Carsten Sticht

Yes-Associated Protein Up-regulates Jagged-1 and Activates the NOTCH Pathway in Human Hepatocellular Carcinoma

BACKGROUND & AIMS Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP activity are unclear. METHODS By using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as a downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAP(S127A)), and measured protein levels in HCC, colorectal and pancreatic tumor samples from patients. RESULTS Human HCC cell lines and mouse hepatocytes that overexpress YAP(S127A) up-regulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEA domain family member 4 (TEAD4); TEAD4 binding required the Mst1/2 but not β-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and correlated with shorter survival times of patients with HCC or colorectal cancer. CONCLUSIONS The transcriptional regulator YAP up-regulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer. Transcript profiling: microarray information was deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jxepvsumwosqkve&acc=GSE35004).

Gremlin 1, frizzled-related protein, and dkk-1 are key regulators of human articular cartilage homeostasis

OBJECTIVE The development of osteoarthritis (OA) may be caused by activation of hypertrophic differentiation of articular chondrocytes. Healthy articular cartilage is highly resistant to hypertrophic differentiation, in contrast to other hyaline cartilage subtypes, such as growth plate cartilage. The purpose of this study was to elucidate the molecular mechanism responsible for the difference in the propensity of human articular cartilage and growth plate cartilage to undergo hypertrophic differentiation. METHODS Whole-genome gene-expression microarray analysis of healthy human growth plate and articular cartilage derived from the same adolescent donors was performed. Candidate genes, which were enriched in the articular cartilage, were validated at the messenger RNA (mRNA) and protein levels and examined for their potential to inhibit hypertrophic differentiation in two models. In addition, we studied a possible genetic association with OA. RESULTS Pathway analysis demonstrated decreased Wnt signaling in articular cartilage as compared to growth plate cartilage. This was at least partly due to increased expression of the bone morphogenetic protein and Wnt antagonists Gremlin 1, Frizzled-related protein (FRP), and Dkk-1 at the mRNA and protein levels in articular cartilage. Supplementation of these proteins diminished terminal hypertrophic differentiation without affecting chondrogenesis in long-bone explant cultures and chondrogenically differentiating human mesenchymal stem cells. Additionally, we found that single-nucleotide polymorphism rs12593365, which is located in a genomic control region of GREM1, was significantly associated with a 20% reduced risk of radiographic hip OA in 2 population-based cohorts. CONCLUSION Taken together, our study identified Gremlin 1, FRP, and Dkk-1 as natural brakes on hypertrophic differentiation in articular cartilage. As hypertrophic differentiation of articular cartilage may contribute to the development of OA, our findings may open new avenues for therapeutic intervention.

miRWalk database for miRNA-target interactions.

miRWalk (http://mirwalk.uni-hd.de/) is a publicly available comprehensive resource, hosting the predicted as well as the experimentally validated microRNA (miRNA)-target interaction pairs. This database allows obtaining the possible miRNA-binding site predictions within the complete sequence of all known genes of three genomes (human, mouse, and rat). Moreover, it also integrates many novel features such as a comparative platform of miRNA-binding sites resulting from ten different prediction datasets, a holistic view of genetic networks of miRNA-gene pathway, and miRNA-gene-Online Mendelian Inheritance in Man disorder interactions, and unique experimentally validated information (e.g., cell lines, diseases, miRNA processing proteins). In this chapter, we describe a schematic workflow on how one can access the stored information from miRWalk and subsequently summarize its applications.

MuRF1-dependent Regulation of Systemic Carbohydrate Metabolism as Revealed from Transgenic Mouse Studies

Under various pathophysiological muscle-wasting conditions, such as diabetes and starvation, a family of ubiquitin ligases, including muscle-specific RING-finger protein 1 (MuRF1), are induced to target muscle proteins for degradation via ubiquitination. We have generated transgenic mouse lines over-expressing MuRF1 in a skeletal muscle-specific fashion (MuRF1-TG mice) in an attempt to identify the in vivo targets of MuRF1. MuRF1-TG lines were viable, had normal fertility and normal muscle weights at eight weeks of age. Comparison of quadriceps from MuRF1-TG and wild type mice did not reveal elevated multi-ubiquitination of myosin as observed in human patients with muscle wasting. Instead, MuRF1-TG mice expressed lower levels of pyruvate dehydrogenase (PDH), a mitochondrial key enzyme in charge of glycolysis, and of its regulator PDK2. Furthermore, yeast two-hybrid interaction studies demonstrated the interaction of MuRF1 with PDH, PDK2, PDK4, PKM2 (all participating in glycolysis) and with phosphorylase beta (PYGM) and glycogenin (both regulating glycogen metabolism). Consistent with the idea that MuRF1 may regulate carbohydrate metabolism, MuRF1-TG mice had twofold elevated insulin blood levels and lower hepatic glycogen contents. To further examine MuRF1s role for systemic carbohydrate regulation, we performed glucose tolerance tests (GTT) in wild type and MuRF1-TG mice. During GTT, MuRF1-TG mice developed striking hyperinsulinaemia and hepatic glycogen stores, that were depleted at basal levels, became rapidly replenished. Taken together, our data demonstrate that MuRF1 expression in skeletal muscle re-directs glycogen synthesis to the liver and stimulates pancreatic insulin secretion, thereby providing a regulatory feedback loop that connects skeletal muscle metabolism with the liver and the pancreas during metabolic stress.

A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling

Significance Plant growth and development depend on the biosynthesis and remodeling of the cell wall. To coordinate these two processes, surveillance mechanisms have evolved to monitor the state of the cell wall. The brassinosteroid (BR) hormone signaling pathway plays an essential role in growth control and regulates the expression of a plethora of cell wall-related genes. We have previously shown that feedback signaling from the wall can modulate the outputs of the BR pathway, ensuring cell wall homeostasis and integrity. Here, we identified a receptor-like protein (RLP44), which mediates the activation of BR signaling through direct interaction with the BR coreceptor BAK1. Thus, RLP44 integrates cell wall surveillance with hormone signaling to control cell wall integrity and growth. The brassinosteroid (BR) signaling module is a central regulator of plant morphogenesis, as indicated by the large number of BR-responsive cell wall-related genes and the severe growth defects of BR mutants. Despite a detailed knowledge of the signaling components, the logic of this auto-/paracrine signaling module in growth control remains poorly understood. Recently, extensive cross-talk with other signaling pathways has been shown, suggesting that the outputs of BR signaling, such as gene-expression changes, are subject to complex control mechanisms. We previously provided evidence for a role of BR signaling in a feedback loop controlling the integrity of the cell wall. Here, we identify the first dedicated component of this feedback loop: a receptor-like protein (RLP44), which is essential for the compensatory triggering of BR signaling upon inhibition of pectin de-methylesterification in the cell wall. RLP44 is required for normal growth and stress responses and connects with the BR signaling pathway, presumably through a direct interaction with the regulatory receptor-like kinase BAK1. These findings corroborate a role for BR in controlling the sensitivity of a feedback signaling module involved in maintaining the physico-chemical homeostasis of the cell wall during cell expansion.

Quantification of glomerular number and size distribution in normal rat kidneys using magnetic resonance imaging

BACKGROUND Glomerular number and size are important risk factors for chronic kidney disease (CKD) and cardiovascular disease and have traditionally been estimated using invasive techniques. Here, we report a novel technique to count and size every glomerulus in the rat kidney using magnetic resonance imaging (MRI). METHODS The ferromagnetic nature of cationized ferritin allowed visualization of single glomeruli in high-resolution susceptibility-weighted MRI. A segmentation algorithm was used to identify and count all glomeruli within the whole kidney. To prove our concept, we estimated total glomerular number and mean glomerular volume of each kidney using design-based stereology. RESULTS The glomerular counts obtained with MRI agreed well with estimates obtained using traditional methods [MRI, 32 785 (3117); stereology, 35 132 (3123)]. For the first time, the glomerular volume distribution for the entire kidney is shown. Additionally, the method is substantially faster than the current methods. CONCLUSIONS MRI provides a new method for measuring these important microanatomical markers of disease risk and leads the way to in vivo analysis of these parameters, including longitudinal studies of animal models of CKD.

Recurrent coamplification of cytoskeleton-associated genes EMS1 and SHANK2 with CCND1 in oral squamous cell carcinoma.

Chromosomal band 11q13 is frequently amplified in oral squamous cell carcinoma (OSCC) and assumed to be critically involved in tumor initiation and progression by proto‐oncogene activation. Though cyclin D1 (CCND1) is supposed to be the most relevant oncogene, several additional putative candidate genes are inside this chromosomal region, for which their actual role in tumorigenesis still needs to be elucidated. To characterize the 11q13 amplicon in detail, 40 OSCCs were analyzed by comparative genomic hybridization to DNA microarrays (matrix‐CGH) containing BAC clones derived from chromosomal band 11q13. This high‐resolution approach revealed a consistent amplicon about 1.7 Mb in size including the CCND1 oncogene. Seven BAC clones covering FGF3, EMS1, and SHANK2 were shown to be frequently coamplified inside the CCND1 amplicon. Subsequent analysis of tissue microarrays by FISH revealed amplification frequencies of 36.8% (88/239) for CCND1, 34.3% (60/175) for FGF3, 37.4% (68/182) for EMS1, and 36.3% (61/168) for SHANK2. Finally, quantitative mRNA expression analysis demonstrated consistent overexpression of CCND1 in all tumors and of EMS1 and SHANK2 in a subset of specimens with 11q13 amplification, but no expression of FGF3 in any of the cases. Our study underlines the critical role of CCND1 in OSCC development and additionally points to the functionally related genes EMS1 and SHANK2, both encoding for cytoskeleton‐associated proteins, which are frequently coamplified with CCND1 and therefore could cooperatively contribute to OSCC pathogenesis.

Sonic Hedgehog-activated engineered blood vessels enhance bone tissue formation

Large bone defects naturally regenerate via a highly vascularized tissue which progressively remodels into cartilage and bone. Current approaches in bone tissue engineering are restricted by delayed vascularization and fail to recapitulate this stepwise differentiation toward bone tissue. Here, we use the morphogen Sonic Hedgehog (Shh) to induce the in vitro organization of an endothelial capillary network in an artificial tissue. We show that endogenous Hedgehog activity regulates angiogenic genes and the formation of vascular lumens. Exogenous Shh further induces the in vitro development of the vasculature (vascular lumen formation, size, distribution). Upon implantation, the in vitro development of the vasculature improves the in vivo perfusion of the artificial tissue and is necessary to contribute to, and enhance, the formation of de novo mature bone tissue. Similar to the regenerating callus, the artificial tissue undergoes intramembranous and endochondral ossification and forms a trabecular-like bone organ including bone-marrow-like cavities. These findings open the door for new strategies to treat large bone defects by closely mimicking natural endochondral bone repair.

Liver sinusoidal endothelium: A microenvironment-dependent differentiation program in rat including the novel junctional protein liver endothelial differentiation-associated protein-1†

Liver sinusoidal endothelium (LSEC) is a prime example of organ‐specific microvascular differentiation and functions. Disease‐associated capillarization of LSEC in vivo and dedifferentiation of LSEC in vitro indicate the importance of the hepatic microenvironment. To identify the LSEC‐specific molecular differentiation program in the rat we used a two‐sided gene expression profiling approach comparing LSEC freshly isolated ex vivo with both lung microvascular endothelial cells (LMEC) and with LSEC cultured for 42 hours. The LSEC signature consisted of 48 genes both down‐regulated in LMEC and in LSEC upon culture (fold change >7 in at least one comparison); quantitative reverse‐transcription polymerase chain reaction confirmation of these genes included numerous family members and signaling pathway‐associated molecules. The LSEC differentiation program comprised distinct sets of growth (Wnt2, Fzd4, 5, 9, Wls, vascular endothelial growth factors [VEGFR] 1, 2, 3, Nrp2) and transcription factors (Gata4, Lmo3, Tcfec, Maf) as well as endocytosis‐related (Stabilin‐1/2, Lyve1, and Ehd3) and cytoskeleton‐associated molecules (Rnd3/RhoE). Specific gene induction in cultured LSEC versus freshly isolated LSEC as well as LMEC (Esm‐1, Aatf) and up‐regulation of gene expression to LMEC levels (CXCR4, Apelin) confirmed true transdifferentiation of LSEC in vitro. In addition, our analysis identified a novel 26‐kDa single‐pass transmembrane protein, liver endothelial differentiation‐associated protein (Leda)‐1, that was selectively expressed in all liver endothelial cells and preferentially localized to the abluminal cell surface. Upon forced overexpression in MDCK cells, Leda‐1 was sorted basolaterally to E‐cadherin‐positive adherens junctions, suggesting functional involvement in cell adhesion and polarity. Conclusion: Comparative microvascular analysis in rat identified a hepatic microenvironment‐dependent LSEC‐specific differentiation program including the novel junctional molecule Leda‐1. HEPATOLOGY 2010

Dynamic Mathematical Modeling of IL13-Induced Signaling in Hodgkin and Primary Mediastinal B-Cell Lymphoma Allows Prediction of Therapeutic Targets

Primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) share a frequent constitutive activation of JAK (Janus kinase)/STAT signaling pathway. Because of complex, nonlinear relations within the pathway, key dynamic properties remained to be identified to predict possible strategies for intervention. We report the development of dynamic pathway models based on quantitative data collected on signaling components of JAK/STAT pathway in two lymphoma-derived cell lines, MedB-1 and L1236, representative of PMBL and cHL, respectively. We show that the amounts of STAT5 and STAT6 are higher whereas those of SHP1 are lower in the two lymphoma cell lines than in normal B cells. Distinctively, L1236 cells harbor more JAK2 and less SHP1 molecules per cell than MedB-1 or control cells. In both lymphoma cell lines, we observe interleukin-13 (IL13)-induced activation of IL4 receptor α, JAK2, and STAT5, but not of STAT6. Genome-wide, 11 early and 16 sustained genes are upregulated by IL13 in both lymphoma cell lines. Specifically, the known STAT-inducible negative regulators CISH and SOCS3 are upregulated within 2 hours in MedB-1 but not in L1236 cells. On the basis of this detailed quantitative information, we established two mathematical models, MedB-1 and L1236 model, able to describe the respective experimental data. Most of the model parameters are identifiable and therefore the models are predictive. Sensitivity analysis of the model identifies six possible therapeutic targets able to reduce gene expression levels in L1236 cells and three in MedB-1. We experimentally confirm reduction in target gene expression in response to inhibition of STAT5 phosphorylation, thereby validating one of the predicted targets.