RESUMEN
Neurofibrillary tangles (NFT) containing tau are a hallmark of neurodegenerative diseases, including Alzheimer's disease (AD). NFT burden correlates with cognitive decline and neurodegeneration in AD. However, little is known about mechanisms that protect against tau-induced neurodegeneration. We used a cross species functional genomic approach to analyze gene expression in multiple brain regions in mouse, in parallel with validation in Drosophila, to identify tau modifiers, including the highly conserved protein puromycin-sensitive aminopeptidase (PSA/Npepps). PSA protected against tau-induced neurodegeneration in vivo, whereas PSA loss of function exacerbated neurodegeneration. We further show that human PSA directly proteolyzes tau in vitro. These data highlight the utility of using both evolutionarily distant species for genetic screening and functional assessment to identify modifiers of neurodegeneration. Further investigation is warranted in defining the role of PSA and other genes identified here as potential therapeutic targets in tauopathy.
Asunto(s)
Aminopeptidasas/metabolismo , Encéfalo/enzimología , Degeneración Nerviosa/enzimología , Tauopatías/genética , Proteínas tau/metabolismo , Animales , Northern Blotting , Western Blotting , Encéfalo/patología , Drosophila , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Transgénicos , Degeneración Nerviosa/patología , Ovillos Neurofibrilares/enzimología , Ovillos Neurofibrilares/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Tauopatías/enzimología , Tauopatías/patología , Proteínas tau/genéticaRESUMEN
Breast cancer cells (BCCs) have preference for the bone marrow (BM). This study used an in vitro coculture of BCCs and BM stroma to represent a model of early breast cancer metastasis to the BM. The overarching hypothesis states that once BCCs are in the BM, microenvironmental factors induce changes in the expression of genes for cytokines and preprotachykinin-I (PPT-I) in both BCCs and stromal cells. Consequently, the expression of both PPT-I and cytokines are altered to facilitate BCC integration within BM stroma. Cytokine and transcription factor arrays strongly suggested that transforming growth factor-beta (TGF-beta) and c-myc regulate the expression of PPT-I so as to facilitate BCC integration among stroma. Northern analyses and TGF-beta bioassays showed that stromal cells and BCCs influence the level of PPT-I and TGF-beta in each other. In cocultures, PPT-I and TGF-beta expressions were significantly (P < 0.05) increased and decreased, respectively. TGF-beta and PPT-I were undetectable in separate stromal cultures but were expressed as cocultures. Two consensus sequences for c-myc in the 5' flanking region of the PPT-I gene were shown to be functional using gel shift and reporter gene assays. Mutagenesis of c-myc sites, neutralization studies with anti-TGF-beta, and transient tranfections all showed that c-myc is required for TGF-beta-mediated induction of PPT-I in BCCs. TGF-beta was less efficient as a mediator of BCC integration within stroma for c-myc-BCCs. Because the model used in this study represents BCC integration within BM stroma, these studies suggest that TGF-beta is important to the regulation of PPT-I in the early events of bone invasion by BCCs.
Asunto(s)
Células de la Médula Ósea/metabolismo , Neoplasias de la Mama/metabolismo , Regulación Neoplásica de la Expresión Génica/fisiología , Genes myc/fisiología , Precursores de Proteínas/genética , Taquicininas/genética , Factor de Crecimiento Transformador beta/biosíntesis , Células de la Médula Ósea/patología , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Técnicas de Cocultivo , Secuencia de Consenso , Citocinas/metabolismo , Genes myc/genética , Humanos , Precursores de Proteínas/biosíntesis , Células del Estroma/metabolismo , Células del Estroma/patología , Taquicininas/biosíntesis , Transfección , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Neurokinin (NK)-1 and NK-2 receptors regulate hematopoiesis by interacting with neurotransmitters that belong to the tachykinin. This report studies the relationship between NK-1 and NK-2 in primary human bone marrow (BM) stroma, which supports hematopoiesis. Use of NK receptor antagonists and deficient stromal cells indicate that the neurotransmitter, substance P (SP), could exert dual hematopoietic effects (inhibitory or stimulatory), depending on the interacting receptor and crosstalk between NK-1 and NK-2. Cloning and identification of the minimal promoter for NK-2 and comparison with NK-1 promoter showed that the hematopoietic functions of NK receptors involve receptor crosstalk and the particular cytokine (IL-3, GM-CSF, TGF-beta or IL-1alpha). Crosstalk between NK-1 and NK-2 adds to communication within neural-hematopoietic axis.
Asunto(s)
Hematopoyesis/fisiología , Regiones Promotoras Genéticas , Receptor Cross-Talk/fisiología , Receptores de Neuroquinina-1/fisiología , Receptores de Neuroquinina-2/química , Receptores de Neuroquinina-2/fisiología , Regiones no Traducidas 5'/efectos de los fármacos , Secuencia de Bases , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , División Celular/fisiología , Células Cultivadas , Clonación Molecular/métodos , Citocinas/farmacología , Regulación hacia Abajo/efectos de los fármacos , Eliminación de Gen , Hematopoyesis/genética , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Humanos , Datos de Secuencia Molecular , Antagonistas del Receptor de Neuroquinina-1 , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/genética , ARN Mensajero/metabolismo , Receptores de Neuroquinina-1/genética , Receptores de Neuroquinina-2/antagonistas & inhibidores , Receptores de Neuroquinina-2/genética , Células del Estroma/efectos de los fármacos , Células del Estroma/metabolismo , Sustancia P/farmacología , Transfección , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Neurokinin 1 (NK-1) is a member of seven transmembrane G protein-coupled receptors. NK-1 interacts with peptides belonging to the tachykinin family and showed preference for substance P (SP). NK-1 is induced in bone marrow (BM) stroma. NK-1-SP interactions could lead to changes in the functions of lymphohematopoietic stem cell (LHSC). This report describes the cloning and characterization of a cDNA clone isolated after screening of three cDNA libraries with an NK-1-specific probe. Based on its expression, the cDNA clone was designated hematopoietic growth factor inducible neurokinin-1 type (HGFIN). Computational analyses predicted that HGFIN is transmembrane with the carboxyl terminal extracellular. Proteomic studies with purified HGFIN and SP showed noncovalent interactions. HGFIN-SP interactions were supported by transient expression of HGFIN in CHO cells. Transient expression of HGFIN in unstimulated BM fibroblasts led to the induction of endogenous NK-1. Since NK-1 expression in BM fibroblasts requires cell stimulation, these studies suggest that there might be intracellular crosstalk between NK-1 and HGFIN. Northern analyses with total RNA from different BM cell subsets showed that HGFIN was preferentially expressed in differentiated cells. This suggests that HGFIN might be involved in the maturation of LHSC. HGFIN was detected in several other tissues, but not in brain where NK-1 is constitutively expressed.
Asunto(s)
Factores de Crecimiento de Célula Hematopoyética/metabolismo , Proteínas de la Membrana/metabolismo , Sustancia P/metabolismo , Animales , Secuencia de Bases , Células de la Médula Ósea/citología , Células CHO , Diferenciación Celular/fisiología , Clonación Molecular , Cricetinae , Sondas de ADN , ADN Complementario/genética , Fibroblastos/metabolismo , Humanos , Glicoproteínas de Membrana , Proteínas de la Membrana/genética , Modelos Moleculares , Datos de Secuencia Molecular , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Ácido Nucleico , Sustancia P/genética , Distribución TisularRESUMEN
OBJECTIVE: The cellular and molecular mechanisms of hematopoietic stimulation have been studied. However, an understanding of negative effects in the hematopoietic system remains elusive. To this end, we studied the effects of vasoactive intestinal peptide (VIP) on bone marrow (BM) progenitors. MATERIALS AND METHODS: Different BM cell subsets were used to perform clonogenic assay for granulocytic (CFU-GM) or erythroid (BFU-E and CFU-E) progenitors with 10(-7)-10(-13) M VIP. The relevant receptor was verified with specific antagonists, or agonists, semi-quantitative RT-PCR, and chemical cross-linking studies with stromal membranes. RESULTS: Assays performed with unfractionated mononuclear cells and enriched CD34(+) cells showed dose-dependent inhibition on BM progenitors with significant inhibition up to 10(-10) M. Nylon wool separated cells, which depleted stroma, reversed the inhibitory effects of VIP between 10 and 20%. Combined experimental evaluation indicated that the effects of VIP on BM functions are mediated through the type 1 receptor (VPAC1). VIP induced the production of TGF-beta and TNF-alpha in BM mononuclear cells and stroma. These cytokines are partly involved in reversing the suppressive effects of VIP on CFU-GM. CONCLUSIONS: The effect of VIP on BM progenitors could be mediated through direct and indirect mechanism. Direct effects were evident by the suppressive effects of VIP on clonogenic assays with highly purified CD34(+) cells. Indirect effects were mediated through putative functions of the stromal cells and the production of TGF-beta and TNF-alpha.