RESUMEN
Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We screened a noncommercial, kinase-focused chemical library which allowed us to identify Sibiriline as a new inhibitor of necroptosis induced by tumor necrosis factor (TNF) in Fas-associated protein with death domain (FADD)-deficient Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in human or mouse cells while not protecting from caspase-dependent apoptosis. By using competition binding assay and recombinant kinase assays, we demonstrated that Sib is a rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is trapped closed to human RIPK1 adenosine triphosphate-binding site in a relatively hydrophobic pocket locking RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis. Finally, Sib protects mice from concanavalin A-induced hepatitis. These results reveal the small-molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of immune-dependent hepatitis.
Asunto(s)
Alcaloides/farmacología , Hepatitis Animal/prevención & control , Factores Inmunológicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Compuestos de Espiro/farmacología , Alcaloides/química , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Caspasa 3/genética , Caspasa 3/inmunología , Línea Celular Transformada , Concanavalina A , Cicloheximida/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Células HT29 , Hepatitis Animal/inducido químicamente , Hepatitis Animal/genética , Hepatitis Animal/inmunología , Humanos , Imidazoles/farmacología , Factores Inmunológicos/química , Indoles/farmacología , Células Jurkat , Masculino , Ratones , Simulación del Acoplamiento Molecular , Necrosis/inducido químicamente , Necrosis/genética , Necrosis/inmunología , Inhibidores de Proteínas Quinasas/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Transducción de Señal , Compuestos de Espiro/química , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
The increased expression of the Disintegrin and Metalloprotease ADAM12 has been associated with human cancers, however its role remain unclear. We have previously reported that ADAM12 expression is induced by the transforming growth factor, TGF-ß and promotes TGF-ß-dependent signaling through interaction with the type II receptor of TGF-ß. Here we explore the implication of ADAM12 in TGF-ß-mediated epithelial to mesenchymal transition (EMT), a key process in cancer progression. We show that ADAM12 expression is correlated with EMT markers in human breast cancer cell lines and biopsies. Using a non-malignant breast epithelial cell line (MCF10A), we demonstrate that TGF-ß-induced EMT increases expression of the membrane-anchored ADAM12L long form. Importantly, ADAM12L overexpression in MCF10A is sufficient to induce loss of cell-cell contact, reorganization of actin cytoskeleton, up-regulation of EMT markers and chemoresistance. These effects are independent of the proteolytic activity but require the cytoplasmic tail and are specific of ADAM12L since overexpression of ADAM12S failed to induce similar changes. We further demonstrate that ADAM12L-dependent EMT is associated with increased phosphorylation of Smad3, Akt and ERK proteins. Conversely, inhibition of TGF-ß receptors or ERK activities reverses ADAM12L-induced mesenchymal phenotype. Together our data demonstrate that ADAM12L is associated with EMT and contributes to TGF-ß-dependent EMT by favoring both Smad-dependent and Smad-independent pathways.
