Biomedical subjects

L Poellinger

Publications and source records attributed to L Poellinger.

At least 73 records · Page 4Linked to original sources

PubMed1991

High level expression of functional full length and truncated glucocorticoid receptor in Chinese hamster ovary cells. Demonstration of ligand-induced down-regulation of expressed receptor mRNA and protein.

Full length human glucocorticoid receptor and truncated receptor derivatives lacking the major amino-terminal trans-activating domain were expressed in stably transfected Chinese hamster ovary (CHO) cells. The receptors were co-expressed together with human metallothionein IIa, and the expression levels were amplified in the presence of increasing concentrations of metal. In amplified cells, both synthesized receptor forms showed the expected molecular weights, as assayed by affinity labeling and immunoblotting. They were expressed at concentrations of about 350,000-520,000 molecules/cell which corresponds to a 10-fold increase in receptor levels as compared to rat liver cells. The hormone (agonist or antagonist) binding properties of the expressed proteins were very similar to those characteristic of authentic glucocorticoid receptors in tissues or cultured cells. Moreover, the expressed proteins specifically recognized a glucocorticoid-response element sequence motif in in vitro protein-DNA binding experiments. The activation of a glucocorticoid-responsive reporter gene by the expressed full length receptor was dramatic (about 75-fold) and strictly ligand-dependent. In contrast, the expressed amino-terminal deletion mutant exhibited considerably weaker functional activity but showed normal hormone-binding properties. Upon exposure to dexamethasone in vivo, the expressed receptor mRNAs and proteins were down-regulated about 2- to 6-fold, indicating that regulatory signals important for autoregulation may be contained within structures corresponding to the ligand and DNA-binding domains. Transcription from the expression vector was not negatively regulated from the hormone, strongly arguing that receptor down-regulation was due to a post-transcriptional mechanism. In conclusion, this expression system should be a useful tool for further structural and functional studies of the receptor, including the biochemistry of its activation from a cryptic to a functional species, and its ligand-dependent autoregulation.

Affinity Labels↗

PubMed1991

Role of the ligand in intracellular receptor function: receptor affinity determines activation in vitro of the latent dioxin receptor to a DNA-binding form.

To reconstitute the molecular mechanisms underlying the cellular response to soluble receptor ligands, we have exploited a cell-free system that exhibits signal- (dioxin-)induced activation of the latent cytosolic dioxin receptor to an active DNA-binding species. The DNA-binding properties of the in vitro-activated form were qualitatively indistinguishable from those of in vivo-activated nuclear receptor extracted from dioxin-treated cells. In vitro activation of the receptor by dioxin was dose dependent and was mimicked by other dioxin receptor ligands in a manner that followed the rank order of their relative affinities for the receptor in vitro and their relative potencies to induce target gene transcription in vivo. Thus, in addition to triggering the initial release of inhibition of DNA binding and presumably allowing nuclear translocation, the ligand appears to play a crucial role in the direct control of the level of functional activity of a given ligand-receptor complex.

Animals↗

PubMed1991

The glucocorticoid receptor binds to a sequence overlapping the TATA box of the human osteocalcin promoter: a potential mechanism for negative regulation.

Expression of the human osteocalcin promoter is negatively regulated by glucocorticoids in vivo. In vitro DNase I and exonuclease III footprinting analysis showed binding of purified glucocorticoid receptor in close proximity to and overlapping with the TATA box of the osteocalcin gene. These results imply competition or interference with binding of the TATA box-binding transcription factor IID as a mechanism of repression of this gene by glucocorticoids. In support of this notion, point mutation analysis of the receptor binding site indicated that flanking nucleotides and not the TATA box motif per se were important for receptor interaction. Moreover, DNA binding competition assays showed specific binding of the receptor only to the TATA box region of the osteocalcin gene and not to the corresponding region of an immunoglobulin heavy-chain promoter.

Base Sequence↗

PubMed1991

Liver cells contain constitutive DNase I-hypersensitive sites at the xenobiotic response elements 1 and 2 (XRE1 and -2) of the rat cytochrome P-450IA1 gene and a constitutive, nuclear XRE-binding factor that is distinct from the dioxin receptor.

Dioxin stimulates transcription from the cytochrome P-450IA1 promoter by interaction with the intracellular dioxin receptor. Upon binding of ligand, the receptor is converted to a form which specifically interacts in vitro with two dioxin-responsive positive control elements located in close proximity to each other about 1 kb upstream of the rat cytochrome P-450IA1 gene transcription start point. In rat liver, the cytochrome P-450IA1 gene is marked at the chromatin level by two DNase I-hypersensitive sites that map to the location of the response elements and exist prior to induction of transcription by the dioxin receptor ligand beta-naphthoflavone. In addition, a DNase I-hypersensitive site is detected near the transcription initiation site and is altered in nuclease sensitivity by induction. The presence of the constitutive DNase I-hypersensitive sites at the dioxin response elements correlates with the presence of a constitutive, labile factor which specifically recognizes these elements in vitro. This factor appears to be distinct from the dioxin receptor, which is observed only in nuclear extract from treated cells. In conclusion, these data suggest that a certain protein-DNA architecture may be maintained at the response elements at different stages of gene expression.

Animals↗

PubMed1990

A hormone response element upstream from the human alcohol dehydrogenase gene ADH2 consists of three tandem glucocorticoid receptor binding sites.

The 5'-flanking region of the human gene encoding beta-alcohol dehydrogenase (ADH2) was shown by DNase I footprinting to contain three tandem binding sites for purified glucocorticoid receptor. The three binding sites lie very close together between nucleotide (nt) positions -245 and -171 with respect to the transcription start point. DNase I footprinting using a rat liver nuclear extract indicated a lack of protection of the glucocorticoid receptor binding sites, but protection of a sequence between nt -209 and -191 which partially overlaps the glucocorticoid receptor binding sites I and II. This site has homology with the known binding site for hepatocyte nuclear factor 1 (HNF1). ADH2 promoter DNA fragments containing various lengths of 5'-flanking sequences were fused upstream from the gene encoding chloramphenicol acetyltransferase (cat) and transfected into the HepG2 human hepatoma cell line. The resulting cat expression was subject to induction by dexamethasone in constructions containing ADH2 DNA between nt -272 and -171. This indicates that the glucocorticoid receptor binding sites identified by footprint analysis function as a glucocorticoid response element (GRE) in a liver cell line. Heterologous ADH-cat fusions, in which the ADH2-GRE was fused to the adenovirus major late promoter, exhibited glucocorticoid induction of cat expression in CV-1B cells when cotransfected with a glucocorticoid receptor expression vector. Glucocorticoid regulation in CV-1B was observed when either all three glucocorticoid receptor binding sites (sites 0, I, II) or the two distal sites (sites 0, I) were present. Overall, these results indicate that the ADH2 gene possesses a functional GRE which can potentially regulate expression transcriptionally.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohol Dehydrogenase↗

PubMed1990

Activation of the dioxin and glucocorticoid receptors to a DNA binding state under cell-free conditions.

The activation in vitro of dioxin and glucocorticoid receptors from a non-DNA binding to a DNA binding state was characterized. Ligand-free dioxin and glucocorticoid receptors were partially co-purified from rat liver cytosol, and both receptors sedimented at 9 S following labeling with the respective ligand. The 9 S forms of the dioxin and glucocorticoid receptors have previously been shown to represent heteromeric complexes containing the Mr approximately equal to 90,000 heat shock protein. The 9 S ligand-free or ligand-bound glucocorticoid receptor was converted to the monomeric 4-5 S form upon exposure to 0.4 M NaCl even in the presence of the stabilizing agent molybdate. Under identical conditions, the 9 S ligand-free and ligand-bound dioxin receptor forms remained essentially intact. However, in the absence of molybdate, the dioxin receptor could be converted to a 4-5 S form upon exposure to high concentrations of salt. These results indicate that the glucocorticoid receptor readily dissociates from the 9 S to the 4-5 S form even in the absence of hormone, whereas both the ligand-free and ligand-occupied 9 S dioxin receptor forms represent more stable species. Gel mobility shift experiments revealed that the 4-5 S glucocorticoid receptor interacted with a glucocorticoid response element both in the absence and presence of ligand. On the other hand, occupation of the dioxin receptor by ligand greatly enhanced the ability of the receptor to be activated to a form that binds to its target enhancer element. Once dissociated, the monomeric form of the dioxin receptor was also able to interact with its DNA target sequences even in the absence of ligand. Thus, ligand binding efficiently facilitates subunit dissociation of the dioxin receptor but is not a prerequisite for DNA binding per se. Given the apparent stability of its non-DNA binding 9 S form, the dioxin receptor system might be a useful model for the investigation of the mechanism of activation of soluble receptor proteins.

Animals↗

PubMed1990

The specific DNA binding activity of the dioxin receptor is modulated by the 90 kd heat shock protein.

The dioxin receptor is a gene regulatory protein which exhibits many structural and functional similarities to steroid hormone receptors. In this study we compare the subunit composition of two forms of the dioxin receptor, sedimenting at approximately 9S and approximately 6S respectively, which are present in nuclear extract from wild-type Hepa 1c1c7 mouse hepatoma cells following treatment in vivo with dioxin. The nuclear approximately 9S receptor form contained the 90 kd heat shock protein, hsp90. As assessed by a gel mobility shift assay, this receptor form did not bind to the xenobiotic response element (XRE) of the target gene cytochrome P-450 IA1. In contrast, the smaller approximately 6S receptor form did not contain any immunochemically detectable hsp90. Moreover, this receptor form specifically bound to the XRE recognition sequence. Thus, the specific DNA binding activity of the dioxin receptor was inhibited by association with hsp90, and the approximately 9S dioxin receptor species could be regarded as a nonactive receptor form. Neither the approximately 9S nor the approximately 6S receptor forms were detected in nuclear extract from a dioxin treated mutant clone of Hepa 1 that expresses a nuclear translocation deficient receptor phenotype. We conclude that activation of the dioxin receptor is, at least, a two step process involving binding of the ligand and dissociation of hsp90 from the ligand-binding receptor protein. Inhibition of the DNA binding activity of transcription factors by protein--protein interaction has also been described for several steroid hormone receptors and for the NF kappa B factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Structural differences between the glucocorticoid, dioxin and oxysterol receptors from rat liver cytosol.

The rat hepatic glucocorticoid, dioxin and oxysterol receptors were subjected to high performance liquid chromatography on size-exclusion and anion-exchange columns. Both the glucocorticoid receptor and the dioxin receptor had a Stokes radius Rs approximately 7.5 nm, expected value for heteromeric complexes containing a dimer of the Mr approximately 90,000 heat shock protein, hsp90 (Rs approximately 7.0 nm). The oxysterol receptor represented a much smaller entity (Rs approximately 6.0 nm). When analyzed on a Mono Q anion-exchange column, the molybdate-stabilized glucocorticoid receptor and dioxin receptor eluted as single peaks at approximately 0.30 M and 0.26-0.28 M NaCl, respectively, whereas the oxysterol receptor represented a less negatively charged species (0.11-0.14 M NaCl). Following washing of the Mono Q column with molybdate-free buffer, the activated monomeric glucocorticoid receptor was detected (0.10-0.12 M NaCl). In contrast, no modification in the elution pattern of the dioxin receptor and the oxysterol receptor was observed. These data demonstrate differences in the physico-chemical properties of the glucocorticoid, dioxin and oxysterol receptors, respectively, which might reflect structural differences.

Animals↗

PubMed1989

Functional cooperativity between protein molecules bound at two distinct sequence elements of the immunoglobulin heavy-chain promoter.

Immunoglobulin heavy-chain gene promoters contain two conserved upstream sequence elements, octamer and heptamer, both of which are required for normal cell type-specific promoter function in vivo. The octamer sequence motif 5'-ATGCAAAT-3', and its precise inverse, are strongly conserved in heavy- and light-chain gene promoters and are important determinants for the lymphoid-specific function of these promoters and of the heavy-chain enhancer. The heptameric sequence element with the consensus 5'-CTCATGA-3' (refs 3 and 4) is also required in addition to the octamer for full lymphoid-specific activity of heavy-chain promoters. Although these two elements have no sequence similarity, they are both recognized in vitro by the ubiquitous octamer transcription factor OTF-1 (reviewed refs 13 and 14) and the lymphoid-specific OTF-2 (reviewed in refs 15 and 16). Here we show that purified OTF-2 binds cooperatively to the immunoglobulin heptamer and octamer elements so that interaction with the octamer element facilitates binding of OTF-2 to the heptamer motif. More important, cooperativity in OTF-2 binding is closely mirrored by functional cooperation between the heptamer and octamer elements in activating transcription from the heavy-chain promoter in vitro.

Base Sequence↗

PubMed1989

Ligand-dependent interaction of the dioxin receptor with target DNA.

Wild type and nuclear transfer deficient mouse hepatoma cell lines were used to study the specific DNA binding of a dioxin inducible factor. This factor interacts with XRE only after dioxin treatment and is absent in receptor mutant containing cells even after treatment. Thus, evidence is provided to substantiate the claim that the dioxin receptor is involved in the specific DNA interaction with dioxin response enhancer elements. It is also shown that the molybdate stabilised dioxin-receptor interacts with hsp90 suggesting that, in similarity to the glucocorticoid receptor, the dioxin receptor is kept in a non-transformed state in the absence of ligand.

Animals↗

PubMed1989

Specific protein-DNA interactions at a xenobiotic-responsive element: copurification of dioxin receptor and DNA-binding activity.

Upon binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (called dioxin or TCDD), the dioxin receptor exhibits increased affinity for the cell nucleus in vivo and for DNA in vitro. To define the recognition sequence of the dioxin receptor and its relationship with that of the glucocorticoid receptor, oligonucleotides derived from dioxin-responsive elements of the rat cytochrome P-450c gene were tested for their ability to form specific protein-DNA complexes in a gel retardation assay. We found that a previously defined sequence motif that is similar to the glucocorticoid-responsive element and exhibits strong enhancer activity in response to dioxin receptor ligands bound a dioxin-inducible factor with high specificity but was not recognized by the DNA-binding domain of the glucocorticoid receptor. Binding to this element was only observed in nuclear extracts of wild-type mouse hepatoma cells in a time- and dose-dependent manner and not in nuclear extracts from a nonresponsive mutant cell line deficient in DNA binding of the dioxin receptor. The specific DNA-binding activity in wild-type nuclear extracts comigrated in a Superose size-exclusion column and cosedimented on sucrose gradients with the in vivo labeled dioxin receptor. These experiments strongly suggest that the dioxin receptor is a sequence-specific DNA-binding protein and is not only biochemically but also functionally similar to the steroid receptor family.

Animals↗

PubMed1989

Octamer transcription factors 1 and 2 each bind to two different functional elements in the immunoglobulin heavy-chain promoter.

Immunoglobulin heavy-chain genes contain two conserved sequence elements 5' to the site of transcription initiation: the octamer ATGCAAAT and the heptamer CTCATGA. Both of these elements are required for normal cell-specific promoter function. The present study demonstrates that both the ubiquitous and lymphoid-cell-specific octamer transcription factors (OTF-1 and OTF-2, respectively) interact specifically with each of the two conserved sequence elements, forming either homo- or heterodimeric complexes. This was surprising, since the heptamer and octamer sequence motifs bear no obvious similarity to each other. Binding of either factor to the octamer element occurred independently. However, OTF interaction with the heptamer sequence appeared to require the presence of an intact octamer motif and occurred with a spacing of either 2 or 14 base pairs between the two elements, suggesting coordinate binding resulting from protein-protein interactions. The degeneracy in sequences recognized by the OTFs may be important in widening the range over which gene expression can be modulated and in establishing cell type specificity.

Base Sequence↗

PubMed1988

Association of the dioxin receptor with the Mr 90,000 heat shock protein: a structural kinship with the glucocorticoid receptor.

The Mr approximately equal to 90,000 heat shock protein, hsp90, readily interacts with the glucocorticoid receptor to form the 9 S, non-DNA-binding receptor complex. This receptor is stabilized in cytosolic preparations by sodium molybdate. In analogy, sodium molybdate stabilizes a 9 S form of the dioxin receptor. Polyclonal antibodies raised against the purified glucocorticoid receptor-associated hsp90 interact with the molybdate-stabilized 9 S dioxin-receptor complex but not with the 4 S dioxin receptor monomer, as assessed by sedimentation shift analysis on sucrose gradients. Thus we conclude that both the dioxin and glucocorticoid receptor can form heteromeric complexes which share a common non-ligand-binding component. These results represent the first demonstration of a structural relationship between the dioxin and glucocorticoid receptors.

Animals↗

PubMed1988

Requirement of hormone for thermal conversion of the glucocorticoid receptor to a DNA-binding state.

A central question arising from the model of eukaryotic gene regulation by steroid hormone receptors is whether or not proteins represent pre-existing gene regulatory proteins that are activated on exposure to the extracellular signal. It has been generally believed that the ligand-binding of steroid hormone receptors triggers an allosteric change in receptor structure, manifested by an increased affinity of the receptor for DNA in vitro and nuclear target elements in vivo, as monitored by nuclear translocation. But this model has been challenged by recent reports indicating that glucocorticoid and progesterone receptors bind specifically in vitro to target DNA sequences even in the absence of hormone. On the other hand, it appears that the hormone induces protection in vivo of the glucocorticoid response element of the tyrosine amino transferase gene. Here we show that under conditions permitting minimal in vitro manipulation, the steroid-free glucocorticoid receptor in crude cytosol associates with the hsp90 heat shock protein (relative molecular mass Mr approximately equal to 90,000) to form a large 300K complex, rather than the 94K liganded receptor monomer. More importantly, we have developed an assay to demonstrate the requirement of hormone to dissociate the 300K complex by heat treatment. Specific DNA-binding activity of the receptor becomes apparent in this process, showing that DNA binding occurs but is inhibited in the large heteromeric complex. We propose a model in which receptor function is repressed by association of the receptor with hsp90. Dissociation of this complex is induced by the binding of steroid and is apparently an irreversible process.

Adrenalectomy↗

PubMed1988

Differential expression of the cytochrome P-450c and P-450d genes in the rat ventral prostate and liver.

Complementary DNA clones specific for cytochromes P-450c and P-450d were used to study the expression of corresponding mRNAs in the rat ventral prostate. Following treatment with beta-naphthoflavone (BNF), an increase in total cellular levels of cytochrome P-450c mRNA was observed in this tissue. The induction kinetics were similar in both rat liver and prostate with regard to the expression of the cytochrome P-450c gene. The mRNA levels reached a maximum at 16 h and decreased to control levels at about 48 h. In contrast, mRNA specific for cytochrome P-450d was detectable only in the liver following BNF treatment (maximum at 24 h). The tissue-specific expression of cytochromes P-450c and P-450d was further investigated using measurements of nuclear transcription in vitro. RNA transcripts specific for cytochrome P-450c were detected in nuclei from both liver and prostate following BNF induction. The rate of cytochrome P-450c transcription was maximal at 4 h and 8-12 h in the liver and prostate, respectively. In the liver, induction by BNF of the rate of transcription of the cytochrome P-450d gene occurred at a slightly later time point as compared to cytochrome P-450c gene expression. No elongation of RNA specific for cytochrome P-450d could be detected in nuclei from the ventral prostate indicating a transcriptional control of cytochrome P-450d gene expression in this tissue.

Animals↗

PubMed1988

DNA binding properties of dioxin receptors in wild-type and mutant mouse hepatoma cells.

The current model of action of 2,3,7,8-tetrachlorodibenzo-p-dioxin (dioxin) entails stimulation of target gene transcription via the formation of dioxin-receptor complexes and subsequent accumulation of the complexes within the cell nucleus. Here, we have analyzed the DNA binding properties of the dioxin receptor in wild-type mouse hepatoma (Hepa 1c1c7) cells and a class of nonresponsive mutant cells which fail to accumulate dioxin-receptor complexes within the nucleus in vivo. In vitro, both the wild-type and mutant [3H]dioxin-receptor complexes exhibited low affinity for DNA-cellulose (5-8% and around 4% retention, respectively) in the absence of prior biochemical manipulations. However, following chromatography on heparin-Sepharose, the wild-type but not the mutant dioxin receptor was transformed to a species with an increased affinity for DNA (40-50% retention on DNA-cellulose). The gross molecular structure of the mutant, non DNA binding dioxin receptor did not appear to be altered as compared to that of the wild-type receptor. These results imply that the primary deficiency in the mutant dioxin receptor form may reside at the DNA binding level and that, in analogy to steroid hormone receptors, DNA binding of the receptor may be an essential step in the regulation of target gene transcription by dioxin.

Animals↗

PubMed1988

The dioxin receptor: a comparison with the glucocorticoid receptor.

The physico-chemical properties of the dioxin and glucocorticoid receptors from rat liver and wild-type and mutant cell lines were investigated and compared. In rat liver, the receptors are virtually indistinguishable. Both are highly asymmetrical proteins with axial ratios of 12-15, have Stokes radii of 6 nm and sedimentation coefficients of approximately 4 S. This results in a calculated apparent mol. wt of approximately 100,000. The dioxin receptor from the mouse hepatoma cell line Hepa 1c1c7 represents an atypical form of the dioxin receptor with a pronounced tendency to aggregate to form Mr approximately equal to 300,000 complexes in high ionic strength and in the absence of sodium molybdate. In the presence of sulphydryl reducing agents, however, the Hepa 1c1c7 dioxin receptor dissociates to an Mr approximately 100,000 species. In analogy to the nt- mutant glucocorticoid receptor in mouse lymphoma cells, there is no gross change in the structure of the nt- dioxin mutant in mouse hepatoma cells compared with the wild-type receptor. The nt- dioxin receptor does, however, have a reduced affinity for DNA.

Animals↗

PubMed1988

The dioxin receptor: characterization of its DNA-binding properties.

The binding of the rat hepatic dioxin and glucocorticoid receptors to the polyanionic matrices heparin-Sepharose and DNA-cellulose in vitro and to cell nuclei in vivo was studied under various conditions. In a non-liganded and non-activated state both receptors eluted from heparin-Sepharose at a low ionic strength and were not retained on DNA-cellulose. Following ligandation and activation in vitro both receptors showed an increased affinity for heparin-Sepharose and were retained on DNA-cellulose. In analogy to these in vitro data, it was found that a high salt concentration (0.4 M KCl) was required to extract in vivo liganded dioxin receptor from purified nuclear preparations in contrast to that previously reported for non-liganded nuclear receptors. Limited proteolysis of both dioxin and glucocorticoid receptors resulted in molecular species of similar binding properties with regard to DNA-cellulose and heparin-Sepharose. We conclude that, in addition to the dioxin and glucocorticoid receptors showing considerable similarities in their physicochemical properties, they may also share a similar structural organization with regard to functional domains.

Animals↗