RESUMEN
The putative protein tyrosine kinase (PTK) inhibitor tyrphostin AG126 has proven beneficial in various models of inflammatory disease. Yet molecular targets and cellular mechanisms remained enigmatic. We demonstrate here that AG126 treatment has beneficial effects in experimental autoimmune encephalomyelitis (EAE), a model for multiple sclerosis. AG126 alleviates the clinical symptoms, diminishes encephalitogenic Th17 differentiation, reduces inflammatory CNS infiltration as well as microglia activation and attenuates myelin damage. We show that AG126 directly inhibits Bruton's tyrosine kinase (BTK), a PTK associated with B cell receptor and Toll-like receptor (TLR) signaling. However, BTK inhibition cannot account for the entire activity spectrum. Effects on TLR-induced proinflammatory cytokine expression in microglia involve AG126 hydrolysis and conversion of its dinitrile side chain to malononitrile (MN). Notably, while liberated MN can subsequently mediate critical AG126 features, full protection in EAE still requires delivery of intact AG126. Its anti-inflammatory potential and especially interference with TLR signaling thus rely on a dual mechanism encompassing BTK and a novel MN-sensitive target. Both principles bear great potential for the therapeutic management of disturbed innate and adaptive immune functions.
Asunto(s)
Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Fármacos Neuroprotectores/farmacología , Tirfostinos/farmacología , Agammaglobulinemia Tirosina Quinasa , Animales , Células Cultivadas , Citocinas/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Encefalomielitis Autoinmune Experimental/fisiopatología , Femenino , Hidrólisis , Factores Inmunológicos/química , Factores Inmunológicos/farmacología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/fisiología , Factor 88 de Diferenciación Mieloide/metabolismo , Fármacos Neuroprotectores/química , Nitrilos/química , Nitrilos/metabolismo , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/efectos de los fármacos , Bazo/citología , Bazo/efectos de los fármacos , Bazo/fisiopatología , Células Th17/efectos de los fármacos , Células Th17/patología , Células Th17/fisiología , Tirfostinos/químicaRESUMEN
Spleen tyrosine kinase Syk and its substrate SLP65 (also called BLNK) are proximal signal transducer elements of the B-cell antigen receptor (BCR). Yet, our understanding of signal initiation and processing is limited owing to the incomplete list of SLP65 interaction partners and our ignorance of their association kinetics. We have now determined and quantified the in vivo interactomes of SLP65 in resting and stimulated B cells by mass spectrometry. SLP65 orchestrated a complex signal network of about 30 proteins that was predominantly based on dynamic interactions. However, a stimulation-independent and constant association of SLP65 with the Cbl-interacting protein of 85 kDa (CIN85) was requisite for SLP65 phosphorylation and its inducible plasma membrane translocation. In the absence of a steady SLP65/CIN85 complex, BCR-induced Ca(2+) and NF-κB responses were abrogated. Finally, live cell imaging and co-immunoprecipitation experiments further confirmed that both SLP65 and CIN85 are key components of the BCR-associated primary transducer module required for the onset and progression phases of BCR signal transduction.