Mechanism underlying berberine's effects on HSP70/TNFα under heat stress: Correlation with the TATA boxes.

Heat stress can stimulate an increase in body temperature, which is correlated with increased expression of heat shock protein 70 (HSP70) and tumor necrosis factor α (TNFα). The exact mechanism underlying the HSP70 and TNFα induction is unclear. Berberine (BBR) can significantly inhibit the temperature rise caused by heat stress, but the mechanism responsible for the BBR effect on HSP70 and TNFα signaling has not been investigated. The aim of the present study was to explore the relationship between the expression of HSP70 and TNFα and the effects of BBR under heat conditions, using in vivo and in vitro models. The expression levels of HSP70 and TNFα were determined using RT-PCR and Western blotting analyses. The results showed that the levels of HSP70 and TNFα were up-regulated under heat conditions (40 °C). HSP70 acted as a chaperone to maintain TNFα homeostasis with rising the temperature, but knockdown of HSP70 could not down-regulate the level of TNFα. Furthermore, TNFα could not influence the expression of HSP70 under normal and heat conditions. BBR targeted both HSP70 and TNFα by suppressing their gene transcription, thereby decreasing body temperature under heat conditions. In conclusion, BBR has a potential to be developed as a therapeutic strategy for suppressing the thermal effects in hot environments.

B1-induced caspase-independent apoptosis in MCF-7 cells is mediated by down-regulation of Bcl-2 via p53 binding to P2 promoter TATA box.

The Bcl-2 family contains a panel of proteins which are conserved regulators of apoptosis in mammalian cells, like the anti-apoptotic protein Bcl-2. According to its significant role in altering susceptibility to apoptosis, the deciphering of the mechanism of Bcl-2 expression modulation may be crucial for identifying therapeutics strategies for cancer. Treatment with naphthalimide-based DNA intercalators, including M2-A and R16, generally leads to a decrease in Bcl-2 intracellular amounts. Whereas the interest for these chemotherapeutics is accompanied by advances in the fundamental understanding of their anticancer properties, the molecular mechanism underlying changes in Bcl-2 expression remains poorly understood. We report here that p53 contributes to Bcl-2 down-regulation induced by B1, a novel naphthalimide-based DNA intercalating agent. Indeed, the decrease in Bcl-2 protein levels observed during B1-induced apoptosis was correlated to the decrease in mRNA levels, as a result of the inhibition of Bcl-2 transcription and promoter activity. In this context, we evaluated p53 contribution in the Bcl-2 transcriptional down-regulation. We found a significant increase of p53 binding to P(2) promoter TATA box in MCF7 cells by chromatin immunoprecipitation. These data suggest that B1-induced caspase-independent apoptosis in MCF-7 cells is associated with the activation of p53 and the down-regulation of Bcl-2. Our study strengthens the links between p53 and Bcl-2 at a transcriptional level, upon naphthalimide-based DNA intercalator treatment.

Phenylephrine requires the TATA box to activate transcription of GLUT1 in neonatal rat cardiac myocytes.

Cardiac hypertrophy and heart failure occur in association to alterations in glucose uptake and metabolism. Phenylephrine, among other hypertrophic agonists, has been reported to increase expression of GLUT1 in neonatal rat cardiac myocytes by activating transcription. However, the specific cis- or trans-acting factors in the GLUT1 gene that are targeted by this agonist remain elusive. Here we describe that the activity of the -99/+134 basal promoter of rat GLUT1 is increased by phenylephrine. Nevertheless, this is not mediated by previously described binding sites (GC-box, MG1E) in the promoter. Rather, the TATA box is required by the agonist to activate transcription from the promoter. Interestingly, The Ras-ERK mitogen-activated protein (MAP) kinase pathway is involved in the actions of phenylephrine on GLUT1 transcription, and the effects of Ras on the activity of the promoter depend on the integrity of the TATA box. Our data indicate that phenylephrine induces the expression of the TBP-associated factor TAF(II)250 mRNA, which increases in parallel to the expression of GLUT1, suggesting that altering the expression of basal transcription factors could be one mechanism by which phenylephrine may regulate the activity of the GLUT1 promoter.

Glutamate-dependent transcriptional regulation of GLAST: role of PKC.

The Na+-dependent glutamate/aspartate transporter GLAST plays a major role in the removal of glutamate from the synaptic cleft. Short-term, as well as long-term changes in transporter activity are triggered by glutamate. An important locus of regulation is the density of transporter molecules present at the plasma membrane. A substrate-dependent change in the translocation rate of the transporter molecules accounts for the short-term effect, whereas the long-term modulation apparently involves transcriptional regulation. Using cultured chick cerebellar Bergmann glial cells, we report here that glutamate receptors activation mediate a substantial reduction in the transcriptional activity of the chglast promoter through the Ca2+/diacylglicerol-dependent protein kinase (PKC) signaling cascade. Overexpression of constitutive active PKC isoforms of mimic the glutamate effect. Accordingly, increased levels of c-Jun or c-Fos, but not Jun-B, Jun-D or Fos-B, lower the chglast promoter activity. Serial deletions and electrophorectic mobility shift assays were used to define a specific region within the 5' proximal region of the chglast promoter, associated with transcriptional repression. A putative glutamate response element could be defined in the proximal promoter stretch more likely between nts -40 and -78. These results demonstrate that GLAST is under glutamate-dependent transcriptional control through PKC, and support the notion of a pivotal role of this neurotransmitter in the regulation of its own removal from the synaptic cleft, thereby modulating, mainly in the long term, glutamatergic transmission.

The antileukemia drug 2-chloro-2'-deoxyadenosine: an intrinsic transcriptional antagonist.

The nucleoside analog 2-chloro-2'-deoxyadenosine (CldAdo; cladribine) is effective in the treatment of hairy cell leukemia and chronic lymphocytic leukemia. CldAdo is phosphorylated and incorporated into cellular DNA but is not an absolute chain terminator. We demonstrated by in vitro gel-shift assays that binding interactions of the human TATA box-binding protein (TBP) were disrupted on 2-chlorodeoxyadenosine monophosphate (CldAMP)-substituted TATA box consensus sequences. We hypothesized that human RNA polymerase II (pol II) transcriptional processes would therefore be affected by 2-chlorodeoxyadenosine triphosphate (CldATP) incorporation into a promoter TATA element. Double-stranded DNA templates containing the adenovirus major late promoter and coding sequences were enzymatically synthesized as control or with site-specific CldAMP residues, incubated with HeLa extract, and the synthesis of radiolabeled 44-base transcripts was assessed. With increasing amounts of HeLa extract, CldAMP substitution for dAMP within the TATA box decreased in vitro pol II transcription by approximately 35% compared with control substrates. Time-course studies showed that transcript production increased in a linear fashion on control substrates. In contrast, transcription on CldAMP-substituted TATA sequences reached a plateau after 20 min. Furthermore, CldAMP-substituted promoter sequences trapped or sequestered TBP, preventing its dissociation from DNA and subsequent binding to additional TATA elements to reinitiate transcription. CldAdo thus represents the first example of a nucleoside analog that acts as a transcriptional antagonist. CldATP incorporation into gene regulatory sequences may provide a novel strategy to modulate specific protein/DNA interactions.

Gal4-VP16 and Gal4-AH increase the orientational and axial specificity of TATA box recognition by TATA box binding protein.

Previous work has shown that binding of the TATA box binding protein (TBP) to the TATA box is a rate-limiting step during pre-initiation complex (PIC) formation. Although the transcription of eukaryotic genes normally proceeds in one direction, studies in solution have shown that TBP lacks the information necessary to orient itself on the TATA box. Instead, yeast TBP binds TATA-containing promoters in two orientations that are related by a 180 rotation about TBP's pseudo-2-fold symmetry axis. Recruitment of PIC components by gene-specific activators is considered a primary mechanism of transcriptional enhancement. Here we ask whether activators might function, at least in part, by increasing the fraction of PICs assembled with TBP bound in the orientation necessary for transcription. We use DNA affinity cleavage and a TBP-phenanthroline-copper conjugate to monitor the orientation of TBP in the presence of the well-studied activators Gal4-VP16 and Gal4-AH. In the absence of a transcriptional activator, only 51% of the TBP x TATA box complexes were bound in the orientation necessary for the initiation of transcription. However, in the presence of saturating Gal4-VP16, 87% of the TBP bound to the TATA box was oriented correctly at equilibrium. This increase in orientational specificity corresponds to a free energy difference (Delta Delta G(obs)) of 1.1 kcal x mol(-1) and was accompanied by a dramatic increase in axial specificity, reminiscent of the effects of transcription factors TFIIB and TFIIA reported previously. Gal4-AH also enhanced the orientational and axial specificity of the TBP x TATA complex, although to a lesser extent. We suggest that these effects on specificity represent a variation of recruitment, since they require direct interactions between the activator and a PIC component but only increase the effective concentration of the correctly oriented PIC component. These findings add to increasing evidence that recruitment may encompass a broad range of mechanisms.

p53 down-regulates human bradykinin B1 receptor gene expression.

The tumor suppressor, p53, has been shown to transcriptionally activate or silence a number of target genes. As an activator, p53 relies on its specific consensus sequence within the promoter. It is not clear whether p53 requires a specific DNA binding site in its action as a gene repressor. This report demonstrates that the human BKB1R gene is a p53 target. Expression of p53 in transiently transfected SV40-transformed IMR90 cells strongly suppressed luciferase reporter activity driven by a 1.8 kb BKB1R promoter as well as its minigene. These down-regulations were p53 dose-dependent. p53 reduced both basal and induced promoter activities of the minigene. Expression of p53 abolished the inducibility of the minigene. Induction of endogenous p53 expression by etoposide also inhibited promoter activity and minigene inducibility. Replacing the region containing both the putative p53 binding site and the TATA-box with a basal adenovirus promoter in the 1.8 kb promoter construct did not prevent p53 from inhibiting BKB1R promoter activity. Thus suppression by p53 is not mediated by competition with the TATA-binding protein and is not through interaction with the putative p53-binding site. p53 also does not appear to suppress BKB1R gene expression through interaction with c-Jun which functions in the inducibility of this gene [Yang et al., 2001].

Controlling gene expression by zinc(II)-macrocyclic tetraamine complexes.

The zinc(II) complexes of 12-membered macrocyclic tetraamines (1,4,7,10-tetraazacyclododecane, cyclen) appended with one or two aryl-methyl group(s) (quinolyl-methyl, naphthyl-methyl, and acridinyl-methyl) selectively bind to thymines in a TATA box of the SV40 early promoter region and thus inhibit the binding of a transcriptional factor, TATA binding protein. These Zn2+-cyclen derivatives also act as inhibitors of DNA-targeted enzymes, type I and type II topoisomerases. They also exhibited strong antimicrobial activities for the gram-positive bacterial strain. These biochemical and biological properties were compared with those of conventionally established AT-recognizing drugs, distamycin A and DAPI. The Zn2+-cyclen complexes are a new type of small molecular, genetic transcriptional regulation factor.

Modeling DNA deformations induced by minor groove binding proteins.

Molecular modeling is used to demonstrate that the major structural deformations of DNA caused by four different minor groove binding proteins, TBP, SRY, LEF-1, and PurR, can all be mimicked by stretching the double helix between two 3'-phosphate groups flanking the binding region. This deformation reproduces the widening of the minor groove and the overall bending and unwinding of DNA caused by protein binding. It also reproduces the principal kinks associated with partially intercalated amino acid side chains, observed with such interactions. In addition, when protein binding involves a local transition to an A-like conformation, phosphate neutralization, via the formation of protein-DNA salt bridges, appears to favor the resulting deformation.

c-sis/platelet-derived growth factor-B promoter requirements for induction during the 12-O-tetradecanoylphorbol-13-acetate-mediated megakaryoblastic differentiation of K562 human erythroleukemia cells.

Platelet-derived growth factor (PDGF), a powerful mitogen and chemoattractant, is composed of two subunits, A and B, which are synthesized by normal megakaryocytes. We have studied the transcriptional regulation of the c-sis/PDGF-B gene in human K562 erythroleukemia cells that have been induced to undergo megakaryoblastic differentiation by treatment with 12-O-tetradecanoylphorbol-13-acetate. Upon differentiation of these cells, c-sis/PDGF-B transcription is increased 50-100-fold. We show here that a minimal c-sis/PDGF-B promoter region, spanning nucleotides -64 to +6, retains full inducibility. Linker scanning mutagenesis within this minimal region identified four segments that were important for expression in differentiating K562 cells: a previously defined sis proximal element (SPE; -64 to -45), the TATA box, the 10 bp immediately downstream of the TATA box [TATA neighboring sequence (TNS); -24 to -15], and the mRNA start site region. Combined mutation of the SPE and TNS resulted in a greater impairment of induction than did mutation of either sequence alone. In contrast, combined mutation of the SPE and the start site or of the TNS and the start site did not lower induction beyond that displayed by the least inducible single mutants. The combination of the SPE and the TNS was sufficient to confer wild-type levels of inducibility to a heterologous promoter. Both the SPE and the TNS were sensitive to alterations in the helical spacing between these elements and the TATA box. Using the electrophoretic mobility shift assay, we demonstrated binding of Sp family members and of two additional unidentified nuclear factors to the TNS in both 12-O-tetradecanoylphorbol-13-acetate-treated and untreated cells. The TNS, therefore, appears to represent a target for a constitutively bound factor(s) that is required for cooperation with a differentiation-specific factor bound at the SPE to drive efficient c-sis/PDGF-B transcription in TPA-treated K562 cells.

Restoration of TATA-dependent transcription in a heat-inactivated extract of tobacco nuclei by recombinant TATA-binding protein (TBP) from tobacco.

We isolated a complementary DNA (cDNA) that encoded a TATA-binding protein (TBP) from a cDNA library of tobacco (Nicotiana tabacum) suspension-cultured cells (BY-2). A comparison among deduced amino acid sequences of plant TBPs revealed the presence of a long conserved region within the amino acid sequence of the TBP. Genomic Southern analysis revealed that tobacco TBP (tTBP) is encoded by only a small number of copies of a gene in the tobacco genome. Addition of recombinant tTBP to an extract of tobacco nuclei (TNE) enhanced the basal transcriptional activity in vitro. This result indicates that the level of tTBP is a rate-limiting factor for basal transcriptional activity in TNE. We subsequently succeeded in the functional complementation of TATA-dependent initiation of transcription that was associated with a plant promoter in a homologous plant system. Addition of bacterially expressed recombinant tTBP to a heat-inactivated TNE restored transcriptional activity, as did the addition of human TBP. Moreover, heating of the recombinant tTBP eliminated its ability to restore transcriptional activity. It appears that the heat inactivation of TNE was caused by the heat inactivation of tTBP in TNE.

Regulation of insulin-like growth factor II P3 promotor by p53: a potential mechanism for tumorigenesis.

Human insulin-like growth factor (IGF)-II mRNA has been shown to be expressed at high levels in a variety of tumors, including rhabdomyosarcomas. In addition, many tumors have alterations in p53 expression. To investigate whether p53 regulates IGF-II gene expression, we transfected wild-type p53 expression vectors and luciferase constructs driven by IGF-II P3 promotors into multiple cell lines. We found that p53 reduced, in a dose-dependent manner, both endogenous IGF-II P3 transcripts and transfected P3 luciferase expression. The inhibition of P3 luciferase expression by p53 was more pronounced in the two cell lines that expressed mutant p53 protein, RD, and HTB114. The element responsible for this inhibition was mapped to the minimal promoter region. We also transfected an HPV-16 E6 expression plasmid into CCL13 cells containing functional p53 and found that E6 up-regulated IGF-II P3 activity. Wild-type, but not mutant, p53 interfered with the binding of TATA-binding protein to the TATA motif of P3, although both could directly associate with human TATA-binding protein. Our results suggest that p53 may play a role in regulation of IGF-II gene expression.

TBP binding to the TATA box induces a specific downstream unwinding site that is targeted by pluramycin.

BACKGROUND: The TATA-binding protein (TBP) is one of the major components of the human TFIID multiprotein complex. It is important in directing the initiation of RNA transcription at a site immediately downstream of the TATA sequence (TATA box) found in many eukaryotic promoters. The crystal structure of TBP complexed with an oligonucleotide containing the TATA box revealed a protein with an approximate two-fold symmetry which apparently has symmetrical interactions with DNA. It is not known how an asymmetric effect involving downstream activation can be produced by an apparent symmetric complex. We set out to examine the state of DNA in the TBP-DNA complex using pluramycin, a small molecular weight probe of DNA accessibility. RESULTS: Binding of TBP to the TATA box facilitates intercalation of pluramycin at a defined site immediately downstream of the TATA sequence through an apparent transient unwinding of the DNA. Pluramycin adducts are detected by the production of DNA strand breakage products upon heating. Incubation of pluramycin with the TBP-DNA complex facilitates the trapping of the specific complex by intercalation. Gel mobility shift and circularization assays reveal that the binding of pluramycin on the 3'-side of the TATA box region considerably stabilizes the TBP-DNA complex. CONCLUSIONS: We propose that the TBP-DNA-pluramycin ternary complex is a 'specific' binding mode in which TBP and pluramycin make compensatory alterations in DNA, accounting for the improved stability of the ternary complex. We also propose a model of the ternary complex that explains the observed asymmetric effect of TBP binding to the TATA box.

Characterization of nuclear factors for elicitor-mediated activation of the promoter of the pea phenylalanine ammonia-lyase gene 1.

The nuclear factors presumably associated with the activation of the gene encoding phenylalanine ammonia-lyase by a fungal elicitor were characterized in pea (Pisum sativum L.) epicotyls. The TATA-proximal region was dissected and putative cis-regulatory elements in the promoter of pea phenylalanine ammonia-lyase gene 1 were examined by gel-mobility shift and in vitro footprinting analyses. Specific binding of the nuclear factors to the promoter-proximal regions of pea phenylalanine ammonia-lyase gene 1 associated with elicitor-mediated activation was detected at a region containing consensus sequence motifs of boxes 2 and 4 and other AT-rich sequences. The analyses of DNA fragments containing the deleted promoter regions suggested that a residue from -183 to -173 (ATTAGTAAGTGAT) was essential for a maximal activity of forming low-mobility complex (LMC) in the gel-mobility shift assay, and synthetic oligonucleotides confirmed the presence of at least one nuclear component associated with the formation of an active LMC. Competition experiments and treatment with Hoechst 33258 provided direct evidence that the formation of LMC with the promoter fragments from genes encoding phenylalanine ammonia-lyase and chalcone synthase in pea contained one or more of the same proteins that recognize AT-rich sequence motifs for binding. It also suggests that common high-mobility group-like proteins might be involved in the regulation of elicitor-inducible genes in pea.

Regulated activity of the distal promoter-like element of the human corticotropin-releasing hormone gene and secondary structural features of its corresponding transcripts.

Corticotropin-releasing hormone (CRH) plays a major role in the coordination of the stress response. Its gene is expressed in multiple brain regions, the peripheral sympathetic system and the placenta, as well as in peripheral inflammatory sites where CRH acts as a pro-inflammatory cytokine. The human (h) CRH gene, in addition to its primary promoter (TATA box I), has a second distal promoter-like structure (TATA box II) and a functional cyclic adenosine monophosphate-responsive element, all of which are preserved in the rat and ovine genes. To examine the functionality of TATA II, we positioned a 881-bp-long segment of the 5' flanking region of the hCRH gene containing TATA II, but lacking TATA I, upstream from a chloramphenicol acetyltransferase (CAT) reporter gene cloned in a pUC vector. We transfected COS-7 cells with this construct and examined responsiveness of CAT activity to potential stimulants and inhibitors. Phorbol ester (TPA) and forskolin had mild but clear stimulatory effects on CAT expression (approximately 1.5- and approximately 1.3-fold, respectively), with a combined effect of approximately 1.9-fold. Dexamethasone (DEX) inhibited TPA-stimulated CAT activity by approximately 2.6-fold. In contrast, in the presence of a co-transfected glucocorticoid receptor cDNA expression plasmid, DEX augmented TPA-stimulated CAT expression by approximately 3.1-fold. The predicted secondary structures of the primary transcripts employing the distal and proximal promoters had significant differences, which could affect their stability and translatability.2

The immunosuppressant FK-506 specifically inhibits mitogen-induced activation of the interleukin-2 promoter and the isolated enhancer elements NFIL-2A and NF-AT1.

The macrolide FK-506, like the cyclic undecapeptide cyclosporin A (CsA), is a potent immunosuppressant that interferes with the transcriptional activation of several early-phase genes in T lymphocytes, including that for interleukin-2 (IL-2). We compared the effects of FK-506 and CsA on transcription from the 5' upstream activating sequences (UAS) of the human IL-2 gene and several cellular and viral UAS to define cis-acting sites which may be responsive to FK-506. The UAS surveyed included the human IL-2 receptor alpha-chain, human metallothionein II, simian virus 40 early, human cytomegalovirus immediate-early, adenovirus major late, and Rous sarcoma virus long terminal repeat UAS. In addition, we studied multimers of several defined promoter elements (NFIL-2A, NF-kappa B, or NF-AT1) which are found in the UAS of the human IL-2 gene and which have been reported to be responsive to CsA when linked to a minimal promoter element (TATA box and transcription start site). Each promoter-regulatory region was fused to the bacterial chloramphenicol acetyltransferase gene and used to transiently transfect Jurkat cells. Quantitative chloramphenicol acetyltransferase assay determinations indicated that the transcriptional activity of each UAS induced upon T-cell activation was (i) completely sensitive, (ii) partially sensitive, or (iii) resistant to inhibition by CsA and FK-506. The induced transcription driven by the IL-2 promoter elements NF-AT1 and NFIL-2A could be blocked completely by FK-506 or CsA. Gel mobility shift assays indicated that the binding activities of the factors specifically interacting with these sequences were detected in activated cells regardless of whether the cells were treated with FK-506 or CsA. The results suggest that FK-506 or CsA inhibits a transacting mechanism(s) without disrupting the binding activities of these transcription factors. The degree to which each UAS was resistant to FK-506 was consistent with the level of transcription induced by phorbol myristate acetate, while UAS which were sensitive to inhibition by FK-506 were dependent on the presence of both phorbol myristate acetate and ionomycin.

Induction of dormant HIV-1 by sodium butyrate: involvement of the TATA box in the activation of the HIV-1 promoter.

Reactivation of latent HIV-1 is believed to play a major role in the pathogenesis of AIDS. Here we show that sodium butyrate (NaB), which can cause gene induction or cell differentiation, reactivates dormant HIV-1 in vitro in chronically infected cells of T-lymphoid and monocytoid origin. The effect of NaB on HIV-1 expression in T-lymphoid cells was apparent 3 h after addition of drug and peaked at 24 h. During this time the proportion of HIV-1 antigen expressing cells increased from less than 0.5 to greater than 90%, and virus production increased by three orders of magnitude. The virus released by the NaB-induced cells was infectious. The extent and kinetics of NaB effects were similar to effects of phorbol 12-myristate 13-acetate in T cells, but not monocytes. Transient expression assays using an indicator gene under the control of the HIV-1 long terminal repeat revealed that mutations which altered the nucleotide sequence in the TATA box significantly reduced the NaB effect. These data show that NaB is a potent inducer of dormant HIV-1 and suggest that the TATA motif is required for this activity.

On the mechanism for efficient repression of the interleukin-6 promoter by glucocorticoids: enhancer, TATA box, and RNA start site (Inr motif) occlusion.

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.