RESUMEN
Therapies reconstituting autologous antiviral immunocompetence may represent an important prophylaxis and treatment for immunosuppressed individuals. Following hematopoietic cell transplantation (HCT), patients are susceptible to Herpesviridae including cytomegalovirus (CMV). We show in a murine model of HCT that macrophage colony-stimulating factor (M-CSF) promoted rapid antiviral activity and protection from viremia caused by murine CMV. M-CSF given at transplantation stimulated sequential myeloid and natural killer (NK) cell differentiation culminating in increased NK cell numbers, production of granzyme B and interferon-γ. This depended upon M-CSF-induced myelopoiesis leading to IL15Rα-mediated presentation of IL-15 on monocytes, augmented by type I interferons from plasmacytoid dendritic cells. Demonstrating relevance to human HCT, M-CSF induced myelomonocytic IL15Rα expression and numbers of functional NK cells in G-CSF-mobilized hematopoietic stem and progenitor cells. Together, M-CSF-induced myelopoiesis triggered an integrated differentiation of myeloid and NK cells to protect HCT recipients from CMV. Thus, our results identify a rationale for the therapeutic use of M-CSF to rapidly reconstitute antiviral activity in immunocompromised individuals, which may provide a general paradigm to boost innate antiviral immunocompetence using host-directed therapies.
Asunto(s)
Infecciones por Citomegalovirus , Trasplante de Células Madre Hematopoyéticas , Humanos , Ratones , Animales , Citomegalovirus , Factor Estimulante de Colonias de Macrófagos , Trasplante de Células Madre Hematopoyéticas/métodos , Infecciones por Citomegalovirus/prevención & control , Hematopoyesis , Antivirales/farmacología , Antivirales/uso terapéutico , Diferenciación CelularRESUMEN
BACKGROUND AND OBJECTIVES: The idiopathic inflammatory myopathy dermatomyositis is an acquired disease that involves muscle, lung, and skin impairments. Patients with dermatomyositis show a wide range of severity of proximal skeletal muscle weakness, associated with inflammatory infiltrates, vasculitis, capillary dropout, and perifascicular myofiber atrophy. Muscles of patients with dermatomyositis show signs of muscle regeneration. Because muscle stem cells (MuSCs) are responsible for myofiber repair, we wondered whether the proliferative properties of MuSCs are altered in dermatomyositis muscle. We investigated the role of type I interferon (IFN-I) in this process because dermatomyositis is associated with sustained inflammation with high IFN-I levels. METHODS: MuSCs isolated from normal muscles and those from adult and juvenile patients with dermatomyositis were grown in culture and analyzed in vitro for their proliferating properties, myogenic capacities, and senescence. Gain- and loss-of-function experiments were performed to assess the role of IFN-I signaling in the proliferative capacities of MuSCs. RESULTS: MuSCs derived from 8 adult patients with dermatomyositis (DM-MuSCs) (5 severe form and 3 mild form, established from histologic evaluation), from 3 patients with juvenile dermatomyositis, and from normal muscle were used to analyze their myogenesis in vitro. DM-MuSCs exhibited strongly reduced proliferating capacities as compared with healthy MuSCs (-31% to -43% for mild and severe dermatomyositis, respectively), leading to poor myotube formation (-36% to -71%). DM-MuSCs were enriched in senescent, ß-galactosidase-positive cells, partly explaining the proliferation defect. Gain- and loss-of-function experiments were performed to assess the role of IFN-I on the proliferative capacity of MuSCs. High concentrations of IFN-I decreased the proliferation of healthy MuSCs. Similarly, conditioned medium from DM-MuSCs decreased the proliferation of healthy MuSCs (-15% to -22%), suggesting the delivery of an autocrine effector. Pharmacologic blockade of IFN signaling (using ruxolitinib or anti-IFN receptor antibodies) in DM-MuSCs rescued their proliferation up to the control values. DISCUSSION: These results show that autocrine IFN-I signaling prevents MuSC expansion, leading to muscle repair deficit. This process may explain the persistent muscle weakness observed in patients with severe dermatomyositis.
Asunto(s)
Dermatomiositis , Interferón Tipo I , Adulto , Proliferación Celular , Dermatomiositis/patología , Humanos , Debilidad Muscular/patología , Músculo Esquelético/patología , Transducción de SeñalRESUMEN
PURPOSE OF REVIEW: The present review describes the interferon (IFN)-signature currently emerging as a tool for the diagnosis of idiopathic inflammatory myopathies (IIMs), and aims at presenting the interests and limitations of this recent tool for the clinics and the research. RECENT FINDINGS: Recent in-vivo and in-vitro transcriptomic studies have evidenced the involvement of IFNs in the pathogenesis of IIMs. A correlation between the IFN-signature and the clinical severity of IIMs has been established. Moreover, studies pointed out differences in the IFN-signature regarding the IIM subgroup (dermatomyositis, polymyositis, inclusion body myositis, anti-synthetase syndrome, immuno-mediated necrotizing myopathies), raising the hypothesis of several pathogenic processes in IIMs. SUMMARY: IIM pathogenesis remains partially understood. IFN-signature represents one of the main recent advances in the field. IFN-signature was identified thanks to transcriptomic analyses of tissues or cells from IIM patients (muscle, skin, blood cells, muscle cells) and should allow to establish new diagnosis and better monitoring of IIM patients. It also provides a tool for investigation of IIM pathogenesis. Nevertheless, IFN-signature still requires accurate definition in order to standardize its use, notably in the clinical practice.
Asunto(s)
Interferones/metabolismo , Miositis/diagnóstico , Perfilación de la Expresión Génica/métodos , Humanos , Interferones/genética , Miositis/genética , Miositis/metabolismoRESUMEN
OBJECTIVE: Juvenile dermatomyositis (JDM) is an inflammatory pediatric myopathy characterized by focal capillary loss in muscle, followed by progressive recovery upon adequate treatment with immunomodulating drugs, although some patients remain refractory to treatment. While the underlying mechanism of capillary depletion remains uncertain, recent studies have identified an up-regulation of type I interferon (IFN) expression specific to JDM. Given that myogenic precursor cells (MPCs) exert proangiogenic activity during normal skeletal muscle regeneration, we hypothesized that they may also modulate vascular remodeling/angiogenesis during JDM. The aim of this study was to investigate that hypothesis. METHODS: Human cell cocultures were used to analyze angiogenic properties in patients with JDM, patients with Duchenne's muscular dystrophy (DMD) (control patients for vascular remodeling), and healthy control subjects. Transcriptome analysis was used to examine muscle-derived MPCs. Histologic analysis of type I IFN in muscle biopsy samples was also performed. RESULTS: Using human cell cocultures, we showed highly angiogenic properties of MPCs from JDM patients in association with the expression of an angiogenic molecular signature. Transcriptome analysis of MPCs freshly isolated from muscle samples revealed type I IFN as the master regulator of the most up-regulated genes in JDM-derived MPCs. Functionally, treatment of normal MPCs with type I IFN recapitulated the molecular pattern and the proangiogenic functions of JDM-derived MPCs. In vivo histologic investigation showed that MPCs synthesized type I IFN and major proangiogenic molecules in JDM muscle. Moreover, MPCs derived from JDM muscles that were characterized by strong vasculopathy produced higher levels of type I IFN, confirming MPCs as a cellular source of type I IFN during JDM, and this correlated with the severity of the disease. CONCLUSION: These results demonstrate a new type I IFN pathway in JDM that activates the production of angiogenic effectors by MPCs, triggering their proangiogenic function to promote vessel recovery and muscle reconstruction.
Asunto(s)
Dermatomiositis/patología , Interferón Tipo I/metabolismo , Músculo Esquelético/patología , Neovascularización Patológica/metabolismo , Células Madre/metabolismo , Técnicas de Cultivo de Célula , Ensayos de Migración Celular , Proliferación Celular , Niño , Preescolar , Dermatomiositis/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Perfilación de la Expresión Génica/métodos , Humanos , Inmunohistoquímica , Masculino , Músculo Esquelético/metabolismo , Distrofia Muscular de Duchenne/patología , Células Madre/patologíaRESUMEN
M-CSF and G-CSF are instructive cytokines that specifically induce differentiation of bipotent myeloid progenitors into macrophages and granulocytes, respectively. Through morphology and colony assay studies, flow cytometry analysis of specific markers, and expression of myeloid transcription factors, we show here that the Eger/Fms cell line is composed of cells whose differentiation fate is instructed by M-CSF and G-CSF, thus representing a good in vitro model of myeloid bipotent progenitors. Consistent with the essential role of ERK1/2 during macrophage differentiation and defects of macrophagic differentiation in native ERK1(-/-) progenitors, ERK signaling is strongly activated in Eger/Fms cells upon M-CSF-induced macrophagic differentiation but only to a very small extent during G-CSF-induced granulocytic differentiation. Previous in vivo studies indicated a key role of Fli-1 in myeloid differentiation and demonstrated its weak expression during macrophagic differentiation with a strong expression during granulocytic differentiation. Here, we demonstrated that this effect could be mediated by a differential regulation of protein kinase Cδ (PKCd) on Fli-1 expression in response to M-CSF and G-CSF. With the use of knockdown of PKCd by small interfering RNA, we demonstrated that M-CSF activates PKCd, which in turn, inhibits Fli-1 expression and granulocytic differentiation. Finally, we studied the connection between ERK and PKCd and showed that in the presence of the MEK inhibitor U0126, PKCd expression is decreased, and Fli-1 expression is increased in response to M-CSF. Altogether, we demonstrated that in bipotent myeloid cells, M-CSF promotes macrophagic over granulocytic differentiation by inducing ERK activation but also PKCd expression, which in turn, down-regulates Fli-1 expression and prevents granulocytic differentiation.
Asunto(s)
Granulocitos/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Células Madre Multipotentes/efectos de los fármacos , Mielopoyesis/efectos de los fármacos , Animales , Butadienos/farmacología , Línea Celular , Ensayo de Unidades Formadoras de Colonias , Activación Enzimática/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/farmacología , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Ratones Noqueados , Proteína Quinasa 3 Activada por Mitógenos/deficiencia , Proteína Quinasa 3 Activada por Mitógenos/fisiología , Mielopoyesis/fisiología , Nitrilos/farmacología , Proteína Quinasa C-delta/genética , Proteína Quinasa C-delta/fisiología , Proteína Proto-Oncogénica c-fli-1/biosíntesis , Proteína Proto-Oncogénica c-fli-1/genética , Interferencia de ARN , ARN Interferente Pequeño/genéticaRESUMEN
Two recent studies shed light on mechanisms underlying muscle dysfunction in age and disease. They reveal that JAK-STAT signaling regulates myogenic differentiation, leading to a reduced reservoir of muscle stem cells. Both genetic and pharmacologic inhibition of STAT3 signaling improve stem cell homeostasis and physiology of aged and dystrophic muscles.
Asunto(s)
Células Madre Adultas/fisiología , Células Satélite del Músculo Esquelético/fisiología , Animales , HumanosRESUMEN
The tyrosine kinase receptor Flt3 (Fms-like tyrosine kinase 3) is almost always expressed in AML (acute myeloid leukemia) cells, and constitutive activation of Flt3 by ITD (internal tandem duplication) mutations is one of the most common molecular alterations known in AML, especially monocytic AML. Furthermore, Flt3-ligand (FL) was shown as an in vitro growth factor for monocytic precursors, pointing to the important role of Flt3 in the regulation of monocyte/macrophage production. To get a relevant model for studying the molecular mechanisms underlying the physiopathological role of Flt3 on monocytic lineage development, we used the IL-3 dependent murine myeloid progenitors FDC-P1 cell line to generate cells stably co-expressing murine Fms (M-CSF receptor) and human Flt3. Wild type (WT)-Flt3 expressing cells could proliferate in an FL-dependent manner, whereas those expressing Flt3-ITD all survived IL-3 deprivation and showed autonomous proliferation, whereas both types of cells could differentiate to monocytic cells in response to M-CSF. Next, by combining phosphoprotein detection or purification, comparative 2D-PAGE and mass spectrometry sequencing, we sought for downstream mediators of Flt3-WT or Flt3-ITD in FD/Fms cell proliferation. Amongst the differentially expressed and/or phosphorylated proteins, 3 showed a specific implication in FD/Fms cell proliferation: Hcls1 and the Pak1/2 in FL-dependent proliferation of Flt3-WT expressing cells and Ezrin in autonomous proliferation of Flt3-ITD expressing cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Sanguíneas/metabolismo , Proliferación Celular , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células Progenitoras Mieloides/metabolismo , Células Madre Neoplásicas/metabolismo , Fosfoproteínas/metabolismo , Proteoma/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismo , Quinasas p21 Activadas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Proteínas Sanguíneas/genética , Línea Celular , Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN/genética , Regulación Leucémica de la Expresión Génica/genética , Humanos , Interleucina-3/genética , Interleucina-3/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Mutación , Células Progenitoras Mieloides/patología , Células Madre Neoplásicas/patología , Fosfoproteínas/genética , Proteoma/genética , Secuencias Repetitivas de Ácidos Nucleicos , Tirosina Quinasa 3 Similar a fms/genética , Quinasas p21 Activadas/genéticaRESUMEN
Epidermal growth factor receptor (EGF-R) is a receptor tyrosine kinase that can be activated by molecules other than its cognate ligands. This form of crosstalk called transactivation is frequently observed in both physiological and pathological cellular responses, yet it involves various mechanisms. Using the RWPE-1 cell line as a model of non-transformed prostate epithelial progenitor cells, we observed that interleukin-6 (IL-6) is able to promote cell proliferation and ERK1/2 activation provided that EGF-R kinase activity is not impaired. Treatment with GM6001, a general matrix metalloprotease inhibitor, indicated that IL-6 activates EGF-R through cleavage and release of membrane-anchored EGF-R ligands. Several inhibitors were used to test implication of "a disintegrin and metalloprotease" ADAM10 and ADAM17. GW280264X that targets both ADAM10 and ADAM17 blocked IL-6-induced proliferation and ERK1/2 phosphorylation with same potency as GM6001. However, ADAM10 inhibitor GI254023X and ADAM17 inhibitor TAPI-2 were less efficient in inhibiting response of RWPE-1 cells to IL-6, indicating possible cooperation of ADAM17 with ADAM10 or other metalloproteases. Accordingly, our findings suggest that IL-6 stimulates shedding of EGF-R ligands and transactivation of EGF-R in normal prostate epithelial cells, which may be an important mechanism to promote cell proliferation in inflammatory prostate.
Asunto(s)
Proliferación Celular/efectos de los fármacos , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Interleucina-6/farmacología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Próstata/metabolismo , Línea Celular , Activación Enzimática , Factor de Crecimiento Epidérmico/farmacología , Células Epiteliales/citología , Humanos , Masculino , Metaloproteasas/fisiología , Próstata/citología , Transducción de Señal , Activación Transcripcional , Familia-src Quinasas/fisiologíaRESUMEN
M-CSF regulates the production, survival, and function of monocytes and macrophages. The MAPKs ERK1/2 are key elements for signal integration downstream of the M-CSFR, and their sustained activation is essential for macrophage differentiation. In this study, we sought to isolate genes whose induction by M-CSF is dependent on persistent MAPK activation, thereby being possibly involved in the commitment of myeloid progenitors to macrophage differentiation. Following SSH between cDNA libraries from FD-Fms cells stimulated by M-CSF for 8 h in the presence or the absence of the MEK inhibitor U0126, we isolated DUSP5. DUSP5 expression is induced by M-CSF in various myeloid cells and acts as a specific negative-feedback regulator of ERK1/2. In FD-Fms cells that proliferate and differentiate toward macrophages in response to M-CSF, overexpression of DUSP5 increased M-CSF-dependent proliferation and strongly decreased differentiation. Similarly, overexpression of DUSP5 in the multipotent EGER-Fms cells not only significantly increased M-CSF-induced proliferation and prevented macrophage differentiation but also favored granulocytic differentiation. Altogether, experiments demonstrated that DUSP5 is implicated in M-CSF signaling and suggested that it may influence myeloid cell fate.
Asunto(s)
Fosfatasas de Especificidad Dual/fisiología , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Células Madre Multipotentes/efectos de los fármacos , Mielopoyesis/efectos de los fármacos , Proteínas ADAM/biosíntesis , Proteínas ADAM/genética , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Butadienos/farmacología , Células Cultivadas/citología , Células Cultivadas/efectos de los fármacos , Fosfatasas de Especificidad Dual/biosíntesis , Fosfatasas de Especificidad Dual/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mielopoyesis/genética , Nitrilos/farmacología , Osteopontina/biosíntesis , Osteopontina/genética , Fosforilación/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Receptor de Factor Estimulante de Colonias de Macrófagos/efectos de los fármacos , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Proteínas Recombinantes de Fusión/efectos de los fármacos , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
The mononuclear phagocyte system (MPS) is a cell population derived from progenitor cells in the bone marrow, and comprising monocytes, macrophages, osteoclasts, dendritic cells, and microglia. Homeostasis of the MPS and response to physiological stress is under the control of signaling molecules and nuclear factors; among them, macrophage-colony-stimulating factor (M-CSF) controls monocyte/macrophage lineage development. Here we discuss the implication of Ifi204, a M-CSF-responsive gene, in the proliferation and differentiation of monocytes/macrophages. Ifi204 is a member of the interferon-inducible p200 family of proteins, and was found to be an important regulator of differentiation of both skeletal and cardiac muscles and osteogenesis. Ifi204 is expressed at the early stages of differentiation of MPS cells and later in the monocyte/macrophage lineage. IFI16, the closest Ifi protein in human, is expressed all along the the monocytic lineage. In MPS cells, Ifi204 expression is induced by interferons but also by various stimuli, independently of the presence of interferon. Enforced expression of p204 in interleukin-3 (IL3)-dependent FD-Fms cell line strongly decreased both IL3- and M-CSF-dependent proliferation and conversely favored macrophage differentiation of FD-Fms cells in response to M-CSF. Altogether, data enlighten a role of Ifi204 as a regulator of monocyte/macrophage differentiation and make possible a connection with other myeloid regulators.
Asunto(s)
Regulación de la Expresión Génica , Interferones/metabolismo , Leucocitos Mononucleares/metabolismo , Proteínas Nucleares/fisiología , Fagocitos/metabolismo , Fosfoproteínas/fisiología , Animales , Proliferación Celular , Humanos , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Ratones , Modelos Biológicos , Transducción de Señal , Distribución TisularRESUMEN
The interface between the blood and the cerebrospinal fluid (CSF) is formed by the choroid plexuses (CPs), which are specialized structures located within the brain ventricles. They are composed of a vascularized stroma surrounded by a tight epithelium that controls molecular and cellular traffic between the blood and the CSF. Cells expressing myeloid markers are present within the choroidal stroma. However, the exact identity, maturation state, and functions of these CP-associated myeloid cells are not fully clarified. We show here that this cell population contains immature myeloid progenitors displaying a high proliferative potential. Thus, in neonate rats and, to a lesser extent, in adult rats, cultured CP stroma cells form large colonies of macrophages, in response to M-CSF or GM-CSF, while, under the same conditions, peripheral blood monocytes do not. In addition, under GM-CSF treatment, free-floating colonies of CD11c(+) monocytic cells are generated which, when restimulated with GM-CSF and IL-4, differentiate into OX62(+)/MHC class II(+) dendritic cells. Interestingly, in CP stroma cultures, myeloid cells are found in close association with fibroblastic-like cells expressing the neural stem-cell marker nestin. Similarly, in the developing brain, macrophages and nestin(+) fibroblastic cells accumulate in vivo within the choroidal stroma. Taken together, these results suggest that the CP stroma represents a niche for myeloid progenitors and may serve as a reservoir for brain macrophages.
Asunto(s)
Plexo Coroideo/citología , Células Dendríticas/citología , Macrófagos/citología , Células Madre/citología , Animales , Animales Recién Nacidos , Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Citometría de Flujo , Factor Estimulante de Colonias de Granulocitos y Macrófagos/farmacología , Antígenos de Histocompatibilidad Clase II/fisiología , Humanos , Interleucina-4/farmacología , Macrófagos/efectos de los fármacos , Ratones , Ratas , Células Madre/efectos de los fármacos , Células del Estroma/citologíaRESUMEN
Receptor tyrosine kinases (RTKs) are involved in the control of fundamental cellular processes in metazoans. In vertebrates, RTK could be grouped in distinct classes based on the nature of their cognate ligand and modular composition of their extracellular domain. RTK with immunoglobulin-like domains (IG-like RTK) encompass several RTK classes and have been found in early metazoans, including sponges. Evolution of IG-like RTK is characterized by extended molecular and functional diversification, which prompted us to study their evolutionary history. For that purpose, a nonredundant data set including annotated protein sequences of IG-like RTK (n = 85) was built, representing 19 species ranging from sponges to humans. Phylogenetic trees were generated from alignment of conserved regions using maximum likelihood approach. Molecular phylogeny strongly suggests that IG-like RTK diversification occurred according to a complex scenario. In particular, we propose that specific cis duplications of a common ancestor to both platelet-derived growth factor receptor (class III) and vascular endothelial growth factor receptor (class V) families preceded two trans duplications. In contrast, other IG-like RTK genes, like Musk and PTK7, apparently did not evolve by duplications, whereas fibroblast growth factor receptors (class IV) evolved through two rounds of trans duplications. The proposed model of IG-like RTK evolution is supported by high bootstrap values and by the clustering of genes encoding class III and class V RTKs at specific chromosomal locations in mouse and human genomes.
Asunto(s)
Evolución Molecular , Proteínas Tirosina Quinasas Receptoras/genética , Animales , Cordados , Eliminación de Gen , Duplicación de Gen , Humanos , Inmunoglobulinas , Familia de Multigenes , Filogenia , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas Receptoras/química , Alineación de Secuencia , Urocordados/genéticaRESUMEN
The interferon-inducible (Ifi)204 gene was isolated as a macrophage-colony stimulating factor (M-CSF)-responsive gene using a gene trap approach in the myeloid interleukin-3 (IL-3)-dependent FD-Fms cell line, which differentiates in macrophages in response to M-CSF. Here, we show that Ifi204 was transcriptionally activated in response to M-CSF, and FD-Fms cells decreased their growth and committed toward a macrophage morphology; this induction was abrogated when the differentiation signal of the M-CSF receptor was blocked; the Ifi204 gene was also induced during macrophage differentiation controlled by leukemia inhibitory factor; and the Ifi204 gene is expressed in different mature monocyte/macrophage cells. Finally, we showed that enforced expression of Ifi204 strongly decreased IL-3- and M-CSF-dependent proliferation and conversely, favored macrophage differentiation of FD-Fms cells in response to M-CSF. Altogether, these results demonstrate that the Ifi204 gene is activated during macrophage development and suggest that the Ifi204 protein may act as a regulator of the balance between proliferation and differentiation. Moreover, this study suggests that other members of the Ifi family might act as regulators of hematopoiesis under the control of hemopoietic cytokines.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/metabolismo , Células Progenitoras Mieloides/metabolismo , Proteínas Nucleares/biosíntesis , Fosfoproteínas/biosíntesis , Animales , Diferenciación Celular/inmunología , Línea Celular , Proliferación Celular/efectos de los fármacos , Regulación de la Expresión Génica/inmunología , Hematopoyesis/genética , Hematopoyesis/inmunología , Humanos , Interleucina-3/inmunología , Interleucina-3/metabolismo , Interleucina-6/inmunología , Interleucina-6/metabolismo , Factor Inhibidor de Leucemia , Activación de Macrófagos/efectos de los fármacos , Activación de Macrófagos/genética , Activación de Macrófagos/inmunología , Factor Estimulante de Colonias de Macrófagos/inmunología , Macrófagos/inmunología , Ratones , Células Progenitoras Mieloides/inmunología , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transducción de Señal/inmunología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/inmunologíaRESUMEN
Macrophage colony-stimulating factor (M-CSF) is a physiological regulator of monocyte-macrophage lineage. Ectopic expression of the M-CSF receptor (M-CSFR, or Fms) in murine myeloid cell line FDC-P1 (FD/Fms cells) results in M-CSF-dependent macrophage differentiation. Previously, we observed that M-CSF induces two temporally distinct phases of mitogen-activated protein kinase (MAPK) phosphorylation. Here we show that levels of phosphorylated MAPK kinase MEK1 follow the same kinetics as MAPK phosphorylation, characterized by an early and transient phase (the first 30 min of M-CSF stimulation) and a late and persistent phase from 4 h of stimulation. The MEK inhibitor U0126 strongly inhibited both phases of MAPK phosphorylation as well as FD/Fms cell differentiation, indicating that MAPK may relay M-CSF differentiation signaling downstream of M-CSFR. Treatment of FD/Fms cells with U0126 during the first hour of M-CSF stimulation reversibly blocked the early phase of MAPK phosphorylation but did not affect differentiation. In contrast, U0126 still inhibited FD/Fms cell differentiation when its addition was delayed by 24 h. This demonstrated that late and persistent MEK activity is specifically required for macrophage differentiation to occur. Furthermore, disrupting Grb2-Sos complexes with a specific blocking peptide did not prevent FD/Fms cells differentiation in response to M-CSF, nor did it abolish MAPK phosphorylation. The role of phosphatidylinositol 3-kinase (PI 3-kinase), another potential regulator of the MAPK pathway, was examined using the specific inhibitor LY294002. This compound could not impede FD/Fms cell commitment to macrophage differentiation and did not significantly affect MAPK phosphorylation in response to M-CSF. Therefore, M-CSF differentiation signaling in myeloid progenitor cells is mediated through persistent MEK activity but it is not strictly dependent upon Grb2-Sos interaction or PI 3-kinase activity.
Asunto(s)
Proteína Adaptadora GRB2/metabolismo , MAP Quinasa Quinasa 1/metabolismo , Factor Estimulante de Colonias de Macrófagos/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteína Son Of Sevenless Drosofila/metabolismo , Animales , Butadienos/farmacología , Línea Celular , Cromonas/farmacología , MAP Quinasa Quinasa 1/antagonistas & inhibidores , Sistema de Señalización de MAP Quinasas , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Morfolinas/farmacología , Células Progenitoras Mieloides/citología , Células Progenitoras Mieloides/metabolismo , Nitrilos/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Unión Proteica , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismoRESUMEN
The production, survival, and function of monocytes and macrophages are regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor. M-CSF receptor activates multiple cytoplasmic pathways in which adaptor and scaffolding proteins play a central role. In this study, we showed that SKAP55-related (SKAP55R) adaptor protein is expressed in myeloid cells and macrophages and is rapidly and transiently tyrosine-phosphorylated in response to M-CSF. M-CSF induced SKAP55R association with other tyrosine-phosphorylated proteins and with actin. When overexpressed in myeloid cells, SKAP55R decreased M-CSF-dependent proliferation without affecting differentiation. Altogether, these results demonstrate that SKAP55R adaptor is implicated in the M-CSF signaling pathway and suggest its role as a negative regulator of growth. Moreover, specific association between SKAP55R and actin support the idea that SKAP55R is implicated in the regulation of actin dynamics under the control of M-CSF.
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Factor Estimulante de Colonias de Macrófagos/metabolismo , Fosfoproteínas/química , Tirosina/metabolismo , Actinas/química , Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Diferenciación Celular , Línea Celular , Linaje de la Célula , Proliferación Celular , Medios de Cultivo Condicionados/metabolismo , Medios de Cultivo Condicionados/farmacología , Citoplasma/metabolismo , Electroforesis en Gel de Poliacrilamida , Citometría de Flujo , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/metabolismo , Sustancias de Crecimiento , Immunoblotting , Inmunoprecipitación , Interleucina-3/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Macrófagos/metabolismo , Ratones , Células Mieloides/citología , Células Mieloides/metabolismo , Fosforilación , Ratas , Retroviridae/genética , Transducción de Señal , Tirosina/químicaRESUMEN
Macrophage colony-stimulating factor (M-CSF) is a growth factor that is known to trigger several signalling pathways through receptor tyrosine kinase activation. We investigated the specific requirements for the activation of phospholipase C gamma 2 (PLC-gamma2) during the differentiation of mouse bone marrow-derived macrophage precursors. M-CSF stimulation induced rapid PLC-gamma2 translocation and phosphorylation from the cytosolic compartment to the cell periphery. Both events were dependent on cytoskeleton integrity and Src kinase activity, but only PLC-gamma2 phosphorylation did not require PI3-kinase activity. Biochemical experiments as well as confocal microscopy analyses indicate that the translocation of PLC-gamma2 is mediated by the direct association of this protein with the actin cytoskeleton. Using GST-fusion proteins containing various deletions of the PLC-gamma2 Src homology region, it was found that PLC-gamma2 binds to F-actin via its SH2 domains, a feature that has equally been found in a co-sedimentation assay. This association, which is increased during actin reorganisation and disrupted by cytoskeleton inhibitors, seems to be a primary means to recruit this enzyme to the cell periphery. These results indicate that, upon M-CSF stimulation, PLC-gamma2 cellular localisation and phosphorylation are strongly dependent on cytoskeleton architecture of the macrophage precursor as well as the PI3-kinase and the Src kinases.
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Actinas/metabolismo , Citoesqueleto/metabolismo , Macrófagos/enzimología , Fosfolipasa C gamma/metabolismo , Familia-src Quinasas/metabolismo , Actinas/química , Animales , Sitios de Unión , Diferenciación Celular , Células Cultivadas , Cromonas/farmacología , Citocalasina D/farmacología , Citoesqueleto/química , Factor Estimulante de Colonias de Macrófagos/farmacología , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Morfolinas/farmacología , Nocodazol/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Transporte de Proteínas , Pirimidinas/farmacología , Factores de Tiempo , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
SH3 domains are protein recognition modules within many adaptors and enzymes. With more than 500 SH3 domains in the human genome, binding selectivity is a key issue in understanding the molecular basis of SH3 domain interactions. The Grb2-like adaptor protein Mona/Gads associates stably with the T-cell receptor signal transducer SLP-76. The crystal structure of a complex between the C-terminal SH3 domain (SH3C) of Mona/Gads and a SLP-76 peptide has now been solved to 1.7 A. The peptide lacks the canonical SH3 domain binding motif P-x-x-P and does not form a frequently observed poly-proline type II helix. Instead, it adopts a clamp-like shape around the circumfence of the SH3C beta-barrel. The central R-x-x-K motif of the peptide forms a 3(10) helix and inserts into a negatively charged double pocket on the SH3C while several other residues complement binding through hydrophobic interactions, creating a short linear SH3C binding epitope of uniquely high affinity. Interestingly, the SH3C displays ion-dependent dimerization in the crystal and in solution, suggesting a novel mechanism for the regulation of SH3 domain functions.
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Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteínas Portadoras/genética , Cristalografía por Rayos X , Dimerización , Humanos , Enlace de Hidrógeno , Técnicas In Vitro , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fosfoproteínas/genética , Filogenia , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Electricidad Estática , Dominios Homologos srcRESUMEN
Receptor Tyrosine Kinases (RTK) are transmembrane receptors specifically found in metazoans. They represent an excellent model for studying evolution of cellular processes in metazoans because they encompass large families of modular proteins and belong to a major family of contingency generating molecules in eukaryotic cells: the protein kinases. Because tyrosine kinases have been under close scrutiny for many years in various species, they are associated with a wealth of information, mainly in mammals. Presently, most categories of RTK were identified in mammals, but in a near future other model species will be sequenced, and will bring us RTKs from other metazoan clades. Thus, collecting RTK sequences would provide a good starting point as a new model for comparative and evolutionary studies applying to multigene families. In this context, we are developing the Receptor Tyrosine Kinase database (RTKdb), which is the only database on tyrosine kinase receptors presently available. In this database, protein sequences from eight model metazoan species are organized under the format previously used for the HOVERGEN, HOBACGEN and NUREBASE systems. RTKdb can be accessed through the PBIL (Pôle Bioinformatique Lyonnais) World Wide Web server at http://pbil.univ-lyon1.fr/RTKdb/, or through the FamFetch graphical user interface available at the same address.
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Bases de Datos de Proteínas , Proteínas Tirosina Quinasas Receptoras/química , Animales , Evolución Molecular , Humanos , Internet , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas Receptoras/clasificación , Proteínas Tirosina Quinasas Receptoras/genética , Interfaz Usuario-ComputadorRESUMEN
Mona, also called Gads, is a molecular adapter that plays a key role in T-cell and platelet signalling by linking the adaptors Src homology 2 domain-containing leukocyte phosphoprotein of 76 kDa (Slp-76) and linker for activation of T cells (LAT) upon T-cell receptor and collagen receptor activation. Platelets express a specific form of Mona mRNA, called 1B, which is transcribed from a megakaryocyte-specific promoter. Mona 1B mRNA differ from 1A transcripts found in T cells and some myeloid cells only by the 5'UTR. We report here that 1B mRNA expressing cells do not express detectable amounts of Mona protein, in contrast to 1A expressing cells, and we show that 1B 5'UTR contains upstream open reading frames (uORFs). Mutating the corresponding uAUG restored efficient Mona translation, or that of an unrelated ORF. This suggested that Mona protein expression in 1B mRNA expressing cells is tightly controlled at the translational level. Accordingly, Mona protein was not detected in resting platelets. Strikingly, platelet activation by thrombin resulted in the rapid induction of Mona protein expression, suggesting that translation inhibition of 1B mRNA may be relieved in activated platelets.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Megacariocitos/fisiología , Sistemas de Lectura Abierta , Biosíntesis de Proteínas , ARN Mensajero/genética , Regiones no Traducidas 5'/genética , Secuencia de Bases , Proteínas Portadoras/sangre , Células Cultivadas , Cartilla de ADN , ADN Complementario , Humanos , Datos de Secuencia Molecular , Procesamiento Postranscripcional del ARN , Transfección , Dominios Homologos srcRESUMEN
BACKGROUND: Macrophages, osteoclasts, dendritic cells, and microglia are highly specialized cells that belong to the mononuclear phagocyte system. Functional and phenotypic heterogeneity within the mononuclear phagocyte system may reveal differentiation plasticity of a common progenitor, but developmental pathways leading to such diversity are still unclear. RESULTS: Mouse bone marrow cells were expanded in vitro in the presence of Flt3-ligand (FL), yielding high numbers of non-adherent cells exhibiting immature monocyte characteristics. Cells expanded for 6 days, 8 days, or 11 days (day 6-FL, day 8-FL, and day 11-FL cells, respectively) exhibited constitutive potential towards macrophage differentiation. In contrast, they showed time-dependent potential towards osteoclast, dendritic, and microglia differentiation that was detected in day 6-, day 8-, and day 11-FL cells, in response to M-CSF and receptor activator of NFkappaB ligand (RANKL), granulocyte-macrophage colony stimulating-factor (GM-CSF) and tumor necrosis factor-alpha (TNFalpha), and glial cell-conditioned medium (GCCM), respectively. Analysis of cell proliferation using the vital dye CFSE revealed homogenous growth in FL-stimulated cultures of bone marrow cells, demonstrating that changes in differential potential did not result from sequential outgrowth of specific precursors. CONCLUSIONS: We propose that macrophages, osteoclasts, dendritic cells, and microglia may arise from expansion of common progenitors undergoing sequential differentiation commitment. This study also emphasizes differentiation plasticity within the mononuclear phagocyte system. Furthermore, selective massive cell production, as shown here, would greatly facilitate investigation of the clinical potential of dendritic cells and microglia.