Rapid Induction of the Grp78 Gene by Cooperative Actions of Okadaic Acid and Heat‐Shock in 9L Rat Brain Tumor Cells

Abstract

We have demonstrated that treatment with 200 nM okadaic acid (OA) for 1 h followed by a 15‐min heat shock (HS) at 45°C (termed OA→HS treatment) leads to a rapid transactivation of grp78, the gene for the 78‐kDa glucose‐regulated protein, in 9L rat brain tumor cells. The level of Grp78 mRNA rose 15‐fold in 60 min after the combined treatment. Nuclear extracts from cells subjected to OA→HS treatment, compared to those of treatment with OA or HS alone, exhibited an increased binding activity toward an oligonucleotide probe containing the cAMP‐responsive element‐like (CRE‐like, TGACGTGA) regulatory element in electrophoretic mobility shift assays (EMSA). The binding resulted in the formation of two protein‐EMSA probe complexes exhibiting different association and dissociation rates in kinetic studies. The protein factors in the upper band (complex I) and lower band (complex II) were identified as the activating transcription factor‐2 (ATF‐2) and the CRE binding factor 1 (CREB‐1), respectively, by antibody interference assays. In addition, the identity of CREB‐1 was confirmed by supershift analysis. The binding activity, as well as the transactivation of the grp78 gene, can be abolished by a 1‐h treatment with the cAMP‐dependent protein kinase (PKA) inhibitor but not with protein kinase C or Ca²⁺/calmodulin‐dependent protein kinase II inhibitors. Accumulation of steady‐state level of ATF‐2 was observed and was also modulated by treatment with H‐89, a PKA inhibitor. From these results, we conclude that the CRE‐like element plays an important role in the rapid transactivation of the grp78 gene and that the PKA signaling pathway is involved. In addition, PKA‐mediated transcriptional regulation of grp78 in OA→HS treatment is through regulation of protein phosphorylation as well as de novo synthesis of ATF‐2.