La migrazione dei neuroblasti è un processo complesso che prevede una serie di eventi cellulari coordinati ed integrati. Alla modulazione del processo partecipano numerosi segnali molecolari, inclusi i neurotrasmettitori e i fattori di crescita e differenziamento. Diversi studi hanno separatamente dimostrato che il sistema neuregulina 1(NRG1)/ErbB4 e i recettori per il glutammato di tipo NMDA (NMDARs) sono coinvolti in svariati aspetti della migrazione neuronale. Il nostro laboratorio ha recentemente dimostrato che: 1) l'espressione stabile del recettore tirosin chinasico ErbB4 induce migrazione nella linea cellulare di progenitori neurali ST14A in seguito a stimolazione con NRG1, 2) questa stimolazione induce un aumento di calcio intracellulare che è coinvolto nel processo di migrazione, 3) l'attivazione del recettore NMDA è in grado di aumentare la migrazione indotta da NRG1 in maniera calcio-dipendente. Nel tessuto cerebrale ErbB4 viene prodotto in 4 isoforme di splicing alternativo. Tali isoforme differiscono nel dominio iuxtra-membrana extracellulare (JMa e JMb) oppure nel dominio intracitoplasmatico (Cyt1 e Cyt2). Nel nostro laboratorio sono disponibili i cloni di ST14A esprimenti le 4 combinazioni di splicing di ErbB4: JMa/Cyt1, JMa/Cyt2, JMb/Cyt1 and JMb/Cyt2. Lo scopo di questo lavoro è stato quello di confrontare i due cloni esprimenti le varianti di ErbB4 che inducono migrazione maggiore in seguito a stimolazione con NRG1. Tali varianti corrispondono all'isoforma JMa/Cyt2, contenente un sito di taglio per la metalloproteasi TACE nella regione iuxtamembrana, e l'isoforma JMb/Cyt1, che presenta un sito di legame intracellulare per la PI3K. Inizialmente abbiamo effettuato un'analisi in western blot che indica come i livelli di ErbB2 ed ErbB3 siano confrontabili tra loro nei due cloni, suggerendo che la presenza di isoforme specifiche di ErbB4 non influenza l'espressione di potenziali partners di dimerizzazione. Saggi di migrazione condotti per tempi relativamente brevi (6 ore) hanno dimostrato che NRG1 induce chemiotassi sulle cellule esprimenti JMb/Cyt1-ErbB4, e chemiocinesi sulle cellule esprimenti JMa/Cyt2-ErbB4. Saggi di migrazione a lungo termine (18 ore) allestiti in presenza di BAPTA-AM, chelante intracellulare del calcio, hanno mostrato una riduzione significativa della migrazione stimolata da NRG1 per entrambe le isoforme, tuttavia la migrazione delle cellule esprimenti JMb/Cyt1-ErbB4 ha dimostrato una minore dipendenza dal calcio. L'aggiunta di NMDA a NRG1 ha indotto un effetto sinergico delle due molecole soltanto nelle cellule esprimenti JMa/Cyt2-ErbB4. Come abbiamo precedentemente dimostrato, tale sinergia è anch'essa calcio-dipendente. Questi dati suggeriscono che le vie dipendenti dal calcio attivate da NRG1 nelle cellule esprimenti le due versioni di splicing di ErbB4 potrebbero essere distinte. Inoltre, mentre l'effetto chemotattico di NRG1 osservato nelle cellule con JMb/Cyt1-ErbB4 potrebbe essere legato ad un meccanismo veloce quale la modificazione del citoscheletro, gli effetti migratori a lungo termine indotti da NRG1 su cellule contenenti JMa/Cyt2-ErbB4 sono compatibili con i tempi di attivazione di trascrizione e traduzione. Esperimenti futuri indagheranno le vie del segnale attivate da queste due isoforme di ErbB4 durante la migrazione delle cellule ST14A.
Neuroblast migration is a complex, integrated process that involves a coordinated sequence of cellular events. Many signalling molecules participate in the modulation of neuronal migration, including molecules classified as neurotransmitters and as growth/differentiation factors. A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. Our laboratory has recently shown that: 1) stable expression of the tyrosine kinase receptor ErbB4 promotes migratory activity in the neural progenitor cell line ST14A upon NRG1 stimulation, 2) this stimulation induces an intracellular calcium increase that is involved in the migratory process, and 3) NMDAR activation can increase NRG1-induced migration in a calcium-dependent manner. In the brain alternative splicing generates four distinct isoforms of ErbB4. These isoforms differ at the extracellular juxtamembrane domain (JMa and JMb isoforms) or at the intracellular cytoplasmic domain (Cyt1 and Cyt2 isoforms). ST14A clones expressing all four ErbB4 splicing combinations are available in our laboratory: JMa/Cyt1, JMa/Cyt2, JMb/Cyt1 and JMb/Cyt2. The aim of this work was to compare the two ErbB4-expressing clones showing the highest increase in migration following NRG1 stimulation. These clones express the JMa/Cyt2 ErbB4 isoform, containing a cleavage site for the metalloprotease TACE in the juxtamembrane region, and the JMb/Cyt1 ErbB4 isoform, that has an intracellular binding site for PI3K. We first observed, by western blot analysis, that both clones express comparable levels of ErbB2 and ErbB3 receptors, suggesting that the presence of a specific ErbB4 isoform does not influence the expression of potential dimerisation partners. Short-term migration assays (6 hrs) showed that NRG1 induces chemotaxis on JMb/Cyt1-ErbB4-expressing ST14 cells, while the NRG1 effect on JMa/Cyt2-ErbB4-expressing cells is mainly chemokinetic. Long-term migration assays (18 hrs) performed in the presence of the intracellular calcium chelator BAPTA-AM showed a significant reduction of NRG1-stimulated migration for both isoforms, however migration of the JMb/Cyt1-ErbB4 expressing cells showed smaller calcium-dependency. When NMDA was added to NRG1, we observed a synergistic effect of these molecules only with JMa/Cyt2-ErbB4 expressing cells. This synergistic effect was previously shown to depend on intracellular Ca2+. These data suggest that the calcium-dependent pathways activated by NRG1 are distinct in JMb/Cyt1-ErbB4 and in JMa/Cyt2-ErbB4 expressing cells. Furthermore, while the NRG1 chemotactic effect observed in JMb/Cyt1-ErbB4 cells after short-term exposure could be related to a fast mechanism of cytoskeletal modification, the long-term migratory effects induced by NRG1 on JMa/Cyt2-ErbB4 expressing cells may involve activation of gene transcription. Future experiments will investigate the signaling pathways activated by these two ErbB4 isoforms during ST14A cell migration.
EFFETTI DELL'NMDA E DELLO SPLICING ALTERNATIVO DI ERBB4 SULLA MIGRAZIONE INDOTTA DA NRG1 NEI PROGENITORI STRIATALI ST14A.
LICHERI, VALENTINA
2010/2011
Abstract
Neuroblast migration is a complex, integrated process that involves a coordinated sequence of cellular events. Many signalling molecules participate in the modulation of neuronal migration, including molecules classified as neurotransmitters and as growth/differentiation factors. A number of studies have separately shown that the neuregulin1 (NRG1)/ErbB4 system and NMDA-type glutamate receptors (NMDARs) are involved in several aspects of neuronal migration. Our laboratory has recently shown that: 1) stable expression of the tyrosine kinase receptor ErbB4 promotes migratory activity in the neural progenitor cell line ST14A upon NRG1 stimulation, 2) this stimulation induces an intracellular calcium increase that is involved in the migratory process, and 3) NMDAR activation can increase NRG1-induced migration in a calcium-dependent manner. In the brain alternative splicing generates four distinct isoforms of ErbB4. These isoforms differ at the extracellular juxtamembrane domain (JMa and JMb isoforms) or at the intracellular cytoplasmic domain (Cyt1 and Cyt2 isoforms). ST14A clones expressing all four ErbB4 splicing combinations are available in our laboratory: JMa/Cyt1, JMa/Cyt2, JMb/Cyt1 and JMb/Cyt2. The aim of this work was to compare the two ErbB4-expressing clones showing the highest increase in migration following NRG1 stimulation. These clones express the JMa/Cyt2 ErbB4 isoform, containing a cleavage site for the metalloprotease TACE in the juxtamembrane region, and the JMb/Cyt1 ErbB4 isoform, that has an intracellular binding site for PI3K. We first observed, by western blot analysis, that both clones express comparable levels of ErbB2 and ErbB3 receptors, suggesting that the presence of a specific ErbB4 isoform does not influence the expression of potential dimerisation partners. Short-term migration assays (6 hrs) showed that NRG1 induces chemotaxis on JMb/Cyt1-ErbB4-expressing ST14 cells, while the NRG1 effect on JMa/Cyt2-ErbB4-expressing cells is mainly chemokinetic. Long-term migration assays (18 hrs) performed in the presence of the intracellular calcium chelator BAPTA-AM showed a significant reduction of NRG1-stimulated migration for both isoforms, however migration of the JMb/Cyt1-ErbB4 expressing cells showed smaller calcium-dependency. When NMDA was added to NRG1, we observed a synergistic effect of these molecules only with JMa/Cyt2-ErbB4 expressing cells. This synergistic effect was previously shown to depend on intracellular Ca2+. These data suggest that the calcium-dependent pathways activated by NRG1 are distinct in JMb/Cyt1-ErbB4 and in JMa/Cyt2-ErbB4 expressing cells. Furthermore, while the NRG1 chemotactic effect observed in JMb/Cyt1-ErbB4 cells after short-term exposure could be related to a fast mechanism of cytoskeletal modification, the long-term migratory effects induced by NRG1 on JMa/Cyt2-ErbB4 expressing cells may involve activation of gene transcription. Future experiments will investigate the signaling pathways activated by these two ErbB4 isoforms during ST14A cell migration.| File | Dimensione | Formato | |
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https://hdl.handle.net/20.500.14240/17979