Abstract
Anoikis is a form of apoptosis induced by cell detachment. Integrin inactivation plays a major role in the process but the exact signalling pathway is ill-defined. Here we identify an anoikis pathway using gliotoxin (GT), a virulence factor of the fungus Aspergillus fumigatus, which causes invasive aspergillosis in humans. GT prevents integrin binding to RGD-containing extracellular matrix components by covalently modifying cysteines in the binding pocket. As a consequence, focal adhesion kinase (FAK) is inhibited resulting in dephosphorylation of p190RhoGAP, allowing activation of RhoA. Sequential activation of ROCK, MKK4/MKK7 and JNK then triggers pro-apoptotic phosphorylation of Bim. Cells in suspension or lacking integrin surface expression are insensitive to GT but are sensitised to ROCK-MKK4/MKK7-JNK-dependent anoikis upon attachment to fibronectin or integrin upregulation. The same signalling pathway is triggered by FAK inhibition or inhibiting integrin αV/β3 with Cilengitide. Thus, GT can target integrins to induce anoikis on lung epithelial cells.
Highlights
Anoikis is a form of apoptosis induced by cell detachment
Since GT causes rapid cell detachment associated with cytoskeletal changes (Supplementary Fig. 1), we looked for an upstream MKK4/MKK7 activator, which is linked to these events
GT qualifies for an anoikis inducer for the following reasons: (i) it induces rapid cell detachment prior to apoptosis induction; (ii) it can directly modify N-terminal cysteines of α and β chains of integrins thereby interfering with their binding to extracellular matrix components; and (iii) it cannot kill suspension cells of the hematopoietic system and does not activate the anoikis signalling pathway in these cells
Summary
Anoikis is a form of apoptosis induced by cell detachment. Integrin inactivation plays a major role in the process but the exact signalling pathway is ill-defined. Described by Frisch et al.[2], anoikis is typically induced by detaching cells with trypsin and preventing their re-attachment to polyHEMA-coated plates This system is artificial as trypsin inappropriately modifies adhesion molecules leading to the activation of signalling pathways that may not reflect physiological ways of anoikis. AKT inhibits Forkhead transcription factors (FOXOs)[23], which are responsible for the transcriptional upregulation of the BH3-only proteins Bim, Puma and Bmf[24] Both ERK-25 and PI3K/AKT-mediated phosphorylation[24] of Bim lead to its proteasomal degradation. It has been unclear if activation of these BH3-only proteins during anoikis is linked to AKT and/or ERK inhibition
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