title: Nuclear factor-kappab activation is associated with glutamate-evoked tissue transglutaminase up-regulation in primary astrocyte cultures.
abstract: 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.
authors.affiliation: Department of Biochemical, Physiological and Nutritional Sciences, University of Messina, Policlinico Universitario, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Caccamo
authors.forename: Agata
authors.initials: A
authors.lastname: Campisi
authors.forename: Monica
authors.initials: M
authors.lastname: Currò
authors.forename: Mohammed
authors.initials: M
authors.lastname: Aguennouz
authors.forename: Giovanni
authors.initials: G
authors.lastname: Li Volti
authors.forename: Roberto
authors.initials: R
authors.lastname: Avola
authors.forename: Riccardo
authors.initials: R
authors.lastname: Ientile
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects* / Q000201:enzymology; D015153:Blotting, Western / Q000379:methods; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D018698:Glutamic Acid / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D011503:Transglutaminases / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects*; D000097782:NF-kappaB-Inducing Kinase
chemical_list: D004791:Enzyme Inhibitors; D018698:Glutamic Acid; D010455:Peptides; D017346:Protein Serine-Threonine Kinases; D012333:RNA, Messenger; D011503:Transglutaminases; D000097782:NF-kappaB-Inducing Kinase; C118258:SN50 peptide