RESUMEN
Somatic cell reprogramming and tissue repair share relevant factors and molecular programs. Here, Dickkopf-3 (DKK3) is identified as novel factor for organ regeneration using combined transcription-factor-induced reprogramming and RNA-interference techniques. Loss of Dkk3 enhances the generation of induced pluripotent stem cells but does not affect de novo derivation of embryonic stem cells, three-germ-layer differentiation or colony formation capacity of liver and pancreatic organoids. However, DKK3 expression levels in wildtype animals and serum levels in human patients are elevated upon injury. Accordingly, Dkk3-null mice display less liver damage upon acute and chronic failure mediated by increased proliferation in hepatocytes and LGR5+ liver progenitor cell population, respectively. Similarly, recovery from experimental pancreatitis is accelerated. Regeneration onset occurs in the acinar compartment accompanied by virtually abolished canonical-Wnt-signaling in Dkk3-null animals. This results in reduced expression of the Hedgehog repressor Gli3 and increased Hedgehog-signaling activity upon Dkk3 loss. Collectively, these data reveal Dkk3 as a key regulator of organ regeneration via a direct, previously unacknowledged link between DKK3, canonical-Wnt-, and Hedgehog-signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Reprogramación Celular/genética , Reprogramación Celular/fisiología , Genómica/métodos , Organogénesis/genética , Organogénesis/fisiología , Animales , Modelos Animales de Enfermedad , Ratones , Ratones Endogámicos C57BL , Regeneración/genética , Regeneración/fisiologíaRESUMEN
Aneuploidy can instigate tumorigenesis. However, mutations in genes that control chromosome segregation are rare in human tumors as these mutations reduce cell fitness. Screening experiments indicate that the knockdown of multiple classes of genes that are not directly involved in chromosome segregation can lead to aneuploidy induction. The possible contribution of these genes to cancer formation remains yet to be defined. Here we identified gene knockdowns that lead to an increase in aneuploidy in checkpoint-deficient human cancer cells. Computational analysis revealed that the identified genes overlap with recurrent mutations in human cancers. The knockdown of the three strongest selected candidate genes (ORP3, GJB3, and RXFP1) enhances the malignant transformation of human fibroblasts in culture. Furthermore, the knockout of Orp3 results in an aberrant expansion of lymphoid progenitor cells and a high penetrance formation of chromosomal instable, pauci-clonal B-cell lymphoma in aging mice. At pre-tumorous stages, lymphoid cells from the animals exhibit deregulated phospholipid metabolism and an aberrant induction of proliferation regulating pathways associating with increased aneuploidy in hematopoietic progenitor cells. Together, these results support the concept that aneuploidy-inducing gene deficiencies contribute to cellular transformation and carcinogenesis involving the deregulation of various molecular processes such as lipid metabolism, proliferation, and cell survival.
Asunto(s)
Aneuploidia , Proteínas de Unión a Ácidos Grasos/deficiencia , Proteínas de Unión a Ácidos Grasos/genética , Técnicas de Silenciamiento del Gen , Linfoma de Células B/genética , Animales , Línea Celular Tumoral , Transformación Celular Neoplásica , Fibroblastos/patología , Humanos , Linfoma de Células B/patología , RatonesRESUMEN
BACKGROUND: Most liver tumors arise on the basis of chronic liver diseases that trigger inflammatory responses. Besides inflammation, subsequent defects in the p53-signaling pathway frequently occurs in liver cancer. In this study, we analyzed the consequences of inflammation and p53 loss in liver carcinogenesis. METHODS: We used inducible liver-specific transgenic mouse strains to analyze the consequences of NF-κB/p65 activation mimicking chronic inflammation and subsequent p53 loss. RESULTS: Ikk2ca driven NF-κB/p65 activation in mice results in liver fibrosis, the formation of ectopic lymphoid structures and carcinogenesis independent of p53 expression. Subsequent deletion of Trp53 led to an increased tumor formation, metastasis and a shift in tumor differentiation towards intrahepatic cholangiocarcinoma. In addition, loss of Trp53 in an inflammatory liver resulted in elevated chromosomal instability and indicated a distinct aberration pattern. CONCLUSIONS: In conclusion, activation of NF-κB/p65 mimicking chronic inflammation provokes the formation of liver carcinoma. Collateral disruption of Trp53 supports tumor progression and influences tumor differentiation and heterogeneity.
RESUMEN
Liver cholestasis is a chronic liver disease and a major health problem worldwide. Cholestasis is characterised by a decrease in bile flow due to impaired secretion by hepatocytes or by obstruction of bile flow through intra- or extrahepatic bile ducts. Thereby cholestasis can induce ductal proliferation, hepatocyte injury and liver fibrosis. Notch signalling promotes the formation and maturation of bile duct structures. Here we investigated the liver regeneration process in the context of cholestasis induced by disruption of the Notch signalling pathway. Liver-specific deletion of recombination signal binding protein for immunoglobulin kappa j region (Rbpj), which represents a key regulator of Notch signalling, induces severe cholestasis through impaired intra-hepatic bile duct (IHBD) maturation, severe necrosis and increased lethality. Deregulation of the biliary compartment and cholestasis are associated with the change of several signalling pathways including a Kyoto Encyclopedia of Genes and Genomes (KEGG) gene set representing the Hippo pathway, further yes-associated protein (YAP) activation and upregulation of SRY (sex determining region Y)-box 9 (SOX9), which is associated with transdifferentiation of hepatocytes. SOX9 upregulation in cholestatic liver injury in vitro is independent of Notch signalling. We could comprehensively address that in vivo Rbpj depletion is followed by YAP activation, which influences the transdifferentiation of hepatocytes and thereby contributing to liver regeneration.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Colestasis/metabolismo , Regeneración Hepática/fisiología , Fosfoproteínas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Conductos Biliares/metabolismo , Conductos Biliares/fisiología , Western Blotting , Proteínas de Ciclo Celular , Transdiferenciación Celular/genética , Transdiferenciación Celular/fisiología , Células Cultivadas , Colestasis/genética , Hepatocitos/citología , Hepatocitos/metabolismo , Regeneración Hepática/genética , Masculino , Ratones , Fosfoproteínas/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Proteínas Señalizadoras YAPRESUMEN
Parkinson's disease is one of the most common neurodegenerative disorders of the elderly and ageing hence described to be a major risk factor. Telomere shortening as a result of the inability to fully replicate the ends of linear chromosomes is one of the hallmarks of ageing. The role of telomere dysfunction in neurological diseases and the ageing brain is not clarified and there is an ongoing discussion whether telomere shortening is linked to Parkinson's disease. Here we studied a mouse model of Parkinson's disease (Thy-1 [A30P] α-synuclein transgenic mouse model) in the background of telomere shortening (Terc knockout mouse model). α-synuclein transgenic mice with short telomeres (αSYN(tg/tg) G3Terc(-/-)) developed an accelerated disease with significantly decreased survival. This accelerated phenotype of mice with short telomeres was characterized by a declined motor performance and an increased formation of α-synuclein aggregates. Immunohistochemical analysis and mRNA expression studies revealed that the disease end-stage brain stem microglia showed an impaired response in αSYN(tg/tg) G3Terc(-/-) microglia animals. These results provide the first experimental data that telomere shortening accelerates α-synuclein pathology that is linked to limited microglia function in the brainstem.
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Tronco Encefálico/metabolismo , Microglía/metabolismo , Trastornos Parkinsonianos/metabolismo , Acortamiento del Telómero/fisiología , alfa-Sinucleína/metabolismo , Animales , Tronco Encefálico/patología , Progresión de la Enfermedad , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/patología , Actividad Motora/fisiología , Trastornos Parkinsonianos/patología , Fenotipo , Equilibrio Postural/fisiología , Agregación Patológica de Proteínas/metabolismo , Agregación Patológica de Proteínas/patología , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
Dynamic regulation of cell-cell adhesion by the coordinated formation and dissolution of E-cadherin-based adherens junctions is crucial for tissue homeostasis. The actin-binding protein cortactin interacts with E-cadherin and enables F-actin accumulation at adherens junctions. Here, we were interested to study the broader functional interactions of cortactin in adhesion complexes. In line with literature, we demonstrate that cortactin binds to E-cadherin, and that a posttranslational modification of cortactin, RhoA-induced phosphorylation by protein kinase D1 (PKD1; also known as PRKD1) at S298, impairs adherens junction assembly and supports their dissolution. Two new S298-phosphorylation-dependent interactions were also identified, namely, that phosphorylation of cortactin decreases its interaction with ß-catenin and the actin-binding protein vinculin. In addition, binding of vinculin to ß-catenin, as well as linkage of vinculin to F-actin, are also significantly compromised upon phosphorylation of cortactin. Accordingly, we found that regulation of cell-cell adhesion by phosphorylation of cortactin downstream of RhoA and PKD1 is vitally dependent on vinculin-mediated protein interactions. Thus, cortactin, unexpectedly, is an important integration node for the dynamic regulation of protein complexes during breakdown and formation of adherens junctions.
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Cadherinas/metabolismo , Cortactina/metabolismo , Canales Catiónicos TRPP/metabolismo , Citoesqueleto de Actina/metabolismo , Uniones Adherentes/metabolismo , Animales , Antígenos CD , Células CACO-2 , Adhesión Celular , Colon/metabolismo , Epitelio/metabolismo , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Biológicos , Fosforilación , Fosfoserina/metabolismo , Vinculina/metabolismoRESUMEN
In addition to performing its canonical function, Telomerase Reverse Transcriptase (TERT) has been shown to participate in cellular processes independent of telomerase activity. Furthermore, although TERT mainly localizes to Cajal bodies, it is also present within the nucleolus. Because the nucleolus is the site of rDNA transcription, we investigated the possible role of telomerase in regulating RNA polymerase I (Pol I). Here we show that TERT binds to rDNA and stimulates transcription by Pol I during liver regeneration and Ras-induced hyperproliferation. Moreover, the inhibition of telomerase activity by TERT- or TERC-specific RNA interference, the overexpression of dominant-negative-TERT, and the application of the telomerase inhibitor imetelstat reduce Pol I transcription and the growth of tumour cells. In vitro, telomerase can stimulate the formation of the transcription initiation complex. Our results demonstrate how non-canonical features of telomerase may direct Pol I transcription in oncogenic and regenerative hyperproliferation.
Asunto(s)
Proliferación Celular/fisiología , ADN Ribosómico/genética , ADN Ribosómico/fisiología , ARN Polimerasa I/fisiología , Telomerasa/fisiología , Transcripción Genética/fisiología , Animales , Línea Celular , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Colorrectales/patología , Humanos , Riñón/citología , Hígado/citología , Regeneración Hepática/genética , Regeneración Hepática/fisiología , Pulmón/citología , Masculino , Ratones , Ratones Endogámicos C57BL , Miofibroblastos/citología , Unión Proteica/fisiología , ARN Polimerasa I/genética , Conejos , Telomerasa/genética , Transcripción Genética/genéticaRESUMEN
BACKGROUND AND AIMS: The cyclin-dependent kinase inhibitor p21 has been implicated as a tumour suppressor. Moreover, recent genetic studies suggest that p21 might be a potential therapeutic target to improve regeneration in chronic diseases. The aim of this study was to delineate the role of p21 in chronic liver injury and to specify its role in hepatocarcinogenesis in a mouse model of chronic cholestatic liver injury. METHODS: The degree of liver injury, regeneration and tumour formation was assessed in Mdr2(-/-) mice and compared with Mdr2/ p21(-/-) mice. Moreover, the role of p21 was evaluated in hepatoma cells in vitro and in human hepatocellular carcinoma (HCC). RESULTS: Mdr2(-/-) mice developed HCCs as a consequence of chronic inflammatory liver injury. In contrast, tumour development was profoundly delayed in Mdr2/ p21(-/-) mice. Delayed tumour development was accompanied by markedly impaired liver regeneration in Mdr2/ p21(-/-) mice. Moreover, the regenerative capacity of the Mdr2/ p21(-/-) livers in response to partial hepatectomy declined with age in these mice. Hepatocyte transplantation experiments revealed that impaired liver regeneration was due to intrinsic factors within the cells and changes in the Mdr2/ p21(-/-) microenvironment. In human HCCs, a subset of tumours expressed p21, which was associated with a significant shorter patient survival. CONCLUSIONS: We provide experimental evidence that p21 is required for sustained liver regeneration and tumour development in chronic liver injury indicating that p21 needs to be tightly regulated in order to balance liver regeneration and cancer risk. Moreover, we identify p21 as a negative prognostic marker in human HCC.
Asunto(s)
Carcinogénesis/metabolismo , Carcinoma Hepatocelular/etiología , Colestasis Intrahepática/complicaciones , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Insuficiencia Hepática/fisiopatología , Neoplasias Hepáticas/etiología , Regeneración Hepática/fisiología , Animales , Biomarcadores/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidad , Línea Celular , Enfermedad Crónica , Progresión de la Enfermedad , Femenino , Hepatectomía , Insuficiencia Hepática/etiología , Insuficiencia Hepática/metabolismo , Insuficiencia Hepática/cirugía , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidad , Masculino , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , PronósticoRESUMEN
BACKGROUND & AIMS: p53 limits the self-renewal of stem cells from various tissues. Loss of p53, in combination with other oncogenic events, results in aberrant self-renewal and transformation of progenitor cells. It is not known whether loss of p53 is sufficient to induce tumor formation in liver. METHODS: We used AlfpCre mice to create mice with liver-specific disruption of Trp53 (AlfpCre(+)Trp53(Δ2-10/Δ2-10) mice). We analyzed colony formation and genomic features and gene expression patterns in liver cells during hepatocarcinogenesis in mice with homozygous, heterozygous, and no disruption of Trp53. RESULTS: Liver-specific disruption of Trp53 consistently induced formation of liver carcinomas that had bilineal differentiation. In nontransformed liver cells and cultured primary liver cells, loss of p53 (but not p21) resulted in chromosomal imbalances and increased clonogenic capacity of liver progenitor cells (LPCs) and hepatocytes. Primary cultures of hepatocytes and LPCs from AlfpCre(+)Trp53(Δ2-10/Δ2-10) mice, but not Cdkn1a(-/-) mice, formed tumors with bilineal differentiation when transplanted into immunocompromised mice. Spontaneous liver tumors that developed in AlfpCre(+)Trp53(Δ2-10/Δ2-10) mice had significant but complex alterations in expression of Rb checkpoint genes compared with chemically induced liver tumors that developed mice with wild-type Trp53. CONCLUSIONS: Deletion of p53 from livers of mice is sufficient to induce tumor formation. The tumors have bilineal differentiation and dysregulation of Rb checkpoint genes.
Asunto(s)
Neoplasias Hepáticas Experimentales/etiología , Hígado/patología , Proteína p53 Supresora de Tumor/fisiología , Envejecimiento , Animales , Diferenciación Celular , Transformación Celular Neoplásica , Inestabilidad Cromosómica , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/fisiología , Genes de Retinoblastoma , Neoplasias Hepáticas Experimentales/genética , Neoplasias Hepáticas Experimentales/patología , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND & AIMS: Ectopic expression of certain transcription factors can reprogram somatic cells to a pluripotent state. Hematopoietic and muscle stem cells can be more efficiently reprogrammed than differentiated blood or muscle cells, yet similar findings have not been shown in other primary organ systems. Moreover, molecular characteristics of the cellular hierarchy of tissues that influence reprogramming capacities need to be delineated. We analyzed the effect of differentiation stage of freshly isolated, mouse liver cells on the reprogramming efficiency. METHODS: Liver progenitor cell (LPC)-enriched cell fractions were isolated from adult (6-8 wk) and fetal (embryonic day 14.5) livers of mice and reprogrammed to become induced pluripotent stem (iPS) cells. Different transcription factors were expressed in liver cells, and markers of pluripotency were examined, along with the ability of iPS cells to differentiate, in vitro and in vivo, into different germ layers. RESULTS: Fetal and adult LPCs had significantly greater reprogramming efficiency after transduction with 3 or 4 reprogramming factors. Transduction efficiency-corrected reprogramming rates of fetal LPCs were 275-fold higher, compared with unsorted fetal liver cells, when 3 reprogramming factors were transduced. The increased reprogramming efficiency of LPCs, compared with differentiated liver cells, occurred independently of proliferation rates, but was associated with endogenous expression of reprogramming factors (Klf4 and c-Myc) and BAF (Brg1/Brm associated factor)-complex members Baf155 and Brg1, which mediate epigenetic changes during reprogramming. Knockdown of BAF complex members negated the increased reprogramming efficiency of LPCs, compared with non-LPCs. CONCLUSIONS: LPCs have intrinsic, cell proliferation-independent characteristics resulting in an increased reprogramming capacity compared to differentiated liver cells.
Asunto(s)
Diferenciación Celular , ADN Helicasas/metabolismo , Hepatocitos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Hígado/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Animales , Biomarcadores/metabolismo , Proliferación Celular , Células Cultivadas , ADN Helicasas/genética , Epigénesis Genética , Regulación del Desarrollo de la Expresión Génica , Genotipo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/metabolismo , Hígado/embriología , Ratones , Proteínas Nucleares/genética , Fenotipo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , Factores de Tiempo , Factores de Transcripción/genética , Transducción Genética , TransfecciónAsunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/fisiología , Miocitos Cardíacos/fisiología , Canales de Potasio Calcio-Activados/fisiología , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Bencimidazoles/farmacología , Miosinas Cardíacas/genética , Miosinas Cardíacas/metabolismo , Proliferación Celular , Forma de la Célula , Células Cultivadas , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Canales Catiónicos Regulados por Nucleótidos Cíclicos/metabolismo , Cuerpos Embrioides , Humanos , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Contracción Miocárdica , Miocitos Cardíacos/metabolismo , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Canales de Potasio , Canales de Potasio Calcio-Activados/agonistas , Canales de Potasio Calcio-Activados/metabolismo , Transcripción GenéticaRESUMEN
UNLABELLED: Telomere shortening impairs liver regeneration in mice and is associated with cirrhosis formation in humans with chronic liver disease. In humans, telomerase mutations have been associated with familial diseases leading to bone marrow failure or lung fibrosis. It is currently unknown whether telomerase mutations associate with cirrhosis induced by chronic liver disease. The telomerase RNA component (TERC) and the telomerase reverse transcriptase (TERT) were sequenced in 1,121 individuals (521 patients with cirrhosis induced by chronic liver disease and 600 noncirrhosis controls). Telomere length was analyzed in patients carrying telomerase gene mutations. Functional defects of telomerase gene mutations were investigated in primary human fibroblasts and patient-derived lymphocytes. An increased incidence of telomerase mutations was detected in cirrhosis patients (allele frequency 0.017) compared to noncirrhosis controls (0.003, P value 0.0007; relative risk [RR] 1.859; 95% confidence interval [CI] 1.552-2.227). Cirrhosis patients with TERT mutations showed shortened telomeres in white blood cells compared to control patients. Cirrhosis-associated telomerase mutations led to reduced telomerase activity and defects in maintaining telomere length and the replicative potential of primary cells in culture. CONCLUSION: This study provides the first experimental evidence that telomerase gene mutations are present in patients developing cirrhosis as a consequence of chronic liver disease. These data support the concept that telomere shortening can represent a causal factor impairing liver regeneration and accelerating cirrhosis formation in response to chronic liver disease.
Asunto(s)
Cirrosis Hepática/genética , Mutación , Telomerasa/genética , Adulto , Anciano , Anciano de 80 o más Años , Enfermedad Crónica , Femenino , Humanos , Cirrosis Hepática/etiología , Hepatopatías/complicaciones , Masculino , Persona de Mediana EdadRESUMEN
BACKGROUND: Ion channels are key determinants for the function of excitable cells, but little is known about their role and involvement during cardiac development. Earlier work identified Ca(2+)-activated potassium channels of small and intermediate conductance (SKCas) as important regulators of neural stem cell fate. Here we have investigated their impact on the differentiation of pluripotent cells toward the cardiac lineage. METHODS AND RESULTS: We have applied the SKCa activator 1-ethyl-2-benzimidazolinone on embryonic stem cells and identified this particular ion channel family as a new critical target involved in the generation of cardiac pacemaker-like cells: SKCa activation led to rapid remodeling of the actin cytoskeleton, inhibition of proliferation, induction of differentiation, and diminished teratoma formation. Time-restricted SKCa activation induced cardiac mesoderm and commitment to the cardiac lineage as shown by gene regulation, protein, and functional electrophysiological studies. In addition, the differentiation into cardiomyocytes was modulated in a qualitative fashion, resulting in a strong enrichment of pacemaker-like cells. This was accompanied by induction of the sino-atrial gene program and in parallel by a loss of the chamber-specific myocardium. In addition, SKCa activity induced activation of the Ras-Mek-Erk signaling cascade, a signaling pathway involved in the 1-ethyl-2-benzimidazolinone-induced effects. CONCLUSIONS: SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.