RAPID INDUCTION OF THE GRP78 GENE BY COOPERATIVE ACTIONS OF OKADAIC ACID AND HEAT-SHOCK IN 9L RAT-BRAIN TUMOR-CELLS - INVOLVEMENT OF A CAMP-RESPONSIVE ELEMENT-LIKE PROMOTER SEQUENCE AND A PROTEIN-KINASE-A SIGNALING PATHWAY

We have demonstrated that treatment with 200 nM okadaic acid (OA) for 1 h followed by a 15-min heat shock (HS) at 45 degrees 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 treatme nt. Nuclear extracts from cells subjected to OA-->HS treatment, compar ed to those of treatment with OA or HS alone, exhibited an increased b inding activity toward an oligonucleotide probe containing the cAMP-re sponsive element-like (CRE-like, TGACGTGA) regulatory element in elect rophoretic mobility shift assays (EMSA). The binding resulted in the f ormation of two protein-EMSA probe complexes exhibiting different asso ciation and dissociation rates in kinetic studies. The protein factors in the upper band (complex I) and lower band (complex II) were identi fied as the activating transcription factor-2 (ATF-2) and the CRE bind ing factor 1 (CREB-1), respectively, by antibody interference assays. In addition, the identity of CREB-1 was confirmed by supershift analys is. The binding activity, as well as the transactivation of the grp78 gene, can be abolished by a 1-h treatment with the cAMP-dependent prot ein kinase (PKA) inhibitor but not with protein kinase C or Ca2+/calmo dulin-dependent protein kinase II inhibitors. Accumulation of steady-s tate level of ATF-2 was observed and was also modulated by treatment w ith H-89, a PKA inhibitor. From these results, we conclude that the CR E-like element plays an important role in the rapid transactivation of the grp78 gene and that the PKA signaling pathway is involved. In add ition, PKA-mediated transcriptional regulation of grp78 in OA-->HS tre atment is through regulation of protein phosphorylation as well as de novo synthesis of ATF-2.