DNA sequence determinants for binding of transformed Ah receptor to a dioxin-responsive enhancer.

We have utilized gel retardation analysis and DNA mutagenesis to examine the specific interaction of transformed guinea pig hepatic cytosolic TCDD.AhR complex with a dioxin-responsive element (DRE). Sequence alignment of the mouse CYPIA1 upstream DREs has identified a common invariant "core" consensus sequence of TNGCGTG flanked by several variable nucleotides. Competitive gel retardation analysis using a series of DRE oligonucleotides containing single or multiple base substitutions has allowed identification of those nucleotides important for TCDD.AhR.DRE complex formation. A putative TCDD.AhR DNA-binding consensus sequence of GCGTGNNA/TNNNC/G has been derived. The four core nucleotides, CGTG, appear to be critical for TCDD-inducible protein-DNA complex formation since their substitution decreased AhR binding affinity by 100-800-fold; the remaining conserved bases are also important, albeit to a lesser degree (3-5-fold). The 5'-ward thymine, present in the invariant core sequence of all the DREs identified to date, appears not to be involved in DNA binding of the AhR. The results obtained here indicate that although the primary interaction of the TCDD.AhR complex with the DRE occurs with the conserved "core" sequence, nucleotides flanking the core also contribute to the specificity of DRE binding.     

Molybdate-stabilized glucocorticoid receptor: evidence for a receptor heteromer

The composition of the molybdate-stabilized glucocorticoid receptor (GR) complex has been investigated with a monoclonal antibody against the steroid-binding Mr 94 000 (94K) GR protein. It was concluded that one antibody molecule binds one 94K GR molecule. This finding constituted the basis for calculating the number of antibodies bound to the molybdate-stabilized nonactivated GR complex, which has an Mr of 302 000 (302K). Gel filtration on Sephacryl S-400 and density gradient centrifugation showed that only one antibody molecule bound to the molybdate-stabilized GR complex (calculated relative molecular mass for the antibody--molybdate-stabilized GR complex, 456 000; relative molecular mass for one antibody molecule, 157 000). Furthermore, experiments performed with a second antibody immunoprecipitation assay in the presence of an excess of both antibody and GR confirmed the above results. The possibility of steric hindrance not allowing more than one antibody molecule to bind to the molybdate-stabilized GR complex could be excluded. These results suggest that the molybdate-stabilized GR complex with an Mr of 302K only contains one steroid-binding 94K GR molecule and therefore represents a heteromeric complex.     

ATP promotes the appearance of two DNA-binding forms of the Ah receptor.

Following the binding of a specific ligand such as 2,3,7,8-tetrachlorodibenzo-p-dioxin, mild heating has previously been shown to convert cytosolic aryl hydrocarbon (Ah) receptor to two DNA-binding forms: peak I, which appears to be an Ah receptor monomer; and peak II, which is larger and resembles nuclear Ah receptor. In rat liver cytosol pretreated with charcoal-dextran, and heated briefly at 22 degrees C, the addition of a physiological (3 mM) concentration of ATP substantially increases the formation of both peak I and peak II receptor. On more extended incubation in the presence of ATP most of the Ah receptor converts to the tighter binding peak II form. The ATP analog 5'-adenylylmethylene diphosphonate (AMPPCP) stimulates the appearance of both DNA-binding forms as effectively as ATP does. In cytosol separated from low molecular weight components by gel filtration prior to incubation, ATP substantially stimulates the appearance of peak II receptor. ATP also increases the amount of peak II receptor formed when peak I receptor is incubated with unlabeled charcoal-treated cytosol. Thus, ATP promotes both the release of Ah receptor monomer from the 9 S complex which cannot bind DNA and the subsequent conversion of that monomer to a form similar or identical to nuclear Ah receptor.     

Induction of the Cyp1a-1 dioxin-responsive enhancer in transgenic mice.

Cyp1a-1, whose product, aryl hydrocarbon hydroxylase, assists in detoxification of polycyclic aromatic hydrocarbons, is the best characterized of the murine cytochrome P450 genes. The Cyp1a-1 dioxin-responsive enhancer region has been previously analyzed in vitro and found to induce expression of heterologous genes upon exposure of transfected cells to various aromatic hydrocarbons. A 2.58 kbp DNA fragment containing the Cyp1a-1 enhancer elements and promoter region was coupled to the chloramphenicol acetyltransferase (CAT) reporter gene and used to create transgenic mice. CAT assays were performed on tissues harvested from three different lines of transgenic mice following mock-induction or induction using the aromatic hydrocarbon, 3-methylcholanthrene. Basal levels of expression were detected in the spleen and small bowel of non-induced mice, with little or no expression detected in the liver. Treatment with 3-methylcholanthrene increased hepatic expression levels by as much as 10,000-fold. More modest levels of induction were also recorded in the spleen, small bowel, kidney, and lung. The results indicate that the dioxin-responsive enhancer region functions as a strongly inducible promoter in vivo. Differences in the response to induction between male and female mice suggest that Cyp1a-1 expression may be governed in a gender related manner.     

Characterization of the Ah receptor from human placental tissue

The rate of thermal inactivation of the unliganded human Ah receptor, studied by sucrose density gradient centrifugation, with respect to loss of ligand binding ability, was found to be greater than those of most rodents at 20°C, but the temperature coefficient of the rate constant was much smaller than for the rodent species. This implies that the unliganded human Ah receptor would be thermally more stable than the rodent analogs at physiological temperatures. The liganded form of the human Ah receptor was found to be less stable with respect to ligand release than the rodent receptors. These differences in behavior between human and rodent Ah receptors underline the difficulties in using rodent data in the development of receptor-based models of dioxin toxicity. Attempts to develop an alternative to sucrose density gradient centrifugation, comparable with the hydroxylapatite adsorption method used to assay rodent hepatic Ah receptor, were unsuccessful.     

Evidence for two functionally distinct forms of the human Ah receptor

The Ah receptor (AhR) was visualized using monoclonal antibody Rpt 1 on protein blots of HeLa cell cytosol; two bands were detected at 104 and 106 kDa. The photoaffinity ligand, 2-azido-3-[¹²⁵I]iodo-7,8-dibromodibenzo-p-dioxin, was added to HeLa cells in culture, and after 1 hour the cells were UV irradiated. Cytosolic and high salt nuclear preparations were isolated and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer of the protein to membrane. The AhR was visualized on the membrane, revealing two bands. Alignment of an autoradiogram with the membrane revealed that only the 106 kDa (upper) band was photoaffinity labeled. The nuclear fraction contained only the photoaffinity-labeled 106 kDa form of the AhR. The 104 kDa AhR does not appear to be a proteolytic product of the 106 kDa form. Cyanogen bromide fragmentation revealed that both forms contain the same size N-terminal fragment. Sucrose density gradient analysis of HeLa cell cytosol indicated that both forms cosedimented at 9 S. Both the 106 and 104 kDa AhR bands were detected in four different human cell lines. Together, these results would indicate that the AhR in human cell lines exists in two distinct forms.     

Disruption of the mu-delta opioid receptor heteromer

The crystal structure of the mu and kappa opioid receptors has revealed dimeric structural arrangements. Mu-delta receptors heteromers also exist and we have identified discrete cytoplasmic regions in each receptor required for oligomer formation. In the carboxyl tail of the delta receptor we identified three glycine residues (-GGG), substitution of any of these residues prevented heteromer formation. In intracellular loop 3 of both mu and delta receptors we identified three residues (-SVR), substitution of any of these residues prevented heteromer formation.     

Characterization of antigen receptor response elements within the interleukin-2 enhancer.

T-cell activation and induction of interleukin-2 (IL-2) expression in human T lymphocytes require both interaction of foreign antigen with the T-cell antigen receptor and protein kinase C (PKC) stimulation. Agents such as phorbol 12-myristate 13-acetate (PMA) that stimulate PKC augment the effects of antigen but are not sufficient for IL-2 activation. By analysis of deletion mutants, we identified three DNA sequences extending from -73 to -89, -217 to -255, and -263 to -279, designated IL-2 sites A, D, and E, respectively, that are required for maximal induction of IL-2 expression. One of these regions, site E, interacted with a protein (NF-IL-2E) present only in the nuclei of cells which have been stimulated. The other two sequences interacted with a protein (NF-IL-2A) that is constitutively expressed in T cells. When multiple tandem copies of either the E site or the A site were placed upstream of the gamma-fibrinogen promoter, they activated expression via this promoter in response to signals initiated at the antigen receptor but not following PMA stimulation. For this reason, we denoted them antigen receptor response elements. The uncoupling of antigen receptor and PKC requirements in these studies indicates that these signal pathways are, at least in part, distinct and integrated at the level of the gene.     

Biosynthesis of promatrix metalloproteinase-9/chondroitin sulphate proteoglycan heteromer involves a Rottlerin-sensitive pathway

Background

Previously we have shown that a fraction of the matrix metalloproteinase-9 (MMP-9) synthesized by the macrophage cell line THP-1 was bound to a chondroitin sulphate proteoglycan (CSPG) core protein as a reduction sensitive heteromer. Several biochemical properties of the enzyme were changed when it was bound to the CSPG.

Methodology/Principal Findings

By use of affinity chromatography, zymography, and radioactive labelling, various macrophage stimulators were tested for their effect on the synthesis of the proMMP-9/CSPG heteromer and its components by THP-1 cells. Of the stimulators, only PMA largely increased the biosynthesis of the heteromer. As PMA is an activator of PKC, we determined which PKC isoenzymes were expressed by performing RT-PCR and Western Blotting. Subsequently specific inhibitors were used to investigate their involvement in the biosynthesis of the heteromer. Of the inhibitors, only Rottlerin repressed the biosynthesis of proMMP-9/CSPG and its two components. Much lower concentrations of Rottlerin were needed to reduce the amount of CSPG than what was needed to repress the synthesis of the heteromer and MMP-9. Furthermore, Rottlerin caused a minor reduction in the activation of the PKC isoenzymes δ, ε, θ and υ (PKD3) in both control and PMA exposed cells.

Conclusions/Significance

The biosynthesis of the proMMP-9/CSPG heteromer and proMMP-9 in THP-1 cells involves a Rottlerin-sensitive pathway that is different from the Rottlerin sensitive pathway involved in the CSPG biosynthesis. MMP-9 and CSPGs are known to be involved in various physiological and pathological processes. Formation of complexes may influence both the specificity and localization of the enzyme. Therefore, knowledge about biosynthetic pathways and factors involved in the formation of the MMP-9/CSPG heteromer may contribute to insight in the heteromers biological function as well as pointing to future targets for therapeutic agents.     

Novel antipeptide antibodies to the human glucocorticoid receptor: recognition of multiple receptor forms in vitro and distinct localization of cytoplasmic and nuclear receptors.

We have synthesized two peptides that correspond to unique regions of the amino-terminus of the human glucocorticoid receptor (GR). Peptides representing amino acids 245-259 and 346-367 (designated 59 and 57, respectively) were chosen on the basis of hydrophobicity/hydrophilicity ratios as well as overall proline content. These peptides were then used as antigens to produce epitope-specific antibodies that recognize and interact with human GR in a variety of physical states. Antiserum directed against each peptide recognizes denatured, [3H] dexamethasone mesylate-labeled GR as well as unliganded receptor on Western blots. In contrast to other antipeptide GR antibodies, these antibodies recognize and form stable complexes with unactivated and molybdate-stabilized forms of the GR, indicating that neither epitope is occluded when the receptor exists in an oligomeric state. Activated, 4S DNA-binding forms of the receptor are also recognized by both antibodies. The interaction of antibodies 59 and 57 with human GR in various states is highly specific based on the observation that preincubation of either antiserum with the appropriate peptide completely precludes the recognition of receptor by antibody. Titration analysis of antisera reveals that an increase in the antibody concentration cause discrete increases in the sedimentation coefficient of GR on sucrose gradients. These shifts occur under high salt conditions and are consistent with the formation of multiple stable antibody-receptor complexes. Interestingly, neither antibody interferes with the ability of the GR to be activated into a DNA-binding form or with the ability of the activated GR to interact with DNA cellulose. Consistent with these observations, both antibodies recognize and form stable complexes with GR when the receptor is associated with DNA fragments that contain specific glucocorticoid-responsive elements. Thus, both antibodies appear to recognize all known forms of the human GR protein. Using immunohistochemical techniques to visualize GR in HeLa S3 cells as well as in Chinese hamster ovary cells that stably express transfected human GR, a cytoplasmic location for receptor is observed in the absence of ligand. In contrast, immunoreactive GR is predominantly nuclear after hormone treatment, further supporting a role for nuclear translocation in GR function.     

Testicular protein kinases. Characterization of multiple forms and ontogeny.

Protein phosphokinase activity from the cytosol (105,000 X g soluble fraction) of testes from sexually mature rats has been resolved be DEAE-cellulose chromatography in three forms of protein kinase, cAMP-dependent protein kinases I and II and cAMP-independent protein kinase III. Adenosine 3':5'-monophosphate-binding activity (cAMP-binding activity) was associated with protein kinases I and II but not with protein kinase III. Protein kinases I, II, and III exhibited different pH optima, cyclic nucleotide dependency, and relative substrate specificity. Protein kinases I and II were inhibited by a heat-stable protein inhibitor from rat skeletal muscle, whereas protein kinase III was not inhibited. According to previously established criteria (Traugh, J. A., Ashby, C.D., and Walsh D. A. (1974) Methods Enzymol. 38, 290-299) protein kinases I and II can be classified as cAMP-dependent holoenzymes consisting of regulatory and catalytic subunits. Protein kinase III is a cAMP-independent protein kinase.