observed. The role of AhR in TCDD-induced death was further assessed by using the antagonists resveratrol and α-naphtoflavone, which readily protected against TCDD toxicity in AhR+/+ CGC cultures. AhR+/+ CGC cultures treated with TCDD showed nuclear fragmentation, DNA laddering, and increased caspase 3 activity, similarly to what was found by the use of staurosporine, a well-established inducer of apoptosis. Finally, the AhR pathway was active in CGC because TCDD could induce the expression of the target gene cytochrome P450 1A2 in AhR+/+ CGC cultures. All together these results support the hypothesis that TCDD toxicity in CGC neurons involves the AhR and that it takes place mainly through an apoptotic process. AhR could be then considered a novel target in neurotoxicity and neurodegeneration whose down-modulation could block certain xenobiotic-related adverse effects in CNS.
Asunto(s)
Apoptosis/efectos de los fármacos , Benzofuranos/toxicidad , Cerebelo/citología , Contaminantes Ambientales/toxicidad , Neuronas/efectos de los fármacos , Receptores de Hidrocarburo de Aril/fisiología , Animales , Caspasa 3/metabolismo , Células Cultivadas , Citocromo P-450 CYP1A2/metabolismo , Relación Dosis-Respuesta a Droga , Indoles , Ratones , Ratones Noqueados , Especies Reactivas de Oxígeno , Receptores de Hidrocarburo de Aril/deficiencia , Sales de Tetrazolio , Tiazoles , Azul de TripanoRESUMEN
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.
Asunto(s)
Apoptosis/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Dioxinas/toxicidad , Factor de Crecimiento Nervioso/farmacología , Neurotoxinas/toxicidad , Análisis de Varianza , Animales , Anexina A5/metabolismo , Caspasa 3/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Citometría de Flujo/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/citología , Indoles , Neuroglía/efectos de los fármacos , Neuronas/efectos de los fármacos , Células PC12/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Zinc is a basic trace element that plays important roles in brain and, consequently, its homeostasis needs to be critically controlled. High zinc concentrations in the interneuron synaptic space may induce neuronal death through mechanisms still partially solved. Undifferentiated pheochromocytoma (PC12) cells have been used to study zinc toxicity. As these cells can be differentiated into neuronal-like cells, the results obtained from differentiated cultures are more useful to understand zinc toxicity in neurons. In this paper, we show by flow cytometry that nerve growth factor (NGF) induces PC12 cells differentiation characterized by cell cycle arrest in the G1/G0 phase, similarly to that observed in serum-deprived cultures. Zinc induces cell death in NGF-differentiated PC12 cultures with an EC(50) value of 143+/-14 microM, which reveals a higher sensitivity with respect to undifferentiated PC12 cultures (EC(50), 308+/-32 microM) and a similar response to that obtained in hippocampal neurons (134+/-12 microM). Thus, the differentiation process appeared responsible for such increase in sensitivity. To further support this tenet, when the NGF differentiation was impaired in presence of 10 microM MK-801, a selective blocker of the N-methyl-d-aspartate (NMDA) receptor that plays a role in the differentiation process, the higher sensitivity to zinc was reverted to an EC(50) value of 241+/-26 microM. Flow cytometry experiments showed that NGF-differentiated PC12 cells in presence of zinc were positive for propidium iodide but not for annexin-V labeling. These results, together with data from fluorescent labeling of nuclear fragmentation, caspase-3 activation, and reactive oxygen species generation, support the view that zinc toxicity in NGF-differentiated PC12 cells takes place mainly through a necrotic process.
Asunto(s)
Ciclo Celular/efectos de los fármacos , Ciclo Celular/fisiología , Factor de Crecimiento Nervioso/metabolismo , Oligoelementos/toxicidad , Sulfato de Zinc/toxicidad , Animales , Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Muerte Celular/fisiología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Células Cultivadas , Maleato de Dizocilpina/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Metalotioneína/metabolismo , Necrosis/inducido químicamente , Necrosis/metabolismo , Neuronas/efectos de los fármacos , Neuronas/fisiología , Células PC12 , Ratas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The dioxin receptor (AhR) modulates cell plasticity and migration, although the signaling involved remains unknown. Here, we report a mechanism that integrates AhR into these cytoskeleton-related functions. Immortalized and mouse embryonic fibroblasts lacking AhR (AhR-/-) had increased cell area due to spread cytoplasms that reverted to wild-type morphology upon AhR re-expression. The AhR-null phenotype included increased F-actin stress fibers, depolarized focal adhesions, and enhanced spreading and adhesion. The cytoskeleton alterations of AhR-/- cells were due to down-regulation of constitutive Vav3 expression, a guanosine diphosphate/guanosine triphosphate exchange factor for Rho/Rac GTPases and a novel transcriptional target of AhR. AhR was recruited to the vav3 promoter and maintained constitutive mRNA expression in a ligand-independent manner. Consistently, AhR-/- fibroblasts had reduced Rac1 activity and increased activation of the RhoA/Rho kinase (Rock) pathway. Pharmacological inhibition of Rac1 shifted AhR+/+ fibroblasts to the null phenotype, whereas Rock inhibition changed AhR-null cells to the AhR+/+ morphology. Knockdown of vav3 transcripts by small interfering RNA induced cytoskeleton defects and changes in adhesion and spreading mimicking those of AhR-null cells. Moreover, vav3-/- MEFs, as AhR-/- mouse embryonic fibroblasts, had increased cell area and enhanced stress fibers. By modulating Vav3-dependent signaling, AhR could regulate cell shape, adhesion, and migration under physiological conditions and, perhaps, in certain pathological states.