Summary The aberrant transcription factor EWS-FLI1 drives Ewing sarcoma yet its molecular function is incompletely understood. We find that EWS-FLI1 reprograms gene regulatory circuits in Ewing sarcoma by directly inducing or repressing enhancers. At GGAA repeat elements, which lack evolutionary conservation and regulatory potential in other cell types, EWS-FLI1 multimers induce chromatin opening and create de novo enhancers that physically interact with target promoters. Conversely, EWS-FLI1 inactivates conserved enhancers containing canonical ETS motifs by displacing wild type ETS transcription factors. These divergent chromatin-remodeling patterns repress tumor suppressors and mesenchymal lineage regulators, while activating oncogenes and new potential therapeutic targets, such as the kinase VRK1. Our findings demonstrate how EWS-FLI1 establishes an oncogenic regulatory program governing both tumor survival and differentiation.

Our results directly demonstrate opening of the TGF-β1 straitjacket by application of mechanical force in the order of magnitude of what can be transmitted by single integrins. For this mechanism to be in place, binding of latent TGF-β1 to LTBP-1 is mandatory. Interfering with mechanical activation of latent TGF-β1 by reducing integrin affinity, cell contractility, and binding of latent TGF-β1 to the ECM provides new possibilities to therapeutically modulate TGF-β1 actions.

The measurement of tissue and cell oxygenation is important for understanding cell metabolism. We have addressed this problem with a novel optical technique, called triplet imaging, that exploits oxygen-induced triplet lifetime changes and is compatible with a variety of fluorophores. A modulated excitation of varying pulse widths allows the extraction of the lifetime of the essentially dark triplet state using a high-fluorescence signal intensity. This enables the monitoring of fast kinetics of oxygen concentration in living cells combined with high temporal and spatial resolution. First, the oxygen-dependent triplet-state quenching of tetramethylrhodamine is validated and then calibrated in an L-ascorbic acid titration experiment demonstrating the linear relation between triplet lifetime and oxygen concentration according to the Stern-Volmer equation. Second, the method is applied to a biological cell system, employing as reporter a cytosolic fusion protein of beta-galactosidase with SNAP-tag labeled with tetramethylrhodamine. Oxygen consumption in single smooth muscle cells A7r5 during an [Arg(8)]-vasopressin-induced contraction is measured. The results indicate a consumption leading to an intracellular oxygen concentration that decays monoexponentially with time. The proposed method has the potential to become a new tool for investigating oxygen metabolism at the single cell and the subcellular level.

Cells with irregular shapes, numerous long thin filaments, and morphological similarities to the gastrointestinal interstitial cells of Cajal (ICCs) have been observed in the wall of some blood vessels. These ICC-like cells (ICC-LCs) do not correspond to the other cell types present in the arterial wall: smooth muscle cells (SMCs), endothelial cells, fibroblasts, inflammatory cells, or pericytes. However, no clear physiological role has as yet been determined for ICC-LCs in the vascular wall. The aim of this study has been to identify and characterize the functional response of ICC-LCs in rat mesenteric arteries. We have observed ICC-LCs and identified them morphologically and histologically in three different environments: isolated artery, freshly dispersed cells, and primary-cultured cells from the arterial wall. Like ICCs but unlike SMCs, ICC-LCs are positively stained by methylene blue. Cells morphologically resembling methylene-blue-positive cells are also positive for the ICC and ICC-LC markers α-smooth muscle actin and desmin. Furthermore, the higher expression of vimentin in ICC-LCs compared with SMCs allows a clear discrimination between these two cell types. At the functional level, the differences observed in the variations of cytosolic free calcium concentration of freshly dispersed SMCs and ICC-LCs in response to a panel of vasoactive molecules show that ICC-LCs, unlike SMCs, do not respond to exogenous ATP and [Arginine](8)-vasopressin.