RESUMEN
INTRODUCTION: The objective of this study is to evaluate the effect of basic fibroblast growth factor (bFGF) on the proliferation and secretion activity of feline adipose-derived mesenchymal stem cells (MSC). METHODS: Feline MSC isolated from the subcutaneous adipose tissue of cats were cultured with or without bFGF. RESULTS: The bFGF addition enhanced the proliferation of feline MSC to a significant great extent compared with that without bFGF, although the cell proliferation tended to increase with the bFGF concentration. In addition, adipogenic and osteogenic staining assay demonstrated that the bFGF addition allowed MSC to maintain the differentiation ability even after the proliferation. Moreover, no change in the surface markers of MSC was observed between the cultures with or without bFGF. A quantitative RT-PCR assay revealed that the HGF and TSG-6 expression significantly increased by the bFGF addition. The highest mRNA expression of MMP-2 was observed for cells cultured in 1000 ng/ml bFGF concentration. CONCLUSIONS: The culture with bFGF is a promising way to enhance the proliferation, and HGF secretion ability of MSC as well as maintain their differentiation ability and immunophenotype nature.
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BACKGROUND: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSH-containing granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)gamma was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kgamma and enhanced PIP5Kgamma enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kgamma knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kgamma, PI(4,5)P(2), and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. CONCLUSIONS/SIGNIFICANCE: Our findings indicated that the control of TSH release by the SIRT1-PIP5Kgamma pathway is important for regulating the metabolism of the whole body.
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Lisina/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Sirtuina 1/metabolismo , Tirotropina/metabolismo , Acetilación , Animales , Western Blotting , Línea Celular , Células Cultivadas , Cromatografía Liquida , Electroporación , Genotipo , Humanos , Inmunoprecipitación , Masculino , Espectrometría de Masas , Ratones , Ratones Noqueados , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Unión Proteica , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sirtuina 1/genéticaAsunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Complejo 2 de Proteína Adaptadora/metabolismo , Subunidades beta de Complejo de Proteína Adaptadora/metabolismo , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Endocitosis/fisiología , Activación Enzimática , Hipocampo/citología , Humanos , Isoenzimas/fisiología , Ratones , Neuronas/metabolismo , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Vesículas Sinápticas/fisiologíaRESUMEN
The remarkably versatile phospholipid, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)], plays crucial roles in signal transduction, actin cytoskeleton reorganization, clathrin-dependent endocytosis, and regulation of membrane morphology. In mammalian cells, PI(4,5)P(2) is synthesized predominantly by phosphatidylinositol 4-phosphate [PI(4)P] 5-kinase (PIP5K) through phosphorylation of PI(4)P at the D-5 position of the inositol ring. PIP5K is composed of three isoforms, PIP5Kalpha, beta, and gamma, and three splicing variants of the gamma isozyme. Although the PIP5Kgamma splicing variant PIP5Kgamma661 appears to be very specifically activated by talin, which plays a crucial role in focal adhesion formation, and the adaptor complex AP-2, the regulation of activities of other PIP5K isozymes is not fully understood at present. To understand the activation mechanism and the physiological function specific to each PIP5K isozyme, it is required to identify a specific activator of each PIP5K isozyme. This chapter describes common assays used to measure interaction and activation of PIP5K isozymes by activators thus far identified. In addition, procedures for preparation of PIP5K isozymes and activators are described.
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Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Animales , Encéfalo/enzimología , Bovinos , Citosol/enzimología , Activación Enzimática , Vectores Genéticos , Homeostasis , Cinética , Fosfotransferasas (Aceptor de Grupo Alcohol)/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Talina/fisiologíaRESUMEN
The phosphoinositide kinase, phosphatidylinositol 4-phosphate 5-kinase (PIP5K), produces the versatile phospholipid phosphatidylinositol 4,5-bisphosphate (PI4,5P(2)), through which PIP5K plays crucial roles in a wide variety of cell functions. So far, three PIP5K isozymes and splicing variants have been identified. We speculate that each PIP5K isozyme or splicing variant is activated in a tempo-spatially different manner, due to the existence of activators or recruiters specific to each isozyme: this tempo-spatially different activation of PIP5K produces PI4,5P(2) at different compartments of the cell at different times, which phenomenon may be responsible for the apparent multifunction of PI4,5P(2)/PIP5K. Accumulating evidence supports this notion that each PIP5K isozyme is activated by its specific activator and plays a crucial role in a unique cell function. In this article, we describe recent advances regarding the PIP5K isozyme-specific activation mechanisms and physiological functions.
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Fosfatidilinositol 4,5-Difosfato/fisiología , Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Transducción de Señal/fisiología , Animales , Activación Enzimática/fisiología , Humanos , Antígenos de Histocompatibilidad Menor , Fracciones Subcelulares/metabolismoRESUMEN
Antigen binding to the B-cell receptor (BCR) induces multiple signaling cascades that ultimately lead to B lymphocyte activation. In addition, the BCR regulates the key trafficking events that allow the antigen to reach endocytic compartments devoted to antigen processing, i.e., that are enriched for major histocompatibility factor class II (MHC II) and accessory molecules such as H2-DM. Here, we analyze the role in antigen processing and presentation of the tyrosine kinase Syk, which is activated upon BCR engagement. We show that convergence of MHC II- and H2-DM-containing compartments with the vesicles that transport BCR-uptaken antigens is impaired in cells lacking Syk activity. This defect in endocytic trafficking compromises the ability of Syk-deficient cells to form MHC II-peptide complexes from BCR-internalized antigens. Altered endocytic trafficking is associated to a failure of Syk-deficient cells to properly reorganize their actin cytoskeleton in response to BCR engagement. We propose that, by modulating the actin dynamics induced upon BCR stimulation, Syk regulates the positioning and transport of the vesicles that carry the molecules required for antigen processing and presentation.
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Actinas/metabolismo , Presentación de Antígeno/inmunología , Endocitosis , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Línea Celular Tumoral , Citoesqueleto/metabolismo , Antígenos de Histocompatibilidad Clase II/metabolismo , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Activación de Linfocitos , Lisosomas/metabolismo , Lisosomas/ultraestructura , Ratones , Péptidos/metabolismo , Transporte de Proteínas , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/ultraestructura , Bazo/citología , Bazo/metabolismo , Quinasa SykRESUMEN
Synaptic vesicles (SVs) are retrieved by clathrin-mediated endocytosis at the nerve terminals. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] drives this event by recruiting the components of the endocytic machinery. However, the molecular mechanisms that result in local generation of PI(4,5)P2 remain unclear. We demonstrate here that AP-2 complex directly interacts with phosphatidylinositol 4-phosphate 5-kinase gamma661 (PIP5Kgamma661), the major PI(4,5)P2-producing enzyme in the brain. The beta2 subunit of AP-2 was found to bind to the C-terminal tail of PIP5Kgamma661 and cause PIP5Kgamma661 activation. The interaction is regulated by PIP5Kgamma661 dephosphorylation, which is triggered by depolarization in mouse hippocampal neurons. Finally, overexpression of the PIP5Kgamma661 C-terminal region in hippocampal neurons suppresses depolarization-dependent SV endocytosis. These findings provide evidence for the molecular mechanism through which PIP5Kgamma661 locally generates PI(4,5)P2 in hippocampal neurons and suggest a model in which the interaction trigger SV endocytosis.
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Complejo 2 de Proteína Adaptadora/metabolismo , Endocitosis/fisiología , Hipocampo/metabolismo , Neuronas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Vesículas Sinápticas/metabolismo , Animales , Activación Enzimática/fisiología , Inmunohistoquímica , Ratones , Modelos Biológicos , Vesículas Sinápticas/fisiologíaAsunto(s)
Fosfotransferasas (Aceptor de Grupo Alcohol)/fisiología , Factores de Ribosilacion-ADP/fisiología , Actinas/metabolismo , Animales , Catálisis , Fenómenos Fisiológicos Celulares , Dendritas/fisiología , Endocitosis , Activación Enzimática , Humanos , Isoenzimas , Ácidos Fosfatidicos/fisiología , Fosfatidilinositol 4,5-Difosfato/fisiología , Fosforilación , Fosfotransferasas (Aceptor de Grupo Alcohol)/química , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Proteínas de Unión al GTP rho/fisiologíaRESUMEN
The mammalian small GTPase ADP-ribosylation factor 6 (ARF6) plays important roles in a wide variety of cellular events, including endocytosis, actin cytoskeletal reorganization, and phosphoinositide metabolism. However, physiological functions for ARF6 have not previously been examined. Here, we described the consequence of ARF6 ablation in mice, which manifests most obviously in the context of liver development. Livers from ARF6-/- embryos are smaller and exhibit hypocellularity, due to the onset of midgestational liver cell apoptosis. Preceding the apoptosis, however, defective hepatic cord formation is observed; the liver cells migrate abnormally upon exiting the primordial hepatic epithelial sheet and clump rather than becoming dispersed. Consistent with this observation, the ability of hepatocyte growth factor/scatter factor (HGF) to induce hepatic cord-like structures from ARF6-/- fetal hepatocytes cultured in vitro in collagen gel matrix is impaired. Finally, we show that endogenous ARF6 in wild-type fetal hepatocytes is activated in response to HGF stimulation. These results provide evidence that ARF6 is an essential component in the signaling pathway coupling HGF signaling to hepatic cord formation.
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Factores de Ribosilacion-ADP/metabolismo , Hígado/embriología , Proteínas de Unión al GTP Monoméricas/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/deficiencia , Animales , Apoptosis , Células Cultivadas , Colágeno/metabolismo , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Exones/genética , Geles , Marcación de Gen , Genotipo , Factor de Crecimiento de Hepatocito/farmacología , Hepatocitos/citología , Hepatocitos/efectos de los fármacos , Hepatocitos/patología , Hígado/anomalías , Hígado/citología , Hígado/patología , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Actin cytoskeletal reorganization and membrane trafficking are important for spine morphogenesis. Here we investigated whether the small GTPase, ADP-ribosylation factor 6 (ARF6), which regulates actin dynamics and peripheral vesicular trafficking, is involved in the regulation of spine formation. The developmental expression pattern of ARF6 in mouse hippocampus was similar to that of the post-synaptic density protein-95, and these molecules colocalized in mouse hippocampal neurons. Overexpression of a constitutively active ARF6 mutant in cultured hippocampal neurons decreased the spine density, whereas a dominant-negative ARF6 mutant increased the density. These results demonstrate a novel function for ARF6 as a key regulator of spine formation.
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Factores de Ribosilacion-ADP/metabolismo , Espinas Dendríticas/metabolismo , Regulación del Desarrollo de la Expresión Génica , Hipocampo/metabolismo , Proteínas de Unión al GTP Monoméricas/metabolismo , Neuronas/metabolismo , Factor 6 de Ribosilación del ADP , Factores de Ribosilacion-ADP/genética , Animales , Western Blotting , Células Cultivadas , Hipocampo/citología , Hipocampo/embriología , Ratones , Microscopía Fluorescente , Mutación , Neuronas/citologíaRESUMEN
The signaling pathway that triggers morphological differentiation of PC12 cells is mediated by extracellular signal-regulated kinase (ERK), the classic mitogen-activated protein (MAP) kinase. However, mediators of the pathway downstream of ERK have not been identified. We show here that phospholipase D2 (PLD2), which generates the pleiotropic signaling lipid phosphatidic acid (PA), links ERK activation to neurite outgrowth in nerve growth factor (NGF)-stimulated PC12 cells. Increased expression of wild type PLD2 (WT-PLD2) dramatically elongated neurites induced by NGF stimulation or transient expression of the active form of MAP kinase-ERK kinase (MEK-CA). The response was activity-dependent, because it was inhibited by pharmacological suppression of the PLD-mediated PA production and by expression of a lipase-deficient PLD2 mutant. Furthermore, PLD2 was activated by MEK-CA, whereas NGF-stimulated PLD2 activation and hypertrophic neurite extension were blocked by an MEK-specific inhibitor. Taken together, these results provide evidence that PLD2 functions as a downstream signaling effector of ERK in the NGF signaling pathway, which leads to neurite outgrowth by PC12 cells.
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Proteínas Quinasas Activadas por Mitógenos/metabolismo , Factor de Crecimiento Nervioso/farmacología , Neuritas , Fosfolipasa D/metabolismo , Actinas/metabolismo , Animales , Activación Enzimática , Células PC12 , Ratas , Transducción de SeñalRESUMEN
The Syk tyrosine kinase is a key molecule in the development of the B cell lineage and the activation of B lymphocytes after Ag recognition by the B cell Ag receptor (BCR). Several genetic studies with chicken B cells have reported that the recruitment of Syk by BCR is essential for activation of a cascade of signaling molecules including phosphatidylinositol 3-kinase, mitogen-activated protein kinases, Ras signaling pathways, phospholipase C-gamma2 activation, and calcium mobilization. The identification of a Syk-deficient mouse IIA1.6/A20 B cell line provided us the opportunity to investigate Syk-mediated signaling in mouse. Surprisingly, phosphatidylinositol 3-kinase, Ras, and mitogen-activated protein kinases were activated upon BCR cross-linking in these Syk-deficient mouse B cells, whereas, as expected from results obtained in chicken B cells, phospholipase C-gamma2 activation and calcium mobilization were impaired as well as the NF-kappaB pathway. These results indicate that BCR signaling is not strictly dependent on Syk expression in mouse IIA1.6/A20 B cells. Thus, B lymphocyte activation may be initiated by Syk-dependent and Syk-independent signaling cascades.
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Precursores Enzimáticos/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Tirosina Quinasas/fisiología , Receptores de Antígenos de Linfocitos B/fisiología , Proteínas ras/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos CD19/fisiología , Calcio/metabolismo , Células Clonales , Activación Enzimática/fisiología , Precursores Enzimáticos/biosíntesis , Precursores Enzimáticos/deficiencia , Precursores Enzimáticos/genética , Interleucina-2/antagonistas & inhibidores , Interleucina-2/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Activación de Linfocitos , Sistema de Señalización de MAP Quinasas/genética , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Datos de Secuencia Molecular , FN-kappa B/antagonistas & inhibidores , FN-kappa B/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Fosfolipasa C gamma , Fosforilación , Proteínas Tirosina Quinasas/biosíntesis , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/genética , Receptores de Antígenos de Linfocitos B/biosíntesis , Transducción de Señal/genética , Transducción de Señal/fisiología , Quinasa Syk , Células Tumorales Cultivadas , Fosfolipasas de Tipo C/metabolismo , Tirosina/metabolismo , Proteínas ras/biosíntesis , Proteínas ras/fisiologíaRESUMEN
Antigen recognition by clonotypic B cell receptor (BcR) is the first step of B lymphocytes differentiation into plasmocytes. This B cell function is dependent on efficient major histocompatibility complex (MHC) class II-restricted presentation of BcR-bound antigens. In this work, we analyzed the subcellular mechanisms underlying antigen presentation after BcR engagement on B cells. In quiescent B cells, we found that MHC class II molecules mostly accumulated at the cell surface and in an intracellular pool of tubulovesicular structures, whereas H2-M molecules were mostly detected in distinct lysosomal compartments devoid of MHC class II. BcR stimulation induced the transient intracellular accumulation of MHC class II molecules in newly formed multivesicular bodies (MVBs), to which H2-M was recruited. The reversible downregulation of cathepsin S activity led to the transient accumulation of invariant chain-MHC class II complexes in MVBs. A few hours after BcR engagement, cathepsin S activity increased, the p10 invariant chain disappeared, and MHC class II-peptide complexes arrived at the plasma membrane. Thus, BcR engagement induced the transient formation of antigen-processing compartments, enabling antigen-specific B cells to become effective antigen-presenting cells.