coculture-analysis-extracellular-protein-interactions-affecting

Coculture Analysis of Extracellular Protein Interactions Affecting Insulin Secretion by Pancreatic Beta Cells

Interactions between cell-surface proteins help coordinate the function of neighboring cells. Pancreatic beta cells are clustered together within ...

University of California, San Diego

Prostate cancer is currently the most common type of neoplasm found in American men, other than skin cancer, and is the second leading cause of cancer death in males. Because cell cycle checkpoint proteins stabilize the genome, the relationship of one such protein, Rad9, to prostate cancer was investigated. We found that four prostate cancer cell lines (CWR22, DU145, LNCaP, and PC-3), relative to PrEC normal prostate cells, have aberrantly high levels of Rad9 protein. The 3'-end region of intron 2 of Rad9 in DU145 cells is hypermethylated at CpG islands, and treatment with 5'-aza-2'-deoxycytidine restores near-normal levels of methylation and reduces Rad9 protein abundance. Southern blot analyses indicate that PC-3 cells contain an amplified Rad9 copy number. Therefore, we provide evidence that Rad9 levels are high in prostate cancer cells due at least in part to aberrant methylation or gene amplification. The effectiveness of small interfering RNA to lower Rad9 protein levels in CWR22, DU145, and PC-3 cells correlated with reduction of tumorigenicity in nude mice, indicating that Rad9 actively contributes to the disease. Rad9 protein levels were high in 153 of 339 human prostate tumor biopsy samples examined and detectable in only 2 of 52 noncancerous prostate tissues. There was a strong correlation between Rad9 protein abundance and cancer stage. Rad9 protein level can thus provide a biomarker for advanced prostate cancer and is causally related to the disease, suggesting the potential for developing novel diagnostic, prognostic, and therapeutic tools based on detection or manipulation of Rad9 protein abundance.

Rad9 has a functional role in human prostate carcinogenesis.

Cancer de la prostate est actuellement le type le plus commun de n&eacute;oplasme trouv&eacute;e chez les hommes am&eacute;ricains, autres que le cancer de la peau et est la deuxi&egrave;me cause de d&eacute;c&egrave;s par cancer chez les hommes. Parce que les prot&eacute;ines de point de contr&ocirc;le de cycle cellulaire stabilisent le g&eacute;nome, la relation d&#39;une telle prot&eacute;ine, Rad9, cancer de la prostate a &eacute;t&eacute; &eacute;tudi&eacute;e. Nous avons constat&eacute; que quatre lign&eacute;es cellulaires cancer de la prostate (CWR22, DU145, LNCaP et PC-3), par rapport aux cellules normales de la prostate PrEC, avaient des niveaux anormalement &eacute;lev&eacute;s de prot&eacute;ine Rad9. La r&eacute;gion de l&#39;extr&eacute;mit&eacute; 3&#39; de l&#39;intron 2 de Rad9 dans les cellules de DU145 est fait aux &icirc;les de CpG, et traitement avec 5&#39;-aza-2&#39;-d&eacute;soxycytidine restaure des niveaux presque normaux de m&eacute;thylation et r&eacute;duit l&#39;abondance des prot&eacute;ines Rad9. Analyses par transfert Southern indiquent que PC-3 cellules contiennent un nombre de copies Rad9 amplifi&eacute;. Par cons&eacute;quent, nous fournir la preuve que les niveaux de Rad9 sont &eacute;lev&eacute;s dans les cellules de cancer de la prostate due au moins en partie au aberrante m&eacute;thylation ou amplification g&eacute;nique. L&#39;efficacit&eacute; des petits ARN interf&eacute;rents pour abaisser des niveaux de prot&eacute;ine de Rad9 dans les cellules CWR22, DU145 et PC-3 en corr&eacute;lation avec la r&eacute;duction de la tumorig&eacute;nicit&eacute; chez la souris nude, indiquant que Rad9 contribue activement &agrave; la maladie. Rad9 taux de prot&eacute;ines &eacute;taient &eacute;lev&eacute;s dans 153 de 339 &eacute;chantillons de biopsie tumeur de la prostate humaine examin&eacute;s et d&eacute;tectables dans seulement deux des 52 tissus non canc&eacute;reuses de la prostate. Il y avait une forte corr&eacute;lation entre l&#39;abondance de prot&eacute;ine Rad9 et stade de cancer. Rad9 niveau de prot&eacute;ine peut donc fournir un biomarqueur pour le cancer de la prostate avanc&eacute; et est directement li&eacute;e &agrave; la maladie, ce qui sugg&egrave;re le potentiel de d&eacute;veloppement de nouveaux outils diagnostiques, pronostiques et th&eacute;rapeutiques bas&eacute;es sur la d&eacute;tection ou la manipulation de l&#39;abondance de prot&eacute;ine Rad9.

Rad9 a un r&ocirc;le fonctionnel dans la canc&eacute;rogen&egrave;se la prostate humaine.

Il cancro della prostata &egrave; attualmente il tipo pi&ugrave; comune di tumore trovato negli uomini americani, diverso dal cancro della pelle ed &egrave; la seconda causa di morte per cancro nei maschi. Perch&eacute; il ciclo cellulare proteine checkpoint stabilizzano il genoma, &egrave; stato studiato il rapporto di una tale proteina, Rad9, al cancro della prostata. Abbiamo trovato che quattro linee di cancro alla prostata cella (CWR22, DU145, LNCaP e PC-3), rispetto al PrEC normali cellule della prostata, hanno modo anomalo alti livelli di proteina Rad9. La regione di estremit&agrave; 3&#39; dell&#39;introne 2 di Rad9 nelle cellule DU145 &egrave; hypermethylated alle isole di CpG e trattamento con 5&#39;-aza-2&#39;-deoxycytidine Ripristina quasi normali livelli di metilazione riduce Rad9 abbondanza di proteine. Southern blot analisi indicano che le cellule PC-3 contengono un numero di copia Rad9 amplificato. Pertanto, forniamo le prove che Rad9 livelli sono elevati in cellule tumorali della prostata dovuto almeno in parte a aberrante metilazione o amplificazione del gene. L&#39;efficacia di piccolo RNA interferente per abbassare i livelli di proteina Rad9 nelle cellule CWR22, DU145 e PC-3 correlato con riduzione del tumorigenicity in topi nudi, indicando che Rad9 contribuisce attivamente alla malattia. Livelli di proteina Rad9 erano alti in 153 del 339 campioni bioptici di tumore prostatico umano esaminati e rilevabili in solo 2 dei 52 tessuti non tumorali della prostata. C&#39;era una forte correlazione tra Rad9 abbondanza della proteina e stadio del tumore. Rad9 livello di proteina pu&ograve; quindi fornire un biomarcatore per il cancro della prostata avanzato e causale &egrave; correlata alla malattia, suggerendo il potenziale per lo sviluppo di strumenti diagnostici, prognostici e terapeutici romanzo basati sul rilevamento o manipolazione di Rad9 abbondanza di proteine.

Rad9 ha un ruolo funzionale nella carcinogenesi della prostata umana.

Rad9 is required for the activity of the genotoxin-induced checkpoint signaling pathway to control cell cycle progress and maintain genomic stability. In the fission yeast S. Pombe and chicken cells, the Rad9 gene is essential for prevention of premature chromosomal condensation (PCC:S/M checkpoint control). However, precise features in the S/M checkpoint controlled by Rad9 in mammalian cells are still not clear. In this study, mouse embryonic stem (ES) cells with a targeted deletion of Mrad9, the counterpart of S. pombe rad9, were used to evaluate the detailed function of Mrad9 in S/M checkpoint control. We found that Mrad9 deletion from ES cells led to failure of S/M checkpoint control across the entire S phase, and was alleviated by introducing mouse or human Rad9 into the Mrad9-deleted cells. We also found that the phosphorylation sites on Tyr28 and the C-terminus of Rad9 are required for this checkpoint control. Moreover, the DNA replication inhibitor hydroxyurea (HU) induced cdc2 Tyr15 phosphorylation and increased 14-3-3sigma protein levels in Mrad9(+)/(+) ES cells, but failed to do so in Mrad9(-)/(-) ES cells. Taken together, these results suggest that phosphorylation of Rad9 plays a critical role in the activation of the S/M checkpoint, and that downstream proteins cdc2 and 14-3-3sigma mediate this function.

Phosphorylation sites on Tyr28 and the C-terminus of Rad9 are required for inhibition of premature chromosomal condensation across the entire S phase.

Rad9 est requis pour l&#39;activit&eacute; de la point de contr&ocirc;le induite par le genotoxin voie pour contr&ocirc;ler la progression du cycle cellulaire et de maintenir la stabilit&eacute; g&eacute;nomique de signalisation. Dans la levure S. Pombe et cellules de poulet, le g&egrave;ne Rad9 est essentiel pour la pr&eacute;vention de la condensation chromosomique pr&eacute;matur&eacute;e (PCC:S / M point de contr&ocirc;le commande). Cependant, des caract&eacute;ristiques pr&eacute;cises dans le point de contr&ocirc;le de S/M contr&ocirc;l&eacute;e par Rad9 en cellules mammif&egrave;res sont toujours pas claires. Dans cette &eacute;tude, les cellules souches embryonnaires (ES) de souris avec une suppression cibl&eacute;e de Mrad9, l&#39;&eacute;quivalent de S. pombe rad9, ont &eacute;t&eacute; utilis&eacute;s pour &eacute;valuer la fonction d&eacute;taill&eacute;e de Mrad9 en S/M point de contr&ocirc;le. Nous avons trouv&eacute; que Mrad9 suppression de cellules d&#39;ES a conduit &agrave; l&#39;&eacute;chec du S/M point de contr&ocirc;le sur toute la phase S et a &eacute;t&eacute; att&eacute;nu&eacute;e par l&#39;introduction de la souris ou Rad9 humaine dans les cellules Mrad9-supprim&eacute;. Nous avons &eacute;galement constat&eacute; que les sites de phosphorylation sur Tyr28 et l&#39;extr&eacute;mit&eacute; C-terminale de Rad9 sont n&eacute;cessaires pour ce point de contr&ocirc;le. En outre, l&#39;ADN r&eacute;plication inhibiteur hydroxyur&eacute;e (HU) induit la phosphorylation de Tyr15 cdc2 et augmentation des niveaux de prot&eacute;ine 14-3-3sigma dans les cellules ES Mrad9(+)/(+), mais n&#39;a pas fait dans les cellules ES Mrad9(-)/(-). Pris ensemble, ces r&eacute;sultats sugg&egrave;rent que la phosphorylation de Rad9 joue un r&ocirc;le crucial dans l&#39;activation de la checkpoint S/M et que les prot&eacute;ines en aval de cdc2 et 14-3-3sigma m&eacute;dier cette fonction.

Sites de phosphorylation sur Tyr28 et l&#39;extr&eacute;mit&eacute; C-terminale de Rad9 sont n&eacute;cessaires pour l&#39;inhibition de la condensation chromosomique pr&eacute;matur&eacute;e &agrave; travers toute la phase S.

Rad9 &egrave; necessaria per l&#39;attivit&agrave; del checkpoint genotoxin-induced signaling pathway per controllare l&#39;avanzamento del ciclo cellulare e mantenere la stabilit&agrave; genomica. Nel lievito di fissione S. Pombe e cellule di pollo, il gene Rad9 &egrave; essenziale per la prevenzione di condensazione cromosomica prematura (PCC:S / controllo del checkpoint M). Precise caratteristiche in S/M checkpoint controllati da Rad9 in cellule di mammifero sono tuttavia ancora non &egrave; chiari. In questo studio, le cellule staminali embrionali (ES) del mouse con un&#39;eliminazione mirata di Mrad9, la controparte di S. pombe rad9, sono state utilizzate per valutare la funzione dettagliata del Mrad9 nel controllo del punto di arresto S/M. Abbiamo trovato Mrad9 cancellazione da cellule ES ha portato al fallimento del controllo del checkpoint S/M in tutta l&#39;intera fase di S che &egrave; stato alleviato introducendo mouse o Rad9 umana nelle cellule del Mrad9 &egrave; stato eliminato. Abbiamo anche trovato che i siti di fosforilazione su Tyr28 e il C-terminale di Rad9 sono necessari per il controllo del punto di arresto. Inoltre, il DNA replica inibitore idrossiurea (HU) indotta da cdc2 Tyr15 fosforilazione e aumentati livelli di proteina 14-3-3sigma in cellule ES Mrad9(+)/(+), ma non &egrave; riuscito a farlo in cellule ES Mrad9(-)/(-). Presi insieme, questi risultati suggeriscono che la fosforilazione di Rad9 svolge un ruolo critico nell&#39;attivazione di checkpoint S/M e che cdc2 proteine a valle e 14-3-3sigma mediare questa funzione.

Siti di fosforilazione su Tyr28 e il C-terminale di Rad9 sono richiesti per l&#39;inibizione della condensazione cromosomica prematura in tutta l&#39;intera fase di S.

Normal glucose-stimulated insulin secretion is dependent on interactions between neighboring Î² cells. Elucidation of the reasons why this cell-to-cell contact is essential will probably yield critical insights into Î² cell maturation and function. In the central nervous system, transcellular protein interactions (i.e. interactions between proteins on the surfaces of different cells) involving neuroligins are key mediators of synaptic functional development. We previously demonstrated that Î² cells express neuroligin-2 and that insulin secretion is affected by changes in neuroligin-2 expression. Here we show that the effect of neuroligin-2 on insulin secretion is mediated by transcellular interactions. Neuroligin-2 binds with nanomolar affinity to a partner on the Î² cell surface and contributes to the increased insulin secretion brought about by Î² cell-to-Î² cell contact. It does so in a manner seemingly independent of interactions with neurexin, a known binding partner. As in the synapse, transcellular neuroligin-2 interactions enhance the functioning of the submembrane exocytic machinery. Also, as in the synapse, neuroligin-2 clustering is important. Neuroligin-2 in soluble form, rather than presented on a cell surface, decreases insulin secretion by rat islets and MIN-6 cells, most likely by interfering with endogenous neuroligin interactions. Prolonged contact with neuroligin-2-expressing cells increases INS-1 Î² cell proliferation and insulin content. These results extend the known parallels between the synaptic and Î² cell secretory machineries to extracellular interactions. Neuroligin-2 interactions are one of the few transcellular protein interactions thus far identified that directly enhance insulin secretion. Together, these results indicate a significant role for transcellular neuroligin-2 interactions in the establishment of Î² cell function.

Transcellular neuroligin-2 interactions enhance insulin secretion and are integral to pancreatic Î² cell function.

S&eacute;cr&eacute;tion d&#39;insuline stimul&eacute;e par le glucose normale d&eacute;pend des interactions entre les cellules &beta; voisines. &Eacute;lucidation des raisons pour lesquelles ce contact cellule-cellule est essentiel donnera probablement un aper&ccedil;u critique de maturation des cellules &beta; et la fonction. Dans le syst&egrave;me nerveux central, interactions prot&eacute;ine transcellulaire (c.-&agrave;-d. les interactions entre les prot&eacute;ines &agrave; la surface des cellules diff&eacute;rentes) impliquant neuroligines sont des m&eacute;diateurs cl&eacute;s de d&eacute;veloppement fonctionnel synaptique. Nous avons pr&eacute;c&eacute;demment d&eacute;montr&eacute; que les cellules &beta; expriment neuroligines-2 et que la s&eacute;cr&eacute;tion d&#39;insuline est affect&eacute;e par des changements dans l&#39;expression neuroligines-2. Ici, nous montrons que l&#39;effet des neuroligines-2 sur la s&eacute;cr&eacute;tion d&#39;insuline est m&eacute;di&eacute; par des interactions transcellulaire. Neuroligines-2 se lie avec affinit&eacute; nanomolar &agrave; un partenaire sur la surface des cellules &beta; et contribue &agrave; la s&eacute;cr&eacute;tion d&#39;insuline accrue provoqu&eacute;e par le contact de cellule-&agrave;-&beta; cellulaire &beta;. Qu&#39;il le fasse d&#39;une mani&egrave;re apparemment ind&eacute;pendante des interactions avec la neurexine, un partenaire de liaison connue. Comme dans la synapse, transcellulaire neuroligines-2 interactions am&eacute;liorent le fonctionnement de la machinerie de s&eacute;cr&eacute;tion de d&eacute;gagement. En outre, comme dans la synapse, neuroligines-2 clusters est important. Neuroligines-2 sous forme soluble, plut&ocirc;t que pr&eacute;sent&eacute;s sur une surface cellulaire, diminue la s&eacute;cr&eacute;tion d&#39;insuline par les &icirc;lots de rat et MIN-6 cellules, tr&egrave;s probables en interf&eacute;rant avec les interactions des neuroligines endog&egrave;ne. Un contact prolong&eacute; avec les cellules exprimant neuroligines-2 augmente INS-1 cellule prolif&eacute;ration et insuline teneur en &beta;. Ces r&eacute;sultats s&#39;&eacute;tendent les parall&egrave;les connues entre la cellule synaptique et &beta; s&eacute;cr&eacute;toires machineries aux interactions extracellulaires. Interactions neuroligines-2 sont une des quelques interactions prot&eacute;ine transcellulaire jusqu&#39;ici identifi&eacute;es qui am&eacute;liorent directement la s&eacute;cr&eacute;tion d&#39;insuline. Ensemble, ces r&eacute;sultats indiquent un r&ocirc;le significatif pour transcellulaire neuroligines-2 interactions dans la mise en place de la fonction cellulaire &beta;.

Transcellulaire neuroligines-2 interactions am&eacute;liorent la s&eacute;cr&eacute;tion d&#39;insuline et font partie int&eacute;grante de la fonction cellulaire &beta; pancr&eacute;atique.

Secrezione di insulina glucosio stimolato normale dipende dalle interazioni tra cellule &beta; adiacenti. Delucidazione dei motivi per cui questo contatto cellula-cellula &egrave; essenziale produrr&agrave; probabilmente critiche intuizioni nella maturazione delle cellule &beta; e funzione. Nel sistema nervoso centrale, transcellular interazioni della proteina (cio&egrave; le interazioni tra proteine sulla superficie delle cellule differenti) che coinvolge neuroligins sono mediatori chiave dello sviluppo funzionale sinaptico. Abbiamo precedentemente dimostrato che le cellule &beta; esprimono neuroligin-2 e che la secrezione di insulina &egrave; influenzata dai cambiamenti nell&#39;espressione di neuroligin-2. Qui ci mostrano che l&#39;effetto di neuroligin-2 sulla secrezione di insulina &egrave; mediata da interazioni transcellular. Neuroligin-2 si lega con nanomolari affinit&agrave; con un partner sulla superficie delle cellule &beta; e contribuisce alla secrezione di insulina aumentata provocata da contatto cellula-&beta; cellula &beta;. Lo fa in maniera apparentemente indipendente di interazioni con neurexin, un noto Associazione partner. Come nelle sinapsi, transcellular neuroligin-2 interazioni migliorano il funzionamento della macchina, exocytic submembrane. Inoltre, come nelle sinapsi, neuroligin-2 clustering &egrave; importante. Neuroligin-2 in forma solubile, piuttosto che presenta sulla superficie delle cellule, diminuisce la secrezione di insulina da isolotti di ratto e MIN-6 celle, molto probabilmente interferendo con le interazioni neuroligin endogena. Prolungato contatto con le cellule che esprimono neuroligin-2 aumenta INS-1 &beta; cellula proliferazione e insulina contenuto. Questi risultati si estendono i noti parallelismi tra la cella sinaptica e &beta; secretive macchinari per interazioni extracellulare. Neuroligin-2 interazioni sono uno dei pochi interazioni proteina transcellular finora identificate che migliorano direttamente la secrezione di insulina. Insieme, questi risultati indicano un ruolo significativo per le interazioni di neuroligin-2 transcellular nello stabilimento della funzione delle cellule &beta;.

Article	Year
Rad9 has a functional role in human prostate carcinogenesis. Cancer research\| PubMed ID: 18316588	2008
Phosphorylation sites on Tyr28 and the C-terminus of Rad9 are required for inhibition of premature chromosomal condensation across the entire S phase. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology\| PubMed ID: 18769057	2008
Transcellular neuroligin-2 interactions enhance insulin secretion and are integral to pancreatic Î² cell function. The Journal of biological chemistry\| PubMed ID: 22528485	2012

Charles Zhang

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