title: cAMP and cAMP-dependent protein kinase regulate the human heat shock protein 70 gene promoter activity.
abstract: The theme of this study is an evaluation of the involvement of cAMP and cAMP-dependent protein kinase (PKA) in the regulation of the human heat shock protein (hsp) 70 gene promoter. Expression of a highly specific protein inhibitor of PKA (pRSVPKI) inhibited the basal as well as heat- and cadmium-induced expression of the cotransfected pHBCAT, a human hsp 70 promoter-driven reporter gene; this inhibition was dependent on the amount of pRSVPKI used. The effect of an expression vector of the RI regulatory subunit of PKA, pMTREV, was similar to that of pRSVPKI; pMTREV inhibited both the basal as well as the heat-induced expression of pHBCAT. The specificity of effects of these expression vectors was demonstrated by the lack of effect of a mutant PKI gene and by the unaffected expression of a reference gene (pRSV beta gal) under these conditions. Analysis of the effects of dibutyryl cAMP (1 mM), forskolin (10 microM), and 8-Br-cAMP (1 mM) on the transient expression of pHBCAT showed that these cAMP-elevating agents stimulated the hsp 70 promoter activity, whereas cAMP (1 mM) was without effect. Chloramphenicol acetyltransferase gene constructs with truncated or mutated hsp 70 promoter were used to define the cis-acting DNA element(s) that confer this cAMP stimulation; the heat induced (42 degrees C) expression was used as a control. Mutation of the adenovirus transcription factor element (pLSN-40/-26) greatly reduced the basal level of expression; forskolin had little or no effect on this adenovirus transcription factor-minus promoter, although the promoter activity was very heat inducible. The absence of a functional heat shock consensus element (HSE) in the construct pLSPNWT rendered the promoter heat insensitive; this construct was forskolin responsive although the magnitude of this stimulation was reduced when compared with that of a control construct with HSE. These results were corroborated by studies using consensus sequence of ATF (ATFE) and HSE as competitors to titrate our cellular factors that may interact with these elements. We showed that cotransfection with ATFE and HSE depressed the basal (37 degrees C) expression of pHBCAT by 25 and 60%, respectively. The heat-induced expression of pHBCAT was not significantly affected by the cotransfection of ATFE and was reduced by 60% when HSE was cotransfected. ATFE and HSE reduced the forskolin-induced pHBCAT expression by 70 and 40%, respectively. The implications of these findings as they relate to the action of cAMP and cAMP-dependent protein kinase in the control of heat shock gene expression are discussed.
authors.affiliation: Department of Biological Sciences, Rutgers-State University of New Jersey, Piscataway 08855-1059.
authors.forename: H S
authors.initials: HS
authors.lastname: Choi
authors.forename: B
authors.initials: B
authors.lastname: Li
authors.forename: Z
authors.initials: Z
authors.lastname: Lin
authors.forename: E
authors.initials: E
authors.lastname: Huang
authors.forename: A Y
authors.initials: AY
authors.lastname: Liu
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D001345:Autoradiography; D003994:Bucladesine / Q000494:pharmacology; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000032:analysis / Q000235:genetics; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000502:physiology*; D004247:DNA / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D005822:Genetic Vectors; D006360:Heat-Shock Proteins / Q000235:genetics*; D006801:Humans; D008168:Lung / Q000166:cytology / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000502:physiology*; D014162:Transfection
chemical_list: D006360:Heat-Shock Proteins; D047428:Protein Kinase Inhibitors; D005576:Colforsin; D015124:8-Bromo Cyclic Adenosine Monophosphate; D003994:Bucladesine; D004247:DNA; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase; D011494:Protein Kinases