title: On the mechanism for efficient repression of the interleukin-6 promoter by glucocorticoids: enhancer, TATA box, and RNA start site (Inr motif) occlusion.
abstract: The feedback inhibition of interleukin-6 (IL-6) gene expression by glucocorticoids represents a regulatory link between the endocrine and immune systems. The mechanism of the efficient repression of the IL-6 promoter by dexamethasone (Dex) was investigated in HeLa cells transiently transfected with plasmid constructs containing different IL-6 promoter elements linked to the herpesvirus thymidine kinase gene (tk) promoter and the bacterial chloramphenicol acetyltransferase gene (cat) and cotransfected with cDNA vectors constitutively expressing either the active wild-type or inactive mutant human glucocorticoid receptor (GR). The induction by interleukin-1, tumor necrosis factor, phorbol ester, or forskolin of IL-6-tk-cat chimeric constructs containing a single copy of the IL-6 DNA segment from -173 to -151 (MRE I) or from -158 to -145 (MRE II), which derive from within the multiple cytokine- and second-messenger-responsive enhancer (MRE) region, was strongly repressed by Dex in a wild-type GR-dependent fashion irrespective of the inducer used. The induction by pseudorabies virus of an IL-6 construct containing the IL-6 TATA box and the RNA start site ("initiator" or Inr element) but not the MRE region was also repressed by Dex in the presence of wild-type GR. DNase I footprinting showed that the purified DNA-binding fragment of GR bound across the MRE, the TATA box, and the Inr site in the IL-6 promoter; this footprint overlapped that produced by proteins present in nuclear extracts from uninduced or induced HeLa cells. Imperfect palindromic nucleotide sequence motifs moderately related to the consensus GR-responsive element (GRE) motif were present at the Inr, the TATA box, and the MRE II site in the IL-6 promoter; although MRE I and a GR-binding site between -201 and -210 in IL-6 both lacked a discernible inverted repeat motif, their sequences showed considerable similarity with negative GRE sequences in other Dex-repressed genes. Surprisingly, chimeric genes containing MRE II, which lacks a recognizable GACGTCA cyclic AMP- and phorbol ester-responsive motif, were strongly induced by both phorbol ester and forskolin, suggesting that MRE II (ACATTGCACAATCT) may be the prototype of a novel cyclic AMP- and phorbol ester-responsive element. Taken together, these observations suggest that ligand-activated GR represses the IL-6 gene by occlusion not only of the inducible IL-6 MRE enhancer region but also of the basal IL-6 promoter elements.
authors.affiliation: Rockefeller University, New York, New York 10021-6399.
authors.forename: A
authors.initials: A
authors.lastname: Ray
authors.forename: K S
authors.initials: KS
authors.lastname: LaForge
authors.forename: P B
authors.initials: PB
authors.lastname: Sehgal
mesh_terms: D001483:Base Sequence; D005576:Colforsin / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology*; D004742:Enhancer Elements, Genetic / Q000187:drug effects*; D005246:Feedback; D015870:Gene Expression / Q000187:drug effects*; D016153:Genes, Suppressor / Q000187:drug effects*; D006367:HeLa Cells / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6 / Q000235:genetics*; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic; D012334:RNA, Neoplasm / Q000187:drug effects / Q000235:genetics; D011965:Receptors, Glucocorticoid / Q000235:genetics / Q000378:metabolism*; D015183:Restriction Mapping; D015290:Second Messenger Systems; D016385:TATA Box / Q000187:drug effects*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D007375:Interleukin-1; D015850:Interleukin-6; D015345:Oligonucleotide Probes; D012334:RNA, Neoplasm; D011965:Receptors, Glucocorticoid; D014409:Tumor Necrosis Factor-alpha; D005576:Colforsin; D003907:Dexamethasone; D013755:Tetradecanoylphorbol Acetate