Up-regulation of TNF-α convertase (TACE/ADAM17) after oxygen–glucose deprivation in rat forebrain slices

Abstract

Tumour necrosis factor-α (TNF-α) is a major immunomodulatory and proinflammatory cytokine which is shed in its soluble form by a membrane-anchored zinc protease, identified as a disintegrin and metalloproteinase (ADAM) called TNF-α convertase (TACE; ADAM17). The role of this protease in the adult nervous system remains poorly understood. During cerebral ischemia and subsequent reperfusion, expression and release of TNF-α have been shown. We have investigated the expression and activity of TACE in an in vitro model of brain ischemia consisting of rat forebrain slices exposed to oxygen–glucose deprivation (OGD). OGD caused the release of TNF-α, an effect which was inhibited by a hydroxamate-based metalloprotease inhibitor, BB-3103, with an IC 50 of 0.1 μM, suggesting that TNF-α release results selectively from TACE activity. Assay of TACE enzymatic activity on a fluorescein-labelled peptide spanning the cleavage site in pro-TNF-α, as well as Western blot and RT–PCR analyses showed that TACE is present in control forebrain and, more interestingly, that TACE expression is increased in OGD-exposed tissue. TACE enzymatic activity from OGD-exposed slices was significantly inhibited by cycloheximide, suggesting that de novo synthesis of TACE contributes to TNF-α release after ischaemia. Moreover, it was also inhibited by bisindolylmaleimide I, indicating that TACE activity is regulated by PKC. These findings posed the question of what was its function therein. Among other actions, TNF-α has been described to be involved in the expression of inducible nitric oxide synthase (iNOS), a high-output NOS isoform associated to cellular damage, but the link between TNF-α release after brain ischaemia and iNOS expression in this condition has not been shown. We have now found that iNOS expression in OGD-subjected brain slices is inhibited by BB-3103 at concentrations below 1 μM, indicating that shedding of TNF-α by TACE plays a necessary part in the induction of this NOS isoenzyme after OGD. Taken together, these data demonstrate that (1) TACE/ADAM17 activity accounts for the majority of TNF-α shedding after OGD in rat forebrain slices, (2) an increase in TACE expression contributes, at least in part, to the rise in TNF-α after OGD and (3) iNOS expression in OGD-subjected brain slices results from TACE activity and subsequent increase in TNF-α levels.