RESUMEN
The Wnts comprise a large class of secreted proteins that control essential developmental processes such as embryonic patterning, cell growth, migration, and differentiation. In the most well-understood "canonical" Wnt signaling pathway, Wnt binding to Frizzled receptors induces beta-catenin protein stabilization and entry into the nucleus, where it complexes with T-cell factor/lymphoid enhancer factor transcription factors to affect the transcription of target genes. In addition to the canonical pathway, evidence for several other Wnt signaling pathways has accumulated, in particular for Wnt5a, which has therefore been classified as a noncanonical Wnt family member. To study the alternative mechanisms by which Wnt proteins signal, we purified the Wnt5a protein to homogeneity. We find that purified Wnt5a inhibits Wnt3a protein-induced canonical Wnt signaling in a dose-dependent manner, not by influencing beta-catenin levels but by downregulating beta-catenin-induced reporter gene expression. The Wnt5a signal is mediated by the orphan tyrosine kinase Ror2, is pertussis toxin insensitive, and does not influence cellular calcium levels. We show that in addition to its inhibitory function, Wnt5a can also activate beta-catenin signaling in the presence of the appropriate Frizzled receptor, Frizzled 4. Thus, this study shows for the first time that a single Wnt ligand can initiate discrete signaling pathways through the activation of two distinct receptors. Based on these and additional observations, we propose a model wherein receptor context dictates Wnt signaling output. In this model, signaling by different Wnt family members is not intrinsically regulated by the Wnt proteins themselves but by receptor availability.
Asunto(s)
Proteínas Proto-Oncogénicas/farmacología , Transducción de Señal/efectos de los fármacos , Factores de Transcripción TCF/metabolismo , Proteínas Wnt/farmacología , beta Catenina/metabolismo , Secuencia de Aminoácidos , Animales , Calcio/química , Calcio/metabolismo , Cationes Bivalentes/química , Línea Celular , Receptores Frizzled/metabolismo , Regulación de la Expresión Génica , Humanos , Ratones , Datos de Secuencia Molecular , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/aislamiento & purificación , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Huérfanos Similares al Receptor Tirosina Quinasa , Proteínas Wnt/química , Proteínas Wnt/aislamiento & purificación , Proteínas Wnt/metabolismo , Proteína Wnt-5aRESUMEN
Wnt signaling has been demonstrated to regulate diverse cell processes throughout the development of the Caenorhabditis elegans embryo. This chapter describes methods that have been used to investigate some of these Wnt-dependent processes: endoderm specification, mitotic spindle orientation, and cell migration.
Asunto(s)
Bioensayo/métodos , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Caenorhabditis elegans/fisiología , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Animales , Caenorhabditis elegans/anatomía & histología , Proteínas de Caenorhabditis elegans/genética , Polaridad Celular , Proteínas Wnt/genéticaRESUMEN
Muscle stem (satellite) cells are relatively resistant to cell-autonomous aging. Instead, their endogenous signaling profile and regenerative capacity is strongly influenced by the aged P-Smad3, differentiated niche, and by the aged circulation. With respect to muscle fibers, we previously established that a shift from active Notch to excessive transforming growth factor-beta (TGF-beta) induces CDK inhibitors in satellite cells, thereby interfering with productive myogenic responses. In contrast, the systemic inhibitor of muscle repair, elevated in old sera, was suggested to be Wnt. Here, we examined the age-dependent myogenic activity of sera TGF-beta1, and its potential cross-talk with systemic Wnt. We found that sera TGF-beta1 becomes elevated within aged humans and mice, while systemic Wnt remained undetectable in these species. Wnt also failed to inhibit satellite cell myogenicity, while TGF-beta1 suppressed regenerative potential in a biphasic fashion. Intriguingly, young levels of TGF-beta1 were inhibitory and young sera suppressed myogenesis if TGF-beta1 was activated. Our data suggest that platelet-derived sera TGF-beta1 levels, or endocrine TGF-beta1 levels, do not explain the age-dependent inhibition of muscle regeneration by this cytokine. In vivo, TGF-beta neutralizing antibody, or a soluble decoy, failed to reduce systemic TGF-beta1 and rescue myogenesis in old mice. However, muscle regeneration was improved by the systemic delivery of a TGF-beta receptor kinase inhibitor, which attenuated TGF-beta signaling in skeletal muscle. Summarily, these findings argue against the endocrine path of a TGF-beta1-dependent block on muscle regeneration, identify physiological modalities of age-imposed changes in TGF-beta1, and introduce new therapeutic strategies for the broad restoration of aged organ repair.
Asunto(s)
Envejecimiento , Senescencia Celular , Músculo Esquelético/metabolismo , Células Madre/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Proteínas Wnt/metabolismo , Animales , Comunicación Celular , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Wnt5a is an important factor patterning many aspects of early development, including the lung. We find pulmonary non-canonical Wnt5a uses Ror2 to control patterning of both distal air and vascular tubulogenesis (alveolarization). Lungs with mis/overexpressed Wnt5a develop with severe pulmonary hypoplasia associated with altered expression patterns of Shh, L-CAM, fibronectin, VEGF and Flk1. This hypoplastic phenotype is rescued by either replacement of the Shh protein or inhibition of fibronectin function. We find that the effect of Wnt5a on vascular patterning is likely to be through fibronectin-mediated VEGF signaling. These results demonstrate the pivotal role of Wnt5a in directing the essential coordinated development of pulmonary airway and vasculature, by affecting fibronectin levels directly, and by affecting the fibronectin pattern of expression through its regulation of Shh. Data herein suggest that Wnt5a functions in mid-pulmonary patterning (during alveolarization), and is distinct from the Wnt canonical pathway which is more important in earlier lung development.
Asunto(s)
Vasos Sanguíneos/embriología , Vasos Sanguíneos/metabolismo , Pulmón/embriología , Sistema Respiratorio/embriología , Sistema Respiratorio/metabolismo , Proteínas Wnt/metabolismo , Animales , Vasos Sanguíneos/citología , Embrión de Pollo , Inmunohistoquímica , Hibridación in Situ , Pulmón/metabolismo , Técnicas de Cultivo de Órganos , Sistema Respiratorio/citologíaRESUMEN
Reciprocal epithelial-mesenchymal interactions shape site-specific development of skin. Here we show that site-specific HOX expression in fibroblasts is cell-autonomous and epigenetically maintained. The distal-specific gene HOXA13 is continually required to maintain the distal-specific transcriptional program in adult fibroblasts, including expression of WNT5A, a morphogen required for distal development. The ability of distal fibroblasts to induce epidermal keratin 9, a distal-specific gene, is abrogated by depletion of HOXA13, but rescued by addition of WNT5A. Thus, maintenance of appropriate HOX transcriptional program in adult fibroblasts may serve as a source of positional memory to differentially pattern the epithelia during homeostasis and regeneration.