RESUMEN
CCS1477 (inobrodib) is a potent, selective EP300/CBP bromodomain inhibitor which induces cell-cycle arrest and differentiation in hematologic malignancy model systems. In myeloid leukemia cells, it promotes rapid eviction of EP300/CBP from an enhancer subset marked by strong MYB occupancy and high H3K27 acetylation, with downregulation of the subordinate oncogenic network and redistribution to sites close to differentiation genes. In myeloma cells, CCS1477 induces eviction of EP300/CBP from FGFR3, the target of the common (4; 14) translocation, with redistribution away from IRF4-occupied sites to TCF3/E2A-occupied sites. In a subset of patients with relapsed or refractory disease, CCS1477 monotherapy induces differentiation responses in AML and objective responses in heavily pre-treated multiple myeloma. In vivo preclinical combination studies reveal synergistic responses to treatment with standard-of-care agents. Thus, CCS1477 exhibits encouraging preclinical and early-phase clinical activity by disrupting recruitment of EP300/CBP to enhancer networks occupied by critical transcription factors.
Asunto(s)
Neoplasias Hematológicas , Proteínas Nucleares , Humanos , Línea Celular Tumoral , Factores de Transcripción , Dominios Proteicos , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/genética , Proteína p300 Asociada a E1ARESUMEN
Pharmacologic inhibition of LSD1 induces molecular and morphologic differentiation of blast cells in acute myeloid leukemia (AML) patients harboring MLL gene translocations. In addition to its demethylase activity, LSD1 has a critical scaffolding function at genomic sites occupied by the SNAG domain transcription repressor GFI1. Importantly, inhibitors block both enzymatic and scaffolding activities, in the latter case by disrupting the protein:protein interaction of GFI1 with LSD1. To explore the wider consequences of LSD1 inhibition on the LSD1 protein complex we applied mass spectrometry technologies. We discovered that the interaction of the HMG-box protein HMG20B with LSD1 was also disrupted by LSD1 inhibition. Downstream investigations revealed that HMG20B is co-located on chromatin with GFI1 and LSD1 genome-wide; the strongest HMG20B binding co-locates with the strongest GFI1 and LSD1 binding. Functional assays demonstrated that HMG20B depletion induces leukemia cell differentiation and further revealed that HMG20B is required for the transcription repressor activity of GFI1 through stabilizing LSD1 on chromatin at GFI1 binding sites. Interaction of HMG20B with LSD1 is through its coiled-coil domain. Thus, HMG20B is a critical component of the GFI1:LSD1 transcription repressor complex which contributes to leukemia cell differentiation block.
Asunto(s)
Histona Demetilasas , Leucemia Mieloide Aguda , Humanos , Diferenciación Celular/genética , Cromatina/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Histona Demetilasas/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Despite absent expression in normal hematopoiesis, the Forkhead factor FOXC1, a critical mesenchymal differentiation regulator, is highly expressed in â¼30% of HOXAhigh acute myeloid leukemia (AML) cases to confer blocked monocyte/macrophage differentiation. Through integrated proteomics and bioinformatics, we find that FOXC1 and RUNX1 interact through Forkhead and Runt domains, respectively, and co-occupy primed and active enhancers distributed close to differentiation genes. FOXC1 stabilizes association of RUNX1, HDAC1, and Groucho repressor TLE3 to limit enhancer activity: FOXC1 knockdown induces loss of repressor proteins, gain of CEBPA binding, enhancer acetylation, and upregulation of nearby genes, including KLF2. Furthermore, it triggers genome-wide redistribution of RUNX1, TLE3, and HDAC1 from enhancers to promoters, leading to repression of self-renewal genes, including MYC and MYB. Our studies highlight RUNX1 and CEBPA transcription factor swapping as a feature of leukemia cell differentiation and reveal that FOXC1 prevents this by stabilizing enhancer binding of a RUNX1/HDAC1/TLE3 transcription repressor complex to oncogenic effect.
Asunto(s)
Diferenciación Celular , Proteínas Co-Represoras/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Factores de Transcripción Forkhead/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Línea Celular Tumoral , Cromatina/metabolismo , Proteínas Co-Represoras/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/química , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Elementos de Facilitación Genéticos , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/genética , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Monocitos/citología , Monocitos/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Dominios Proteicos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Regulación hacia ArribaRESUMEN
The histone demethylase lysine-specific demethylase 1 (LSD1 or KDM1A) has emerged as a candidate therapeutic target in acute myeloid leukaemia (AML); tranylcypromine-derivative inhibitors induce loss of clonogenic activity and promote differentiation, in particular in the MLL-translocated molecular subtype of AML. In AML, the use of drugs in combination often delivers superior clinical activity. To identify genes and cellular pathways that collaborate with LSD1 to maintain the leukaemic phenotype, and which could be targeted by combination therapies, we performed a genome-wide CRISPR-Cas9 dropout screen. We identified multiple components of the amino acid sensing arm of mTORC1 signalling-RRAGA, MLST8, WDR24 and LAMTOR2-as cellular sensitizers to LSD1 inhibition. Knockdown of mTORC1 components, or mTORC1 pharmacologic inhibition, in combination with LSD1 inhibition enhanced differentiation in both cell line and primary cell settings, in vitro and in vivo, and substantially reduced the frequency of clonogenic primary human AML cells in a modelled minimal residual disease setting. Synergistic upregulation of a set of transcription factor genes associated with terminal monocytic lineage differentiation was observed. Thus, dual mTORC1 and LSD1 inhibition represents a candidate combination approach for enhanced differentiation in MLL-translocated AML which could be evaluated in early phase clinical trials.
Asunto(s)
Everolimus/farmacología , Histona Demetilasas/antagonistas & inhibidores , N-Metiltransferasa de Histona-Lisina/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Diana Mecanicista del Complejo 1 de la Rapamicina/antagonistas & inhibidores , Proteína de la Leucemia Mieloide-Linfoide/genética , Translocación Genética , Tranilcipromina/farmacología , Animales , Antidepresivos/farmacología , Antineoplásicos/farmacología , Apoptosis , Proliferación Celular , Quimioterapia Combinada , Femenino , Regulación Leucémica de la Expresión Génica , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acute myeloid leukemia (AML) is an aggressive clonal disorder of hematopoietic stem cells (HSCs) and primitive progenitors that blocks their myeloid differentiation, generating self-renewing leukemic stem cells (LSCs). Here, we show that the mRNA m6A reader YTHDF2 is overexpressed in a broad spectrum of human AML and is required for disease initiation as well as propagation in mouse and human AML. YTHDF2 decreases the half-life of diverse m6A transcripts that contribute to the overall integrity of LSC function, including the tumor necrosis factor receptor Tnfrsf2, whose upregulation in Ythdf2-deficient LSCs primes cells for apoptosis. Intriguingly, YTHDF2 is not essential for normal HSC function, with YTHDF2 deficiency actually enhancing HSC activity. Thus, we identify YTHDF2 as a unique therapeutic target whose inhibition selectively targets LSCs while promoting HSC expansion.
Asunto(s)
Leucemia Mieloide Aguda/terapia , Células Madre Neoplásicas/fisiología , Proteínas de Unión al ARN/metabolismo , Animales , Autorrenovación de las Células , Hematopoyesis , Células Madre Hematopoyéticas , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Interferente Pequeño/genética , Proteínas de Unión al ARN/genética , Células THP-1RESUMEN
Pharmacologic inhibition of KDM1A (Lysine Demethylase 1A) induces differentiation in certain subtypes of acute myeloid leukemia. Our recent studies reveal this is dependent upon drug-induced disruption of the GFI1 (Growth Factor Independent 1) transcription repressor complex, leading to activation of enhancers distributed close to genes controlling monocytic lineage differentiation.
RESUMEN
Pharmacologic inhibition of LSD1 promotes blast cell differentiation in acute myeloid leukemia (AML) with MLL translocations. The assumption has been that differentiation is induced through blockade of LSD1's histone demethylase activity. However, we observed that rapid, extensive, drug-induced changes in transcription occurred without genome-wide accumulation of the histone modifications targeted for demethylation by LSD1 at sites of LSD1 binding and that a demethylase-defective mutant rescued LSD1 knockdown AML cells as efficiently as wild-type protein. Rather, LSD1 inhibitors disrupt the interaction of LSD1 and RCOR1 with the SNAG-domain transcription repressor GFI1, which is bound to a discrete set of enhancers located close to transcription factor genes that regulate myeloid differentiation. Physical separation of LSD1/RCOR1 from GFI1 is required for drug-induced differentiation. The consequent inactivation of GFI1 leads to increased enhancer histone acetylation within hours, which directly correlates with the upregulation of nearby subordinate genes.
Asunto(s)
Proteínas de Unión al ADN/antagonistas & inhibidores , Histona Demetilasas/antagonistas & inhibidores , Leucemia Mieloide Aguda/tratamiento farmacológico , Factores de Transcripción/antagonistas & inhibidores , Diferenciación Celular/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The Iroquois homeodomain transcription factor gene IRX3 is expressed in the developing nervous system, limb buds, and heart, and transcript levels specify obesity risk in humans. We now report a functional role for IRX3 in human acute leukemia. Although transcript levels are very low in normal human bone marrow cells, high IRX3 expression is found in â¼30% of patients with acute myeloid leukemia (AML), â¼50% with T-acute lymphoblastic leukemia, and â¼20% with B-acute lymphoblastic leukemia, frequently in association with high-level HOXA gene expression. Expression of IRX3 alone was sufficient to immortalize hematopoietic stem and progenitor cells (HSPCs) in myeloid culture and induce lymphoid leukemias in vivo. IRX3 knockdown induced terminal differentiation of AML cells. Combined IRX3 and Hoxa9 expression in murine HSPCs impeded normal T-progenitor differentiation in lymphoid culture and substantially enhanced the morphologic and phenotypic differentiation block of AML in myeloid leukemia transplantation experiments through suppression of a terminal myelomonocytic program. Likewise, in cases of primary human AML, high IRX3 expression is strongly associated with reduced myelomonocytic differentiation. Thus, tissue-inappropriate derepression of IRX3 contributes significantly to the block in differentiation, which is the pathognomonic feature of human acute leukemias.
Asunto(s)
Proteínas de Homeodominio/genética , Leucemia Mieloide Aguda/genética , Factores de Transcripción/genética , Animales , Diferenciación Celular , Humanos , Leucemia Mieloide Aguda/patología , RatonesRESUMEN
Inhibition of lysine specific demethylase 1 (LSD1) has been shown to induce the differentiation of leukemia stem cells in acute myeloid leukemia (AML). Irreversible inhibitors developed from the nonspecific inhibitor tranylcypromine have entered clinical trials; however, the development of effective reversible inhibitors has proved more challenging. Herein, we describe our efforts to identify reversible inhibitors of LSD1 from a high throughput screen and subsequent in silico modeling approaches. From a single hit (12) validated by biochemical and biophysical assays, we describe our efforts to develop acyclic scaffold-hops from GSK-690 (1). A further scaffold modification to a (4-cyanophenyl)glycinamide (e.g., 29a) led to the development of compound 32, with a Kd value of 32 nM and an EC50 value of 0.67 µM in a surrogate cellular biomarker assay. Moreover, this derivative does not display the same level of hERG liability as observed with 1 and represents a promising lead for further development.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Glicina/análogos & derivados , Histona Demetilasas/antagonistas & inhibidores , Leucemia/tratamiento farmacológico , Compuestos de Espiro/farmacología , Biomarcadores , Línea Celular Tumoral , Simulación por Computador , Diseño de Fármacos , Descubrimiento de Drogas , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Glicina/síntesis química , Glicina/farmacología , Ensayos Analíticos de Alto Rendimiento , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Compuestos de Espiro/síntesis química , Relación Estructura-Actividad , Tranilcipromina/análogos & derivados , Tranilcipromina/química , Tranilcipromina/farmacologíaRESUMEN
As part of our ongoing efforts to develop reversible inhibitors of LSD1, we identified a series of 4-(pyrrolidin-3-yl)benzonitrile derivatives that act as successful scaffold-hops of the literature inhibitor GSK-690. The most active compound, 21g, demonstrated a Kd value of 22nM and a biochemical IC50 of 57nM. In addition, this compound displayed improved selectivity over the hERG ion channel compared to GSK-690, and no activity against the related enzymes MAO-A and B. In human THP-1 acute myeloid leukaemia cells, 21g was found to increase the expression of the surrogate cellular biomarker CD86. This work further demonstrates the versatility of scaffold-hopping asa method to develop structurally diverse, potent inhibitors of LSD1.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Histona Demetilasas/antagonistas & inhibidores , Nitrilos/química , Nitrilos/farmacología , Sitios de Unión , Línea Celular Tumoral , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Histona Demetilasas/metabolismo , Humanos , Concentración 50 Inhibidora , Simulación del Acoplamiento Molecular , Nitrilos/síntesis química , Estructura Terciaria de Proteína , Pirrolidinas/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A collaborative high throughput screen of 1.35 million compounds against mutant (R132H) isocitrate dehydrogenase IDH1 led to the identification of a novel series of inhibitors. Elucidation of the bound ligand crystal structure showed that the inhibitors exhibited a novel binding mode in a previously identified allosteric site of IDH1 (R132H). This information guided the optimization of the series yielding submicromolar enzyme inhibitors with promising cellular activity. Encouragingly, one compound from this series was found to induce myeloid differentiation in primary human IDH1 R132H AML cells in vitro.
Asunto(s)
Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Isocitrato Deshidrogenasa/antagonistas & inhibidores , Leucemia Mieloide Aguda/enzimología , Regulación Alostérica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/química , Humanos , Isocitrato Deshidrogenasa/aislamiento & purificación , Isocitrato Deshidrogenasa/metabolismo , Leucemia Mieloide Aguda/patología , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Through in silico and other analyses, we identified FOXC1 as expressed in at least 20% of human AML cases, but not in normal hematopoietic populations. FOXC1 expression in AML was almost exclusively associated with expression of the HOXA/B locus. Functional experiments demonstrated that FOXC1 contributes to a block in monocyte/macrophage differentiation and enhances clonogenic potential. In in vivo analyses, FOXC1 collaborates with HOXA9 to accelerate significantly the onset of symptomatic leukemia. A FOXC1-repressed gene set identified in murine leukemia exhibited quantitative repression in human AML in accordance with FOXC1 expression, and FOXC1(high) human AML cases exhibited reduced morphologic monocytic differentiation and inferior survival. Thus, FOXC1 is frequently derepressed to functional effect in human AML.
Asunto(s)
Factores de Transcripción Forkhead/genética , Leucemia Mieloide Aguda/genética , Animales , Diferenciación Celular/genética , Factores de Transcripción Forkhead/metabolismo , Hematopoyesis/genética , Proteínas de Homeodominio/metabolismo , Humanos , Leucemia Mieloide Aguda/metabolismo , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Monocitos/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismoRESUMEN
Given the importance of deregulated phosphoinositide (PI) signaling in leukemic hematopoiesis, genes coding for proteins that regulate PI metabolism may have significant and as yet unappreciated roles in leukemia. We performed a targeted knockdown (KD) screen of PI modulator genes in human acute myeloid leukemia (AML) cells and identified candidates required to sustain proliferation or prevent apoptosis. One of these, the lipid kinase phosphatidylinositol-5-phosphate 4-kinase, type II, alpha (PIP4K2A) regulates cellular levels of phosphatidylinositol-5-phosphate (PtsIns5P) and phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). We found PIP4K2A to be essential for the clonogenic and leukemia-initiating potential of human AML cells, and for the clonogenic potential of murine MLL-AF9 AML cells. Importantly, PIP4K2A is also required for the clonogenic potential of primary human AML cells. Its KD results in accumulation of the cyclin-dependent kinase inhibitors CDKN1A and CDKN1B, G1 cell cycle arrest and apoptosis. Both CDKN1A accumulation and apoptosis were partially dependent on activation of the mTOR pathway. Critically, however, PIP4K2A KD in normal hematopoietic stem and progenitor cells, both murine and human, did not adversely impact either clonogenic or multilineage differentiation potential, indicating a selective dependency that we suggest may be the consequence of the regulation of different transcriptional programs in normal versus malignant cells. Thus, PIP4K2A is a novel candidate therapeutic target in myeloid malignancy.
Asunto(s)
Técnicas de Silenciamiento del Gen , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Fosfatidilinositoles/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Animales , Apoptosis , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular/genética , Análisis por Conglomerados , Activación Enzimática , Perfilación de la Expresión Génica , Humanos , Espacio Intracelular/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Transgénicos , Células Madre Neoplásicas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Ensayo de Tumor de Célula MadreRESUMEN
Using a mouse model of human MLL-AF9 leukemia, we identified the lysine-specific demethylase KDM1A (LSD1 or AOF2) as an essential regulator of leukemia stem cell (LSC) potential. KDM1A acts at genomic loci bound by MLL-AF9 to sustain expression of the associated oncogenic program, thus preventing differentiation and apoptosis. In vitro and in vivo pharmacologic targeting of KDM1A using tranylcypromine analogs active in the nanomolar range phenocopied Kdm1a knockdown in both murine and primary human AML cells exhibiting MLL translocations. By contrast, the clonogenic and repopulating potential of normal hematopoietic stem and progenitor cells was spared. Our data establish KDM1A as a key effector of the differentiation block in MLL leukemia, which may be selectively targeted to therapeutic effect.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Histona Demetilasas/fisiología , Leucemia/genética , Células Madre Neoplásicas/enzimología , Oxidorreductasas N-Desmetilantes/fisiología , Animales , Apoptosis/genética , Diferenciación Celular/genética , Epigénesis Genética , Técnicas de Silenciamiento del Gen , Histona Demetilasas/genética , Humanos , Leucemia/enzimología , Leucemia/patología , Ratones , Proteína de la Leucemia Mieloide-Linfoide/genética , Células Madre Neoplásicas/patología , Proteínas de Fusión Oncogénica/genética , Oxidorreductasas N-Desmetilantes/genéticaRESUMEN
The genetic programs that promote retention of self-renewing leukemia stem cells (LSCs) at the apex of cellular hierarchies in acute myeloid leukemia (AML) are not known. In a mouse model of human AML, LSCs exhibit variable frequencies that correlate with the initiating MLL oncogene and are maintained in a self-renewing state by a transcriptional subprogram more akin to that of embryonic stem cells (ESCs) than to that of adult stem cells. The transcription/chromatin regulatory factors Myb, Hmgb3, and Cbx5 are critical components of the program and suffice for Hoxa/Meis-independent immortalization of myeloid progenitors when coexpressed, establishing the cooperative and essential role of an ESC-like LSC maintenance program ancillary to the leukemia-initiating MLL/Hox/Meis program. Enriched expression of LSC maintenance and ESC-like program genes in normal myeloid progenitors and poor-prognosis human malignancies links the frequency of aberrantly self-renewing progenitor-like cancer stem cells (CSCs) to prognosis in human cancer.
Asunto(s)
Células Madre Embrionarias/fisiología , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Células Madre Neoplásicas/fisiología , Transcripción Genética , Animales , Células Cultivadas , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Modelos Animales de Enfermedad , Células Madre Embrionarias/citología , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Proteína HMGB3/genética , Proteína HMGB3/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Proteína de la Leucemia Mieloide-Linfoide/genética , Células Madre Neoplásicas/citología , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Oncogénicas v-myb/genética , Proteínas Oncogénicas v-myb/metabolismo , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismoRESUMEN
Bone is a complex, evolving tissue, architecturally defined by the activities of osteoclasts and osteoblasts that continually resorb and replace the mineralised matrix. Numerous regulatory mechanisms exist to control bone remodelling and the maintenance of bone mass. The consequences of inappropriate or uncoupled bone resorption and formation are significant and invariably lead to different disease states, the most prevalent being osteoporosis. In recent years, much attention has focused on unravelling the systemic and local signalling interactions that influence the differentiation and function of bone cells with a view to developing our understanding of bone biology and identifying potential new targets for therapeutic intervention. Several lines of evidence indicate that neurotransmitters and neuromodulators have influential roles to play in the regulation of bone remodelling and much of this research has involved analysis of the excitatory amino acid glutamate. This review will summarise current understanding of glutamate signalling in bone cells, addressing specifically the function of N-methyl-D-aspartate (NMDA)-type glutamate receptor signalling mechanisms, and will address the functional significance and future prospects for this area of research.
Asunto(s)
Remodelación Ósea/fisiología , Ácido Glutámico/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Transducción de Señal , Animales , Ácido Glutámico/metabolismo , Humanos , Modelos Biológicos , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoclastos/citología , Osteoclastos/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
Reports implicating Wnt signalling in the regulation of bone mass have prompted widespread interest in the use of Wnt mimetics for the treatment of skeletal disorders. To date much of this work has focused on their anabolic effects acting on cells of the osteoblast lineage. In this study we provide evidence that Wnts also regulate osteoclast formation and bone resorption, through a mechanism involving transcriptional repression of the gene encoding the osteoclastogenic cytokine receptor activator of NFkappaB ligand (RANKL or TNFSF11) expressed by osteoblasts. In co-cultures of mouse mononuclear spleen cells and osteoblasts, inhibition of GSK3beta with LiCl or exposure to Wnt3a inhibited the formation of tartrate-resistant acid phosphatase-positive multinucleated cells compared with controls. However, these treatments had no consistent effect on the differentiation, survival or activity of osteoclasts generated in the absence of supporting stromal cells. Activation of Wnt signalling downregulated RANKL mRNA and protein expression, and overexpression of fulllength beta-catenin, but not transcriptionally inactive beta-catenin DeltaC(695-781), inhibited RANKL promoter activity. Since previous studies have demonstrated an absence of resorptive phenotype in mice lacking LRP5, we determined expression of a second Wnt co-receptor LRP6 in human osteoblasts, CD14(+) osteoclast progenitors and mature osteoclasts. LRP5 expression was undetectable in CD14-enriched cells and mature human osteoclasts, although LRP6 was expressed at high levels by these cells. Our evidence of Wnt-dependent regulation of osteoclastogenesis adds to the growing complexity of Wnt signalling mechanisms that are now known to influence skeletal function and highlights the requirement to develop novel therapeutics that differentially target anabolic and catabolic Wnt effects in bone.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación de la Expresión Génica , Glicoproteínas de Membrana/metabolismo , Osteoblastos/metabolismo , Osteoclastos/fisiología , Transducción de Señal , Proteínas Wnt/metabolismo , Fosfatasa Ácida/metabolismo , Animales , Animales Recién Nacidos , Proteínas Portadoras/genética , Diferenciación Celular , Línea Celular Tumoral , Núcleo Celular/metabolismo , Células Cultivadas , Células Clonales , Técnicas de Cocultivo , Medios de Cultivo Condicionados , Proteínas del Citoesqueleto , Relación Dosis-Respuesta a Droga , Regulación hacia Abajo , Proteínas de Unión al GTP/metabolismo , Humanos , Isoenzimas/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Cloruro de Litio/farmacología , Proteína-5 Relacionada con Receptor de Lipoproteína de Baja Densidad , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Masculino , Glicoproteínas de Membrana/genética , Monocitos/citología , Proteínas Nucleares , Osteoblastos/citología , Osteoclastos/citología , Osteoclastos/metabolismo , Regiones Promotoras Genéticas , Ligando RANK , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptor Activador del Factor Nuclear kappa-B , Bazo/citología , Fosfatasa Ácida Tartratorresistente , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
Through their broad differentiation potential, mesenchymal stem cells (MSCs) are candidates for a range of therapeutic applications, but the precise signaling pathways that determine their differentiated fate are not fully understood. Evidence is emerging that developmental signaling cues may be important in regulating stem cell self-renewal and differentiation programs. Here we have identified a consistent expression profile of Wnt signaling molecules in MSCs and provide evidence that an endogenous canonical Wnt pathway functions in these cells. Wnts bind to Frizzled (Fz) receptors and subsequent canonical signaling inhibits glycogen synthase kinase-3beta (GSK-3beta), causing beta-catenin translocation into the nucleus to induce target gene expression. In human MSCs isolated from bone marrow of different donors, we appear to have identified a common Wnt/Fz expression profile using reverse transcriptase polymerase chain reaction (RT-PCR). Associated Wnt signaling components, including low-density lipoprotein receptor-related protein-5 (LRP-5), kremen-1, dickkopf-1 (Dkk-1), secreted Frizzled-related peptide (sFRP)-2, sFRP3, sFRP4, Disheveled (Dvl), GSK-3beta, adenomatous polyposis coli (APC), beta-catenin,T-cell factor (TCF)-1, and TCF-4, were also identified. Nuclear beta-catenin was observed in 30%-40% of MSCs, indicative of endogenous Wnt signaling. Exposure to both Wnt3a and Li+ ions, which promotes canonical Wnt signaling by inhibiting GSK-3beta, reduced phosphorylation of beta-catenin in MSCs and increased beta-catenin nuclear translocation approximately threefold over that of the controls. Our findings indicate that autocrine Wnt signaling operates in primitive MSC populations and supports previous evidence that Wnt signaling regulates mesenchymal lineage specification. The identification of a putative common Wnt/Fz molecular signature in MSCs will contribute to our understanding of the molecular mechanisms that regulate self-renewal and lineage-specific differentiation.
Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Madre Mesenquimatosas/metabolismo , Transducción de Señal/genética , Transporte Activo de Núcleo Celular/efectos de los fármacos , Transporte Activo de Núcleo Celular/genética , Animales , Células de la Médula Ósea/metabolismo , Línea Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica/genética , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Glucógeno Sintasa Quinasa 3/metabolismo , Glucógeno Sintasa Quinasa 3 beta , Humanos , Litio/farmacología , Células Madre Mesenquimatosas/citología , Ratones , Fosforilación/efectos de los fármacos , Proteínas/metabolismo , Proteínas/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Proteínas Wnt , Proteína Wnt3 , Proteína Wnt3ARESUMEN
Recent work has demonstrated that neurotransmitters, signalling molecules primarily associated with the nervous system, can have profound effects on the skeleton. Bone cells express a broad range of neurotransmitter receptors and transporters, and respond to receptor activation by initiating diverse intracellular signalling pathways, which modulate cellular function. Evidence of neuronal innervation in skeletal tissues, neurotransmitter release directly from bone cells and functional effects of pharmacological manipulation support the existence of a complex and functionally significant neurotransmitter-mediated signalling network in bone. This review aims to concisely summarise our current understanding of how neurotransmitters affect the skeletal system, focusing on their origin, cellular targets and functional effects in bone.
Asunto(s)
Huesos/inervación , Neuronas/fisiología , Transducción de Señal , Animales , Huesos/citología , Humanos , Neurotransmisores/metabolismo , Neurotransmisores/fisiología , Receptores de Neurotransmisores/metabolismo , Receptores de Neurotransmisores/fisiologíaRESUMEN
BACKGROUND: The adaptive response of bone cells to mechanical strain is a primary determinant of skeletal architecture and bone mass. In vivo mechanical loading induces new bone formation and increases bone mineral density whereas disuse, immobilisation and weightlessness induce bone loss. The potency of mechanical strain is such that a single brief period of loading at physiological strain magnitude is able to induce a long-lasting osteogenic response that lasts for days. Although the process of mechanotransduction in bone is incompletely understood, observations that responses to mechanical strain outlast the duration of stimulation necessitate the existence of a form of cellular memory through which transient strain episodes are recorded, interpreted and remembered by bone cells. Recent evidence supports the existence of a complex multicellular glutamate-signalling network in bone that shares functional similarities to glutamatergic neurotransmission in the central nervous system. In neurones, these signalling molecules coordinate synaptic communication required to support learning and memory formation, through a complex process of long-term potentiation. PRESENTATION OF THE HYPOTHESIS: We hypothesise that osteoblasts use a cellular mechanism similar or identical to neuronal long-term potentiation in the central nervous system to mediate long-lasting changes in osteogenesis following brief periods of mechanical strain. TESTING THE HYPOTHESIS: N-methyl-D-aspartate (NMDA) receptor antagonism should inhibit the saturating response of mechanical strain and reduce the enhanced osteogenicity of segregated loading to that of an equivalent period of uninterrupted loading. Changes in alpha-amino-3-hydroxy-5-methyl-isoxazole propionate (AMPA) receptor expression, localisation and electrophysiological responses should be induced by mechanical strain and inhibited by modulators of neuronal long-term potentiation. IMPLICATIONS OF THE HYPOTHESIS: If true, this hypothesis would provide a mechanism through which the skeleton could be pharmacologically primed to enhance or retrieve the normal osteogenic response to exercise. This would form a basis through which novel therapies could be developed to target osteoporosis and other prevalent bone disorders associated with low bone mass.