RESUMEN
During development, many neurons display calcium-dependent migration, but the role of this messenger in regulating gene expression leading to this event has not yet been elucidated. Among the decoders of calcium signals is calcineurin, a Ca(2+)/calmodulin serine/threonine phosphatase that has been involved in both short-term and long-term cellular changes. By using immortalized GnRH-secreting neurons, we now show that, in vitro, Ca(2+)-dependent gene expression, proceeding via calcineurin and the transcription factor nuclear factor of activated T cells, is a key player controlling the chemomigratory potential of developing GnRH-secreting neurons. Furthermore, our data highlight the switch nature of this phosphatase, whose activation or inactivation guides cells to proceed from one genetic program to the next.
Asunto(s)
Calcineurina/fisiología , Quimiotaxis/fisiología , Hormona Liberadora de Gonadotropina/fisiología , Neuronas/fisiología , Sistemas Neurosecretores/fisiología , Transporte Biológico , Inhibidores de la Calcineurina , Señalización del Calcio , Línea Celular , Ciclosporina/farmacología , Activación Enzimática , Humanos , Microscopía Fluorescente , Factores de Transcripción NFATC/fisiología , Neuronas/enzimología , Sistemas Neurosecretores/citología , Sistemas Neurosecretores/enzimología , Sirolimus/farmacología , Tacrolimus/farmacologíaRESUMEN
During CNS development neurons undergo directional migration to achieve their adult localizations. To study neuronal migration, we used a model cell line of immortalized murine neurons (gonadotropin-releasing hormone expressing neurons; GN11), enriched with caveolins and caveolae invaginations that show in vitro chemotaxis upon serum exposure. Cholesterol depletion with methyl-beta-cyclodextrin induced the loss of caveolae and the inhibition of chemotaxis, thus suggesting that GN11 migration depends upon the structural integrity of caveolae. Polarization of proteins and organelles is a hallmark of cell migration. Accordingly, GN11 cells transmigrating through filter pores exhibited a polarized distribution of caveolin-1 isoform (cav-1) in the leading processes. In contrast, during two-dimensional migration cav-1 and caveolae polarized at the trailing edge. As caveolae are enriched with signaling molecules, we suggest that asymmetrical localization of caveolae may spatially orient GN11 neurons to incoming migratory signals thereby transducing them into directional migration.
Asunto(s)
Caveolas/metabolismo , Caveolinas/metabolismo , Movimiento Celular/fisiología , Neuronas/fisiología , Animales , Línea Celular Transformada , Movimiento Celular/efectos de los fármacos , Quimiotaxis/efectos de los fármacos , Quimiotaxis/fisiología , Colesterol/metabolismo , Hormona Liberadora de Gonadotropina/metabolismo , Indoles , Ratones , Microscopía Electrónica de Transmisión/métodos , Neuronas/efectos de los fármacos , Neuronas/ultraestructura , Sales de Tetrazolio , Tiazoles , beta-Ciclodextrinas/farmacologíaRESUMEN
Platelet-enriched plasma (PRP) is used in therapy as a source of growth factors in bone fracture and wound healing; however, few data exist on its role in the different aspects of the healing process. The effect of PRP and of the two main growth factors present in this preparation (platelet-derived growth factor [PDGF] and transforming growth factor-beta [TGF-beta]) was evaluated in vitro using the human osteoblastic cell line SaOS-2, which was shown by reverse transcription-polymerase chain reaction to express both PDGF-alpha and -beta receptors. Batroxobine-activated PRP was added in different concentrations to SaOS-2 cells to assess cell migration (by a microchemotaxis assay) and cell proliferation (by [3H]-thymidine incorporation into the DNA). Immunoneutralization with anti-PDGF-beta or anti-TGF-beta antibodies allowed the assessment of the specific role of these growth factors. The overall results obtained indicate that PRP dose-dependently stimulates both chemotaxis and cell proliferation. PDGF and TGF-beta appear to exert distinct effects on the two parameters, the former involved in stimulating cell migration and the latter in inhibiting cell proliferation. It is concluded that the different growth factors present in activated PRP can specifically contribute to the main processes of tissue regeneration.
Asunto(s)
Plaquetas/fisiología , Osteoblastos/efectos de los fármacos , Factor de Crecimiento Derivado de Plaquetas/farmacología , Factor de Crecimiento Transformador beta/farmacología , Análisis de Varianza , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Quimiotaxis , Humanos , Recuento de Plaquetas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Neuroendocrine control of physiological functions needs a complex developmental organisation of the hypothalamic parvicellular neurons, which synthesise and release hypophysiotropic hormones. Among the hypothalamic neuroendocrine cells, Gonadotropin-releasing hormone (GnRH) neurons represent a unique class; they are generated in the olfactory placode and, during embryonic life, migrate to the septo/hypothalamic region along terminal and vomeronasal nerves. At this level GnRH neurons undergo terminal differentiation and start to release GnRH to modulate the secretion of pituitary gonadotropins. All these steps are under the strict control of several developmental cues and their defect might represent a cause of clinical disorders. A number of factors have been proposed to be involved in the migration of GnRH neurons, but their role is still unclear. By using gene knockout techniques it has been found that mice carrying a targeted deletion of Ebf2 gene, a component of Olf/Ebf bHLH transcription factors, show a defective migration of GnRH neurons, providing the first evidence of a mouse model of such defect. Since the investigation of GnRH neurons is hindered by their peculiar anatomical distribution, other studies has been forwarded by the availability of immortalized GnRH-expressing neurons (GN11 cells) that retain a strong chemomigratory response "in vitro". Among the factors analysed, we found that hepatocyte growth factor/scatter factor (HGF/SF) and vascular endothelial growth factor (VEGF) induce specific chemotaxis of GN 11 neurons, suggesting that migratory signals can arise from nasal mesenchyme and from the concomitant vasculogenesis. Finally, anosmin-1 (the product of the gene responsible of the X-linked form of Kallmann's disease) was found to induce a significant chemotactic response of GN11 cells, confirming a permissive/instructive role of KAL1 gene product in the migratory behaviour of GnRH neurons. In conclusion, the migration of the GnRH neurons appears to be a complex process, which involves the interplay of multiple molecular cues. These studies may provide new insights on the etiopathogenesis of the large proportion of reproductive dysfunctions that affect humans and could provide novel insights on common biochemical events controlling neuronal development and migration.