Ralf Mrowka

Molecular insights into reprogramming-initiation events mediated by the OSKM gene regulatory network.

Somatic cells can be reprogrammed to induced pluripotent stem cells by over-expression of OCT4, SOX2, KLF4 and c-MYC (OSKM). With the aim of unveiling the early mechanisms underlying the induction of pluripotency, we have analyzed transcriptional profiles at 24, 48 and 72 hours post-transduction of OSKM into human foreskin fibroblasts. Experiments confirmed that upon viral transduction, the immediate response is innate immunity, which induces free radical generation, oxidative DNA damage, p53 activation, senescence, and apoptosis, ultimately leading to a reduction in the reprogramming efficiency. Conversely, nucleofection of OSKM plasmids does not elicit the same cellular stress, suggesting viral response as an early reprogramming roadblock. Additional initiation events include the activation of surface markers associated with pluripotency and the suppression of epithelial-to-mesenchymal transition. Furthermore, reconstruction of an OSKM interaction network highlights intermediate path nodes as candidates for improvement intervention. Overall, the results suggest three strategies to improve reprogramming efficiency employing: 1) anti-inflammatory modulation of innate immune response, 2) pre-selection of cells expressing pluripotency-associated surface antigens, 3) activation of specific interaction paths that amplify the pluripotency signal.

Indirubin Derivatives Modulate TGFβ/BMP Signaling at Different Levels and Trigger Ubiquitin-Mediated Depletion of Nonactivated R-Smads

Regulatory Smads (R-Smads), Smad1/5/8 and Smad2/3, are the central mediators of TGFβ and BMP signaling pathways. Here, we screened indirubin derivatives, known kinase inhibitors, and observed strong interference with BMP signaling. We found that indirubin derivative E738 inhibited both TGFβ and BMP pathways through ubiquitin-proteasome-mediated depletion of total R-Smad pools, although phospho-R-Smad levels were initially stabilized by GSK3β and cyclin-dependent kinase inhibition. E738 also enhanced p38 and JNK phosphorylation, involved in Smad-independent TGFβ/BMP signaling. Additionally, using a small siRNA screen, we showed that depletion of ubiquitin proteases USP9x and USP34 significantly reduced total R-Smad levels, mimicking E738 treatment. In fact, both USP9x and USP34 levels were significantly reduced in E738-treated cells. Our findings not only describe the complex activity profile of the indirubin derivative E738, but also reveal a mechanism for controlling TGFβ/BMP signaling, the control of R-Smad protein levels through deubiquitination.

Identification of 2-[4-[(4-Methoxyphenyl)methoxy]-phenyl]acetonitrile and Derivatives as Potent Oct3/4 Inducers

Reprogramming somatic cells into induced-pluripotent cells (iPSCs) provides new access to all somatic cell types for clinical application without any ethical controversy arising from the use of embryonic stem cells (ESCs). Established protocols for iPSCs generation based on viral transduction with defined factors are limited by low efficiency and the risk of genetic abnormality. Several small molecules have been reported as replacements for defined transcriptional factors, but a chemical able to replace Oct3/4 allowing the generation of human iPSCs is still unavailable. Using a cell-based High Throughput Screening (HTS) campaign, we identified that 2-[4-[(4-methoxyphenyl)methoxy]phenyl]acetonitrile (1), termed O4I1, enhanced Oct3/4 expression. Structural verification and modification by chemical synthesis showed that O4I1 and its derivatives not only promoted expression and stabilization of Oct3/4 but also enhanced its transcriptional activity in diverse human somatic cells, implying the possible benefit from using this class of compounds in regenerative medicine.

Ethyl 2-((4-Chlorophenyl)amino)thiazole-4-carboxylate and Derivatives Are Potent Inducers of Oct3/4

The octamer-binding transcription factor 4 (Oct3/4) is a master gene in the transcriptional regulatory network of pluripotent cells. Repression of Oct3/4 in embryonic stem cells (ESCs) is associated with cell differentiation and loss of pluripotency, whereas forced overexpression in cooperation with other transcriptional factors, such as Nanog, Sox2, and Lin28, can reprogram somatic cells back into pluripotent cells, termed induced pluripotent stem cells (iPSCs). However, random integration and potential tumorigenic transformation caused by viral transduction limit the clinical application of iPSCs. By performing a cell-based high throughput screening (HTS) campaign, we identified several potential small molecules as inducers of Oct3/4 expression. Here we report a lead structure ethyl 2-((4-chlorophenyl)amino)-thiazole-4-carboxylate, termed O4I2, showing high activity in enforcing Oct3/4 expression. On the basis of chemical expansion, we further identified derivatives having increased activities toward Oct3/4 induction. Thus, O4I2 and its derivatives should provide a new class of small molecules suitable for iPSC generation.

Evaluation of in vivo and in vitro models of toxicity by comparison of toxicogenomics data with the literature

Toxicity affecting humans is studied by observing the effects of chemical substances in animal organisms (in vivo) or in animal and human cultivated cell lines (in vitro). Toxicogenomics studies collect gene expression profiles and histopathology assessment data for hundreds of drugs and pollutants in standardized experimental designs using different model systems. These data are an invaluable source for analyzing genome-wide drug response in biological systems. However, a problem remains that is how to evaluate the suitability of heterogeneous in vitro and in vivo systems to model the many different aspects of human toxicity. We propose here that a given model system (cell type or animal organ) is supported to appropriately describe a particular aspect of human toxicity if the set of compounds associated in the literature with that aspect of toxicity causes a change in expression of genes with a particular function in the tested model system. This approach provides candidate genes to explain the toxicity effect (the differentially expressed genes) and the compounds whose effect could be modeled (the ones producing both the change of expression in the model system and that are associated with the human phenotype in the literature). Here we present an application of this approach using a computational pipeline that integrates compound-induced gene expression profiles (from the Open TG-GATEs database) and biomedical literature annotations (from the PubMed database) to evaluate the suitability of (human and rat) in vitro systems as well as rat in vivo systems to model human toxicity.

Human microRNA-299-3p decreases invasive behavior of cancer cells by downregulation of Oct4 expression and causes apoptosis

Purpose Oct4 was reported to be one of the most important pluripotency transcription factors in the biology of stem cells including cancer stem cells, and progressed malignant cells. Here we report the investigation of gene expression control of Oct4 by selected human microRNAs and the physiological effect of Oct4 silencing in invasive cancer cells. Methods and results High throughput luciferase activity assay revealed the microRNA-299-3p to be the most effective in reducing gene expression of Oct4, which was confirmed by Western blot analysis and Oct4 promoter activity in a target luciferase assay. Furthermore, it could be demonstrated that downregulation of Oct4 by microRNAs-299-3p in breast cancer and fibrosarcoma cells lead to a decreased invasiveness in a microfluidic chip assay. Additionally, microRNA-299-3p causes apoptosis in cancer cells. Comparison with Oct4 specific siRNA transfection confirmed that this effect is primary due to the blockade of Oct4 expression. Conclusion The results suggest that microRNA-299-3p is an interesting target for potential clinical use. It may be able to decrease invasive behaviour of carcinoma cells; or even kill these cells by causing apoptosis.

Similarity in targets with REST points to neural and glioblastoma related miRNAs

There are groups of genes that need coordinated repression in multiple contexts, for example if they code for proteins that work together in a pathway or in a protein complex. Redundancy of biological regulatory networks implies that such coordinated repression might occur at both the pre- and post-transcriptional level, though not necessarily simultaneously or under the same conditions. Here, we propose that such redundancy in the global regulatory network can be detected by the overlap between the putative targets of a transcriptional repressor, as identified by a ChIP-seq experiment, and predicted targets of a microRNA (miRNA). To test this hypothesis, we used publicly available ChIP-seq data of the neural transcriptional repressor RE1 silencing transcription factor (REST) from 15 different cell samples. We found 20 miRNAs, each of which shares a significant amount of predicted targets with REST. The set of predicted associations between these 20 miRNAs and the overlapping REST targets is enriched in known miRNA targets. Many of the detected miRNAs have functions related to neural identity and glioblastoma, which could be expected from their overlap in targets with REST. We propose that the integration of experimentally determined transcription factor binding sites with miRNA-target predictions provides functional information on miRNAs.

ExActa: blood pressure.

High blood pressure plays a prominent role in the development of cardiovascular disease such as stroke and myocardial infarction. On the other hand, low blood pressure may also predict cardiovascular disease risk in subpopulations such as haemodialysis patients (Anker et al. 2016) according to a recent study in almost 5000 patients in a European cohort. In the past, researchers focused typically on knockout models when addressing genetic alterations in animals. It might be evident that fine-tuning and imbalanced gene regulation is of great importance too, but it is difficult to address. These aspects would include, for example, mechanisms that modify transcriptional and translational activity, splicing and post-translational mechanisms such as folding, protein modification, storage and other processing. These mechanisms might be determined genetically but also by non-genetic epigenetic factors. An example of a genetically determined modification found in a noncoding region is a single nucleotide polymorphism (SNP) that increases the FOXA2-bound enhancer activity and increases the risk of another complex condition, namely type II diabetes, which was observed in a ‘brute force-like’ fine mapping approach (Gaulton et al. 2015). Non-coding RNA might also contribute to gene regulation, and SNPs therein may confer increased susceptibility to the development of high blood pressure as it was suggested for the non-coding RNA CDKN2B-AS1 (Bayoglu et al. 2016). The cardiovascular system is one of the most addressed systems in modern pharmaceutical therapies. A number of feedback mechanisms are involved in the regulation of the arterial blood pressure. The regulation of blood pressure is therefore complex, and if one of the systems is out of balance, others may take a greater role. A number of interesting articles appeared in recent issues of Acta Physiologica dealing with the regulation of blood pressure at various levels; some of them include aspects of gene regulation. Knockout models are used extensively in cardiovascular research. The genetic background of the animal strain may greatly modify the phenotype. This may explain in part conflicting results in knockout studies in general. This issue was addressed by Kim et al. (2015) for the A1AR deficiency on cardiovascular and renal functional parameters. The A1 adenosine receptor (A1AR) has been proposed to be important in the tubuloglomerular feedback (TGF) as determined by stop-flow pressure measurements. The authors investigated blood pressure and heart rate and plasma renin concentrations and TGF for homozygous A1AR deficiency in the C57Bl/6 and SWR/J background. The authors find that a high NaCl intake increased arterial pressure in A1AR / mice on C57Bl/6 background, while there was less or no salt sensitivity in the SWR/J background. Plasma renin activity was not altered by A1AR deficiency in any of the investigated strains. The TGF was found to be absent in A1AR-deficient mice with SWR/J genetic background. Salt sensitivity of blood pressure is one of the enigmas in the field and may depend on ethnic background in humans (Farquhar et al. 2015). In a study conducted by Isaksson et al. (2014), the relation between plasma renin concentration, arterial blood pressure and glomerular filtration rate (GFR) depending on sodium intake was addressed in a rat model. The authors find a markedly log-linear relationship between the entities; for example, an increase in sodium intake by a factor of 10 was followed by a blood pressure increase by 5 mmHg and GFR increase by 1.4 mL min . Surprisingly, under steady state conditions plasma renin levels appear independent of renal nerves in this model, as shown by pharmacological intervention and denervation experiments. Khan et al. (2015) found that intact renal nerves are required for the following: feeding a high-fat diet causes hypertension associated with dysregulation of the arterial and cardiopulmonary baroreflexes in rats. They conclude that in obese states, neural signals arising from the kidney may contribute to a deranged autonomic control (Khan et al. 2015). Hypoxia is a key event in development of vascular beds in the normal development and also under pathological conditions such as tumour growth and is governed by gene regulatory mechanisms. The effect of high-altitude-induced hypoxia on blood pressure was investigated in humans by Calbet et al. (2014). The authors find an increase of arterial blood pressure by 13% at an altitude of about 4500 m above sea level. Vasodilatory responses after infusion of adenosine and ATP were reduced in high altitude, which might contribute to the effect of higher blood pressure. An interesting study towards the establishment of urinary proteomic biomarkers for essential hypertension was conducted by Damkjaer et al. (2014). An intrarenal mechanism important for sodium balance

Monitoring cytochrome P450 activity in living hepatocytes by chromogenic substrates in response to drug treatment or during cell maturation

The metabolic activity of hepatocytes is a central prerequisite for drug activity and a key element in drug–drug interaction. This central role in metabolism largely depends on the activity of the cytochrome P450 (CYP450) enzyme family, which is not only dependent on liver cell maturation but is also controlled in response to drug and chemical exposure. Here, we report the use of VividDye fluorogenic CYP450 substrates to directly measure and continuously monitor metabolic activity in living hepatocytes. We observed time- and dose-dependent correlation in response to established and putative CYP450 inducers acting through the aryl hydrocarbon receptor and drug combinations. Using repetitive addition of VividDye fluorogenic substrate on a daily basis, we demonstrated the new application of VividDye for monitoring the maturation and dedifferentiation of hepatic cells. Despite a lack of high specificity for individual CYP450 isoenzymes, our approach enables continuous monitoring of metabolic activity in living cells with no need to disrupt cultivation. Our assay can be integrated in in vitro liver-mimetic models for on-line monitoring and thus should enhance the reliability of these tissue model systems.

Microfluidic devices for stem-cell cultivation, differentiation and toxicity testing

The development of new drugs is time-consuming, extremely expensive and often promising drug candidates fail in late stages of the development process due to the lack of suitable tools to either predict toxicological effects or to test drug candidates in physiologically relevant environments prior to clinical tests. We therefore try to develop diagnostic multiorgan microfluidic chips based on patient specific induced pluripotent stem cell (iPS) technology to explore liver dependent toxic effects of drugs on individual human tissues such as liver or kidney cells. Based initially on standardized microfluidic modules for cell culture, we have developed integrated microfluidic devices which contain different chambers for cell/tissue cultivation. The devices are manufactured using injection molding of thermoplastic polymers such as polystyrene or cyclo-olefin polymer. In the project, suitable surface modification methods of the used materials had to be explored. We have been able to successfully demonstrate the seeding, cultivation and further differentiation of modified iPS, as shown by the use of differentiation markers, thus providing a suitable platform for toxicity testing and potential tissue-tissue interactions.