HSP90 chaperones are transducer proteins of many signaling pathways in cells. Using a highly specific inhibitor, geldanamycin (GA), an increasing number of the HSP90 client proteins have been identified. Nevertheless, there is little information on the differential transactivation of the two isoforms of the hsp90 genes, hsp90alpha and beta, in cells under stress conditions. Here, we demonstrate the differential expression of the HSP90 isoforms, HSP90alpha and beta, in rat gliosarcoma 9L cells using a modified SDS-PAGE system that allowed us to distinguish the isoforms. We subsequently assessed the transcriptional controls involving the transcription elements located in the promoter regions of the hsp90 genes. At the protein level, HSP90alpha is more responsive to GA in terms of rate of de novo synthesis and amount of accumulation, as shown by metabolic-labeling and Western-blotting analyses. Upregulation of the hsp90 genes was demonstrated by real-time qPCR. The promoter elements hsp90alpha-HSE2 and hsp90beta-HSE1 were also identified to be the major transcription elements involved in GA-activated gene expression, as shown by EMSA, whereas the results of supershift showed that the transcription factor HSF1 is also involved. Moreover, EMSA results of analysis of the GC box showed differences in both the initial amounts and inductive response of hsp90s transcripts, whereas analysis of the TATA box showed GA responsiveness in hsp90alpha only. Collectively, these results indicate that GA exerts its regulatory effects through transcription elements including heat-shock elements (HSEs), GC boxes and TATA boxes, resulting in differential transactivation of hsp90alpha and hsp90beta in rat gliosarcoma 9L cells.
2008 Wiley-Liss, Inc.