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.

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.

Autosomal polycystic kidney disease (ADPKD) is a frequent monogenic renal disease, characterised by fluid-filled cysts that are thought to result from multiple deregulated pathways such as cell proliferation and apoptosis. MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of many genes associated with such biological processes and human pathologies. To explore the possible regulatory role of miRNAs in PKD, the PKD/Mhm (cy/+) rat, served as a model to study human ADPKD. A parallel microarray-based approach was conducted to profile the expression changes of mRNAs and miRNAs in PKD/Mhm rats. 1,573 up- and 1,760 down-regulated genes were differentially expressed in PKD/Mhm. These genes are associated with 17 pathways (such as focal adhesion, cell cycle, ECM-receptor interaction, DNA replication and metabolic pathways) and 47 (e.g., cell proliferation, Wnt and Tgfβ signaling) Gene Ontologies. Furthermore, we found the similar expression patterns of deregulated genes between PKD/Mhm (cy/+) rat and human ADPKD, PKD1L3/L3, PKD1−/−, Hnf1α-deficient, and Glis2lacZ/lacZ models. Additionally, several differentially regulated genes were noted to be target hubs for miRNAs. We also obtained 8 significantly up-regulated miRNAs (rno-miR-199a-5p, −214, −146b, −21, −34a, −132, −31 and −503) in diseased kidneys of PKD/Mhm rats. Additionally, the binding site overrepresentation and pathway enrichment analyses were accomplished on the putative targets of these 8 miRNAs. 7 out of these 8 miRNAs and their possible interactions have not been previously described in ADPKD. We have shown a strong overlap of functional patterns (pathways) between deregulated miRNAs and mRNAs in the PKD/Mhm (cy/+) rat model. Our findings suggest that several miRNAs may be associated in regulating pathways in ADPKD. We further describe novel miRNAs and their possible targets in ADPKD, which will open new avenues to understand the pathogenesis of human ADPKD. Furthermore they could serve as a useful resource for anti-fibrotic therapeutics.

We used human fetal bone marrow-derived mesenchymal stromal cells (hfMSCs) differentiating towards chondrocytes as an alternative model for the human growth plate (GP). Our aims were to study gene expression patterns associated with chondrogenic differentiation to assess whether chondrocytes derived from hfMSCs are a suitable model for studying the development and maturation of the GP. hfMSCs efficiently formed hyaline cartilage in a pellet culture in the presence of TGFβ3 and BMP6. Microarray and principal component analysis were applied to study gene expression profiles during chondrogenic differentiation. A set of 232 genes was found to correlate with in vitro cartilage formation. Several identified genes are known to be involved in cartilage formation and validate the robustness of the differentiating hfMSC model. KEGG pathway analysis using the 232 genes revealed 9 significant signaling pathways correlated with cartilage formation. To determine the progression of growth plate cartilage formation, we compared the gene expression profile of differentiating hfMSCs with previously established expression profiles of epiphyseal GP cartilage. As differentiation towards chondrocytes proceeds, hfMSCs gradually obtain a gene expression profile resembling epiphyseal GP cartilage. We visualized the differences in gene expression profiles as protein interaction clusters and identified many protein clusters that are activated during the early chondrogenic differentiation of hfMSCs showing the potential of this system to study GP development.

OBJECTIVES: We analyzed the effects of anti-hedgehog signaling on the 18F-FDG uptake of pancreatic cancer xenografts (PCXs) using a clinically implemented positron emission tomography (PET)-computer tomography (CT) scanner with high-resolution reconstruction. METHODS: PCXs from two pancreatic cancer cell lines were developed subcutaneously in nude mice and injected intraperitoneally with a low dose of cyclopamine for 1 week. 18F-FDG PET-CT was performed using a new-generation clinical PET-CT scanner with minor modifications of the scanning protocol to adapt for small-animal imaging. The data set was reconstructed and quantified using a three-dimensional workstation. RESULTS: MiaPaCa-2 cells, which respond to cyclopamine, showed decreased 18F-FDG uptake without a change in tumor size. For hip tumors, the maximum standardized uptake value (SUVmax) was reduced by -24.5 ± 9.2%, the average SUV (SUVavg) by -33.5 ± 7.0%, and the minimum SUV (SUVmin) by -54.4 ± 11.5% (P < .05). For shoulder tumors, SUVmax was reduced by -14.7 ± 7.5%, SUVavg by -12.6 ± 6.3, and SUVmin by -30.3 ± 16.7% (P < .05). Capan-1 cells, which do not respond to cyclopamine, did not show significant SUV changes. CONCLUSIONS: The new generations of clinically implemented PET-CT scanners with high-resolution reconstruction detect a minimal response of PCX to low-dose short-term cyclopamine therapy without changes in tumor size and offer potential for preclinical translational imaging.

Background

TGF-β plays a dual role in the progression of human cancer. During the early stages of carcinogenesis, TGF-β functions as a tumor suppressor. During the late stages of tumor development, however, TGF-β can promote tumor growth and metastasis. A shift in Smad2/3 phosphorylation from the carboxy terminus to linker sites is a key event determining biological function of TGF-β in colorectal and hepatocellular carcinoma. In the present study, we investigated the potential role of differential Smad2/3 phosphorylation in gastric adenocarcinoma.

Methodology

Immunohistochemical staining with anti-P-Smad2/3C and P-Smad2/3L antibodies was performed on 130 paraffin-embedded gastric adenocarcinoma specimens. The relationship between P-Smad2/3C and P-Smad2/3L immunohistochemical score and clinicopathologic characteristics of patients was analyzed. Real time PCR was used to measure mRNA expression of Smad2 and Smad3 in cancer and surrounding non-tumor tissue.

Principal Findings

No significant P-Smad2L and/or P-Smad3L positive staining was detected in the majority of specimens (positive staining in 18/130 samples). Positive P-Smad2/3L staining was not associated with a decrease in carboxyterminal phosphorylation staining. Loss of P-Smad2C remarkably correlated with depth of tumor infiltration and poor differentiation of cancer cells in patients with gastric cancer. No correlation was detectable between P-Smad3C and clinicopathologic characteristics of gastric adenocarcinoma. However, co-staining analysis revealed that P-Smad3C co-localised with α-SMA and collagen I in gastric cancer cells, indicating a potential link between P-Smad3C and epithelial-to-mesenchymal transition of cancer. Real time PCR demonstrated reduced mRNA expression of Smad2 in gastric cancer when compared with surrounding non-tumor tissue in 15/16 patients.

Conclusions

Loss of P-Smad2C tightly correlated with cancer invasion and poor differentiation in gastric cancer. Contrary to colorectal and hepatocellular carcinoma, canonical carboxy-terminal phosphorylation, but not linker phosphorylation, of Smad2 is critical for gastric cancer.

Heparin binding epidermal growth factor (HBEGF) is expressed in podocytes and was shown to play a role in glomerular physiology. MicroRNA binding sites on the 3′UTR of HBEGF were predicted using miRWalk algorithm and followed by DNA sequencing in 103 patients diagnosed with mild or severe glomerulopathy. A single nucleotide polymorphism, miRSNP C1936T (rs13385), was identified at the 3′UTR of HBEGF that corresponds to the second base of the hsa-miR-1207-5p seed region. When AB8/13 undifferentiated podocytes were transfected with miRNA mimics of hsa-miR-1207-5p, the HBEGF protein levels were reduced by about 50%. A DNA fragment containing the miRSNP allele-1936C was cloned into the pMIR-Report Luciferase vector and co-transfected with miRNA mimics of hsa-miR-1207-5p into AB8/13 podocytes. In agreement with western blot data, this resulted in reduced luciferase expression demonstrating the ability of hsa-miR-1207-5p to directly regulate HBEGF expression. On the contrary, in the presence of the miRSNP 1936T allele, this regulation was abolished. Collectively, these results demonstrate that variant 1936T of this miRSNP prevents hsa-miR-1207-5p from down-regulating HBEGF in podocytes. We hypothesized that this variant has a functional role as a genetic modifier. To this end, we showed that in a cohort of 78 patients diagnosed with CFHR5 nephropathy (also known as C3-glomerulopathy), inheritance of miRSNP 1936T allele was significantly increased in the group demonstrating progression to chronic renal failure on long follow-up. No similar association was detected in a cohort of patients with thin basement membrane nephropathy. This is the first report associating a miRSNP as genetic modifier to a monogenic renal disorder.

Quantitative analysis of time-resolved data in primary erythroid progenitor cells reveals that a dual negative transcriptional feedback mechanism underlies the ability of STAT5 to respond to the broad spectrum of physiologically relevant Epo concentrations.

A mathematical dual feedback model of the Epo-induced JAK2/STAT5 signaling pathway was calibrated with extensive time-resolved quantitative data sets from immunoblotting, mass spectrometry and qRT–PCR experiments in primary erythroid progenitor cells.We show that the amount of nuclear phosphorylated STAT5 integrated for 60 min post Epo stimulation directly correlates with the fraction of surviving cells 24 h later.CIS and SOCS3 were identified as the most relevant transcriptional feedback regulators of JAK2/STAT5 signaling in primary erythroid progenitor cells. Applying the model, we revealed that CIS-mediated inhibitory effects are most important at low ligand concentrations, whereas SOCS3 inhibition is more effective at high ligand doses.The distinct modes of inhibition of CIS and SOCS3 at various Epo concentrations provide a strategy for achieving control of JAK2/STAT5 signaling over the entire range of physiological Epo concentrations.

Cells interpret information encoded by extracellular stimuli through the activation of intracellular signaling networks and translate this information into cellular decisions. A prime example for a system that is exposed to extremely variable ligand concentrations is the erythroid lineage. The key regulator Erythropoietin (Epo) facilitates continuous renewal of erythrocytes at low basal levels but also secures compensation in case of, e.g., blood loss through an up to 1000-fold increase in hormone concentration. The Epo receptor (EpoR) is expressed on erythroid progenitor cells at the colony forming unit erythroid (CFU-E) stage. Stimulation of these cells with Epo leads to rapid but transient activation of receptor and JAK2 phosphorylation followed by phosphorylation of the latent transcription factor STAT5. Although STAT5 is known to be an essential regulator of survival and differentiation of erythroid progenitor cells, a quantitative link between the dynamic properties of STAT5 signaling and survival decisions remained unknown. STAT5-mediated responses in CFU-E cells are modulated by multiple attenuation mechanisms that operate on different time scales. Fast-acting mechanisms such as depletion of Epo by rapid receptor turnover and recruitment of the phosphatase SHP-1 control the initial signal amplitude at the receptor level. Transcriptional feedback regulators such as suppressor of cytokine signaling (SOCS) family members CIS and SOCS3 operate at a slower time scale. Despite the ample knowledge of the individual components involved, only little is known about the specific contributions of these regulators in controlling dynamic properties of STAT5 in response to a broad range of input signals. Therefore, dynamic pathway modeling is required to understand the complex regulatory network of feedback regulators.

To address these questions, we established a dual negative feedback model of JAK2/STAT5 signaling in primary erythroid progenitor cells isolated from mouse fetal livers. We provide a large data set of JAK2/STAT5 signaling dynamics employing quantitative immunoblotting, mass spectrometry and quantitative RT–PCR measured under different perturbation conditions to calibrate our model (Figure 3). The structure of our model was constructed to comprise the minimal number of parameters necessary to explain the data. Thereby, we aimed at a model with fully identifiable parameters that are essential to obtain high predictive power. Parameter identifiability was analyzed by the profile likelihood approach. Applying this method, we could establish a dual negative feedback model of JAK2-STAT5 signaling with structurally and in most cases practically identifiable parameters.

A major bottle-neck in combining signal transduction events with cellular phenotypes is the discrepancy in the time scale and stimuli concentrations that are applied in the different experiments. The sensitivity of biochemical assays to determine phosphorylation events within minutes or hours after stimulation is usually lower than the threshold of sensitivity in assays to determine the physiological response after one or more days. Facilitated by the model, we were able to compute the integrated response of JAK2/STAT5 signaling components for experimentally unaddressable Epo concentrations. Our results demonstrate that the integrated response of pSTAT5 in the nucleus accurately correlates with the experimentally determined survival of CFU-E cells. This provides a quantitative link of the dependency of primary CFU-E cells on pSTAT5 activation dynamics. By correlation analysis, we could identify the early signaling phase (⩽1 h) of STAT5 to be the most predictive for the fraction of surviving cells, which was determined ∼24 h later. Thus, we hypothesize that as a general principle in apoptotic decisions, ligand concentrations translated into kinetic-encoded information of early signaling events downstream of receptors can be predictive for survival decisions 24 h later.

After the first hour of stimulation, it is important to constrain signaling to a residual steady-state level. Constitutive phosphorylation of the JAK2/STAT5 pathway has a crucial role in the onset of polycythemia vera (PV), a disease associated with Epo-independent erythroid differentiation. The two identified transcriptional feedback proteins, CIS and SOCS3, are responsible for adjusting the phosphorylation level of STAT5 after 1 h of stimulation. Since the Epo input signal can vary over a broad range of ligand concentrations, we asked how CIS and SOCS3 can facilitate control of STAT5 long-term phosphorylation levels over the entire physiological relevant hormone concentrations. By using model simulations, we revealed that the two feedbacks are most effective at different Epo concentration ranges. Predicted by our mathematical model, the major role of CIS in modulating STAT5 phosphorylation levels is at low, basal Epo concentrations, whereas SOCS3 is essential to control the STAT5 phosphorylation levels at high Epo doses (Figure 6). As a potential molecular mechanism of this dose-dependent inhibitory effect, we could identify the quantity of pJAK2 relative to pEpoR that increases with higher Epo concentrations. Since SOCS3 can inhibit JAK2 directly via its KIR domain to attenuate downstream STAT5 activation, SOCS3 becomes more effective with the relative increase of JAK2 activation. Hence, CIS and SOCS3 act in a concerted manner to ensure tight regulation of STAT5 responses over the broad physiological range of Epo concentrations.

In summary, our mathematical approach provided new insights into the specific function of feedback regulation in STAT5-mediated life or death decisions of primary erythroid cells. We dissected the roles of the transcriptionally induced proteins CIS and SOCS3 that operate as dual feedback with divided function thereby facilitating the control of STAT5 activation levels over the entire range of physiological Epo concentrations. The detailed understanding of the molecular processes and control distribution of Epo-induced JAK/STAT signaling can be further applied to gain insights into alterations promoting malignant hematopoietic diseases.

Cellular signal transduction is governed by multiple feedback mechanisms to elicit robust cellular decisions. The specific contributions of individual feedback regulators, however, remain unclear. Based on extensive time-resolved data sets in primary erythroid progenitor cells, we established a dynamic pathway model to dissect the roles of the two transcriptional negative feedback regulators of the suppressor of cytokine signaling (SOCS) family, CIS and SOCS3, in JAK2/STAT5 signaling. Facilitated by the model, we calculated the STAT5 response for experimentally unobservable Epo concentrations and provide a quantitative link between cell survival and the integrated response of STAT5 in the nucleus. Model predictions show that the two feedbacks CIS and SOCS3 are most effective at different ligand concentration ranges due to their distinct inhibitory mechanisms. This divided function of dual feedback regulation enables control of STAT5 responses for Epo concentrations that can vary 1000-fold in vivo. Our modeling approach reveals dose-dependent feedback control as key property to regulate STAT5-mediated survival decisions over a broad range of ligand concentrations.

Background

The β-amyloid precursor protein (APP) and the related β-amyloid precursor-like proteins (APLPs) undergo complex proteolytic processing giving rise to several fragments. Whereas it is well established that Aβ accumulation is a central trigger for Alzheimer's disease, the physiological role of APP family members and their diverse proteolytic products is still largely unknown. The secreted APPsα ectodomain has been shown to be involved in neuroprotection and synaptic plasticity. The γ-secretase-generated APP intracellular domain (AICD) functions as a transcriptional regulator in heterologous reporter assays although its role for endogenous gene regulation has remained controversial.

Results

To gain further insight into the molecular changes associated with knockout phenotypes and to elucidate the physiological functions of APP family members including their proposed role as transcriptional regulators, we performed DNA microarray transcriptome profiling of prefrontal cortex of adult wild-type (WT), APP knockout (APP-/-), APLP2 knockout (APLP2-/-) and APPsα knockin mice (APPα/α) expressing solely the secreted APPsα ectodomain. Biological pathways affected by the lack of APP family members included neurogenesis, transcription, and kinase activity. Comparative analysis of transcriptome changes between mutant and wild-type mice, followed by qPCR validation, identified co-regulated gene sets. Interestingly, these included heat shock proteins and plasticity-related genes that were both down-regulated in knockout cortices. In contrast, we failed to detect significant differences in expression of previously proposed AICD target genes including Bace1, Kai1, Gsk3b, p53, Tip60, and Vglut2. Only Egfr was slightly up-regulated in APLP2-/- mice. Comparison of APP-/- and APPα/α with wild-type mice revealed a high proportion of co-regulated genes indicating an important role of the C-terminus for cellular signaling. Finally, comparison of APLP2-/- on different genetic backgrounds revealed that background-related transcriptome changes may dominate over changes due to the knockout of a single gene.

Conclusion

Shared transcriptome profiles corroborated closely related physiological functions of APP family members in the adult central nervous system. As expression of proposed AICD target genes was not altered in adult cortex, this may indicate that these genes are not affected by lack of APP under resting conditions or only in a small subset of cells.

Vasoregression is a hallmark of vascular eye diseases but the mechanisms involved are still largely unknown. We have recently characterized a rat ciliopathy model which develops primary photoreceptor degeneration and secondary vasoregression. To improve the understanding of secondary vasoregression in retinal neurodegeneration, we used microarray techniques to compare gene expression profiles in this new model before and after retinal vasoregression. Differential gene expression was validated by quantitative RT-PCR, Western blot and immunofluorescence. Of the 157 genes regulated more than twofold, the MHC class II invariant chain CD74 yielded the strongest upregulation, and was allocated to activated microglial cells close to the vessels undergoing vasoregression. Pathway clustering identified genes of the immune system including inflammatory signaling, and components of the complement cascade upregulated during vasoregression. Together, our data suggest that microglial cells involved in retinal immune response participate in the initiation of vasoregression in the retina.

Background

External stimulations of cells by hormones, cytokines or growth factors activate signal transduction pathways that subsequently induce a re-arrangement of cellular gene expression. The analysis of such changes is complicated, as they consist of multi-layered temporal responses. While classical analyses based on clustering or gene set enrichment only partly reveal this information, matrix factorization techniques are well suited for a detailed temporal analysis. In signal processing, factorization techniques incorporating data properties like spatial and temporal correlation structure have shown to be robust and computationally efficient. However, such correlation-based methods have so far not be applied in bioinformatics, because large scale biological data rarely imply a natural order that allows the definition of a delayed correlation function.

Results

We therefore develop the concept of graph-decorrelation. We encode prior knowledge like transcriptional regulation, protein interactions or metabolic pathways in a weighted directed graph. By linking features along this underlying graph, we introduce a partial ordering of the features (e.g. genes) and are thus able to define a graph-delayed correlation function. Using this framework as constraint to the matrix factorization task allows us to set up the fast and robust graph-decorrelation algorithm (GraDe). To analyze alterations in the gene response in IL-6 stimulated primary mouse hepatocytes, we performed a time-course microarray experiment and applied GraDe. In contrast to standard techniques, the extracted time-resolved gene expression profiles showed that IL-6 activates genes involved in cell cycle progression and cell division. Genes linked to metabolic and apoptotic processes are down-regulated indicating that IL-6 mediated priming renders hepatocytes more responsive towards cell proliferation and reduces expenditures for the energy metabolism.

Conclusions

GraDe provides a novel framework for the decomposition of large-scale 'omics' data. We were able to show that including prior knowledge into the separation task leads to a much more structured and detailed separation of the time-dependent responses upon IL-6 stimulation compared to standard methods. A Matlab implementation of the GraDe algorithm is freely available at http://cmb.helmholtz-muenchen.de/grade.

CXCL13 is a key B cell chemoattractant and marker of disease activity in patients with SLE; however, the mechanism of its induction has not been identified yet. Here, we have shown that the proteoglycan biglycan triggers CXCL13 expression via TLR2/4 in macrophages and dendritic cells. In vivo, levels of biglycan were markedly elevated in the plasma and kidneys of human SLE patients and lupus-prone (MRL/lpr) mice. Overexpression of soluble biglycan in MRL/lpr mice raised plasma and renal levels of CXCL13 and caused accumulation of B cells with an enhanced B1/B cell ratio in the kidney, worsening of organ damage, and albuminuria. Importantly, biglycan also triggered CXCL13 expression and B cell infiltration in the healthy kidney. Conversely, biglycan deficiency improved systemic and renal outcome in lupus-prone mice, with lower levels of autoantibodies, less enlargement of the spleen and lymph nodes, and reduction in renal damage and albuminuria. This correlated with a marked decline in circulating and renal CXCL13 and a reduction in the number of B cells in the kidney. Collectively, our results describe what we believe to be a novel mechanism for the regulation of CXCL13 by biglycan, a host-derived ligand for TLR2/4. Blocking biglycan-TLR2/4 interactions might be a promising strategy for the management of SLE and other B cell–mediated inflammatory disease entities.

Background

Polycystic Kidney Disease is characterized by the formation of large fluid-filled cysts that eventually destroy the renal parenchyma leading to end-stage renal failure. Although remarkable progress has been made in understanding the pathologic mechanism of the disease, the precise orchestration of the early events leading to cyst formation is still unclear. Abnormal cellular proliferation was traditionally considered to be one of the primary irregularities leading to cyst initiation and growth. Consequently, many therapeutic interventions have focused on targeting this abnormal proliferation, and some have even progressed to clinical trials. However, the role of proliferation in cyst development was primarily examined at stages where cysts are already visible in the kidneys and therefore at later stages of disease development.

Methods

In this study we focused on the cystic phenotype since birth in an attempt to clarify the temporal contribution of cellular proliferation in cyst development. Using a PKD2 transgenic rat model (PKD2 (1-703)) of different ages (0-60 days after birth) we performed gene expression profiling and phenotype analysis by measuring various kidney parameters.

Results

Phenotype analysis demonstrated that renal cysts appear immediately after birth in the PKD2 transgenic rat model (PKD2 (1-703)). On the other hand, abnormal proliferation occurs at later stages of the disease as identified by gene expression profiling. Interestingly, other pathways appear to be deregulated at early stages of the disease in this PKD model. Specifically, gene expression analysis demonstrated that at day 0 the RAS system is involved. This is altered at day 6, when Wnt signaling and focal adhesion pathways are affected. However, at and after 24 days, proliferation, apoptosis, altered ECM signaling and many other factors become involved.

Conclusions

Our data suggest that cystogenesis precedes deregulation of proliferation-related pathways, suggesting that proliferation abnormalities may contribute in cyst growth rather than cyst formation.

Background

Cytokines such as TNF-alpha and IL-1beta are known for their contribution to inflammatory processes in liver. In contrast, the cytokine IL-17 has not yet been assigned a role in liver diseases. IL-17 can cooperate with TNF-alpha to induce a synergistic response on several target genes in different cell lines, but no data exist for primary hepatocytes. To enhance our knowledge on the impact of IL-17 alone and combined with TNF-alpha in primary murine hepatocytes a comprehensive microarray study was designed. IL-1beta was included as this cytokine is suggested to act in a similar manner as the combination of TNF-alpha and IL-17, especially with respect to its role in mRNA stabilization.

Results

The present microarray analysis demonstrates that primary murine hepatocytes responded to IL-17 stimulation by upregulation of chemokines and genes, which are functionally responsible to increase and sustain inflammation. Cxcl2, Nfkbiz and Zc3h12a were strongly induced, whereas the majority of the genes were only very moderately up-regulated. Promoter analysis revealed involvement of NF-kappaB in the activation of many genes. Combined stimulation of TNF-alpha/IL-17 resulted in enhanced induction of gene expression, but significantly synergistic effects could be applied only to a few genes, such as Nfkbiz, Cxcl2, Zc3h12 and Steap4. Comparison of the gene expression profile obtained after stimulation of TNF-alpha/IL-17 versus IL-1beta proposed an "IL-1beta-like effect" of the latter cytokine combination. Moreover, evidence was provided that modulation of mRNA stability may be a major mechanism by which IL-17 regulates gene expression in primary hepatocytes. This assumption was exemplarily proven for Nfkbiz mRNA for the first time in hepatocytes. Our studies also suggest that RNA stability can partially be correlated to the existence of AU rich elements, but further mechanisms like the RNase activity of the up-regulated Zc3h12a have to be considered.

Conclusions

Our microarray analysis gives new insights in IL-17 induced gene expression in primary hepatocytes highlighting the crosstalk with the NF-kappaB signaling pathway. Gene expression profile suggests IL-17 alone and in concert with TNF-alpha a role in sustaining liver inflammatory processes. IL-17 might exceed this function by RNA stabilization.

Background

Neuronal damage is correlated with vascular dysfunction in the diseased retina, but the underlying mechanisms remain controversial because of the lack of suitable models in which vasoregression related to neuronal damage initiates in the mature retinal vasculature. The aim of this study was to assess the temporal link between neuronal damage and vascular patency in a transgenic rat (TGR) with overexpression of a mutant cilia gene polycystin-2.

Methods

Vasoregression, neuroglial changes and expression of neurotrophic factors were assessed in TGR and control rats in a time course. Determination of neuronal changes was performed by quantitative morphometry of paraffin-embedded vertical sections. Vascular cell composition and patency were assessed by quantitative retinal morphometry of digest preparations. Glial activation was assessed by western blot and immunofluorescence. Expression of neurotrophic factors was detected by quantitative PCR.

Findings

At one month, number and thickness of the outer nuclear cell layers (ONL) in TGR rats were reduced by 31% (p<0.001) and 17% (p<0.05), respectively, compared to age-matched control rats. Furthermore, the reduction progressed from 1 to 7 months in TGR rats. Apoptosis was selectively detected in the photoreceptor in the ONL, starting after one month. Nevertheless, TGR and control rats showed normal responses in electroretinogram at one month. From the second month onwards, TGR retinas had significantly increased acellular capillaries (p<0.001), and a reduction of endothelial cells (p<0.01) and pericytes (p<0.01). Upregulation of GFAP was first detected in TGR retinas after 1 month in glial cells, in parallel with an increase of FGF2 (fourfold) and CNTF (60 %), followed by upregulation of NGF (40 %) at 3 months.

Interpretation

Our data suggest that TGR is an appropriate animal model for vasoregression related to neuronal damage. Similarities to experimental diabetic retinopathy render this model suitable to understand general mechanisms of maturity-onset vasoregression.

Background

MicroRNAs (miRNAs) play key roles in mammalian gene expression and several cellular processes, including differentiation, development, apoptosis and cancer pathomechanisms. Recently the biological importance of primary cilia has been recognized in a number of human genetic diseases. Numerous disorders are related to cilia dysfunction, including polycystic kidney disease (PKD). Although involvement of certain genes and transcriptional networks in PKD development has been shown, not much is known how they are regulated molecularly.

Results

Given the emerging role of miRNAs in gene expression, we explored the possibilities of miRNA-based regulations in PKD. Here, we analyzed the simultaneous expression changes of miRNAs and mRNAs by microarrays. 935 genes, classified into 24 functional categories, were differentially regulated between PKD and control animals. In parallel, 30 miRNAs were differentially regulated in PKD rats: our results suggest that several miRNAs might be involved in regulating genetic switches in PKD. Furthermore, we describe some newly detected miRNAs, miR-31 and miR-217, in the kidney which have not been reported previously. We determine functionally related gene sets, or pathways to reveal the functional correlation between differentially expressed mRNAs and miRNAs.

Conclusion

We find that the functional patterns of predicted miRNA targets and differentially expressed mRNAs are similar. Our results suggest an important role of miRNAs in specific pathways underlying PKD.

Background

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts that destroy the kidney architecture resulting in end-stage renal failure. Mutations in genes PKD1 and PKD2 account for nearly all cases of ADPKD. Increased cell proliferation is one of the key features of the disease. Several studies indicated that polycystin-1 regulates cellular proliferation through various signaling pathways, but little is known about the role played by polycystin-2, the product of PKD2. Recently, it was reported that as with polycystin-1, polycystin-2 can act as a negative regulator of cell growth by modulating the levels of the cyclin-dependent kinase inhibitor, p21 and the activity of the cyclin-dependent kinase 2, Cdk2.

Methods

Here we utilized different kidney cell-lines expressing wild-type and mutant PKD2 as well as primary tubular epithelial cells isolated from a PKD transgenic rat to further explore the contribution of the p21/Cdk2 pathway in ADPKD proliferation.

Results

Surprisingly, over-expression of wild-type PKD2 in renal cell lines failed to inactivate Cdk2 and consequently had no effect on cell proliferation. On the other hand, expression of mutated PKD2 augmented proliferation only in the primary tubular epithelial cells of a rat model but this was independent of the STAT-1/p21 pathway. On the contrary, multiple approaches revealed unequivocally that expression of the cyclin-dependent kinase inhibitor, p57KIP2, is downregulated, while p21 remains unchanged. This p57 reduction is accompanied by an increase in Cdk2 levels.

Conclusion

Our results indicate the probable involvement of p57KIP2 on epithelial cell proliferation in ADPKD implicating a new mechanism for mutant polycystin-2 induced proliferation. Most importantly, contrary to previous studies, abnormal proliferation in cells expressing mutant polycystin-2 appears to be independent of STAT-1/p21.

Background

Post-puberty deterioration of kidneys is more rapid in males than in females. To reveal the underlying molecular mechanisms for this difference, we analyzed gender-dependent gene expression in kidneys of three groups of 36 day-old rats.

Results

The number of genes exhibiting gender-dependent expression was highly influenced by the genetic background of the rat group examined. 373, 288 and 79 genes showed differential gene expression between males and females (p = 0.001) in US, Mhm and Mhm*BN rats, respectively. Of all gender dependently expressed genes, only 39 genes were differentially expressed in all tested groups and the direction of expression change was the same for those genes for all groups. The gene expression profile suggests higher metabolic and transport activities, enhanced cell proliferation, elevated oxidative stress, and altered vascular biology in males. Furthermore, elevated levels of superoxide anion (two- to three-fold) in males compared to females were detected at early puberty, but neither at pre-puberty nor at late puberty/early adulthood.

Conclusion

Our data suggest that early puberty, with gender-related elevation in oxidative stress in males, is a key compromising factor on kidneys in males.

MicroRNAs (miRNAs) constitute a recently discovered class of small non-coding RNAs that regulate expression of target genes either by decreasing the stability of the target mRNA or by translational inhibition. They are involved in diverse processes, including cellular differentiation, proliferation and apoptosis. Recent evidence also suggests their importance for cancerogenesis. By far the most important model systems in cancer research are mammalian organisms. Thus, we decided to compile comprehensive information on mammalian miRNAs, their origin and regulated target genes in an exhaustive, curated database called Argonaute (). Argonaute collects latest information from both literature and other databases. In contrast to current databases on miRNAs like miRBase::Sequences, NONCODE or RNAdb, Argonaute hosts additional information on the origin of an miRNA, i.e. in which host gene it is encoded, its expression in different tissues and its known or proposed function, its potential target genes including Gene Ontology annotation, as well as miRNA families and proteins known to be involved in miRNA processing. Additionally, target genes are linked to an information retrieval system that provides comprehensive information from sequence databases and a simultaneous search of MEDLINE with all synonyms of a given gene. The web interface allows the user to get information for a single or multiple miRNAs, either selected or uploaded through a text file. Argonaute currently has information on 839 miRNAs from human, mouse and rat.

Background

Existing animal models provide only indirect information about the pathogenesis of infections caused by indigenous gastrointestinal microflora and the kinetics of bacterial translocation. The aim of this study was to develop a novel animal model to assess bacterial translocation and intestinal barrier function in vivo.

Methods

In anaesthetized male Wistar rats, 0.5 ml of a suspension of green fluorescent protein-transfected E. coli was administered by intraluminal injection in a model of small bowel obstruction. Animals were randomly subjected to non-ischemic or ischemic bowel obstruction. Ischemia was induced by selective clamping of the terminal mesenteric vessels feeding the obstructed bowel loop. Time intervals necessary for translocation of E. coli into the submucosal stroma and the muscularis propria was assessed using intravital microscopy.

Results

Bacterial translocation into the submucosa and muscularis propria took a mean of 36 ± 8 min and 80 ± 10 min, respectively, in small bowel obstruction. Intestinal ischemia significantly accelerated bacterial translocation into the submucosa (11 ± 5 min, p < 0.0001) and muscularis (66 ± 7 min; p = 0.004). Green fluorescent protein-transfected E. coli were visible in frozen sections of small bowel, mesentery, liver and spleen taken two hours after E. coli administration.

Conclusions

Intravital microscopy of fluorescent bacteria is a novel approach to study bacterial translocation in vivo. We have applied this technique to define minimal bacterial transit time as a functional parameter of intestinal barrier function.