Abstract
HSP86 and HSP84 are mammalian heat shock proteins identified to be the inducible and constitutive isoform of HSP90, respectively. However, the regulatory mechanism of hsp86 upon heavy metal and antitumor drugs stress are still unclear. In this study, rat hsp86 promoter region was used to investigate the mechanism of hsp86 expression induced under stress. The full length promoter region of rat hsp86 was used to search the putative transcription elements in hsp86 promoter from transcription element database. We also conducted sequence analysis between promoters of several hsp86 in mammals. The sequence comparison results showed that the -270 to +1 region of mammalian hsp86 promoters is almost identical, suggesting the importance of functional conservation. To test whether the -270 to +1 region is sufficient and to characterize the individual cis-acting elements, full length and serial deletions of the predicted hsp86 promoter isolated from genomic library were constructed into firefly luciferase reporter plasmid. The functional analyses of these promoter constructs were first verified under normal condition and then tested under the stimulation of cadmium chloride, geldanamycin, 17-allylamino, 17-demethoxygeldanamycin, and radicicol in 9L rat brain tumor (RBT) cells. The same minimal essential promoter region of rat hsp86 located within the –471 to +1 region is identified under normal condition, and stimulation of the heavy metal cadmium, and three anticancer drugs. Our experimental results identified two more regulatory regions than the hypothetic result from conserved sequence analysis data of the -270 to +1 region. The functional analysis indicated, consistently, that within the hypothetic -270 to +1 region, the heat shock element (HSE) containing region from –137 to +1 plays the main transactivation role, and the GC containing region (from –265 to –137) exhibits a positive regulatory activity. However, the nuclear factor kappa chain transcription B cells (NF-kappaB) containing region (from -471 to –265) has a newly found negative regulatory activity and a distal region containing cAMP responsive element (CRE) from –1418 to –1177 is identified as a possible cadmium-specific, responsive region. To detect the secondary important elements, HSE was specifically mutated from the deletion constructs and the results identified NF-kappaB as the secondary effective element during cadmium treatment. No obvious secondary element was found under normal condition indicating the NF-kappaB element is required but not sufficient for the basal level expression of hsp86.