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 inin 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 11) were identified as the activating transcription factor-2 (ATF-2) and the CRE binding factor 1 (CREB-I), 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 I-h treatment with the CAMP-dependent protein kinase (PKA) inhibitor but not with protein kinase C or Ca'+/calmodulin-dependent protein kinase I1 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.

Exposure of 9L rat brain tumor cells to 40-100 microM CdCl2 for 2 h leads to an induction of a wide spectrum of heat shock proteins (HSPs). We have demonstrated that induction of the 70-kDa HSP (HSP70) and enhanced expression of its cognate (HSC70) by cadmium are concentration dependent and that the induction kinetics of these HSP70s are different. The increased synthesis of the HSP70s is accompanied by the increase in hsp70 and hsc70 mRNA levels, indicative of transcriptional regulation of the heat shock genes. Electrophoretic mobility shift assay (EMSA) using probes encompassing heat shock element (HSE), TATA, GC, and CCAAT boxes derived from the promoter regions of the heat shock genes shows distinguished binding patterns between hsp70 and hsc70 genes in both control and cadmium-treated cells. The results indicate that, in addition to the HSEs, the basal transcription elements are important in the regulation of the heat shock genes. The binding patterns of the corresponding transcription factors of these elements are examined by EMSA by using extended promoter fragments from respective heat shock genes with sequential addition of excess oligonucleotides encompassing individual transcription elements. Taken together, our results show that the differential induction of hsp70 and hsc70 involves multiple transcription factors that interact with HSE, TATA, GC, and CCAAT boxes.