Sp1/Sp3 and DNA-methylation contribute to basal transcriptional activation of human podoplanin in MG63 versus Saos-2 osteoblastic cells

BACKGROUND: Podoplanin is a membrane mucin that, among a series of tissues, is expressed on late osteoblasts and osteocytes. Since recent findings have focussed on podoplanin's potential role as a tumour progression factor, we aimed at identifying regulatory elements conferring PDPN promoter activity. Here, we characterized the molecular mechanism controlling basal PDPN transcription in human osteoblast-like MG63 versus Saos-2 cells. RESULTS: We cloned and sequenced 2056 nucleotides from the 5'-flanking region of the PDPN gene and a computational search revealed that the TATA and CAAT box-lacking promoter possesses features of a growth-related gene, such as a GC-rich 5' region and the presence of multiple putative Sp1, AP-4 and NF-1 sites. Reporter gene assays demonstrated a functional promoter in MG63 cells exhibiting 30-fold more activity than in Saos-2 cells. In vitro DNase I footprinting revealed eight protected regions flanked by DNaseI hypersensitive sites within the region bp -728 to -39 present in MG63, but not in Saos-2 cells. Among these regions, mutation and supershift electrophoretic mobility shift assays (EMSA) identified four Sp1/Sp3 binding sites and two binding sites for yet unknown transcription factors. Deletion studies demonstrated the functional importance of two Sp1/Sp3 sites for PDPN promoter activity. Overexpression of Sp1 and Sp3 independently increased the stimulatory effect of the promoter and podoplanin mRNA levels in MG63 and Saos-2 cells. In SL2 cells, Sp3 functioned as a repressor, while Sp1 and Sp3 acted positively synergistic. Weak PDPN promoter activity of Saos-2 cells correlated with low Sp1/Sp3 nuclear levels, which was confirmed by Sp1/Sp3 chromatin immunoprecipitations in vivo. Moreover, methylation-sensitive Southern blot analyses and bisulfite sequencing detected strong methylation of CpG sites upstream of bp -464 in MG63 cells, but hypomethylation of these sites in Saos-2 cells. Concomitantly, treatment with the DNA methyltransferase inhibitor 5-azaCdR in combination with trichostatin A (TSA) downregulated podoplanin mRNA levels in MG63 cells, and region-specific in vitro methylation of the distal promoter suggested that DNA methylation rather enhanced than hindered PDPN transcription in both cell types. CONCLUSION: These data establish that in human osteoblast-like MG63 cells, Sp1 and Sp3 stimulate basal PDPN transcription in a concerted, yet independent manner, whereas Saos-2 cells lack sufficient nuclear Sp protein amounts for transcriptional activation. Moreover, a highly methylated chromatin conformation of the distal promoter region confers cell-type specific podoplanin upregulation versus Saos-2 cells

Colon cancer cell-derived 12(S)-HETE induces the retraction of cancer-associated fibroblast via MLC2, RHO/ROCK and Ca2+ signalling

Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca2+ levels were measured and pharmacological-or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca2+, Ca(2+)calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca2+ release. Thus, Ca2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction

The Tandem-L Mission Proposal: Monitoring Earth’s Dynamics with High Resolution SAR Interferometry

Tandem-L is a proposal for an innovative interferometric and polarimetric radar mission that enables the systematic monitoring of dynamic processes on the Earth surface. Important mission objectives are global forest height and biomass inventories, large scale measurements of millimetric displacements due to tectonic shifts, and systematic observations of glacier movements. The innovative mission concept and the high data acquisition capacity of Tandem-L provide a unique data source to observe, analyze and quantify the dynamics of a wide range of mutually interacting processes in the bio-, litho-, hydro- and cryosphere. By this, Tandem-L will be an essential step to advance our understanding of the Earth system and its intricate dynamics. This paper provides an overview of the Tandem-L mission concept and its main application areas. Performance predictions show the great potential of Tandem-L to acquire a wide range of bio- and geophysical parameters with high accuracy on a global scale. Innovative aspects like the employment of advanced digital beamforming techniques to improve performance and coverage are discussed in detail

Pursuing the diffraction limit with Nano-LED scanning transmission optical microscopy

Recent research into miniaturized illumination sources has prompted the development of alternative microscopy techniques. Although they are still being explored, emerging nano-light-emitting-diode (nano-LED) technologies show promise in approaching the optical resolution limit in a more feasible manner. This work presents the exploration of their capabilities with two different prototypes. In the first version, a resolution of less than 1 µm was shown thanks to a prototype based on an optically downscaled LED using an LED scanning transmission optical microscopy (STOM) technique. This research demonstrates how this technique can be used to improve STOM images by oversampling the acquisition. The second STOM-based microscope was fabricated with a 200 nm GaN LED. This demonstrates the possibilities for the miniaturization of on-chip-based microscopes

Xanthohumol attenuates tumour cell-mediated breaching of the lymphendothelial barrier and prevents intravasation and metastasis

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In vitro characterisation of the anti-intravasative properties of the marine product heteronemin

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ApoB100-LDL Acts as a Metabolic Signal from Liver to Peripheral Fat Causing Inhibition of Lipolysis in Adipocytes

International audienceBACKGROUND: Free fatty acids released from adipose tissue affect the synthesis of apolipoprotein B-containing lipoproteins and glucose metabolism in the liver. Whether there also exists a reciprocal metabolic arm affecting energy metabolism in white adipose tissue is unknown. METHODS AND FINDINGS: We investigated the effects of apoB-containing lipoproteins on catecholamine-induced lipolysis in adipocytes from subcutaneous fat cells of obese but otherwise healthy men, fat pads from mice with plasma lipoproteins containing high or intermediate levels of apoB100 or no apoB100, primary cultured adipocytes, and 3T3-L1 cells. In subcutaneous fat cells, the rate of lipolysis was inversely related to plasma apoB levels. In human primary adipocytes, LDL inhibited lipolysis in a concentration-dependent fashion. In contrast, VLDL had no effect. Lipolysis was increased in fat pads from mice lacking plasma apoB100, reduced in apoB100-only mice, and intermediate in wild-type mice. Mice lacking apoB100 also had higher oxygen consumption and lipid oxidation. In 3T3-L1 cells, apoB100-containing lipoproteins inhibited lipolysis in a dose-dependent fashion, but lipoproteins containing apoB48 had no effect. ApoB100-LDL mediated inhibition of lipolysis was abolished in fat pads of mice deficient in the LDL receptor (Ldlr(-/-)Apob(100/100)). CONCLUSIONS: Our results show that the binding of apoB100-LDL to adipocytes via the LDL receptor inhibits intracellular noradrenaline-induced lipolysis in adipocytes. Thus, apoB100-LDL is a novel signaling molecule from the liver to peripheral fat deposits that may be an important link between atherogenic dyslipidemias and facets of the metabolic syndrome

TanDEM-X Performance Analysis

TanDEM-X (TerraSAR-X add-on for Digital Elevation Measurement) is a space borne earth observation mis-sion with the goal of generating a global Digital Elevation Model (DEM) with an unprecedented accuracy corre-sponding to the HRTI-3 specifications. TanDEM-X will fly in a special orbit formation (HELIX formation) with the fully compatible TerraSAR-X (TSX-1) satellite in order to collect interferometric SAR data. It is planned to cover the earth surface twice with different baselines, in order to ease phase unwrapping. Within a mission dura-tion of only three years, data for a global HRTI-3 DEM shall be acquired. The paper is divided into two parts. Starting with the TanDEM-X mission objective, the height performance model is presented, taking into account several error sources, like thermal decorrelation, quantization errors, range- and azimuth-ambiguities, misregistration as well as volume decorrelation. The analysis is extended to dif-ferent terrain-types, showing a significant impact on the height accuracy. Based on a global terrain classification map, a first rough estimate of the global DEM performance is given. The second part concentrates on an analysis of the impact of the block adaptive quantiser (BAQ) in terrain with high dynamic range in backscatter power (e.g. urban areas). Simulation results based on TerraSAR-X measure-ment data will be presented

TanDEM-X Performance Optimization

Abstract TanDEM-X (TerraSAR-X add-on for Digital Elevation Measurement) is a space borne earth observation mission with the goal of generating a global Digital Elevation Model (DEM) with an unprecedented accuracy corresponding to the HRTI-3 specifications. TanDEM-X will fly in a special orbit formation (HELIX formation) with the fully compatible TerraSAR-X (TSX-1) satellite in order to collect interferometric SAR data. It is planned to cover the earth surface twice with different baselines. Presented in this paper is an optimization strategy, with the goal of a quasi-homogeneous height accuracy, which meets the HRTI-3 standard. The optimization has a major impact on the data acquisition plan. The acquisition of a DEM on a global scale requires an optimized mission plan, since the time, where both satellites are in orbit simultaneously, is limited to three years. Within this time the landmass is imaged in strip map mode twice, which allows for multi baseline phase unwrapping techniques. Using 167 satellite orbits, the region to be imaged has an across-track extension of about 240km at the equator. This region is subdivided into several swaths, so called data takes. The swath-width is determined by the timing, but also by the pattern of the SAR antenna. The shape of the antenna beam and SAR system parameters, like the pulse repetition frequency (PRF) or the transmitted bandwidth, are in the focus of the optimization. The quantity, which is a measure for the quality in any DEM, is the height accuracy. Therefore the optimization concentrates directly on this parameter, rather than trying to maximize a single parameter, e.g. the signal to noise ratio (SNR). In the first part of the paper, a height performance model is presented, taking into account several error sources, like thermal decorrelation, quantization errors, range- and azimuth-ambiguities, misregistration as well as volume decorrelation. The analysis is extended to different terrain-types and terrain-slopes, showing a significant impact on the height accuracy. The second part is dedicated to the optimization strategy. It has to be mentioned, that this optimization approach is a heuristic method, rather than a mathematical procedure with a cost function to be minimized. Since the parameters under consideration are in complex connection, the main challenge is to reduce the number of variables and still yield an accurate optimization result. Moreover a method is presented, how to fuse several independent interferometric data acquisitions to a single DEM, further increasing the height accuracy