Stratification of pancreatic tissue samples for molecular studies: RNA-based cellular annotation procedure

Abstract Background/objectives Meaningful profiling of pancreatic cancer samples is particularly challenging due to their complex cellular composition. Beyond tumor cells, surgical biopsies contain desmoplastic stroma with infiltrating inflammatory cells, adjacent normal parenchyma, and "non-pancreatic tissues". The risk of misinterpretation rises when the heterogeneous cancer tissues are sub-divided into smaller fragments for multiple analytic procedures. Pre-analytic histological evaluation is the best option to characterize pancreatic tissue samples. Our aim was to develop a complement or alternative procedure to determine the cellular composition of pancreatic cancerous biopsies, basing on intra-analytic molecular annotation. A standard process for sample stratification at a molecular level does not yet exist. Particularly in the case of retrospective or data depository-based studies, when hematoxylin-eosin stained sections are not available, it supports the correct interpretation of expression profiles. Methods A five-gene transcriptional signature ( RNA CellStrat) was defined that allows cell type-specific stratification of pancreatic tissues. Testing biopsy material from biobanks with this procedure demonstrated high correspondence of molecular (qRT-PCR and microarray) and histologic (hematoxylin-eosin stain) evaluations. Results Notably, about a quarter of randomly selected samples (tissue fragments) were exposed as inappropriate for subsequent clinico-pathological interpretation. Conclusions Via immediate intra-analytical procedure, our RNA-based stratification RNA CellStrat increases the accuracy and reliability of the conclusions drawn from diagnostic and prognostic molecular information

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

Genome-Wide Profiling of MicroRNAs in Adipose Mesenchymal Stem Cell Differentiation and Mouse Models of Obesity

In recent years, there has been accumulating evidence that microRNAs are key regulator molecules of gene expression. The cellular processes that are regulated by microRNAs include e.g. cell proliferation, programmed cell death and cell differentiation. Adipocyte differentiation is a highly regulated cellular process for which several important regulating factors have been discovered, but still not all are known to fully understand the underlying mechanisms. In the present study, we analyzed the expression of 597 microRNAs during the differentiation of mouse mesenchymal stem cells into terminally differentiated adipocytes by real-time RT-PCR. In total, 66 miRNAs were differentially expressed in mesenchymal stem cell-derived adipocytes compared to the undifferentiated progenitor cells. To further study the regulation of these 66 miRNAs in white adipose tissue in vivo and their dependence on PPARγ activity, mouse models of genetically or diet induced obesity as well as a mouse line expressing a dominant negative PPARγ mutant were employed

ReishiMax, mushroom based dietary supplement, inhibits adipocyte differentiation, stimulates glucose uptake and activates AMPK

<p>Abstract</p> <p>Background</p> <p>Obesity is a health hazard which is closely associated with various complications including insulin resistance, hypertension, dyslipidemia, atherosclerosis, type 2 diabetes and cancer. In spite of numerous preclinical and clinical interventions, the prevalence of obesity and its related disorders are on the rise demanding an urgent need for exploring novel therapeutic agents that can regulate adipogenesis. In the present study, we evaluated whether a dietary supplement ReishiMax (RM), containing triterpenes and polysaccharides extracted from medicinal mushroom <it>Ganoderma lucidum</it>, affects adipocyte differentiation and glucose uptake in 3T3-L1 cells.</p> <p>Methods</p> <p>3T3-L1 pre-adipocytes were differentiated into adipocytes and treated with RM (0-300 μg/ml). Adipocyte differentiation/lipid uptake was evaluated by oil red O staining and triglyceride and glycerol concentrations were determined. Gene expression was evaluated by semi-quantitative RT-PCR and Western blot analysis. Glucose uptake was determined with [³H]-glucose.</p> <p>Results</p> <p>RM inhibited adipocyte differentiation through the suppresion of expression of adipogenic transcription factors peroxisome proliferator-activated receptor-γ (PPAR-γ), sterol regulatory element binding element protein-1c (SREBP-1c) and CCAAT/enhancer binding protein-α (C/EBP-α). RM also suppressed expression of enzymes and proteins responsible for lipid synthesis, transport and storage: fatty acid synthase (FAS), acyl-CoA synthetase-1 (ACS1), fatty acid binding protein-4 (FABP4), fatty acid transport protein-1 (FATP1) and perilipin. RM induced AMP-activated protein kinase (AMPK) and increased glucose uptake by adipocytes.</p> <p>Conclusion</p> <p>Our study suggests that RM can control adipocyte differentiation and glucose uptake. The health benefits of ReishiMax warrant further clinical studies.</p

Cross species comparison of C/EBPα and PPARγ profiles in mouse and human adipocytes reveals interdependent retention of binding sites

<p>Abstract</p> <p>Background</p> <p>The transcription factors peroxisome proliferator activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα) are key transcriptional regulators of adipocyte differentiation and function. We and others have previously shown that binding sites of these two transcription factors show a high degree of overlap and are associated with the majority of genes upregulated during differentiation of murine 3T3-L1 adipocytes.</p> <p>Results</p> <p>Here we have mapped all binding sites of C/EBPα and PPARγ in human SGBS adipocytes and compared these with the genome-wide profiles from mouse adipocytes to systematically investigate what biological features correlate with retention of sites in orthologous regions between mouse and human. Despite a limited interspecies retention of binding sites, several biological features make sites more likely to be retained. First, co-binding of PPARγ and C/EBPα in mouse is the most powerful predictor of retention of the corresponding binding sites in human. Second, vicinity to genes highly upregulated during adipogenesis significantly increases retention. Third, the presence of C/EBPα consensus sites correlate with retention of both factors, indicating that C/EBPα facilitates recruitment of PPARγ. Fourth, retention correlates with overall sequence conservation within the binding regions independent of C/EBPα and PPARγ sequence patterns, indicating that other transcription factors work cooperatively with these two key transcription factors.</p> <p>Conclusions</p> <p>This study provides a comprehensive and systematic analysis of what biological features impact on retention of binding sites between human and mouse. Specifically, we show that the binding of C/EBPα and PPARγ in adipocytes have evolved in a highly interdependent manner, indicating a significant cooperativity between these two transcription factors.</p

Reducing Glycosphingolipid Content in Adipose Tissue of Obese Mice Restores Insulin Sensitivity, Adipogenesis and Reduces Inflammation

Adipose tissue is a critical mediator in obesity-induced insulin resistance. Previously we have demonstrated that pharmacological lowering of glycosphingolipids and subsequently GM3 by using the iminosugar AMP-DNM, strikingly improves glycemic control. Here we studied the effects of AMP-DNM on adipose tissue function and inflammation in detail to provide an explanation for the observed improved glucose homeostasis. Leptin-deficient obese (LepOb) mice were fed AMP-DNM and its effects on insulin signalling, adipogenesis and inflammation were monitored in fat tissue. We show that reduction of glycosphingolipid biosynthesis in adipose tissue of LepOb mice restores insulin signalling in isolated ex vivo insulin-stimulated adipocytes. We observed improved adipogenesis as the number of larger adipocytes was reduced and expression of genes like peroxisome proliferator-activated receptor (PPAR) γ, insulin responsive glucose transporter (GLUT)-4 and adipsin increased. In addition, we found that adiponectin gene expression and protein were increased by AMP-DNM. As a consequence of this improved function of fat tissue we observed less inflammation, which was characterized by reduced numbers of adipose tissue macrophages (crown-like structures) and reduced levels of the macrophage chemo attractants monocyte-chemoattractant protein-1 (Mcp-1/Ccl2) and osteopontin (OPN). In conclusion, pharmacological lowering of glycosphingolipids by inhibition of glucosylceramide biosynthesis improves adipocyte function and as a consequence reduces inflammation in adipose tissue of obese animals

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