Iromycin A

Resolving the temporal dynamics of cell signaling pathways is essential for regulating numerous downstream functions, from gene expression to cellular responses. Mapping these signaling pathways requires the exposure of cells to time-varying chemical signals; these are difficult to generate and control over a wide temporal range. Herein, we present an acoustofluidic chemical signal generator based on a sharp-edge-based micromixing strategy. The device, simply by modulating the driving signals of an acoustic transducer including the ON/OFF switching frequency, actuation time and duty cycle, is capable of generating both single-pulse and periodic chemical signals that are temporally controllable in terms of stimulation period, stimulation duration and duty cycle. We also demonstrate the device's applicability and versatility for cell signaling studies by probing the calcium (Ca2+) release dynamics of three different types of cells stimulated by ionomycin signals of different shapes. Upon short single-pulse ionomycin stimulation (~100 ms) generated by our device, we discover that cells tend to dynamically adjust the intracellular level of Ca2+ through constantly releasing and accepting Ca2+ to the cytoplasm and from the extracellular environment, respectively. With advantages such as simple fabrication and operation, compact device design, and reliability and versatility, our device will enable decoding of the temporal characteristics of signaling dynamics for various physiological processes.

Interleukin-6 (IL-6) and interleukin-11 (IL-11) are two important pleiotropic cytokines, both of which signal through a homodimer of the β-receptor gp130. Specificity is gained through the unique, nonsignaling α-receptors IL-6R and IL-11R. Soluble variants of IL-6R and IL-11R also exist. Both membrane-bound receptors can be cleaved by the metalloprotease ADAM10. Here, we use ten different chimeric receptors consisting of different parts of IL-6R and IL-11R and analyze their susceptibility toward cleavage by ADAM10. As expected, all chimeras are substrates of ADAM10. However, we observed that cleavage of chimeric receptors containing the stalk region of the IL-11R could be blocked by the protease inhibitor GI (selective for ADAM10), but not by the protease inhibitor GW (selective for both ADAM10 and ADAM17), suggesting that another protease besides ADAM10 is involved in cleavage of these chimeras.

Aims/hypothesis:Existing thiazolidinedione (TZD) drugs for diabetes have severe side effects. The aim of this study is to develop alternative peroxisome proliferator-activated receptor γ (PPARγ) ligands that retain the benefits in improving insulin resistance but with reduced side effects.Methods:We used AlphaScreen assay to screen for new PPARγ ligands from compound libraries. In vitro biochemical binding affinity assay and in vivo cell-based reporter assay were used to validate ionomycin as a partial ligand of PPARγ. A mouse model of diabetes was used to assess the effects of ionomycin in improving insulin sensitivity. Crystal structure of PPARγ complexed with ionomycin revealed the unique binding mode of ionomycin, which elucidated the molecular mechanisms allowing the discrimination of ionomycin from TZDs.Results:We found that the antibiotic ionomycin is a novel modulating ligand for PPARγ. Both the transactivation and binding activity of PPARγ by ionomycin can be blocked by PPARγ specific antagonist GW9662. Ionomycin interacts with the PPARγ ligand-binding domain in a unique binding mode with properties and epitopes distinct from those of TZD drugs. Ionomycin treatment effectively improved hyperglycaemia and insulin resistance, but had reduced side effects compared with TZDs in the mouse model of diabetes. In addition, ionomycin effectively blocked the phosphorylation of PPARγ at Ser273 by cyclin-dependent kinase 5 both in vitro and in vivo.Conclusions/interpretation:Our studies suggest that ionomycin may represent a unique template for designing novel PPARγ ligands with advantages over current TZD drugs.