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1.  Ataxin-10 is part of a cachexokine cocktail triggering cardiac metabolic dysfunction in cancer cachexia 
Molecular Metabolism  2015;5(2):67-78.
Cancer cachexia affects the majority of tumor patients and significantly contributes to high mortality rates in these subjects. Despite its clinical importance, the identity of tumor-borne signals and their impact on specific peripheral organ systems, particularly the heart, remain mostly unknown.
Methods and results
By combining differential colon cancer cell secretome profiling with large-scale cardiomyocyte phenotyping, we identified a signature panel of seven “cachexokines”, including Bridging integrator 1, Syntaxin 7, Multiple inositol-polyphosphate phosphatase 1, Glucosidase alpha acid, Chemokine ligand 2, Adamts like 4, and Ataxin-10, which were both sufficient and necessary to trigger cardiac atrophy and aberrant fatty acid metabolism in cardiomyocytes. As a prototypical example, engineered secretion of Ataxin-10 from non-cachexia-inducing cells was sufficient to induce cachexia phenotypes in cardiomyocytes, correlating with elevated Ataxin-10 serum levels in murine and human cancer cachexia models.
As Ataxin-10 serum levels were also found to be elevated in human cachectic cancer patients, the identification of Ataxin-10 as part of a cachexokine cocktail now provides a rational approach towards personalized predictive, diagnostic and therapeutic measures in cancer cachexia.
•Cancer cachexia induces remodeling of the heart.•Tumor-borne secreted factors mediate cardiomyocyte atrophy.•Selective cachexokines, including Bin1, Stx7, Minpp1, Gaa, Ccl2, Adamtsl4, and Atxn10 provoke aberrant cardiac FA metabolism.•Ataxin-10 levels are elevated under cachectic conditions in mice and tumor patients.
PMCID: PMC4735665  PMID: 26909315
Cancer cachexia; Ataxin-10; Cardiac dysfunction; Fatty acid metabolism
2.  Measures of kidney function by minimally invasive techniques correlate with histological glomerular damage in SCID mice with adriamycin-induced nephropathy 
Scientific Reports  2015;5:13601.
Maximising the use of preclinical murine models of progressive kidney disease as test beds for therapies ideally requires kidney function to be measured repeatedly in a safe, minimally invasive manner. To date, most studies of murine nephropathy depend on unreliable markers of renal physiological function, exemplified by measuring blood levels of creatinine and urea, and on various end points necessitating sacrifice of experimental animals to assess histological damage, thus counteracting the principles of Replacement, Refinement and Reduction. Here, we applied two novel minimally invasive techniques to measure kidney function in SCID mice with adriamycin-induced nephropathy. We employed i) a transcutaneous device that measures the half-life of intravenously administered FITC-sinistrin, a molecule cleared by glomerular filtration; and ii) multispectral optoacoustic tomography, a photoacoustic imaging device that directly visualises the clearance of the near infrared dye, IRDye 800CW carboxylate. Measurements with either technique showed a significant impairment of renal function in experimental animals versus controls, with significant correlations with the proportion of scarred glomeruli five weeks after induction of injury. These technologies provide clinically relevant functional data and should be widely adopted for testing the efficacies of novel therapies. Moreover, their use will also lead to a reduction in experimental animal numbers.
PMCID: PMC4556979  PMID: 26329825
3.  Transcriptional co-factor Transducin beta-like (TBL) 1 acts as a checkpoint in pancreatic cancer malignancy 
EMBO Molecular Medicine  2015;7(8):1048-1062.
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer fatalities in Western societies, characterized by high metastatic potential and resistance to chemotherapy. Critical molecular mechanisms of these phenotypical features still remain unknown, thus hampering the development of effective prognostic and therapeutic measures in PDAC. Here, we show that transcriptional co-factor Transducin beta-like (TBL) 1 was over-expressed in both human and murine PDAC. Inactivation of TBL1 in human and mouse pancreatic cancer cells reduced cellular proliferation and invasiveness, correlating with diminished glucose uptake, glycolytic flux, and oncogenic PI3 kinase signaling which in turn could rescue TBL1 deficiency-dependent phenotypes. TBL1 deficiency both prevented and reversed pancreatic tumor growth, mediated transcriptional PI3 kinase inhibition, and increased chemosensitivity of PDAC cells in vivo. As TBL1 mRNA levels were also found to correlate with PI3 kinase levels and overall survival in a cohort of human PDAC patients, TBL1 was identified as a checkpoint in the malignant behavior of pancreatic cancer and its expression may serve as a novel molecular target in the treatment of human PDAC.
PMCID: PMC4551343  PMID: 26070712
gemcitabine; pancreatic cancer; PI3 kinase; TBL1; tumor metabolism
4.  Homo sapiens exhibit a distinct pattern of CNV genes regulation: an important role of miRNAs and SNPs in expression plasticity 
Scientific Reports  2015;5:12163.
Gene expression regulation is a complex and highly organized process involving a variety of genomic factors. It is widely accepted that differences in gene expression can contribute to the phenotypic variability between species, and that their interpretation can aid in the understanding of the physiologic variability. CNVs and miRNAs are two major players in the regulation of expression plasticity and may be responsible for the unique phenotypic characteristics observed in different lineages. We have previously demonstrated that a close interaction between these two genomic elements may have contributed to the regulation of gene expression during evolution. This work presents the molecular interactions between CNV and non CNV genes with miRNAs and other genomic elements in eight different species. A comprehensive analysis of these interactions indicates a unique nature of human CNV genes regulation as compared to other species. By using genes with short 3′ UTR that abolish the “canonical” miRNA-dependent regulation, as a model, we demonstrate a distinct and tight regulation of human genes that might explain some of the unique features of human physiology. In addition, comparison of gene expression regulation between species indicated that there is a significant difference between humans and mice possibly questioning the effectiveness of the latest as experimental models of human diseases.
PMCID: PMC4503977  PMID: 26178010
5.  Alteration of mRNA and microRNA expression profiles in rat muscular type vasculature in early postnatal development 
Scientific Reports  2015;5:11106.
The vascular system is characterized by a high degree of plasticity. In particular, functional and structural remodeling of the arterial system takes place during early postnatal development. However, the mechanisms providing such alterations in the rapidly growing organisms are poorly understood, especially for the peripheral vasculature. To explore this, we performed mRNA- and miRNA microarray analysis on muscular type saphenous arteries of young (10–12 days) and adult (2–3 months) rats. Thirty-eight significant pathways (such as oxidative phosphorylation, MAPK signaling, metabolism, cell cycle, DNA replication and focal adhesion) were obtained on differentially regulated genes during postnatal development. Many differentially regulated genes were determined as target- and miRNA-hubs. We also found 92 miRNAs differentially expressed in arteries of young and adult rats. Several significantly regulated pathways were found on these regulated miRNAs. Interestingly, these biological cascades also contain those significantly enriched pathways that were previously identified based on the differently expressed genes. Our data indicate that the expression of many genes involved in the regulation of pathways that are relevant for different functions in arteries may be under the control of miRNAs and these miRNAs regulate the functional, and structural remodeling occurring in the vascular system during early postnatal development.
PMCID: PMC4466593  PMID: 26073182
6.  Loss of the Mechanotransducer Zyxin Promotes a Synthetic Phenotype of Vascular Smooth Muscle Cells 
Exposure of vascular smooth muscle cells (VSMCs) to excessive cyclic stretch such as in hypertension causes a shift in their phenotype. The focal adhesion protein zyxin can transduce such biomechanical stimuli to the nucleus of both endothelial cells and VSMCs, albeit with different thresholds and kinetics. However, there is no distinct vascular phenotype in young zyxin-deficient mice, possibly due to functional redundancy among other gene products belonging to the zyxin family. Analyzing zyxin function in VSMCs at the cellular level might thus offer a better mechanistic insight. We aimed to characterize zyxin-dependent changes in gene expression in VSMCs exposed to biomechanical stretch and define the functional role of zyxin in controlling the resultant VSMC phenotype.
Methods and Results
DNA microarray analysis was used to identify genes and pathways that were zyxin regulated in static and stretched human umbilical artery–derived and mouse aortic VSMCs. Zyxin-null VSMCs showed a remarkable shift to a growth-promoting, less apoptotic, promigratory and poorly contractile phenotype with ≈90% of the stretch-responsive genes being zyxin dependent. Interestingly, zyxin-null cells already seemed primed for such a synthetic phenotype, with mechanical stretch further accentuating it. This could be accounted for by higher RhoA activity and myocardin-related transcription factor-A mainly localized to the nucleus of zyxin-null VSMCs, and a condensed and localized accumulation of F-actin upon stretch.
At the cellular level, zyxin is a key regulator of stretch-induced gene expression. Loss of zyxin drives VSMCs toward a synthetic phenotype, a process further consolidated by exaggerated stretch.
PMCID: PMC4599528  PMID: 26071033
gene expression; hypertension; remodeling; vascular smooth muscle cells; zyxin
7.  Tbx4 Interacts With the Short Stature Homeobox Gene Shox2 in Limb Development 
The short stature homeodomain transcription factors SHOX and SHOX2 play key roles in limb formation. To gain more insight into genes regulated by Shox2 during limb development, we analyzed expression profiles of WT and Shox2−/− mouse embryonic limbs and identified the T-Box transcription factor Tbx4 as a potential downstream target. Tbx4 is known to exert essential functions in skeletal and muscular hindlimb development. In humans, haploinsufficiency of TBX4 causes small patella syndrome, a skeletal dysplasia characterized by anomalies of the knee, pelvis, and foot.
Here, we demonstrate an inhibitory regulatory effect of Shox2 on Tbx4 specifically in the forelimbs. We also show that Tbx4 activates Shox2 expression in fore- and hindlimbs, suggesting Shox2 as a feedback modulator of Tbx4. Using EMSA studies, we find that Tbx4/TBX4 is able to bind to distinct T-box binding sites within the mouse and human Shox2/SHOX2 promoter.
Our data identifies Tbx4 as a novel transcriptional activator of Shox2 during murine fore- and hindlimb development. Tbx4 is also regulated by Shox2 specifically in the forelimb bud possibly via a feedback mechanism. These data extend our understanding of the role and regulation of Tbx4 and Shox2 in limb development and limb associated diseases.
PMCID: PMC4232222  PMID: 24347445
8.  Brown Adipose Tissue Harbors a Distinct Sub-Population of Regulatory T Cells 
PLoS ONE  2015;10(2):e0118534.
Regulatory T (Treg) cells are critical determinants of both immune responses and metabolic control. Here we show that systemic ablation of Treg cells compromised the adaptation of whole-body energy expenditure to cold exposure, correlating with impairment in thermogenic marker gene expression and massive invasion of pro-inflammatory macrophages in brown adipose tissue (BAT). Indeed, BAT harbored a unique sub-set of Treg cells characterized by a unique gene signature. As these Treg cells respond to BAT activation upon cold exposure, this study defines a BAT-specific Treg sub-set with direct implications for the regulation of energy homeostasis in response to environmental stress.
PMCID: PMC4340926  PMID: 25714366
9.  A Pilot Study to Assess the Feasibility of Transcutaneous Glomerular Filtration Rate Measurement Using Fluorescence-Labelled Sinistrin in Dogs and Cats 
PLoS ONE  2014;9(11):e111734.
In dogs and cats an assessment of renal function is often needed, however, existing methods including urine and plasma clearances are invasive, cumbersome and time consuming. This pilot study evaluated the feasibility of a transcutaneous glomerular filtration rate (GFR) measurement in dogs and cats. Additionally the optimal dose and location for the transcutaneous measurement device were investigated. Renal elimination of fluorescein-isothiocyanate-labelled sinistrin (FITC-S) was measured transcutaneously for 4 hours. The procedures were performed in awake, freely moving animals using escalating doses of FITC-S (10 mg/kg, 30 mg/kg, 50 mg/kg) with a wash-out period of at least 24 h in between. Multiple devices were placed on each animal. The resulting FITC-S disappearance curves were visually assessed to determine the most suitable location and the appropriate dose to reach an adequate transcutaneous peak signal for kinetic analysis. In both species 30 mg/kg were adequate for kinetic calculation. The most suitable place for the device was the lateral thoracic wall in dogs and the ventral abdominal wall in cats, respectively. Transcutaneous FITC-S clearance was then repeated using the optimal dose and location and in parallel with an additional plasma sinistrin clearance. Plasma elimination half-lives [min] were 26, 31 and 35, and corresponding transcutaneous elimination half-lives [min] were 26, 34 and 55, respectively in the dogs. Plasma elimination half-lives [min] were 51, 60 and 61, and corresponding transcutaneous elimination half-lives [min] were 75, 96 and 83, respectively in the cats. In conclusion, transcutaneous FITC-S clearance is a feasible method for the assessment of GFR in awake dogs and cats. It is noninvasive, well tolerated and easy to perform even in a clinical setting with results being readily available. A dose of 30 mg/kg of FITC-S seems adequate for kinetic assessment. Further studies are now needed to establish reference values and evaluate transcutaneous renal clearance in various conditions.
PMCID: PMC4244090  PMID: 25423195
10.  CaM Kinase II mediates maladaptive post-infarct remodeling and pro-inflammatory chemoattractant signaling but not acute myocardial ischemia/reperfusion injury 
EMBO Molecular Medicine  2014;6(10):1231-1245.
CaMKII was suggested to mediate ischemic myocardial injury and adverse cardiac remodeling. Here, we investigated the roles of different CaMKII isoforms and splice variants in ischemia/reperfusion (I/R) injury by the use of new genetic CaMKII mouse models. Although CaMKIIδC was upregulated 1 day after I/R injury, cardiac damage 1 day after I/R was neither affected in CaMKIIδ-deficient mice, CaMKIIδ-deficient mice in which the splice variants CaMKIIδB and C were re-expressed, nor in cardiomyocyte-specific CaMKIIδ/γ double knockout mice (DKO). In contrast, 5 weeks after I/R, DKO mice were protected against extensive scar formation and cardiac dysfunction, which was associated with reduced leukocyte infiltration and attenuated expression of members of the chemokine (C-C motif) ligand family, in particular CCL3 (macrophage inflammatory protein-1α, MIP-1α). Intriguingly, CaMKII was sufficient and required to induce CCL3 expression in isolated cardiomyocytes, indicating a cardiomyocyte autonomous effect. We propose that CaMKII-dependent chemoattractant signaling explains the effects on post-I/R remodeling. Taken together, we demonstrate that CaMKII is not critically involved in acute I/R-induced damage but in the process of post-infarct remodeling and inflammatory processes.
PMCID: PMC4287929  PMID: 25193973
apoptosis; Ca2+/calmodulin-dependent protein kinase II; cardiac remodeling; gene replacement; ischemia/reperfusion injury
11.  Neisseria meningitidis elicits a pro-inflammatory response involving IκBζ in a human blood-cerebrospinal fluid barrier model 
The human-specific, Gram-negative bacterium Neisseria meningitidis (Nm) is a leading cause of bacterial meningitis worldwide. The blood-cerebrospinal fluid barrier (BCSFB), which is constituted by the epithelial cells of the choroid plexus (CP), has been suggested as one of the potential entry sites of Nm into the CSF and can contribute to the inflammatory response during infectious diseases of the brain. Toll-like receptors (TLRs) are involved in mediating signal transduction caused by the pathogens.
Using a recently established in vitro model of the human BCSFB based on human malignant CP papilloma (HIBCPP) cells we investigated the cellular response of HIBCPP cells challenged with the meningitis-causing Nm strain, MC58, employing transcriptome and RT-PCR analysis, cytokine bead array, and enzyme-linked immunosorbent assay (ELISA). In comparison, we analyzed the answer to the closely related unencapsulated carrier isolate Nm α14. The presence of TLRs in HIBCPP and their role during signal transduction caused by Nm was studied by RT-PCR and the use of specific agonists and mutant bacteria.
We observed a stronger transcriptional response after infection with strain MC58, in particular with its capsule-deficient mutant MC58siaD−, which correlated with bacterial invasion levels. Expression evaluation and Gene Set Enrichment Analysis pointed to a NFκB-mediated pro-inflammatory immune response involving up-regulation of the transcription factor IκBζ. Infected cells secreted significant levels of pro-inflammatory chemokines and cytokines, including, among others, IL8, CXCL1-3, and the IκBζ target gene product IL6. The expression profile of pattern recognition receptors in HIBCPP cells and the response to specific agonists indicates that TLR2/TLR6, rather than TLR4 or TLR2/TLR1, is involved in the cellular reaction following Nm infection.
Our data show that Nm can initiate a pro-inflammatory response in human CP epithelial cells probably involving TLR2/TLR6 signaling and the transcriptional regulator IκBζ.
Electronic supplementary material
The online version of this article (doi:10.1186/s12974-014-0163-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4172843  PMID: 25347003
Blood-cerebrospinal fluid barrier; Cellular immune response; Choroid plexus; Host-pathogen interactions; Microarray; Neisseria meningitidis; Toll-like receptors; Transcriptomics
12.  Inhibition of Comt with tolcapone slows progression of polycystic kidney disease in the more severely affected PKD/Mhm (cy/+) substrain of the Hannover Sprague-Dawley rat 
Nephrology Dialysis Transplantation  2013;28(8):2045-2058.
Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common human inherited diseases. Modifier genes seem to modulate the disease progression and might therefore be promising drug targets. Although a number of modifier loci have been already identified, no modifier gene has been proven to be a real modifier yet.
Gene expression profiling of two substrains of the Han:SPRD rat, namely PKD/Mhm and PKD/US, both harboring the same mutation, was conducted in 36-day-old animals. Catechol-O-methyltransferase (Comt) was identified as a potential modifier gene. A 3-month treatment with tolcapone, a selective inhibitor of Comt, was carried out in PKD/Mhm and PKD/US (cy/+) animals.
Comt is localized within a known modifier locus of PKD (MOP2). The enzyme encoding gene was found upregulated in the more severely affected PKD/Mhm substrain and was hence presumed to be a putative modifier gene of PKD. The treatment with tolcapone markedly attenuated the loss of renal function, inhibited renal enlargement, shifted the size distribution of renal cysts and retarded cell proliferation, apoptosis, inflammation and fibrosis development in affected (cy/+) male and female PKD/Mhm and PKD/US rats.
Comt has been confirmed to be the first reported modifier gene for PKD and tolcapone offers a promising drug for treating PKD.
PMCID: PMC3983428  PMID: 23543593
catechol-O-methyltransferase; gene expression profiling; modifier; polycystic kidney disease; tolcapone
13.  Systemic Treatment with Erythropoietin Protects the Neurovascular Unit in a Rat Model of Retinal Neurodegeneration 
PLoS ONE  2014;9(7):e102013.
Rats expressing a transgenic polycystic kidney disease (PKD) gene develop photoreceptor degeneration and subsequent vasoregression, as well as activation of retinal microglia and macroglia. To target the whole neuroglialvascular unit, neuro- and vasoprotective Erythropoietin (EPO) was intraperitoneally injected into four –week old male heterozygous PKD rats three times a week at a dose of 256 IU/kg body weight. For comparison EPO-like peptide, lacking unwanted side effects of EPO treatment, was given five times a week at a dose of 10 µg/kg body weight. Matched EPO treated Sprague Dawley and water-injected PKD rats were held as controls. After four weeks of treatment the animals were sacrificed and analysis of the neurovascular morphology, glial cell activity and pAkt localization was performed. The number of endothelial cells and pericytes did not change after treatment with EPO or EPO-like peptide. There was a nonsignificant reduction of migrating pericytes by 23% and 49%, respectively. Formation of acellular capillaries was significantly reduced by 49% (p<0.001) or 40% (p<0.05). EPO-treatment protected against thinning of the central retina by 10% (p<0.05), a composite of an increase of the outer nuclear layer by 12% (p<0.01) and in the outer segments of photoreceptors by 26% (p<0.001). Quantification of cell nuclei revealed no difference. Microglial activity, shown by gene expression of CD74, decreased by 67% (p<0.01) after EPO and 36% (n.s.) after EPO-like peptide treatment. In conclusion, EPO safeguards the neuroglialvascular unit in a model of retinal neurodegeneration and secondary vasoregression. This finding strengthens EPO in its protective capability for the whole neuroglialvascular unit.
PMCID: PMC4094460  PMID: 25013951
14.  Progression of GFR reduction determined in conscious Dahl S hypertensive rats 
Hypertension  2013;62(1):85-90.
Sequential changes in glomerular filtration rate (GFR) during development of hypertension in the conscious Dahl salt-sensitive (SS) rat were determined using a new method for measurement. Utilizing a miniaturized device, disappearance curves of fluorescein isothiocyanate (FITC)-sinistrin were measured by transcutaneous excitation and real time detection of the emitted light through the skin. Rats with implanted femoral venous catheters (dye injection and sampling) and carotid catheters (mean arterial pressure (MAP) by telemetry) were studied while maintained on a 0.4% NaCl diet and on days 2,5,7,14 and 21 after switching to 4.0% (HS) diet. A separate group of rats were maintained on 0.4% for 21 days as a time control. MAP rose progressively from the last day of 0.4% (130±2 mmHg) reaching significance by day 5 of HS and averaged 162±7 mmHg by day 21. Urine albumin excretion was significantly elevated (3×) by day 7 of HS in SS rats. GFR became reduced on day 14 of HS falling from 1.53±0.06 ml/min/100g bwgt to 1.27±0.04. By day 21, GFR had fallen 28% to 1.1±0.04 ml/min/100g bwgt (t1/2 28.4±1.1 min.) No significant reductions of creatinine clearance (Ccre) were observed throughout the study in response to HS demonstrating the insensitivity of Ccre measurements even with creatinine measured using mass spectrometry. We conclude that the observed reduction of GFR was a consequence and not a cause of the hypertension and that this non-invasive approach could be used in these conscious SS rats for a longitudinal assessment of renal function.
PMCID: PMC3806646  PMID: 23630946
GFR; Dahl S rat; salt-sensitive hypertension; creatinine clearance
15.  Identification of Novel SHOX Target Genes in the Developing Limb Using a Transgenic Mouse Model 
PLoS ONE  2014;9(6):e98543.
Deficiency of the human short stature homeobox-containing gene (SHOX) has been identified in several disorders characterized by reduced height and skeletal anomalies such as Turner syndrome, Léri-Weill dyschondrosteosis and Langer mesomelic dysplasia as well as isolated short stature. SHOX acts as a transcription factor during limb development and is expressed in chondrocytes of the growth plates. Although highly conserved in vertebrates, rodents lack a SHOX orthologue. This offers the unique opportunity to analyze the effects of human SHOX expression in transgenic mice. We have generated a mouse expressing the human SHOXa cDNA under the control of a murine Col2a1 promoter and enhancer (Tg(Col2a1-SHOX)). SHOX and marker gene expression as well as skeletal phenotypes were characterized in two transgenic lines. No significant skeletal anomalies were found in transgenic compared to wildtype mice. Quantitative and in situ hybridization analyses revealed that Tg(Col2a1-SHOX), however, affected extracellular matrix gene expression during early limb development, suggesting a role for SHOX in growth plate assembly and extracellular matrix composition during long bone development. For instance, we could show that the connective tissue growth factor gene Ctgf, a gene involved in chondrogenic and angiogenic differentiation, is transcriptionally regulated by SHOX in transgenic mice. This finding was confirmed in human NHDF and U2OS cells and chicken micromass culture, demonstrating the value of the SHOX-transgenic mouse for the characterization of SHOX-dependent genes and pathways in early limb development.
PMCID: PMC4041798  PMID: 24887312
16.  Yes-Associated Protein Upregulates Jagged-1 and Activates the NOTCH Pathway in Human Hepatocellular Carcinoma 
Gastroenterology  2013;144(7):1530-1542.e12.
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 are unclear.
Using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as 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 (YAPS127A), and measured protein levels in HCC and colorectal and pancreatic tumor samples from patients.
Human HCC cell lines and mouse hepatocytes that overexpress YAPS127A upregulated 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 TEAD4; TEAD4 binding required Mst1/2, but not WNT-β-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and shorter survival times of patients with HCC or colorectal cancer.
The transcriptional regulator YAP upregulates 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.
PMCID: PMC3665638  PMID: 23419361
transcriptional regulators; liver cancer; colorectal cancer; pancreatic cancer
17.  MicroRNA profiling of rats with ochratoxin A nephrotoxicity 
BMC Genomics  2014;15(1):333.
Nephrotoxicity is the most prominent one among the various toxicities of ochratoxin A (OTA). MicroRNAs (miRNAs) are small non-coding RNAs that have an impact on a wide range of biological processes by regulating gene expression at post-transcriptional level or protein systhesis level. The objective of this study is to analyze miRNA profiling in the kidneys of rats gavaged with OTA.
To profile miRNAs in the kidneys of rats with OTA nephrotoxicity, high-throughput sequencing and bioinformatics approaches were applied to analyze the miRNAs in the kidney of rats following OTA treatment. A total of 409 known miRNAs and 8 novel miRNAs were identified in the kidney and the levels of the novel miRNAs were varied in response to different doses of OTA. Expression of miR-129, miR-130a, miR-130b, miR-141, miR-218b and miR-3588 were uniquely suppressed in mid dose but then elevated in high dose, with opposite expression to their target genes. The expression pattern was closely related with the “MAPK signaling pathway”. Dicer1 and Drosha were significantly suppressed, indicating an impairment of miRNA biogenesis in response to OTA.
The abrogation of miRNA maturation process suggests a new target of OTA toxicity. Moreover, the identification of the differentially expressed miRNAs provides us a molecular insight into the nephrtoxicity of OTA.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-333) contains supplementary material, which is available to authorized users.
PMCID: PMC4035064  PMID: 24885635
High throughput sequencing; Ochratoxin A; miRNA biogenesis; miRNA expression; Nephrotoxicity
18.  Forced arm use is superior to voluntary training for motor recovery and brain plasticity after cortical ischemia in rats 
Background and purpose
Both the immobilization of the unaffected arm combined with physical therapy (forced arm use, FAU) and voluntary exercise (VE) as model for enriched environment are promising approaches to enhance recovery after stroke. The genomic mechanisms involved in long-term plasticity changes after different means of rehabilitative training post-stroke are largely unexplored. The present investigation explored the effects of these physical therapies on behavioral recovery and molecular markers of regeneration after experimental ischemia.
42 Wistar rats were randomly treated with either forced arm use (FAU, 1-sleeve plaster cast onto unaffected limb at 8/10 days), voluntary exercise (VE, connection of a freely accessible running wheel to cage), or controls with no access to a running wheel for 10 days starting at 48 hours after photothrombotic stroke of the sensorimotor cortex. Functional outcome was measured using sensorimotor test before ischemia, after ischemia, after the training period of 10 days, at 3 and 4 weeks after ischemia. Global gene expression changes were assessed from the ipsi- and contralateral cortex and the hippocampus.
FAU-treated animals demonstrated significantly improved functional recovery compared to the VE-treated group. Both were superior to cage control. A large number of genes are altered by both training paradigms in the ipsi- and contralateral cortex and the hippocampus. Overall, the extent of changes observed correlated well with the functional recovery obtained. One category of genes overrepresented in the gene set is linked to neuronal plasticity processes, containing marker genes such as the NMDA 2a receptor, PKC ζ, NTRK2, or MAP 1b.
We show that physical training after photothrombotic stroke significantly and permanently improves functional recovery after stroke, and that forced arm training is clearly superior to voluntary running training. The behavioral outcomes seen correlate with patterns and extent of gene expression changes in all brain areas examined. We propose that physical training induces a fundamental change in plasticity-relevant gene expression in several brain regions that enables recovery processes. These results contribute to the debate on optimal rehabilitation strategies, and provide a valuable source of molecular entry points for future pharmacological enhancement of recovery.
PMCID: PMC3937028  PMID: 24528872
19.  Gene expression changes in spinal motoneurons of the SOD1G93A transgenic model for ALS after treatment with G-CSF 
Background: Amyotrophic lateral sclerosis (ALS) is an incurable fatal motoneuron disease with a lifetime risk of approximately 1:400. It is characterized by progressive weakness, muscle wasting, and death ensuing 3–5 years after diagnosis. Granulocyte-colony stimulating factor (G-CSF) is a drug candidate for ALS, with evidence for efficacy from animal studies and interesting data from pilot clinical trials. To gain insight into the disease mechanisms and mode of action of G-CSF, we performed gene expression profiling on isolated lumbar motoneurons from SOD1G93A mice, the most frequently studied animal model for ALS, with and without G-CSF treatment.
Results: Motoneurons from SOD1G93A mice present a distinct gene expression profile in comparison to controls already at an early disease stage (11 weeks of age), when treatment was initiated. The degree of deregulation increases at a time where motor symptoms are obvious (15 weeks of age). Upon G-CSF treatment, transcriptomic deregulations of SOD1G93A motoneurons were notably restored. Discriminant analysis revealed that SOD1 mice treated with G-CSF has a transcriptom close to presymptomatic SOD1 mice or wild type mice. Some interesting genes modulated by G-CSF treatment relate to neuromuscular function such as CCR4-NOT or Prss12.
Conclusions: Our data suggest that G-CSF is able to re-adjust gene expression in symptomatic SOD1G93A motoneurons. This provides further arguments for G-CSF as a promising drug candidate for ALS.
PMCID: PMC4299451  PMID: 25653590
motoneuron; ALS; G-CSF; neurodegeneration; mouse model; gene expression; laser microdissection
20.  CNVs-microRNAs Interactions Demonstrate Unique Characteristics in the Human Genome. An Interspecies in silico Analysis 
PLoS ONE  2013;8(12):e81204.
MicroRNAs (miRNAs) and copy number variations (CNVs) represent two classes of newly discovered genomic elements that were shown to contribute to genome plasticity and evolution. Recent studies demonstrated that miRNAs and CNVs must have co-evolved and interacted in an attempt to maintain the balance of the dosage sensitive genes and at the same time increase the diversity of dosage non-sensitive genes, contributing to species evolution. It has been previously demonstrated that both the number of miRNAs that target genes found in CNV regions as well as the number of miRNA binding sites are significantly higher than those of genes found in non-CNV regions. These findings raise the possibility that miRNAs may have been created under evolutionary pressure, as a mechanism for increasing the tolerance to genome plasticity. In the current study, we aimed in exploring the differences of miRNAs-CNV functional interactions between human and seven others species. By performing in silico whole genome analysis in eight different species (human, chimpanzee, macaque, mouse, rat, chicken, dog and cow), we demonstrate that miRNAs targeting genes located within CNV regions in humans have special functional characteristics that provide an insight into the differences between humans and other species.
PMCID: PMC3846834  PMID: 24312536
21.  Simultaneous Measurement of Kidney Function by Dynamic Contrast Enhanced MRI and FITC-Sinistrin Clearance in Rats at 3 Tesla: Initial Results 
PLoS ONE  2013;8(11):e79992.
Glomerular filtration rate (GFR) is an essential parameter of kidney function which can be measured by dynamic contrast enhanced magnetic resonance imaging (MRI-GFR) and transcutaneous approaches based on fluorescent tracer molecules (optical-GFR). In an initial study comparing both techniques in separate measurements on the same animal, the correlation of the obtained GFR was poor. The goal of this study was to investigate if a simultaneous measurement was feasible and if thereby, the discrepancies in MRI-GFR and optical-GFR could be reduced. For the experiments healthy and unilateral nephrectomised (UNX) Sprague Dawley (SD) rats were used. The miniaturized fluorescent sensor was fixed on the depilated back of an anesthetized rat. A bolus of 5 mg/100 g b.w. of FITC-sinistrin was intravenously injected. For dynamic contrast enhanced perfusion imaging (DCE-MRI) a 3D time-resolved angiography with stochastic trajectories (TWIST) sequence was used. By means of a one compartment model the excretion half-life (t1/2) of FITC-sinistrin was calculated and converted into GFR. GFR from DCE-MRI was calculated by fitting pixel-wise a two compartment renal filtration model. Mean cortical GFR and GFR by FITC-sinistrin were compared by Bland-Altman plots and pair-wise t-test. Results show that a simultaneous GFR measurement using both techniques is feasible. Mean optical-GFR was 4.34±2.22 ml/min (healthy SD rats) and 2.34±0.90 ml/min (UNX rats) whereas MRI-GFR was 2.10±0.64 ml/min (SD rats) and 1.17±0.38 ml/min (UNX rats). Differences between healthy and UNX rats were significant (p<0.05) and almost equal percentage difference (46.1% and 44.3%) in mean GFR were assessed with both techniques. Overall mean optical-GFR values were approximately twice as high compared to MRI-GFR values. However, compared to a previous study, our results showed a higher agreement. In conclusion, the possibility to use the transcutaneous method in MRI may have a huge impact in improving and validating MRI methods for GFR assessment in animal models.
PMCID: PMC3832374  PMID: 24260332
22.  In-Silico Algorithms for the Screening of Possible microRNA Binding Sites and Their Interactions 
Current Genomics  2013;14(2):127-136.
MicroRNAs (miRNAs) comprise a recently discovered class of small, non-coding RNA molecules of 21-25 nucleotides in length that regulate the gene expression by base-pairing with the transcripts of their targets i.e. protein-coding genes, leading to down-regulation or repression of the target genes. However, target gene activation has also been described. miRNAs are involved in diverse regulatory pathways, including control of developmental timing, apoptosis, cell proliferation, cell differentiation, modulation of immune response to macrophages, and organ development and are associated with many diseases, such as cancer. Computational prediction of miRNA targets is much more challenging in animals than in plants, because animal miRNAs often perform imperfect base-pairing with their target sites, unlike plant miRNAs which almost always bind their targets with near perfect complementarity. In the past years, a large number of target prediction programs and databases on experimentally validated information have been developed for animal miRNAs to fulfil the need of experimental scientists conducting miRNA research. In this review we first succinctly describe the prediction criteria (rules or principles) adapted by prediction algorithms to generate possible miRNA binding site interactions and introduce most relevant algorithms, and databases. We then summarize their applications with the help of some previously published studies. We further provide experimentally validated functional binding sites outside 3’-UTR region of target mRNAs and the resources which offer such predictions. Finally, the issue of experimental validation of miRNA binding sites will be briefly discussed.
PMCID: PMC3637677  PMID: 24082822
microRNAs; miRWalk; Target prediction; Promoter; CDS; UTR; Prediction algorithm; Database.
23.  Neuronal Expression of Glucosylceramide Synthase in Central Nervous System Regulates Body Weight and Energy Homeostasis 
PLoS Biology  2013;11(3):e1001506.
Body weight and energy homeostasis are regulated by leptin receptor interactions with gangliosides, a class of plasma membrane lipids, in forebrain neurons of mice.
Hypothalamic neurons are main regulators of energy homeostasis. Neuronal function essentially depends on plasma membrane-located gangliosides. The present work demonstrates that hypothalamic integration of metabolic signals requires neuronal expression of glucosylceramide synthase (GCS; UDP-glucose:ceramide glucosyltransferase). As a major mechanism of central nervous system (CNS) metabolic control, we demonstrate that GCS-derived gangliosides interacting with leptin receptors (ObR) in the neuronal membrane modulate leptin-stimulated formation of signaling metabolites in hypothalamic neurons. Furthermore, ganglioside-depleted hypothalamic neurons fail to adapt their activity (c-Fos) in response to alterations in peripheral energy signals. Consequently, mice with inducible forebrain neuron-specific deletion of the UDP-glucose:ceramide glucosyltransferase gene (Ugcg) display obesity, hypothermia, and lower sympathetic activity. Recombinant adeno-associated virus (rAAV)-mediated Ugcg delivery to the arcuate nucleus (Arc) significantly ameliorated obesity, specifying gangliosides as seminal components for hypothalamic regulation of body energy homeostasis.
Author Summary
Obesity is a growing health threat that affects nearly half a billion people worldwide, and its incidence rates in lower income countries are rising dramatically. As obesity is a major risk factor for type II diabetes and cardiovascular disease, significant effort has been put into the exploration of causes, prevention, and potential treatment. Recent research has demonstrated that a region of the brain called the hypothalamus is a major integrator of metabolic and nutrient signals, adapting food intake and energy expenditure to current metabolic needs. Leptin or insulin receptors located in the plasma cell membrane of neurons sense energy signals from the body. They transmit this information inside the cell, which then regulates neuronal function. In this study, we show that leptin receptors interact with gangliosides, a class of plasma membrane lipids. This interaction is a prerequisite for proper receptor activation. Consequently, ganglioside loss in hypothalamic neurons inhibits leptin receptor signal transduction in response to energy metabolites. Furthermore, mice lacking gangliosides in distinct forebrain areas, amongst them the hypothalamus, develop progressive obesity and hypothermia. Our results suggest a previously unknown regulatory mechanism of plasma membrane lipids for hypothalamic control of body weight.
PMCID: PMC3595213  PMID: 23554574
24.  Islet1 is a direct transcriptional target of the homeodomain transcription factor Shox2 and rescues the Shox2-mediated bradycardia 
Basic Research in Cardiology  2013;108(2):339.
The heart’s rhythm is initiated and regulated by a group of specialized cells in the sinoatrial node (SAN), the primary pacemaker of the heart. Abnormalities in the development of the SAN can result in irregular heart rates (arrhythmias). Although several of the critical genes important for SAN formation have been identified, our understanding of the transcriptional network controlling SAN development remains at a relatively early stage. The homeodomain transcription factor Shox2 is involved in the specification and patterning of the SAN. While the Shox2 knockout in mice results in embryonic lethality due to severe cardiac defects including improper SAN development, Shox2 knockdown in zebrafish causes a reduced heart rate (bradycardia). In order to gain deeper insight into molecular pathways involving Shox2, we compared gene expression levels in right atria of wildtype and Shox2−/− hearts using microarray experiments and identified the LIM homeodomain transcription factor Islet1 (Isl1) as one of its putative target genes. The downregulation of Isl1 expression in Shox2−/− hearts was confirmed and the affected region narrowed down to the SAN by whole-mount in situ hybridization. Using luciferase reporter assays and EMSA studies, we identified two specific SHOX2 binding sites within intron 2 of the ISL1 locus. We also provide functional evidence for Isl1 as a transcriptional target of Shox2 by rescuing the Shox2-mediated bradycardia phenotype with Isl1 using zebrafish as a model system. Our findings demonstrate a novel epistatic relationship between Shox2 and Isl1 in the heart with important developmental consequences for SAN formation and heart beat.
Electronic supplementary material
The online version of this article (doi:10.1007/s00395-013-0339-z) contains supplementary material, which is available to authorized users.
PMCID: PMC3597335  PMID: 23455426
Arrhythmia; Gene regulation; Islet1; Shox2; Sinoatrial node; Transcription factors
25.  TSC22D4 is a molecular output of hepatic wasting metabolism 
EMBO Molecular Medicine  2013;5(2):294-308.
In mammals, proper storage and distribution of lipids in and between tissues is essential for the maintenance of energy homeostasis. Here, we show that tumour growth triggers hepatic metabolic dysfunction as part of the cancer cachectic phenotype, particularly by reduced hepatic very-low-density-lipoprotein (VLDL) secretion and hypobetalipoproteinemia. As a molecular cachexia output pathway, hepatic levels of the transcription factor transforming growth factor beta 1-stimulated clone (TSC) 22 D4 were increased in cancer cachexia. Mimicking high cachectic levels of TSC22D4 in healthy livers led to the inhibition of hepatic VLDL release and lipogenic genes, and diminished systemic VLDL levels under both normal and high fat dietary conditions. Liver-specific ablation of TSC22D4 triggered hypertriglyceridemia through the induction of hepatic VLDL secretion. Furthermore, hepatic TSC22D4 expression levels were correlated with the degree of body weight loss and VLDL hypo-secretion in cancer cachexia, and TSC22D4 deficiency rescued tumour cell-induced metabolic dysfunction in hepatocytes. Therefore, hepatic TSC22D4 activity may represent a molecular rationale for peripheral energy deprivation in subjects with metabolic wasting diseases, including cancer cachexia.
PMCID: PMC3569644  PMID: 23307490
cancer cachexia; lipid metabolism; liver; transcription; TSC22D4

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