S.6.1 β-catenin is a central mediator in SSc

Background. β-catenin is the central integrator of canonical Wnt signalling. Since recent evidence suggests a central role of Wnts in fibrosis, we examined the β-catenin/Wnt pathway in SSc and focused on the role of β-catenin in fibroblast activation. Methods. We performed qPCR for several Wnt ligands and axin-2 to examine Wnt expression in SSc skin. We further studied protein levels of Wnt-1, -4, -10b and β-catenin by IHC. To establish the effects of β-catenin/Wnt signalling on collagen release, we created mice with fibroblast-specific stabilization of β-catenin (dEx3 β-catenin (wt/fl) × Col1a2; Cre-ER) as well as mice carrying fibroblast-specific deletion of β-catenin [Ctnnb1(fl/fl) × Col1a2; Cre-ER]. Summary of the results. We could demonstrate mRNA overexpression of Wnt-1, -2, -9a, -9b, -10a, -10b and -16 in SSc skin. Wnt-1, -4 and -10b consistently showed strong expression in SSc skin when compared with healthy skin. On protein level, however, Wnt-4 was indistinguishable between SSc patients and healthy controls, whereas Wnt-1 and Wnt-10b protein levels were increased in SSc skin. The overexpression of Wnt-1 and Wnt-10b resulted in a prominent nuclear accumulation of β-catenin in fibroblasts. Finally, increased mRNA levels of the target gene axin-2 confirmed the activation of canonical Wnt signalling. In dEx3 β-catenin (wt/ex) mice, we addressed the consequences of enhanced Wnt signalling and increased accumulation of β-catenin in SSc. We selectively targeted β-catenin in fibroblasts. Cre-activated dEx3 β-catenin (wt/fl) × Col1a2; Cre-ER mice showed massive and spontaneous dermal thickening even 2 weeks after Cre activation. Eight weeks after Cre-activation, skin thickening cumulated at 102.6% (P < 0.001). In line with the dermal thickening, hydroxyproline content and myofibroblast counts showed strong increases. To test the therapeutic potential of targeting β-catenin/Wnt signaling, we created Ctnnb1(fl/fl) x Col1a2;Cre-ER mice to specifically delete β-catenin in fibroblasts. After Cre activation and β-catenin deletion in fibroblasts, mice were challenged with bleomycin subcutaneously for 4 weeks. We found that Cre-activated Ctnnb1(fl/fl) × Col1a2; Cre-ER mice were protected from bleomycin-induced dermal with a reduction of skin thickening by 71% (P < 0.05). Conclusions. We demonstrated a prominent activation of canonical Wnt signalling in SSc with nuclear accumulation of β-catenin in fibroblasts and activation of the target gene axin-2. Our results showed that fibroblast-specific stabilization of β-catenin resulted in enhanced collagen release, whereas deletion of β-catenin potently reduced collagen production. Together, our findings highlight a key role of β-catenin in fibroblast activation and fibrosis. Thus, β-catenin may be promising molecular target for anti-fibrotic therapie

Mesodermal fate decisions of a stem cell: the Wnt switch

Stem cells are a powerful resource for cell-based transplantation therapies in osteodegenerative disorders, but before some kinds of stem cells can be applied clinically, several aspects of their expansion and differentiation need to be better controlled. Wnt molecules and members of the Wnt signaling cascade have been ascribed a role in both these processes in vitro as well as normal development in vivo. However some results are controversial. In this review we will present the hypothesis that both canonical and non-canonical signaling are involved in mesenchymal cell fate regulation, such as adipogenesis, chondrogenesis and osteogenesis, and that in vitro it is a timely switch between the two that specifies the identity of the differentiating cell. We will specifically focus on the in vitro differentiation of adipocytes, chondrocytes and osteoblasts contrasting embryonic and mesenchymal stem cells as well as the role of Wnts in mesenchymal fate specification during embryogenesis

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)1.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field

Omentum and bone marrow: how adipocyte‐rich organs create tumour microenvironments conducive for metastatic progression

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/134464/1/obr12450_am.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/134464/2/obr12450.pd