RESUMEN
The cellular mechanisms required to ensure homeostasis of the hematopoietic niche and the ability of this niche to support hematopoiesis upon stress remain elusive. We here identify Wnt5a in Osterix+ mesenchymal progenitor and stem cells (MSPCs) as a critical factor for niche-dependent hematopoiesis. Mice lacking Wnt5a in MSPCs suffer from stress-related bone marrow (BM) failure and increased mortality. Niche cells devoid of Wnt5a show defective actin stress fiber orientation due to an elevated activity of the small GTPase CDC42. This results in incorrect positioning of autophagosomes and lysosomes, thus reducing autophagy and increasing oxidative stress. In MSPCs from patients from BM failure states which share features of peripheral cytopenia and hypocellular BM, we find similar defects in actin stress fiber orientation, reduced and incorrect colocalization of autophagosomes and lysosomes, and CDC42 activation. Strikingly, a short pharmacological intervention to attenuate elevated CDC42 activation in vivo in mice prevents defective actin-anchored autophagy in MSPCs, salvages hematopoiesis and protects against lethal cytopenia upon stress. In summary, our study identifies Wnt5a as a restriction factor for niche homeostasis by affecting CDC42-regulated actin stress-fiber orientation and autophagy upon stress. Our data further imply a critical role for autophagy in MSPCs for adequate support of hematopoiesis by the niche upon stress and in human diseases characterized by peripheral cytopenias and hypocellular BM.
Asunto(s)
Autofagia , Trastornos de Fallo de la Médula Ósea/metabolismo , Hematopoyesis , Células Madre Mesenquimatosas/citología , Animales , Células Cultivadas , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Estrés Oxidativo , Proteína Wnt-5a/metabolismoRESUMEN
Alimentary lymphomas arising from T cells are rare and aggressive malignancies in humans. In comparison, they represent the most common anatomical form of lymphoma in cats. Due to the low prevalence in humans, the underlying pathomechanism for these diseases is poorly characterised, limiting experimental analysis and therapeutic exploration. To date, activating mutations of the JAK/STAT core cancer pathway and particularly the STAT5B oncoprotein have been identified in human enteropathy-associated T cell lymphoma. Here, we describe a high homology of human and feline STAT3 and STAT5B proteins and strong conservation at the genomic level. Analysis of 42 samples of feline T cell alimentary lymphoma reveals broad activation of STAT3 and STAT5B. Screening for known activating mutations in STAT3 or STAT5B identifies the presence of the STAT5BN642H driver mutation in feline enteropathy-associated T cell lymphoma in 7 out of 42 (16.67%) samples in total. Regarding lymphoma subtypes, the majority of mutations with 5 out of 17 (29.41%) cases were found in feline enteropathy-associated lymphoma type II (EATL II). This identification of an oncogenic STAT5B driver mutation in felines recapitulates the genetic situation in the corresponding human disease, thereby establishing the cat as a potential new model for a rare and incurable human T cell disease.
RESUMEN
Functional intestinal disorders constitute major, potentially lethal health problems in humans. Consequently, research focuses on elucidating the underlying pathobiological mechanisms and establishing therapeutic strategies. In this context, intestinal organoids have emerged as a potent in vitro model as they faithfully recapitulate the structure and function of the intestinal segment they represent. Interestingly, human-like intestinal diseases also affect dogs, making canine intestinal organoids a promising tool for canine and comparative research. Therefore, we generated organoids from canine duodenum, jejunum and colon, and focused on simultaneous long-term expansion and cell differentiation to maximize applicability. Following their establishment, canine intestinal organoids were grown under various culture conditions and then analyzed with respect to cell viability/apoptosis and multi-lineage differentiation by transcription profiling, proliferation assay, cell staining, and transmission electron microscopy. Standard expansion medium supported long-term expansion of organoids irrespective of their origin, but inhibited cell differentiation. Conversely, transfer of organoids to differentiation medium promoted goblet cell and enteroendocrine cell development, but simultaneously induced apoptosis. Unimpeded stem cell renewal and concurrent differentiation was achieved by culturing organoids in the presence of tyrosine kinase ligands. Our findings unambiguously highlight the characteristic cellular diversity of canine duodenum, jejunum and colon as fundamental prerequisite for accurate in vitro modelling.
Asunto(s)
Diferenciación Celular , Intestinos/citología , Organoides/citología , Animales , Biomarcadores/metabolismo , Linaje de la Célula , Células Cultivadas , Medios de Cultivo , Perros , Células Enteroendocrinas/citología , Femenino , Células Caliciformes/citología , Masculino , Organoides/crecimiento & desarrollo , Organoides/ultraestructuraRESUMEN
The use of transgenic mouse models has revolutionized the study of many human diseases. However, murine models are limited in their representation of spontaneously arising tumors and often lack key clinical signs and pathological changes. Thus, a closer representation of complex human diseases is of high therapeutic relevance. Given the high failure rate of drugs at the clinical trial phase (i.e., around 90%), there is a critical need for additional clinically relevant animal models. Companion animals like cats and dogs display chronic inflammatory or neoplastic diseases that closely resemble the human counterpart. Cat and dog patients can also be treated with clinically approved inhibitors or, if ethics and drug safety studies allow, pilot studies can be conducted using, e.g., inhibitors of the evolutionary conserved JAK-STAT pathway. The incidence by which different types of cancers occur in companion animals as well as mechanisms of disease are unique between humans and companion animals, where one can learn from each other. Taking advantage of this situation, existing inhibitors of known oncogenic STAT3/5 or JAK kinase signaling pathways can be studied in the context of rare human diseases, benefitting both, the development of drugs for human use and their application in veterinary medicine.
RESUMEN
Epithelial cell adhesion molecule EpCAM is expressed in pluripotent embryonic stem cells (ESC) in vitro, but is repressed in differentiated cells, except epithelia and carcinomas. Molecular functions of EpCAM, possibly imposing such repression, were primarily studied in malignant cells and might not apply to non-pathologic differentiation. Here, we comprehensively describe timing and rationale for EpCAM regulation in early murine gastrulation and ESC differentiation using single cell RNA-sequencing datasets, in vivo and in vitro models including CRISPR-Cas9-engineered ESC-mutants. We demonstrate expression of EpCAM in inner cell mass, epiblast, primitive/visceral endoderm, and strict repression in the most primitive, nascent Flk1+ mesoderm progenitors at E7.0. Selective expression of EpCAM was confirmed at mid-gestation and perinatal stages. The rationale for strict patterning was studied in ESC differentiation. Gain/loss-of-function demonstrated supportive functions of EpCAM in achieving full pluripotency and guided endodermal differentiation, but repressive functions in mesodermal differentiation as exemplified with cardiomyocyte formation. We further identified embryonic Ras (ERas) as novel EpCAM interactor of EpCAM and an EpCAM/ERas/AKT axis that is instrumental in differentiation regulation. Hence, spatiotemporal patterning of EpCAM at the onset of gastrulation, resulting in early segregation of interdependent EpCAM+ endodermal and EpCAM-/vimentin+ mesodermal clusters represents a novel regulatory feature during ESC differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Células Madre Embrionarias/metabolismo , Endodermo/metabolismo , Molécula de Adhesión Celular Epitelial/metabolismo , Mesodermo/metabolismo , Animales , Línea Celular , Linaje de la Célula/fisiología , Gastrulación/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Estratos Germinativos/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Madre Pluripotentes/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The maintenance of hematopoietic stem cells (HSCs) during ex vivo culture is an important prerequisite for their therapeutic manipulation. However, despite intense research, culture conditions for robust maintenance of HSCs are still missing. Cultured HSCs are quickly lost, preventing their improved analysis and manipulation. Identification of novel factors supporting HSC ex vivo maintenance is therefore necessary. Coculture with the AFT024 stroma cell line is capable of maintaining HSCs ex vivo long-term, but the responsible molecular players remain unknown. Here, we use continuous long-term single-cell observation to identify the HSC behavioral signature under supportive or nonsupportive stroma cocultures. We report early HSC survival as a major characteristic of HSC-maintaining conditions. Behavioral screening after manipulation of candidate molecules revealed that the extracellular matrix protein dermatopontin (Dpt) is involved in HSC maintenance. DPT knockdown in supportive stroma impaired HSC survival, whereas ectopic expression of the Dpt gene or protein in nonsupportive conditions restored HSC survival. Supplementing defined stroma- and serum-free culture conditions with recombinant DPT protein improved HSC clonogenicity. These findings illustrate a previously uncharacterized role of Dpt in maintaining HSCs ex vivo.
Asunto(s)
Proteoglicanos Tipo Condroitín Sulfato/metabolismo , Proteínas de la Matriz Extracelular/metabolismo , Células Madre Hematopoyéticas/metabolismo , Animales , Técnicas de Cultivo de Célula , Línea Celular , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Proteoglicanos Tipo Condroitín Sulfato/genética , Proteoglicanos Tipo Condroitín Sulfato/farmacología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/farmacología , Células Madre Hematopoyéticas/citología , Masculino , Ratones , Ratones Transgénicos , Células del Estroma/citología , Células del Estroma/metabolismo , Factores de TiempoRESUMEN
Sfrp2 is overexpressed in stromal cells which maintain hematopoietic stem cells (HSCs) during in vitro culture. We here showed, that coculture of hematopoetic cells with stromal cells with reduced expression of Sfrp2 increases the number lineage-negative Kit(+) Sca-1(+) (LSK) and progenitor cells in vitro. The LSK cells from these cocultures showed activation of canonical Wnt signaling, higher levels of Ki-67, BrdU incorporation, and the number of γH2A.X positive foci. Total repopulating activity of these cultures was, however, diminished, indicating loss of HSC. To extend these in vitro data, we modelled stress in vivo, i.e., by aging, or 5-FU treatment in Sfrp2(-) (/) (-) mice, or replicative stress in regeneration of HSCs in Sfrp2(-) (/) (-) recipients. In all three in vivo stress situations, we noted an increase of LSK cells, characterized by increased levels of ß-catenin and cyclin D1. In the transplantation experiments, the increase in LSK cells in primary recipients was subsequently associated with a progressive loss of HSCs in serial transplantations. Similar to the in vitro coculture stress, in vivo genotoxic stress in 5-FU-treated Sfrp2(-) (/) (-) mice increased cell cycle activity of LSK cells with higher levels of BrdU incorporation, increased expression of Ki-67, and canonical Wnt signaling. Importantly, as noted in vitro, increased cycling of LSKs in vivo was accompanied by a defective γH2A.X-dependent DNA damage response and depolarized localization of acetylated H4K16. Our experiments support the view that Sfrp2 expression in the niche is required to maintain the HSC pool by limiting stress-induced DNA damage and attenuating canonical Wnt-mediated HSC activation. Stem Cells 2016;34:2381-2392.
Asunto(s)
Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Proteínas de la Membrana/deficiencia , Regeneración , Nicho de Células Madre , Estrés Fisiológico , Envejecimiento/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Microambiente Celular/efectos de los fármacos , Senescencia Celular/efectos de los fármacos , Técnicas de Cocultivo , Daño del ADN , Fluorouracilo/farmacología , Hematopoyesis/efectos de los fármacos , Trasplante de Células Madre Hematopoyéticas , Células Madre Hematopoyéticas/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Regeneración/efectos de los fármacos , Nicho de Células Madre/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismoRESUMEN
BACKGROUND: The Wnt inhibitor Dickkopf-1 (DKK-1) has been linked to the progression of malignant bone disease by impairing osteoblast activity. In addition, there is increasing data to suggest direct tumor promoting effects of DKK-1. The prognostic role of DKK-1 expression in prostate cancer remains unclear. METHODS: A prostate cancer tissue microarray (n = 400) was stained for DKK-1 and DKK-1 serum levels were measured in 80 patients with prostate cancer. The independent prognostic value of DKK-1 expression was assessed using multivariate analyses. RESULTS: DKK-1 tissue expression was significantly increased in prostate cancer compared to benign disease, but was not correlated with survival. However, high DKK-1 serum levels at the time of the diagnosis were associated with a significantly shorter overall and disease-specific survival. Multivariate analyses defined high serum levels of DKK-1 as an independent prognostic marker in prostate cancer (HR 3.73; 95%CI 1.44-9.66, p = 0.007). CONCLUSION: High DKK-1 serum levels are associated with a poor survival in patients with prostate cancer. In light of current clinical trials evaluating the efficacy of anti-DKK-1 antibody therapies in multiple myeloma and solid malignancies, the measurement of DKK-1 in prostate cancer may gain clinical relevance.
Asunto(s)
Biomarcadores de Tumor/sangre , Péptidos y Proteínas de Señalización Intercelular/sangre , Neoplasias de la Próstata/sangre , Neoplasias de la Próstata/diagnóstico , Anciano , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , Neoplasias de la Próstata/patología , Análisis de Supervivencia , Análisis de Matrices TisularesRESUMEN
Myogenic regulatory factors such as MyoD and Myf5 lie at the core of vertebrate muscle differentiation. However, E-boxes, the cognate binding sites for these transcription factors, are not restricted to the promoters/enhancers of muscle cell-specific genes. Thus, the specificity in myogenic transcription is poorly defined. Here we describe the transcription factor Ebf3 as a new determinant of muscle cell-specific transcription. In the absence of Ebf3 the lung does not unfold at birth, resulting in respiratory failure and perinatal death. This is due to a hypercontractile diaphragm with impaired Ca(2+) efflux-related muscle functions. Expression of the Ca(2+) pump Serca1 (Atp2a1) is downregulated in the absence of Ebf3, and its transgenic expression rescues this phenotype. Ebf3 binds directly to the promoter of Atp2a1 and synergises with MyoD in the induction of Atp2a1. In skeletal muscle, the homologous family member Ebf1 is strongly expressed and together with MyoD induces Atp2a1. Thus, Ebf3 is a new regulator of terminal muscle differentiation in the diaphragm, and Ebf factors cooperate with MyoD in the induction of muscle-specific genes.
Asunto(s)
Relajación Muscular/fisiología , Proteína MioD/fisiología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/fisiología , Factores de Transcripción/fisiología , Animales , Ratones , Ratones Noqueados , Insuficiencia Respiratoria/genética , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/genéticaRESUMEN
Genetic redundancy poses a major problem to the analysis of gene function. RNA interference allows the down-regulation of several genes simultaneously, offering the possibility to overcome genetic redundancy, something not easily achieved with traditional genetic approaches. Previously we have used a polycistronic miR155-based framework to knockdown expression of three genes of the early B cell factor family in cultured cells. Here we develop the system further by generating transgenic mice expressing the RNAi construct in vivo in an inducible manner. Expression of the transgene from the strong CAG promoter is compatible with a normal function of the basal miRNA/RNAi machinery, and the miR155 framework readily allows inducible expression from the Rosa26 locus as shown by Gfp. However, expression of the transgene in hematopoietic cells does not lead to changes in B cell development and neuronal expression does not affect cerebellar architecture as predicted from genetic deletion studies. Protein as well as mRNA levels generated from Ebf genes in hetero- and homozygous animals are comparable to wild-type levels. A likely explanation for the discrepancy in the effectiveness of the RNAi construct between cultured cells and transgenic animals lies in the efficiency of the sequences used, possibly together with the complexity of the transgene. Since new approaches allow to overcome efficiency problems of RNAi sequences, the data lay the foundation for future work on the simultaneous knockdown of several genes in vivo.
Asunto(s)
Regulación de la Expresión Génica , Silenciador del Gen , Familia de Multigenes , Interferencia de ARN , Animales , Línea Celular , Cerebelo/metabolismo , Regulación hacia Abajo , Células Madre Embrionarias/metabolismo , Expresión Génica , Orden Génico , Marcación de Gen , Ratones , Ratones Transgénicos , Células Precursoras de Linfocitos B/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , TransgenesRESUMEN
Hematopoiesis requires the interaction of hematopoietic stem cells (HSCs) with various stromal microenvironments. Here, we examine the role of early B cell factor 2 (Ebf2), a transcription factor expressed in a subset of immature osteoblastic cells. Ebf2(-/-) mice show decreased frequencies of HSCs and lineage-committed progenitors. This defect is cell nonautonomous, as shown by the fact that transplantation of Ebf2-deficient bone marrow into wild-type hosts results in normal hematopoiesis. In coculture experiments, Ebf2(-/-) osteoblastic cells have reduced potential to support short-term proliferation of HSCs. Expression profiling of sorted Ebf2(-/-) osteoblastic cells indicated that several genes implicated in the maintenance of HSCs are downregulated relative to Ebf2(+/-) cells, whereas genes encoding secreted frizzled-related proteins are upregulated. Moreover, wild-type HSCs cocultured with Ebf2(-/-) osteoblastic cells show a reduced Wnt response relative to coculture with Ebf2(+/-) cells. Thus, Ebf2 acts as a transcriptional determinant of an osteoblastic niche that regulates the maintenance of hematopoietic progenitors, in part by modulating Wnt signaling.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Osteoblastos/citología , Animales , Linfocitos B/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular , Linaje de la Célula , Regulación de la Expresión Génica , Ratones , Mutación , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Communication between bone-depositing osteoblasts and bone-resorbing osteoclasts is required for bone development and homeostasis. Here, we identify EBF2, a member of the early B cell factor (EBF) family of transcription factors that is expressed in osteoblast progenitors, as a regulator of osteoclast differentiation. We find that mice homozygous for a targeted inactivation of Ebf2 show reduced bone mass and an increase in the number of osteoclasts. These defects are accompanied by a marked downregulation of the osteoprotegerin (Opg) gene, encoding a RANK decoy receptor. EBF2 binds to sequences in the Opg promoter and transactivates the Opg promoter in synergy with the Wnt-responsive LEF1/TCF:beta-catenin pathway. Taken together, these data identify EBF2 as a regulator of RANK-RANKL signaling and osteoblast-dependent differentiation of osteoclasts.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Diferenciación Celular , Osteoblastos/metabolismo , Osteoclastos/citología , Animales , Desarrollo Óseo , Resorción Ósea , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/fisiología , Regulación de la Expresión Génica , Glicoproteínas/genética , Glicoproteínas/metabolismo , Células HeLa , Homocigoto , Humanos , Hibridación in Situ , Factor de Unión 1 al Potenciador Linfoide/genética , Factor de Unión 1 al Potenciador Linfoide/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Noqueados , Osteogénesis , Osteoprotegerina , Regiones Promotoras Genéticas/genética , Ligando RANK , Receptor Activador del Factor Nuclear kappa-B , Receptores Citoplasmáticos y Nucleares/genética , Receptores Citoplasmáticos y Nucleares/metabolismo , Receptores del Factor de Necrosis Tumoral/genética , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Factor 1 de Transcripción de Linfocitos T/genética , Factor 1 de Transcripción de Linfocitos T/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Activación Transcripcional , Proteínas Wnt , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Protein inhibitors of activated STATs (PIAS) represent a small family of nuclear proteins that modulate the activity of many transcription factors and act as E3 ligases for covalent modification of proteins with the small ubiquitin-like modifier (SUMO). In particular, PIASy has been shown to inhibit the activation of gene expression by the IFN-responsive transcription factor STAT1 and the Wnt-responsive transcription factor LEF1. To assess the function of PIASy in vivo, we generated and analyzed mice carrying a targeted mutation of the Piasy gene. We find that homozygous mutant mice have no obvious morphological defects and have a normal distribution of lymphocyte populations. Molecular analysis of signaling in response to IFN-gamma and Wnt agonists revealed a modest reduction in the activation of endogenous and transfected target genes. Two-dimensional analysis of total proteins and SUMO-modified proteins in transformed pre-B cells showed no significant differences between wild-type mice and homozygous mutant mice. Taken together, our data indicate that PIASy has a modest effect on cytokine and Wnt signaling, suggesting a redundancy with other members of the family of PIAS proteins.
Asunto(s)
Interferones/fisiología , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal/genética , Animales , Línea Celular Transformada , Células Cultivadas , Citocinas/fisiología , Electroforesis en Gel Bidimensional , Regulación del Desarrollo de la Expresión Génica/genética , Silenciador del Gen , Hematopoyesis/genética , Hematopoyesis/inmunología , Homocigoto , Interferón gamma/farmacología , Péptidos y Proteínas de Señalización Intracelular/fisiología , Operón Lac , Ratones , Ratones Noqueados , Mutagénesis Insercional/métodos , Proteínas Inhibidoras de STAT Activados , Transducción de Señal/inmunología , Proteínas WntRESUMEN
The generation of B lymphocyte precursors is dependent on the combinatorial action of the transcription factors PU.1, Ikaros, E2A, EBF, and Pax-5. Loss of PU.1 results in a severe reduction in Flk2+, IL-7R+ lymphoid progenitors as well as impaired expression of EBF and Pax-5. Restoration of EBF expression facilitates rapid generation of pro-B cells from PU.1-/- progenitors. Molecular analysis suggests that PU.1 directly participates in regulation of the EBF gene. Although PU.1 is dispensable for expression of most early B lineage genes, it is required for CD45R/B220. Using EBF-/- mutant progenitors, we show that EBF induces Pax-5 and the early program of B lineage gene expression. Importantly, Pax-5 does not rescue B cell development from either PU.1-/- or EBF-/- progenitors. Pax-5 expression and function are contingent on EBF. Based on these results, we propose a hierarchical regulatory network for specification and commitment to the B cell fate.