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

Direct Visualization by Cryo-EM of the Mycobacterial Capsular Layer: A Labile Structure Containing ESX-1-Secreted Proteins

The cell envelope of mycobacteria, a group of Gram positive bacteria, is composed of a plasma membrane and a Gram-negative-like outer membrane containing mycolic acids. In addition, the surface of the mycobacteria is coated with an ill-characterized layer of extractable, non-covalently linked glycans, lipids and proteins, collectively known as the capsule, whose occurrence is a matter of debate. By using plunge freezing cryo-electron microscopy technique, we were able to show that pathogenic mycobacteria produce a thick capsule, only present when the cells were grown under unperturbed conditions and easily removed by mild detergents. This detergent-labile capsule layer contains arabinomannan, α-glucan and oligomannosyl-capped glycolipids. Further immunogenic and proteomic analyses revealed that Mycobacterium marinum capsule contains high amounts of proteins that are secreted via the ESX-1 pathway. Finally, cell infection experiments demonstrated the importance of the capsule for binding to cells and dampening of pro-inflammatory cytokine response. Together, these results show a direct visualization of the mycobacterial capsular layer as a labile structure that contains ESX-1-secreted proteins

Rapid, Nongenomic Stimulation of Multidrug Resistance Protein 2 (Mrp2) Activity by Glucocorticoids in Renal Proximal Tubule

In renal proximal tubule, multidrug resistance protein 2 (Mrp2) actively transports many organic anions into urine, including drugs and metabolic wastes. Upon exposure to nephrotoxicants or during endotoxemia, both Mrp2 activity and expression are up-regulated. This may result from induced de novo synthesis of Mrp2 or post-transcriptional events involving specific signaling pathways. Here, we investigated glucocorticoid signaling to Mrp2 in killifish renal proximal tubules, a model system in which transport activity can be measured using a fluorescent substrate and confocal imaging. Exposure of tubules to dexamethasone rapidly increased Mrp2-mediated fluorescein methotrexate transport. Other glucocorticoid receptor (GR) ligands, cortisol and triamcinolone acetonide, also stimulated Mrp2-mediated transport. The GR antagonist, mifepristone 17β-hydroxy-11β-[4-dimethylamino phenyl]-17α-[1-propynyl]estra-4,9-dien-3-one (RU486), abolished stimulation by all three ligands, whereas the mineralocorticoid receptor antagonist, spironolactone, was ineffective. Consistent with action through a nongenomic mechanism, dexamethasone stimulation of Mrp2-mediated transport was insensitive to cycloheximide and actinomycin D, and immunohistochemistry revealed no alterations in Mrp2 expression at the luminal membrane. (9S-(9α,10β,12α))-2,3,9,10,11,12-hexahydro-10-hydroxy-10-(methoxycarbonyl)-9-methyl-9,12-epoxy-1H-diindolo[1,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-i][1,6]benzodiazocin-1-one (K252a), an inhibitor of the tyrosine receptor kinase subfamily, reduced the dexamethasone effect, as did the specific hepatocyte growth factor receptor (c-Met) tyrosine kinase inhibitor, (2R)-1-[[5-[(Z)-[5-[[(2,6-dichlorophenyl)methyl]sulfonyl]-1,2-dihydro-2-oxo-3H-indol-3-ylidene]methyl]-2,4-dimethyl-1H-pyrrol-3-yl]carbonyl]-2-(1-pyrrolidinylmethyl)pyrrolidine (PHA-665752). Hepatocyte growth factor (HGF), an endogenous ligand for c-Met, stimulated Mrp2-mediated transport. This effect was reversed by PHA-665752 but not by RU486. Inhibition of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK 1/2) also abolished the effects of dexamethasone and HGF. Our results disclose a novel mechanism by which glucocorticoids acting through GR, c-Met, and MEK1/2 cause rapid, nongenomic stimulation of Mrp2-mediated transport in renal proximal tubules. This up-regulation may be nephroprotective, enhancing efflux of metabolic wastes and toxicants during cell and tissue stress