RESUMEN
DNA damage response signaling is crucial for genome maintenance in all organisms and is corrupted in cancer. In an RNA interference (RNAi) screen for (de)ubiquitinases and sumoylases modulating the apoptotic response of embryonic stem (ES) cells to DNA damage, we identified the E3 ubiquitin ligase/ISGylase, ariadne homologue 1 (ARIH1). Silencing ARIH1 sensitized ES and cancer cells to genotoxic compounds and ionizing radiation, irrespective of their p53 or caspase-3 status. Expression of wild-type but not ubiquitinase-defective ARIH1 constructs prevented sensitization caused by ARIH1 knockdown. ARIH1 protein abundance increased after DNA damage through attenuation of proteasomal degradation that required ATM signaling. Accumulated ARIH1 associated with 4EHP, and in turn, this competitive inhibitor of the eukaryotic translation initiation factor 4E (eIF4E) underwent increased nondegradative ubiquitination upon DNA damage. Genotoxic stress led to an enrichment of ARIH1 in perinuclear, ribosome-containing regions and triggered 4EHP association with the mRNA 5' cap as well as mRNA translation arrest in an ARIH1-dependent manner. Finally, restoration of DNA damage-induced translation arrest in ARIH1-depleted cells by means of an eIF2 inhibitor was sufficient to reinstate resistance to genotoxic stress. These findings identify ARIH1 as a potent mediator of DNA damage-induced translation arrest that protects stem and cancer cells against genotoxic stress.
Asunto(s)
Proteínas Portadoras/metabolismo , Daño del ADN , Factor 4E Eucariótico de Iniciación/metabolismo , Proteínas de Unión a Caperuzas de ARN/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas Portadoras/genética , Caspasa 3/metabolismo , Línea Celular , Línea Celular Tumoral , Células Madre Embrionarias/metabolismo , Humanos , Ratones , Biosíntesis de Proteínas , Interferencia de ARN , ARN Mensajero/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
In pluripotent stem cells, DNA damage triggers loss of pluripotency and apoptosis as a safeguard to exclude damaged DNA from the lineage. An intricate DNA damage response (DDR) signaling network ensures that the response is proportional to the severity of the damage. We combined an RNA interference screen targeting all kinases, phosphatases, and transcription factors with global transcriptomics and phosphoproteomics to map the DDR in mouse embryonic stem cells treated with the DNA cross-linker cisplatin. Networks derived from canonical pathways shared in all three data sets were implicated in DNA damage repair, cell cycle and survival, and differentiation. Experimental probing of these networks identified a mode of DNA damage-induced Wnt signaling that limited apoptosis. Silencing or deleting the p53 gene demonstrated that genotoxic stress elicited Wnt signaling in a p53-independent manner. Instead, this response occurred through reduced abundance of Csnk1a1 (CK1α), a kinase that inhibits ß-catenin. Together, our findings reveal a balance between p53-mediated elimination of stem cells (through loss of pluripotency and apoptosis) and Wnt signaling that attenuates this response to tune the outcome of the DDR.
Asunto(s)
Quinasa de la Caseína I/metabolismo , Daño del ADN , Células Madre Embrionarias/enzimología , Células Madre Pluripotentes/enzimología , Biología de Sistemas , Vía de Señalización Wnt , Animales , Apoptosis/genética , Quinasa de la Caseína I/genética , Línea Celular , Células Madre Embrionarias/citología , Ratones , Células Madre Pluripotentes/citología , Interferencia de ARN , Transcriptoma/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Activation of the protein kinase A (PKA) pathway with dibutyryl cyclic adenosine monophosphate (db-cAMP) was recently shown to enhance osteogenic differentiation of human mesenchymal stromal cells (hMSCs) in vitro and bone formation in vivo. The major drawback of this compound is its inhibitory effect on proliferation of hMSCs. Therefore, we investigated whether fine-tuning of the dose and timing of PKA activation could enhance bone formation even further, with minimum effects on proliferation. To test this, we selected two different PKA activators (8-bromo-cAMP (8-br-cAMP) and forskolin) and compared their effects on proliferation and osteogenic differentiation with those of db-cAMP. We found that all three compounds induced alkaline phosphatase levels, bone-specific target genes, and secretion of insulin-like growth factor-1, although 8-br-cAMP induced adipogenic differentiation in long-term cultures and was thus considered unsuitable for further in vivo testing. All three compounds inhibited proliferation of hMSCs in a dose-dependent manner, with forskolin inhibiting proliferation most. The effect of forskolin on in vivo bone formation was tested by pretreating hMSCs before implantation, and we observed greater amounts of bone using forskolin than db-cAMP. Our data show forskolin to be a novel agent that can be used to increase bone formation and also suggests a role for PKA in the delicate balance between adipogenic and osteogenic differentiation.
Asunto(s)
Colforsina/farmacología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/metabolismo , Osteogénesis/efectos de los fármacos , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Animales , Antígenos de Diferenciación/biosíntesis , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Humanos , Ratones , Trasplante HeterólogoRESUMEN
Previously, we demonstrated that protein kinase A (PKA) activation using dibutyryl-cAMP in human mesenchymal stem cells (hMSCs) induces in vitro osteogenesis and bone formation in vivo. To further investigate the physiological role of PKA in hMSC osteogenesis, we tested a selection of G-protein-coupled receptor ligands which signal via intracellular cAMP production and PKA activation. Treatment of hMSCs with parathyroid hormone, parathyroid hormone-related peptide, melatonin, epinephrine, calcitonin or calcitonin gene-related peptide did not result in accumulation of cAMP or induction of alkaline phosphatase (ALP) expression. The only ligand that did induce cAMP, prostaglandin E2, even inhibited ALP expression and mineralization, suggesting that physiological levels of cAMP may inhibit osteogenesis. Furthermore, intermittent exposure of hMSCs to dibutyryl-cAMP inhibited ALP expression, whereas we did not observe an inhibitive effect at low dibutyryl-cAMP concentrations. Taken together, our results demonstrate that cAMP can either stimulate or inhibit osteogenesis in hMSCs, depending on the duration, rather than the strength, of the signal provided.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , AMP Cíclico/farmacología , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Osteogénesis/efectos de los fármacos , AMP Cíclico/metabolismo , Dinoprostona/farmacología , Humanos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Ligandos , Hormona Paratiroidea/farmacología , Proteína Relacionada con la Hormona Paratiroidea/farmacología , Factores de TiempoRESUMEN
We previously demonstrated that cAMP-mediated protein kinase A (PKA) activation induces in vitro osteogenesis and in vivo bone formation by human mesenchymal stem cells (hMSCs). To analyze the species-specific response of this phenomenon and to translate our findings into a clinical trial, suitable animal models and cell lines are desirable. In this report, we assessed whether PKA plays a similar proosteogenic role played by two commonly used PKA activators-N6,2'-O-dibutyryl-cAMP (db-cAMP) and 8-bromo cAMP (8b-cAMP)-in a number of model systems. To this end, we treated MC3T3-E1 cells, mouse calvarial osteoblasts, mouse MSCs, and rat MSCs with cAMP. We demonstrate that cAMP inhibits osteogenesis in rodent cell types, evidenced by inhibition of osteogenic markers such as alkaline phosphatase (ALP), osteocalcin (BGLAP), and collagen type 1 (COL1A1). In support of this, ex vivo-cultured mouse calvaria exposed to db-cAMP showed a reduction in bone volume. Interestingly, cAMP even stimulated adipogenic differentiation in rat MSCs. Taken together, our data demonstrate that cAMP inhibits osteogenesis in vitro and bone formation ex vivo in rodent models in contrast to our earlier findings in hMSCs. The species discrepancy in response to various osteogenic signals is a critical need to be tested in clinically relevant models to translate the fundamental findings in lower species level to clinical applications.
Asunto(s)
Diferenciación Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Osteogénesis , Transducción de Señal , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Adipogénesis/efectos de los fármacos , Fosfatasa Alcalina/antagonistas & inhibidores , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Bucladesina/farmacología , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Regulación de la Expresión Génica/efectos de los fármacos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/metabolismo , Ratones , Modelos Animales , Osteogénesis/efectos de los fármacos , Osteogénesis/genética , Ratas , Transducción de Señal/efectos de los fármacosRESUMEN
Tissue engineering of large bone defects is approached through implantation of autologous osteogenic cells, generally referred to as multipotent stromal cells or mesenchymal stem cells (MSCs). Animal-derived MSCs successfully bridge large bone defects, but models for ectopic bone formation as well as recent clinical trials demonstrate that bone formation by human MSCs (hMSCs) is inadequate. The expansion phase presents an attractive window to direct hMSCs by pharmacological manipulation, even though no profound effect on bone formation in vivo has been described so far using this approach. We report that activation of protein kinase A elicits an immediate response through induction of genes such as ID2 and FosB, followed by sustained secretion of bone-related cytokines such as BMP-2, IGF-1, and IL-11. As a consequence, PKA activation results in robust in vivo bone formation by hMSCs derived from orthopedic patients.
Asunto(s)
Huesos/metabolismo , AMP Cíclico/metabolismo , Células Madre Mesenquimatosas/citología , Ingeniería de Tejidos/métodos , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Citocinas/metabolismo , Humanos , Proteína 2 Inhibidora de la Diferenciación/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Interleucina-11/metabolismo , Modelos Biológicos , Osteogénesis , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
The use of multipotent human mesenchymal stem cells (hMSCs) for tissue engineering has been a subject of extensive research. The donor variation in growth, differentiation and in vivo bone forming ability of hMSCs is a bottleneck for standardization of therapeutic protocols. In this study, we isolated and characterized hMSCs from 19 independent donors, aged between 27 and 85 years, and investigated the extent of heterogeneity of the cells and the extent to which hMSCs can be expanded without loosing multipotency. Dexamethasone-induced ALP expression varied between 1.2- and 3.7-fold, but no correlation was found with age, gender, or source of isolation. The cells from donors with a higher percentage of ALP-positive cells in control and dexamethasone-induced groups showed more calcium deposition than cells with lower percentage of ALP positive cells. Despite the variability in osteogenic gene expression among the donors tested, ALP, Collagen type 1, osteocalcin, and S100A4 showed similar trends during the course of osteogenic differentiation. In vitro expansion studies showed that hMSCs can be effectively expanded up to four passages (approximately 10-12 population doublings from a P0 culture) while retaining their multipotency. Our in vivo studies suggest a correlation between in vitro ALP expression and in vivo bone formation. In conclusion, irrespective of age, gender, and source of isolation, cells from all donors showed osteogenic potential. The variability in ALP expression appears to be a result of sampling method and cellular heterogeneity among the donor population.
Asunto(s)
Sustitutos de Huesos , Células Madre Mesenquimatosas/fisiología , Células Madre Multipotentes/fisiología , Donantes de Tejidos/clasificación , Ingeniería de Tejidos/métodos , Acetábulo/citología , Adulto , Anciano , Anciano de 80 o más Años , Fosfatasa Alcalina/análisis , Fosfatasa Alcalina/genética , Animales , Diferenciación Celular/efectos de los fármacos , Dexametasona/farmacología , Femenino , Perfilación de la Expresión Génica , Variación Genética , Humanos , Ilion/citología , Masculino , Células Madre Mesenquimatosas/citología , Ratones , Persona de Mediana Edad , Células Madre Multipotentes/citología , OsteogénesisRESUMEN
Bone tissue engineering using human mesenchymal stem cells (hMSCs) is a multidisciplinary field that aims to treat patients with trauma, spinal fusion and large bone defects. Cell-based bone tissue engineering encompasses the isolation of multipotent hMSCs from the bone marrow of the patient, in vitro expansion and seeding onto porous scaffold materials. In vitro pre-differentiation of hMSCs into the osteogenic lineage augments their in vivo bone forming capacity. Differentiation of hMSCs into bone forming osteoblasts is a multi-step process regulated by various molecular signaling pathways, which warrants a thorough understanding of these signaling cues for the efficient use of hMSCs in bone tissue engineering. Recently, there has been a surge of knowledge on the molecular cues regulating osteogenic differentiation but extrapolation to hMSC differentiation is not guaranteed, because of species- and cell-type specificity. In this review, we describe a number of key osteogenic signaling pathways, which directly or indirectly regulate osteogenic differentiation of hMSCs. We will discuss how and to what extent the process is different from that in other cell types with special emphasis on applications in bone tissue engineering.
Asunto(s)
Huesos/fisiología , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas/fisiología , Osteogénesis , Ingeniería de Tejidos , Animales , Diferenciación Celular , Humanos , Células Madre Mesenquimatosas/ultraestructura , Osteoblastos/fisiología , Transducción de SeñalRESUMEN
Human mesenchymal stem cells (hMSCs) from the bone marrow represent a potential source of pluripotent cells for autologous bone tissue engineering. We previously discovered that over activation of the Wnt signal transduction pathway by either lithium or Wnt3A stimulates hMSC proliferation while retaining pluripotency. Release of Wnt3A or lithium from porous calcium phosphate scaffolds, which we use for bone tissue engineering, could provide a mitogenic stimulus to implanted hMSCs. To define the proper release profile, we first assessed the effect of Wnt over activation on osteogenic differentiation of hMSCs. Here, we report that both lithium and Wnt3A strongly inhibit dexamethasone-induced expression of the osteogenic marker alkaline phosphatase (ALP). Moreover, lithium partly inhibited mineralization of hMSCs whereas Wnt3A completely blocked it. Time course analysis during osteogenic differentiation revealed that 4 days of Wnt3A exposure before the onset of mineralization is sufficient to block mineralization completely. Gene expression profiling in Wnt3A and lithium-exposed hMSCs showed that many osteogenic and chondrogenic markers, normally expressed in proliferating hMSCs, are downregulated upon Wnt stimulation. We conclude that Wnt signaling inhibits dexamethasone-induced osteogenesis in hMSCs. In future studies, we will try to limit release of lithium or Wnt3A from calcium phosphate scaffolds to the proliferative phase of osteogenesis.