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Transcription-independent effects of TGF-B on mitochondrial Ca2+ homeostasis

Siviero, Roberta (2011) Transcription-independent effects of TGF-B on mitochondrial Ca2+ homeostasis. PhD Thesis , Università degli Studi di Ferrara.

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      Abstract

      The rapid response of mitochondria to cellular Ca2+ signals depends on their close proximity to the ER, allowing them to sense microdomains of high [Ca2+] meeting the low affinity of the mitochondrial Ca2+ uniporter (MCU) of the inner membrane. Recent work has demonstrated that the subcellular fraction denominated Mitochondria-Associated Membranes (MAMs) may correspond to this signaling domain, as in electron micrographs it shows the apposition of the two organelles and it is enriched in Ca2+ channels and regulatory proteins. The Transforming Growth Factor-β (TGF-β) family consists of more than 30 different but structurally related polypeptides, which are known to have crucial roles in the regulation of cell proliferation, differentiation and apoptosis. The downstream effectors of TGF-β signaling are intracellular proteins called Smads that hetero-oligomerize after phosphorylation and subsequently migrate into the nucleus to influence gene expression. While much progress has been made in understanding TGF-β regulation of gene expression, the subcellular distribution of Smad proteins and their nuclearindependent activity are still incompletely understood. We have investigated the effect of the TGF-β signaling on intracellular Ca2+ homeostasis. The results showed that Smad2/3, both in HeLa cells and in liver preparations, are present in mitochondria, with a specific enrichment in the MAM fraction. Such a distribution may underlie a direct, transcription-independent role in the modulation of the ER/mitochondria Ca2+ cross-talk. This possibility has been directly investigated using aequorin-based recombinant probes targeted to the mitochondria, the ER and the cytosol. Specifically, we observed that treatment with inhibitors of the TGF-β receptor family, such as SB431542 and Dorsomorphin (Compound C) reduces agonistdependent increases of mitochondrial Ca2+ concentration [Ca2+]m, while leaving the cytosolic responses unaffected. The effects were observed also upon inhibition of protein synthesis, thus ruling out the possibility that they are due to alterations of the expression levels of Ca2+ transporters. The same effects were observed by shRNA silencing of Smads, thus involving these TGF-β transducers in the mitochondrial effects. Work is currently under way to identify the mechanism of the Ca2+ signalling alterations (intrinsic desensitization of the MCU, reduction of the electrochemical driving force, etc.). Altogether, these data demonstrate that also the TGF-β signaling pathway converges on mitochondrial checkpoint, clustering intracellular transducers in critical signaling domains and modulating Ca2+ loading and the sensitivity to growth-promoting and apoptotic challenges.

      Item Type:Thesis (PhD Thesis)
      Date:14 March 2011
      Tutor:Rimessi, Alessandro
      Coordinator:Cuneo, Antonio
      Institution:Università degli Studi di Ferrara
      PhD:XXIII Anno 2008 > FARMACOLOGIA E ONCOLOGIA MOLECOLARE
      Divisions:Dipartimento > Scienze biomediche e terapie avanzate
      Subjects:Area 06 - Scienze mediche > MED/04 Patologia generale
      Deposited on:13 Apr 2012 14:59

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