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Caparros-Martin J.A.,Institute Investigaciones Biomedicas Of Madrid Csic Uam | Caparros-Martin J.A.,Institute Salud Carlos III | Valencia M.,Institute Investigaciones Biomedicas Of Madrid Csic Uam | Reytor E.,Institute Investigaciones Biomedicas Of Madrid Csic Uam | And 11 more authors.
Human Molecular Genetics | Year: 2013

Hedgehog (Hh) signaling is involved in patterning and morphogenesis of most organs in the developing mammalian embryo. Despite many advances in understanding core components of the pathway, little is known about how the activity of the Hh pathway is adjusted in organ- and tissue-specific developmental processes. Mutations in EVC or EVC2 disrupt Hh signaling in tooth and bone development. Using mouse models, we show here that Evc and Evc2 are mutually required for localizing to primary cilia and also for maintaining their normal protein levels. Consistent with Evc and Evc2 functioning as a complex, the skeletal phenotypes in either single or double homozygous mutant mice are virtually indistinguishable. Smo translocation to the cilium was normal in Evc2-deficient chondrocytes following Hh activation with the Smo-agonist SAG. However, Gli3 recruitment to cilia tips was reduced and Sufu/Gli3 dissociation was impaired. Interestingly, we found Smo to co-precipitate with Evc/Evc2, indicating that in some cells Hh signaling requires direct interaction of Smo with the Evc/Evc2 complex. Expression of a dominantly acting Evc2 mutation previously identified in Weyer's acrodental dysostosis (Evc2δ43) caused mislocalization of Evc/Evc2δ43 within the cilium and also reproduced the Gli3-related molecular defects observed in Evc2-/- chondrocytes. Moreover, Evc silencing in Sufu-/- cells attenuated the output of the Hh pathway, suggesting that Evc/Evc2 also promote Hh signaling in the absence of Sufu. Together our data reveal that the Hh pathway involves Evc/Evc2-dependent modulations that are necessary for normal endochondral bone formation. © The Author 2012. Published by Oxford University Press. All rights reserved.

Carrillo J.,Institute Investigaciones Biomedicas Of Madrid Csic Uam | Carrillo J.,CIBER ISCIII | Gonzalez A.,Institute Investigaciones Biomedicas Of Madrid Csic Uam | Gonzalez A.,CIBER ISCIII | And 6 more authors.
Clinical and Translational Oncology | Year: 2014

Background: Dyskeratosis congenita (DC) is a rare inherited bone marrow failure syndrome with high clinical heterogeneity. Various mutations have been reported in DC patients, affecting genes that code for components of H/ACA ribonucleoproteins, proteins of the telomerase complex and components of the shelterin complex. Objectives: We aim to clarify the role of ribosome biogenesis failure in senescence induction in X-DC since some studies in animal models have reported a decrease in ribosome biogenesis as a major role in the disease. Methods: Dyskerin was depleted in normal human fibroblasts by expressing two DKC1 shRNAs. Common changes in gene expression profile between these dyskerin-depleted cells and X-DC fibroblasts were analyzed. Results: Dyskerin depletion induced early activation of the p53 pathway probably secondary to ribosome biogenesis failure. However, the p53 pathway in the fibroblasts from X-DC patients was activated only after an equivalent number of passes to AD-DC fibroblasts, in which telomere attrition in each division rendered shorter telomeres than control fibroblasts. Indeed, no induction of DNA damage was observed in dyskerin-depleted fibroblasts in contrast to X-DC or AD-DC fibroblasts suggesting that DNA damage induced by telomere attrition is responsible for p53 activation in X-DC and AD-DC fibroblasts. Moreover, p53 depletion in senescent DC fibroblasts rescued their proliferative capacity and reverted the morphological changes produced after prolonged culture. Conclusions: Our data indicate that ribosome biogenesis do not seem to play an important role in dyskeratosis congenita, conversely increasing DNA damage and activation of p53 pathway triggered by telomere shortening is the main activator of cell senescence. © Federación de Sociedades Españolas de Oncología (FESEO) 2013.

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