Novel mechanisms for regulating the TGF‐β‐/Smad‐signalling pathway

  Aberrant transforming growth factor‐β (TGF‐β) signalling is responsible for a number of developmental disorders, human cancers and fibrotic disease. Signalling is accomplished via cell‐surface serine/threonine kinase receptors and intracellular Smad transcription factors. A key regulatory step in the pathway involves phosphorylation of Smads by Ca 2+ ‐dependent kinases such as Cam kinase II (Wicks et al . 2000; Abdel‐Wahab et al . 2001). In addition, specific ubiquitination by Smurfs and other ubiquitin ligases mediates the proteosomal degradation of Smads. The aims of this study were to functionally analyse the regulation of Smads by phosphorylation in response to decorin‐mediated Ca 2+ mobilization and to use modified yeast‐2 hybrid systems for the identification of novel Smad regulatory proteins. Methods  Phosphorylation sites in Smad‐2 were mapped in vitro using Smad‐2–GST fusion proteins incubated with purified kinases in the presence of 32 P‐γ‐ATP. Sites were confirmed by site‐directed mutagenesis, and phosphopeptide‐specific antisera were used to study in vivo phosphorylation in decorin‐treated human mesangial cells. Yeast‐2 hybrid screening was performed using Smad3 1−240 as bait and a mouse brain cDNA library. Interactions were then verified by co‐expression and co‐immunoprecipitation of epitope‐tagged proteins in HEK‐293 cells. Results  In this study, we provide evidence that decorin, a naturally occurring inhibitor of the TGF‐β‐dependent fibrotic response, can disrupt glucose‐ and TGF‐β/Smad‐dependent transcriptional events in human mesangial cells through a mechanism that involves an increase in Ca 2+ signalling, the activation of Cam kinase II and ensuing phosphorylation of Smad‐2 at serine‐240. We show that decorin also induces serine‐240 phospho‐Smad hetero‐oligomerization with Smad‐4 and the nuclear localization of this complex, independently of TGF‐β receptor activation. Thus, in human mesangial cells, the mechanism of decorin‐mediated inhibition of TGF‐β signalling may involve activation of Ca 2+ signalling, the subsequent phosphorylation of Smad‐2 at a key regulatory site and the sequestration of Smad‐4 in the nucleus. In other studies, we have used two‐hybrid screening approaches to identify Smad‐specific regulatory proteins. Data will be presented on a novel interaction between Smad‐3 and a ubiquitin c‐termninal hydrolase that is likely to compete with Smurf‐mediated ubiquitination and subsequent Smad turnover. Conclusions  In summary, we conclude that an important mechanism by which decorin can play a functional role in regulating TGF‐β signalling is through decorin‐induced Ca 2+ signalling and subsequent inhibition of the Smad pathway. In addition, we have identified a novel interaction that occurs between the Smad‐3 transcription factor and a ubiquitin C‐terminal hydrolase that could lead to stabilization of the Smad‐3 protein and potentiation of TGF‐β signalling by reversal of ubiquitin‐mediated proteosomal degradation. Overall, our present findings may lead to the development of new approaches in the treatment of fibrotic diseases.