RESUMEN
A key property of hematopoietic stem and progenitor cells (HSPCs) regarding differentiation from the self-renewing quiescent to the proliferating stage is their adhesion to the bone marrow (BM) niche. An important molecule involved in proliferation and pool size of HSPCs in the BM is the hypoxia-induced urokinase-type plasminogen activator receptor (uPAR). Here, we show that the soluble form (sLR11) of LR11 (also called SorLA or SORL1) modulates the uPAR-mediated attachment of HSPCs under hypoxic conditions. Immunohistochemical and mRNA expression analyses revealed that hypoxia increased LR11 expression in hematological c-Kit(+) Lin(-) cells. In U937 cells, hypoxia induced a transient rise in LR11 transcription, production of cellular protein, and release of sLR11. Attachment to stromal cells of c-Kit(+) Lin(-) cells of lr11(-/-) mice was reduced by hypoxia much more than of lr11(+/+) animals. sLR11 induced the adhesion of U937 and c-Kit(+) Lin(-) cells to stromal cells. Cell attachment was increased by sLR11 and reduced in the presence of anti-uPAR antibodies. Furthermore, the fraction of uPAR co-immunoprecipitated with LR11 in membrane extracts of U937 cells was increased by hypoxia. CoCl2, a chemical inducer of HIF-1α, enhanced the levels of LR11 and sLR11 in U937 cells. The decrease in hypoxia-induced attachment of HIF-1α-knockdown cells was largely prevented by exogenously added sLR11. Finally, hypoxia induced HIF-1α binding to a consensus binding site in the LR11 promoter. Thus, we conclude that sLR11 regulates the hypoxia-enhanced adhesion of HSPCs via an uPAR-mediated pathway that stabilizes the hematological pool size by controlling cell attachment to the BM niche.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Receptores de LDL/metabolismo , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Nicho de Células Madre/fisiología , Animales , Anticuerpos/farmacología , Antimutagênicos/farmacología , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Hipoxia de la Célula/efectos de los fármacos , Hipoxia de la Célula/fisiología , Cobalto/farmacología , Células Madre Hematopoyéticas/citología , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas de Transporte de Membrana/genética , Ratones , Ratones Noqueados , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , Receptores de LDL/genética , Receptores del Activador de Plasminógeno Tipo Uroquinasa/genética , Elementos de Respuesta/fisiología , Células U937RESUMEN
Granulocyte colony-stimulating factor (G-CSF) induces the mobilization of leukocytes from the bone marrow (BM) to the circulation by a yet incompletely understood mechanism. Here, we describe that the membrane-bound receptor LR11 is highly expressed in human myeloid cells and that the shed soluble form of LR11 (sLR11) is a modifier of myeloid cell migration. In the process of leukocyte mobilization by G-CSF treatment, circulating sLR11 levels are transiently elevated in humans and mice. Moreover, following G-CSF treatment, the sLR11 levels in patients show significant positive correlation with the numbers of mobilized leukocytes. The changes of LR11 levels in BM cells and of sLR11 released into the BM fluid of mice correlate tightly with the changes in circulating sLR11 levels. G-CSF dose-dependently enhanced sLR11 release from HL-60 cells, which in turn accelerated cell migration. Finally, cooperatively with tumor necrosis factor-α (TNF-α) and G-CSF, sLR11 increased the attachment of floating cells (HL-60 and U937) to endothelial cells. We propose that sLR11 is a novel candidate modifier of G-CSF-mediated mobilization of hematologic cells. Identification of sLR11 as a regulatory component of G-CSF-mediated hematologic cell mobilization may facilitate further improvement of hematologic stem cell collection for clinical applications.
Asunto(s)
Médula Ósea/fisiología , Movimiento Celular/fisiología , Factor Estimulante de Colonias de Granulocitos/administración & dosificación , Movilización de Célula Madre Hematopoyética/métodos , Proteínas Relacionadas con Receptor de LDL/sangre , Proteínas de Transporte de Membrana/sangre , Células Mieloides/fisiología , Animales , Biomarcadores/sangre , Médula Ósea/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Femenino , Células HL-60 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Inyecciones Subcutáneas , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Células Mieloides/efectos de los fármacos , Células U937RESUMEN
In mammals, the expression of 5-10% of genes occurs with circadian fluctuation in various organs and tissues. This cyclic transcription is thought to be directly or indirectly regulated through circadian transcriptional/translational feedback loops consisting of a set of clock genes. Among the clock genes in mammals, expression of the Dbp mRNA robustly oscillates both in vivo and in culture cells. Here, we present circadian enhancer detection strategy using prokaryotic transposon system. The mDbp promoter drives reporter gene expression in robust circadian cycles in rat-1 fibroblasts. To identify the circadian enhancer generating this robust rhythm, we developed a prokaryotic transposon-based enhancer detecting vector for in vitro transposition. Using this system, we identified a strong circadian enhancer region containing the CATGTG sequence in the 5' flanking region of the mDbp gene; this enhancer region is critical for the ability of the mDbp promoter to drive robust oscillation in living cells. This enhancer is classified as a CANNTG type non-canonical E-box. These findings strongly suggest that CANNTG-type non-canonical E-boxes may contribute, at least in part, to the regulation of robust circadian gene expression. Furthermore, these data may help explain the wider effects of the CLOCK/BMAL1 complex in control of clock output genes.
Asunto(s)
Ritmo Circadiano/genética , Elementos Transponibles de ADN , Proteínas de Unión al ADN/genética , Elementos E-Box , Regulación de la Expresión Génica , Vectores Genéticos , Factores de Transcripción/genética , Factores de Transcripción ARNTL , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas CLOCK , Células Cultivadas , Genes Reporteros , Luciferasas/análisis , Luciferasas/genética , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Regiones Promotoras Genéticas , Ratas , Transactivadores/metabolismo , Activación TranscripcionalRESUMEN
Post-transcriptional/translational mechanisms regulate the circadian clock system of many organisms, including mammals. The level of the essential clock protein mPER2 daily oscillates in peripheral cells as well as in neurons of the master oscillator in the suprachiasmatic nucleus (SCN). Post-translational modifications of mPER2, such as phosphorylation and ubiquitination, are likely involved in the regulation of its stability and intracellular accumulation rhythms, which in turn create an approximately 2-4 h delay from the rhythm of mPer2 mRNA. However, there are no direct evidences linking the above biochemical processes to the generation of the mPER2 protein cycle itself. Here, we show that multiple circadian waves of bioluminescence are detectable in cells constitutively expressing an mPer2-luciferase fusion mRNA. This suggests that a post-transcriptional/translational mechanism itself is capable of generating the circadian mPER2 accumulation cycle, and thus this type of regulation may function in the circadian clock system in mammals.
Asunto(s)
Relojes Biológicos/genética , Ritmo Circadiano/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Procesamiento Proteico-Postraduccional/genética , Procesamiento Postranscripcional del ARN/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Bioensayo/métodos , Proteínas de Ciclo Celular , Línea Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica/genética , Luciferasas/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas Circadianas Period , ARN Mensajero/metabolismo , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Factores de TiempoRESUMEN
LR11, also known as SorLA or SORL1, is a type-I membrane protein from which a large extracellular part, soluble LR11 (sLR11), is released by proteolytic shedding on cleavage with a disintegrin and metalloproteinase 17 (ADAM17). A shedding mechanism is presumed to have a key role in the functions of LR11, but the evidence for this has not yet been demonstrated. Tetraspanin CD9 has been recently shown to regulate the ADAM17-mediated shedding of tumor necrosis factor-α and intercellular adhesion molecule-1 on the cell surface. Here, we investigated the role of CD9 on the shedding of LR11 in leukocytes. LR11 was not expressed in THP-1 monocytes, but it was expressed and released in phorbol 12-myristate 13-acetate (PMA)-induced THP-1 macrophages (PMA/THP-1). Confocal microscopy showed colocalization of LR11 and CD9 proteins on the cell surface of PMA/THP-1. Ectopic neo-expression of CD9 in CCRF-SB cells, which are LR11-positive and CD9-negative, reduced the amount of sLR11 released from the cells. In contrast, incubation of LR11-transfected THP-1 cells with neutralizing anti-CD9 monoclonal antibodies increased the amount of sLR11 released from the cells. Likewise, the PMA-stimulated release of sLR11 increased in THP-1 cells transfected with CD9-targeted shRNAs, which was negated by treatment with the metalloproteinase inhibitor GM6001. These results suggest that the tetraspanin CD9 modulates the ADAM17-mediated shedding of LR11 in various leukemia cell lines and that the association between LR11 and CD9 on the cell surface has an important role in the ADAM17-mediated shedding mechanism.