RESUMEN
There is evidence suggesting that N-cadherin expression on osteoblast lineage cells regulates hematopoietic stem cell (HSC) function and quiescence. To test this hypothesis, we conditionally deleted N-cadherin (Cdh2) in osteoblasts using Cdh2(flox/flox) Osx-Cre mice. N-cadherin expression was efficiently ablated in osteoblast lineage cells as assessed by mRNA expression and immunostaining of bone sections. Basal hematopoiesis is normal in these mice. In particular, HSC number, cell cycle status, long-term repopulating activity, and self-renewal capacity were normal. Moreover, engraftment of wild-type cells into N-cadherin-deleted recipients was normal. Finally, these mice responded normally to G-CSF, a stimulus that mobilizes HSCs by inducing alterations to the stromal micro-environment. In conclusion, N-cadherin expression in osteoblast lineage cells is dispensable for HSC maintenance in mice.
Asunto(s)
Cadherinas/metabolismo , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Animales , Secuencia de Bases , Enfermedades Óseas Metabólicas/etiología , Cadherinas/deficiencia , Cadherinas/genética , Linaje de la Célula/genética , Linaje de la Célula/fisiología , Femenino , Fluorouracilo/farmacología , Factor Estimulante de Colonias de Granulocitos/farmacología , Hematopoyesis/genética , Hematopoyesis/fisiología , Movilización de Célula Madre Hematopoyética , Células Madre Hematopoyéticas/efectos de los fármacos , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/farmacologíaRESUMEN
Severe congenital neutropenia is associated with a marked propensity to develop myelodysplasia or acute myeloid leukemia (AML). Truncation mutations of CSF3R, encoding the granulocyte colony-stimulating factor receptor (G-CSFR), are associated with development of myelodysplasia/AML in severe congenital neutropenia. However, a causal relationship between CSF3R mutations and leukemic transformation has not been established. Herein, we show that truncated G-CSFR cooperates with the PML-RARα oncogene to induce AML in mice. Expression of truncated G-CSFR significantly shortens the latency of AML in a G-CSF-dependent fashion and it is associated with a distinct AML presentation characterized by higher blast counts and more severe myelosuppression. Basal and G-CSF-induced signal transducer and activator of transcription 3, signal transducer and activator of transcription 5, and extracellular signal-regulated kinase 1/2 phosphorylation were highly variable but similar in leukemic blasts expressing wild-type and truncated G-CSFR. These data provide new evidence suggesting a causative role for CSF3R mutations in human AML.
Asunto(s)
Transformación Celular Neoplásica/genética , Codón sin Sentido , Leucemia Mieloide Aguda/genética , Proteínas de Fusión Oncogénica/fisiología , Receptores de Factor Estimulante de Colonias de Granulocito/genética , Animales , Cruzamientos Genéticos , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/fisiología , Técnicas de Sustitución del Gen , Genotipo , Factor Estimulante de Colonias de Granulocitos/farmacología , Factor Estimulante de Colonias de Granulocitos/toxicidad , Humanos , Ratones , Ratones Endogámicos C57BL , Fenotipo , Polietilenglicoles/farmacología , Polietilenglicoles/toxicidad , Receptores de Factor Estimulante de Colonias de Granulocito/química , Receptores de Factor Estimulante de Colonias de Granulocito/fisiología , Factores de Transcripción STAT/fisiologíaRESUMEN
Granulocyte colony-stimulating factor (G-CSF), the prototypical mobilizing cytokine, induces hematopoietic stem and progenitor cell (HSPC) mobilization from the bone marrow in a cell-nonautonomous fashion. This process is mediated, in part, through suppression of osteoblasts and disruption of CXCR4/CXCL12 signaling. The cellular targets of G-CSF that initiate the mobilization cascade have not been identified. We use mixed G-CSF receptor (G-CSFR)-deficient bone marrow chimeras to show that G-CSF-induced mobilization of HSPCs correlates poorly with the number of wild-type neutrophils. We generated transgenic mice in which expression of the G-CSFR is restricted to cells of the monocytic lineage. G-CSF-induced HSPC mobilization, osteoblast suppression, and inhibition of CXCL12 expression in the bone marrow of these transgenic mice are intact, demonstrating that G-CSFR signals in monocytic cells are sufficient to induce HSPC mobilization. Moreover, G-CSF treatment of wild-type mice is associated with marked loss of monocytic cells in the bone marrow. Finally, we show that bone marrow macrophages produce factors that support the growth and/or survival of osteoblasts in vitro. Together, these data suggest a model in which G-CSFR signals in bone marrow monocytic cells inhibit the production of trophic factors required for osteoblast lineage cell maintenance, ultimately leading to HSPC mobilization.
Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/metabolismo , Movilización de Célula Madre Hematopoyética/métodos , Monocitos/metabolismo , Receptores de Factor Estimulante de Colonias de Granulocito/metabolismo , Análisis de Varianza , Animales , Quimiocina CXCL12/metabolismo , Quimera/metabolismo , Cartilla de ADN/genética , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Regulación de la Expresión Génica/inmunología , Factor Estimulante de Colonias de Granulocitos/farmacología , Macrófagos/metabolismo , Ratones , Ratones Transgénicos , Monocitos/efectos de los fármacos , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Receptores de Factor Estimulante de Colonias de Granulocito/deficiencia , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
There is considerable interest in the potential of cell-based approaches to mediate therapeutic angiogenesis for acute and chronic vascular syndromes. Using a mouse model of HLI, we showed previously that adoptive transfer of a small number of donor monocytes enhanced revascularization significantly. Herein, we provide data suggesting that the BM resident monocytes sense systemic signals that influence their future functional capacity. Specifically, following induction of distant ischemia, the angiogenic capacity of BM resident monocytes is reduced markedly. We provide evidence that G-CSF and IL-6 represent such "conditioning" signals. Systemic levels of G-CSF and IL-6 are increased significantly following induction of HLI. Accordingly, BM resident monocytes from ischemic mice exhibited increased pSTAT3 and STAT3 target gene expression. Finally, G-CSFR(-/-) and IL-6(-/-) mice were resistant to the deleterious effects of ischemic conditioning on monocyte angiogenic potential. RNA expression profiling suggested that ischemia-conditioned monocytes in the BM up-regulate the well-described M2 polarization markers Chi3l4 and Lrg1. Consistent with this observation, M2-skewed monocytes from SHIP(-/-) mice also had impaired angiogenic capacity. Collectively, these data show that G-CSF and IL-6 provide signals that determine the angiogenic potential of BM resident monocytes.
Asunto(s)
Células de la Médula Ósea/fisiología , Factor Estimulante de Colonias de Granulocitos/fisiología , Interleucina-6/fisiología , Monocitos/fisiología , Neovascularización Fisiológica , Traslado Adoptivo , Animales , Miembro Posterior/irrigación sanguínea , Isquemia , Ratones , Ratones Endogámicos C57BL , Factor de Transcripción STAT3/metabolismoRESUMEN
Shwachman-Diamond syndrome (SDS) is a rare multisystem disorder characterized by exocrine pancreatic insufficiency, multilineage hematopoietic dysfunction, and metaphyseal chondrodysplasia. Bone marrow dysfunction is present in nearly all patients with SDS, with neutropenia being the most common abnormality. The majority of patients with SDS have mutations in the Shwachman Bodian Diamond syndrome (SBDS) gene. We have developed a strategy to examine the consequences of lentiviral-mediated RNA interference (RNAi) of Sbds on hematopoiesis. Here, we report that both Sbds RNA and protein expression can be efficiently inhibited in primary murine hematopoietic cells using lentiviral-mediated RNAi. Inhibition of Sbds results in a defect in granulocytic differentiation in vitro and impairs myeloid progenitor generation in vivo. In addition, short-term hematopoietic engraftment was impaired, which is due in part to reduced homing of hematopoietic progenitors to the bone marrow. Finally, we show that inhibition of Sbds is associated with a decrease in circulating B lymphocytes, despite evidence of normal B lymphopoiesis. These data provide the first evidence that loss of Sbds is sufficient to induce abnormalities in hematopoiesis.
Asunto(s)
Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Lentivirus/genética , Proteínas/metabolismo , Interferencia de ARN , Animales , Linfocitos B/citología , Linfocitos B/metabolismo , Secuencia de Bases , Trasplante de Médula Ósea , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Regulación de la Expresión Génica , Granulocitos/citología , Granulocitos/metabolismo , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Células Mieloides/citología , Células Mieloides/metabolismo , Proteínas/genética , Factores de TiempoRESUMEN
Shwachman-Diamond Syndrome (SDS) is a rare multisystem disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction, and metaphyseal chondrodysplasia. Recent studies show that mutations of SBDS, a gene of unknown function, are present in the majority of patients with SDS. In the present study, we show that most, but not all, patients classified based on rigorous clinical criteria as having SDS had compound heterozygous mutations of SBDS. Full-length SBDS protein was not detected in leukocytes of SDS patients with the most common SBDS mutations, consistent with a loss-of-function mechanism. In contrast, SBDS protein was expressed at normal levels in SDS patients without SBDS mutations. These data confirm the absence of SBDS mutations in this subgroup of patients and suggest that SDS is a genetically heterogeneous disorder. The presence (or absence) of SBDS mutations may define subgroups of patients with SDS who share distinct clinical features or natural history.