Nuclear factor-kappab 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 astrocytecultures. Furthermore, glutamate exposure activated the nuclear factor (NF)-jB pathway, and its effects were significantly reduced by antioxidants. Here, we investigated the possible involvement of activated NF-jB pathway in glutamate-evoked tissue transglutaminase up-regulationin primary astrocytes. The presence of DNA binding activity by NF-kB in nuclear extracts of astrocytes, treated for 24 hr with glutamate (500 lM) or untreated, was assessedby EMSA, using an oligonucleotide probe containing the NF-kB consensus sequence present in the tissue transglutaminase promoter. Supershifting with monoclonal antibodies revealed that activated NF-jB dimer complexes were composed of p50 and p65 subunits.Interestingly, the specific NF-jB inhibitor SN50 (but notits inactive analogue SN50M), when added to cell cultures30 min prior to glutamate treatment, was ablegradually to reduce glutamate-induced NF-kB activation.Western blot analysis confirmed the reduction ofthe p50 amount in nuclear extracts. Notably, the preincubation with SN50 also diminished glutamate-increasedtissue transglutaminase expression, as showedby both RT-PCR and Western blotting. Competition experiments, carried out with an excess of a probe containing the NF-jB consensus sequence present in the jlight-chain promoter, demonstrated a preferential binding of the tissue transglutaminase specific NF-jB probe in the nuclear extracts of glutamate-treated astrocytes compared with untreated astrocytes. These preliminary data suggest that NF-jB 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 upregulation.