Nuclear factor‐κb activation is associated with glutamate‐evoked tissue transglutaminase up‐regulation in primary astrocyte cultures

We have previously demonstrated that alterations of cell redox state, evoked by glutamate, are associated with tissue transglutaminase increases in primary astrocyte cultures. Furthermore, glutamate exposure activated the nuclear factor (NF)-kappaB pathway, and its effects were significantly reduced by antioxidants. Here, we investigated the possible involvement of activated NF-kappaB pathway in glutamate-evoked tissue transglutaminase up-regulation in primary astrocytes. The presence of DNA binding activity by NF-kappaB in nuclear extracts of astrocytes, treated for 24 hr with glutamate (500 microM) or untreated, was assessed by EMSA, using an oligonucleotide probe containing the NF-kappaB consensus sequence present in the tissue transglutaminase promoter. Supershifting with monoclonal antibodies revealed that activated NF-kappaB dimer complexes were composed of p50 and p65 subunits. Interestingly, the specific NF-kappaB inhibitor SN50 (but not its inactive analogue SN50M), when added to cell cultures 30 min prior to glutamate treatment, was able gradually to reduce glutamate-induced NF-kappaB activation. Western blot analysis confirmed the reduction of the p50 amount in nuclear extracts. Notably, the preincubation with SN50 also diminished glutamate-increased tissue transglutaminase expression, as showed by both RT-PCR and Western blotting. Competition experiments, carried out with an excess of a probe containing the NF-kappaB consensus sequence present in the kappa-light-chain promoter, demonstrated a preferential binding of the tissue transglutaminase specific NF-kappaB probe in the nuclear extracts of glutamate-treated astrocytes compared with untreated astrocytes. These preliminary data suggest that NF-kappaB activation, which has been demonstrated to be involved in astrocyte response to glutamate, could also be associated with the molecular pathway leading to glutamate-evoked tissue transglutaminase up-regulation.