RESUMEN
During vertebrate gastrulation, canonical Wnt signaling induces the formation of neural plate border (NPB). Wnt is also thought to be required for the subsequent specification of neural crest (NC) lineage at the NPB, but the direct evidence is lacking. We found previously that the disintegrin metalloproteinase ADAM13 is required for Wnt activation and NC induction in Xenopus Here, we report that knockdown of ADAM13 or its close paralog ADAM19 severely downregulates Wnt activity at the NPB, inhibiting NC specification without affecting earlier NPB formation. Surprisingly, ADAM19 functions nonproteolytically in NC specification by interacting with ADAM13 and inhibiting its proteasomal degradation. Ectopic expression of stabilized ADAM13 mutants that function independently of ADAM19 can induce the NC marker/specifier snail2 in the future epidermis via Wnt signaling. These results unveil the essential roles of a novel protease-protease interaction in regulating a distinct wave of Wnt signaling, which directly specifies the NC lineage.
Asunto(s)
Proteínas ADAM/metabolismo , Tipificación del Cuerpo/fisiología , Cresta Neural/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus/metabolismo , Animales , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/metabolismo , Cresta Neural/embriología , Placa Neural/metabolismo , Transducción de Señal , Vía de Señalización Wnt/fisiología , Xenopus/embriologíaRESUMEN
The effect of methionine deprivation (methionine stress) on the proliferation, survival, resistance to chemotherapy, and regulation of gene and protein expression in pancreatic tumor lines is examined. Methionine stress prevents successful mitosis and promotes cell cycle arrest and accumulation of cells with multiple micronuclei with decondensed chromatin. Inhibition of mitosis correlates with CDK1 down-regulation and/or inhibition of its function by Tyr(15) phosphorylation or Thr(161) dephosphorylation. Inhibition of cell cycle progression correlates with loss of hyperphosphorylated Rb and up-regulation of p21 via p53 and/or transforming growth factor-beta (TGF-beta) activation depending on p53 status. Although methionine stress-induced toxicity is not solely dependent on p53, the gain in p21 and loss in CDK1 transcription are more enhanced in wild-type p53 tumors. Up-regulation of SMAD7, a TGF-beta signaling inhibitor, suggests that SMAD7 does not restrict the TGF-beta-mediated induction of p21, although it may prevent up-regulation of p27. cDNA oligoarray analysis indicated a pleiotropic response to methionine stress. Cell cycle and mitotic arrest is in agreement with up-regulation of NF2, ETS2, CLU, GADD45alpha, GADD45beta, and GADD45gamma and down-regulation of AURKB, TOP2A, CCNA, CCNB, PRC1, BUB1, NuSAP, IFI16, and BRCA1. Down-regulation of AREG, AGTR1, M-CSF, and EGF, IGF, and VEGF receptors and up-regulation of GNA11 and IGFBP4 signify loss of growth factor support. PIN1, FEN1, and cABL up-regulation and LMNB1, AREG, RhoB, CCNG, TYMS, F3, and MGMT down-regulation suggest that methionine stress sensitizes the tumor cells to DNA-alkylating drugs, 5-fluorouracil, and radiation. Increased sensitivity of pancreatic tumor cell lines to temozolomide is shown under methionine stress conditions and is attributed in part to diminished O(6)-methylguanine-DNA methyltransferase and possibly to inhibition of the cell cycle progression.
Asunto(s)
Adenocarcinoma/metabolismo , Proteínas de Ciclo Celular/metabolismo , Expresión Génica , Metionina/deficiencia , Neoplasias Pancreáticas/metabolismo , Adenocarcinoma/tratamiento farmacológico , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos Alquilantes/farmacología , Western Blotting , Ciclo Celular/fisiología , Línea Celular Tumoral , Proliferación Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Dacarbazina/análogos & derivados , Dacarbazina/farmacología , Fluorouracilo/farmacología , Perfilación de la Expresión Génica , Humanos , Proteínas de Neoplasias/análisis , O(6)-Metilguanina-ADN Metiltransferasa/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Neoplasias Pancreáticas/tratamiento farmacológico , Fosforilación/efectos de los fármacos , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Temozolomida , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Cell adhesion molecules such as cadherins alternate their expression throughout cranial neural crest (CNC) development, yet our understanding of the role of these molecules during CNC migration remains incomplete. The "mesenchymal" cadherin-11 is expressed in the CNC during migration yet prevents migration when overexpressed in the embryo, suggesting that a defined level of cadherin-11-mediated cell adhesion is required for migration. Here we show that members of the meltrin subfamily of ADAM metalloproteases cleave the extracellular domain of cadherin-11 during CNC migration. We show that a fragment corresponding to the putative shed form of cadherin-11 retains biological activity by promoting CNC migration in vivo, in a non-cell-autonomous manner. Additionally, cleavage of cadherin-11 does not affect binding to beta-catenin and downstream signaling events. We propose that ADAM cleavage of cadherin-11 promotes migration by modifying its ability to support cell-cell adhesion while maintaining the membrane-bound pool of beta-catenin associated with the cadherin-11 cytoplasmic domain.
Asunto(s)
Proteínas ADAM/metabolismo , Cadherinas/metabolismo , Movimiento Celular/fisiología , Isoenzimas/metabolismo , Cresta Neural , Xenopus laevis , Proteínas ADAM/genética , Animales , Cadherinas/genética , Células Cultivadas , Hibridación in Situ , Isoenzimas/genética , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Morfogénesis/fisiología , Cresta Neural/citología , Cresta Neural/fisiología , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Xenopus laevis/anatomía & histología , Xenopus laevis/embriologíaRESUMEN
ADAM19 is a member of the meltrin subfamily of ADAM metalloproteases. In Xenopus, ADAM19 is present as a maternal transcript. Zygotic expression starts during gastrulation and is apparent in the dorsal blastopore lip. ADAM19 expression through neurulation and tailbud formation becomes enriched in dorsal structures such as the neural tube, the notochord and the somites. Using morpholino knock-down, we show that a reduction of ADAM19 protein in gastrula stage embryos results in a decrease of Brachyury expression in the notochord concomitant with an increase in the dorsal markers, Goosecoid and Chordin. These changes in gene expression are accompanied by a decrease in phosphorylated AKT, a downstream target of the EGF signaling pathway, and occur while the blastopore closes at the same rate as the control embryos. During neurulation and tailbud formation, ADAM19 knock-down induces a reduction of the neural markers N-tubulin and NRP1 but not Sox2. In the somitic mesoderm, the expression of MLC is also decreased while MyoD is not. ADAM19 knockdown also reduces neural crest markers prior to cell migration. Neural crest induction is also decreased in embryos treated with an EGF receptor inhibitor suggesting that this pathway is necessary for neural crest cell induction. Using targeted knock-down of ADAM19 we show that the reduction of neural and neural crest markers is cell autonomous and that the migration if the cranial neural crest is perturbed. We further show that ADAM19 protein reduction affects somite organization, reduces 12-101 expression and perturbs fibronectin localization at the intersomitic boundary.