RESUMEN
The contractile phenotype of smooth muscle cells (SMCs) is transcriptionally controlled by a complex of the DNA-binding protein SRF and the transcriptional co-activator MYOCD. The pathways that activate expression of Myocd and of SMC structural genes in mesenchymal progenitors are diverse, reflecting different intrinsic and extrinsic signaling inputs. Taking the ureter as a model, we analyzed whether Notch signaling, a pathway previously implicated in vascular SMC development, also affects visceral SMC differentiation. We show that mice with a conditional deletion of the unique Notch mediator RBPJ in the undifferentiated ureteric mesenchyme exhibit altered ureter peristalsis with a delayed onset, and decreased contraction frequency and intensity at fetal stages. They also develop hydroureter 2 weeks after birth. Notch signaling is required for precise temporal activation of Myocd expression and, independently, for expression of a group of late SMC structural genes. Based on additional expression analyses, we suggest that a mesenchymal JAG1-NOTCH2/NOTCH3 module regulates visceral SMC differentiation in the ureter in a biphasic and bimodal manner, and that its molecular function differs from that in the vascular system.
Asunto(s)
Diferenciación Celular , Miocitos del Músculo Liso/metabolismo , Transducción de Señal , Uréter/metabolismo , Actinas/genética , Actinas/metabolismo , Animales , Diferenciación Celular/efectos de los fármacos , Diaminas/farmacología , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/deficiencia , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Proteína Jagged-1/genética , Proteína Jagged-1/metabolismo , Masculino , Ratones , Ratones Noqueados , Miocitos del Músculo Liso/citología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Receptores Notch/metabolismo , Transducción de Señal/efectos de los fármacos , Tiazoles/farmacología , Transactivadores/genética , Transactivadores/metabolismo , Uréter/citología , Uréter/crecimiento & desarrollo , Vísceras/citología , Vísceras/metabolismoRESUMEN
The TBX18 transcription factor regulates patterning and differentiation programs in the primordia of many organs yet the molecular complexes in which TBX18 resides to exert its crucial transcriptional function in these embryonic contexts have remained elusive. Here, we used 293 and A549 cells as an accessible cell source to search for endogenous protein interaction partners of TBX18 by an unbiased proteomic approach. We tagged endogenous TBX18 by CRISPR/Cas9 targeted genome editing with a triple FLAG peptide, and identified by anti-FLAG affinity purification and subsequent LC-MS analysis the ZMYM2 protein to be statistically enriched together with TBX18 in both 293 and A549 nuclear extracts. Using a variety of assays, we confirmed the binding of TBX18 to ZMYM2, a component of the CoREST transcriptional corepressor complex. Tbx18 is coexpressed with Zmym2 in the mesenchymal compartment of the developing ureter of the mouse, and mutations in TBX18 and in ZMYM2 were recently linked to congenital anomalies in the kidney and urinary tract (CAKUT) in line with a possible in vivo relevance of TBX18-ZMYM2 protein interaction in ureter development.
Asunto(s)
Núcleo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteómica/métodos , Transducción de Señal/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/metabolismo , Células A549 , Animales , Proteínas de Unión al ADN/genética , Desarrollo Embrionario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Técnicas de Sustitución del Gen , Humanos , Ratones , Mutación , Embarazo , Unión Proteica , Proteínas de Dominio T Box/genética , Factores de Transcripción/genética , Transfección , Uréter/embriología , Uréter/metabolismo , Anomalías Urogenitales/genética , Anomalías Urogenitales/metabolismo , Reflujo Vesicoureteral/genética , Reflujo Vesicoureteral/metabolismoRESUMEN
The urothelium is a stratified epithelium that lines the inner surface of the components of the urinary drainage system. It is composed of a layer of basal cells, one or several layers of intermediate cells, and a layer of large luminal superficial or umbrella cells. In the mouse, only a small set of markers is available that allows easy molecular distinction of these urothelial cell types. Here, we analyzed expression of S100A1, a member of the S100 family of calcium-binding proteins, in the urothelium of the two major organs of the murine urinary tract, the ureter and the bladder. Using RNA in situ hybridization analysis, we found exclusive expression of S100a1 mRNA in luminal cells of the ureter from embryonic day (E)17.5 onwards and of the bladder from E15.5 to adulthood. Immunofluorescence analysis showed that expression of S100A1 protein is confined to terminally differentiated superficial cells of both the ureter and bladder where it localized to the nucleus and cytoplasm. We conclude that S100A1 is a suitable marker for mature superficial cells in the urothelial lining of the drainage system of the developing and mature mouse.
Asunto(s)
Proteínas S100/metabolismo , Uréter , Urotelio , Animales , Proteínas de Unión al Calcio/análisis , Diferenciación Celular , Ratones , ARN/análisis , ARN Mensajero/metabolismo , Uréter/citología , Uréter/metabolismo , Vejiga Urinaria , Urotelio/citologíaRESUMEN
From fly to mammals, the Smaug/Samd4 family of prion-like RNA-binding proteins control gene expression by destabilizing and/or repressing the translation of numerous target transcripts. However, the regulation of its activity remains poorly understood. We show that Smaug's protein levels and mRNA repressive activity are downregulated by Hedgehog signaling in tissue culture cells. These effects rely on the interaction of Smaug with the G-protein coupled receptor Smoothened, which promotes the phosphorylation of Smaug by recruiting the kinase Fused. The activation of Fused and its binding to Smaug are sufficient to suppress its ability to form cytosolic bodies and to antagonize its negative effects on endogenous targets. Importantly, we demonstrate in vivo that HH reduces the levels of smaug mRNA and increases the level of several mRNAs downregulated by Smaug. Finally, we show that Smaug acts as a positive regulator of Hedgehog signaling during wing morphogenesis. These data constitute the first evidence for a post-translational regulation of Smaug and reveal that the fate of several mRNAs bound to Smaug is modulated by a major signaling pathway.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Proteínas de Unión al ARN/genética , Receptores Acoplados a Proteínas G/genética , Proteínas Represoras/metabolismo , Receptor Smoothened/genéticaRESUMEN
Tumours are complex ecosystems composed of different types of cells that communicate and influence each other. While the critical role of stromal cells in affecting tumour growth is well established, the impact of mutant cancer cells on healthy surrounding tissues remains poorly defined. Here, using mouse intestinal organoids, we uncover a paracrine mechanism by which intestinal cancer cells reactivate foetal and regenerative YAP-associated transcriptional programmes in neighbouring wildtype epithelial cells, rendering them adapted to thrive in the tumour context. We identify the glycoprotein thrombospondin-1 (THBS1) as the essential factor that mediates non-cell-autonomous morphological and transcriptional responses. Importantly, Thbs1 is associated with bad prognosis in several human cancers. This study reveals the THBS1-YAP axis as the mechanistic link mediating paracrine interactions between epithelial cells in intestinal tumours.