Molybdate inhibition of glucocorticoid receptor inactivation and transformation.

The inactivation of glucocorticoid receptors that occurs when cytosol is heated at 25 degrees C is blocked reversibly by molybdate and slowed by some other phosphatase inhibitors such as fluoride and glucose 1-phosphate. Molybdate is also capable of preventing nonenzymatic inactivation of unbound receptors caused by exposure to salt or precipitation with ammonium sulfate at 0 degrees C. Inactivation of unbound receptors caused by Sephadex G-50 gel filtration is prevented by all three inhibitors. Both molybdate and tungstate block temperature-dependent transformation of glucocorticoid.receptor complexes to the DNA-binding state, where fluoride and glucose 1-phosphate have no effect. Transformation brought about at 0 degrees C by salt, ammonium sulfate precipitation, or gel filtration is also blocked by both molybdate and tungstate. Tungstate differs from molybdate in that it has little or no effect on receptor inactivation. Fluoride and glucose 1-phosphate do not inhibit transformation. These observations support the proposal that molybdate and tungstate are interacting through a reversible association with the glucocorticoid receptor itself. We propose that they may act by forming a complex with a phosphate moiety on the receptor.     

Leupeptin inhibits the transformation of glucocorticoid receptor

The effect of leupeptin upon the transformation of the glucocorticoid receptor was tested. When the labeled receptor was treated with heat or high salt in the presence of leupeptin, the binding to DNA-cellulose decreased in a dose-dependent manner. We observed 50% inhibition with about 40 mM leupeptin. The addition of leupeptin after the transformation procedures did not inhibit the binding to DNA-cellulose. In gradient centrifugation, 40 mM leupeptin retained approximately 10S, untransformed form. Elution profiles from DEAE-cellulose showed the preservation of the peak eluted with 0.2 M KCl, corresponding to the untransformed form. These results indicate that leupeptin might have the similar effects to molybdate in regard to blocking the transformation of rat liver glucocorticoid receptor, though the effects with leupeptin were not as great as those seen with molybdate.     

Stability and transformation of the glucocorticoid receptor under acidic conditions

The [3H]triamcinolone acetonide ([3H]TA)-binding ability of the rat liver glucocorticoid receptor (GR) was investigated under acidic conditions, ranging from pH 2 to 7.3. Both in the presence and absence of 10 mM molybdate, the [3H]TA-binding ability decreased below pH 6.5 and was almost completely lost below pH 5, pH 5.9 +/- 0.1 giving 50% [3H]TA-binding. The binding ability was recovered when the pH of the cytosol was reversed to 7.3 or the precipitate obtained on acidification was dissolved in a buffer of pH 7.3. Moreover, in the absence of molybdate, the [3H]TA-GR complexes formed at pH 7.3 remained unchanged until pH 5. Then they decreased, pH 3.9 +/- 0.1 giving 50% binding, and completely disappeared at pH 3. [3H]TA-binding activity recovered from the precipitate also decreased in a similar pH region (a 50% decrease in binding being observed at pH 4.2 +/- 0.04). These results suggest that rat liver GR is rather resistant under acidic conditions and that it exists in a peculiar state below pH 5.9 to approximately 4 as to its ligand binding property: unoccupied GR has no [3H]TA-binding ability but [3H]TA-GR complexes once formed at neutral pH do not dissociate. [3H]TA-GR complexes recovered from the precipitate at pH 5 had a Stokes radius of 7.5 nm, little DNA-cellulose-binding ability and sedimented at 8.6S on glycerol gradient centrifugation, indicating that the receptor existed in a nontransformed state. In addition, both occupied and unoccupied GR were transformed at about pH 4, their being 50% transformation. This transformation was accompanied by irreversible denaturation of the receptor.(ABSTRACT TRUNCATED AT 250 WORDS)     

A possible role for dephosphorylation in glucocorticoid receptor transformation

Addition of bovine intestinal alkaline phosphatase to mouse AtT-20 cell cytosol increases the rate of glucocorticoid receptor transformation, as evidenced by a change in sedimentation rate from 9.1S to 5.2S. Acid phosphatases are completely ineffective in this regard. Alkaline phosphatase-promoted receptor transformation is both time- and dose-dependent. A variety of phosphatase inhibitors are effective in inhibiting this process, the most potent being transition metal oxyanions such as molybdate, tungstate, and arsenate. The ability of the various inhibitors to suppress alkaline phosphatase-promoted receptor transformation does not correspond well with their potencies for inhibiting para-nitrophenyl phosphate hydrolysis. However, a better correspondence between the inhibition of endogenous receptor transformation and total cytosolic phosphatase activity is observed, and both sodium fluoride and glucose-1-phosphate inhibit endogenous receptor transformation. The protease inhibitors phenyl-methylsulfonyl fluoride and antipain have no effect on receptor transformation. Surprisingly, leupeptin is effective in inhibiting alkaline phosphatase-promoted receptor transformation. Although this raises the possibility of a contaminating protease activity in the alkaline phosphatase enzyme preparation, treatment of covalently affinity-labeled receptor with the enzyme shows no proteolysis of the receptor or any other non-specifically labeled cytosolic protein. Thus, it is possible that a novel action of leupeptin, unrelated to its protease-inhibitory activity, may be involved in the suppression of receptor transformation. The studies presented here suggest that dephosphorylation of some component in cytosol is involved in the destabilization of receptor subunit interactions, resulting in glucocorticoid receptor transformation.     

Aluminum fluoride inhibition of cabbage phospholipase D by a phosphate-mimicking mechanism

The intermediate CuI-semiquinone radical species in the catalytic mechanism of copper-amine oxidase from Lens esculenta and Pisum sativum seedlings has been studied by optical, Raman resonance and ESR spectroscopies and by stopped-flow and temperature-jump measurements. Treatment of highly purified enzyme preparations with good, poor or suicide substrates, under anaerobic and aerobic conditions, at different pH values and temperatures, makes it possible to generate, detect and characterize this free radical intermediate.     

Nontransformed rabbit liver glucocorticoid receptor: purification, characterization and transformation

The molybdate-stabilized nontransformed form of the glucocorticoid receptor from rabbit liver has been purified approximately 8,000-fold by a three-step procedure. The first step involved protamine sulfate precipitation which allowed a 5-6-fold purification with 85% yield. The second step, affinity chromatography using a N-(12-dodecyl-amino) 9 alpha-fluoro-16 alpha-methyl-11 beta, 17 alpha-dihydroxy-3-oxo-1,4-androstadiene-17 beta-carboxamide substituted Sepharose gel, purified the receptor 1,500-2,000-fold as calculated by specific radioactivity. The third step involved high performance liquid chromatography resulting in overall purification near 8,000-fold. The final glucocorticoid receptor appeared about 60% pure. The purified nontransformed glucocorticoid receptor had a sedimentation coefficient of 9 S in 0.16 M phosphate containing 5-20% sucrose gradients and the Stokes radius was 6.1-6.3 nm as determined by low pressure gel filtration and HPLC. Binding specificity of the purified receptor was identical to that previously reported in crude rabbit liver cytosol. Isoelectricfocusing and ion-exchange chromatography showed that the purification procedure affected the net charge of the receptor protein. This phenomenon could be related to interactions between the glucocorticoid receptor and cytosolic factors. SDS polyacrylamide gel electrophoresis showed a major Mr = 94,000 protein band which is in good agreement with previously reported values for glucocorticoid receptors. Transformation of the purified receptor was achieved after removal of molybdate by exposure at 25 degrees C to 0.4 M KCl. Characterization of the molecular forms was performed by means of incorporation into isolated nuclei, affinity towards polyanionic exchangers and high pressure size exclusion chromatography. Results show that about 40% of the receptor is in the transformed state.     

Melatonin prevents glucocorticoid inhibition of cell proliferation and toxicity in hippocampal cells by reducing glucocorticoid receptor nuclear translocation

Glucocorticoids are the main product of the adrenal cortex and participate in multiple cell functions as immunosupressors and modulators of neural function. Within the brain, glucocorticoid activity is mediated by high-affinity mineralocorticoid and low-affinity glucocorticoid receptors. Among brain cells, hippocampal cells are rich in glucocorticoid receptors where they regulate excitability and morphology. Also, elevated glucocorticoid levels suppress hippocampal neurogenesis in adults. The pineal neuroindole, melatonin, reduces the affinity of glucocorticoid receptors in rat brain and prevents glucocorticoid-induced apoptosis. Here, the ability of melatonin to prevent glucocorticoid-induced cell death in hippocampal HT22 cells was investigated in the presence of neurotoxins. Results showed that glucocorticoids reduce cellular growth and also enhance sensitivity to neurotoxins. We found a G(1) cell cycle arrest mediated by an increase of cyclin/cyclin-dependent kinase inhibitor p21(WAF1/CIP1) protein after dexamethasone treatment and incremental change in amyloid beta protein and glutamate toxicity. Melatonin prevents glucocorticoids inhibition of cell proliferation and reduces the toxicity caused by glucocorticoids when cells were treated with dexamethasone in combination with neurotoxins. Although, melatonin does not reduce glucocorticoid receptor mRNA or protein levels, it decreases receptor translocation to nuclei in these cells.     

糖皮质激素受体及其选择性调节剂研究进展

糖皮质激素（glucocorticoids,GCs）是临床上广泛使用的一类抗炎药物,在体内主要通过糖皮质激素受体（glucocorticoid receptor,GR）发挥生理和药理作用。GR是核受体超家族的成员之一,为配体激活的转录因子,在机体的多种生理和病理活动中扮演重要的角色。随着对GR信号通路的深入研究,寻找针对糖皮质激素受体的新型调节剂,以期将抗炎作用和现有糖皮质激素的副作用相分离,已经成为新药发现的研究热点。本文对近年来GR的分子结构、生物学作用及其选择性调节剂的研究进展作一简要的介绍。     

The role of sulfhydryl groups in permitting transformation and DNA binding of the glucocorticoid receptor

Treatment of rat liver cytosol containing temperature-transformed, [3H]dexamethasone-bound receptors at 0 degree C with the sulfhydryl-modifying reagent methyl methanethiosulfonate (MMTS) inhibits the DNA-binding activity of the receptor, and DNA-binding activity is restored after addition of dithiothreitol (DTT). When cytosol containing untransformed receptors is heated at 25 degrees C in the presence of MMTS, the 90-kDa heat shock protein dissociates from the receptor in the same manner as in the absence of MMTS, and the receptor will bind to DNA-cellulose if DTT is added subsequently at 0 degree C. These observations are consistent with the conclusion of Bodwell et al. (Bodwell, J. E., Holbrook. N. J. and Munck, A. (1984) Biochemistry 23, 1392-1398) that sulfhydryl moieties on the receptor are absolutely required for the receptor to bind to DNA, and they show that the sulfhydryl-modifying reagent does not inhibit the temperature-mediated dissociation of the heteromeric receptor complex that accompanies transformation to the DNA-binding state. When steroid-receptor complexes that are prebound to DNA-cellulose are exposed to MMTS, the steroid rapidly dissociates, but the receptor remains bound to DNA. Thus, the presence of steroid is not required for the receptor to remain bound to DNA in a high affinity manner. Treatment of cytosol containing transformed glucocorticoid-receptor complexes at 0 degrees C with 20 mM hydrogen peroxide also inactivates the DNA-binding activity of the receptor. The peroxide-induced inactivation is reversed by DTT. Incubation of rat liver cytosol containing untransformed glucocorticoid-receptor complexes at 25 degrees C with hydrogen peroxide prevents their transformation to the DNA-binding form as shown by their inability to bind to DNA-cellulose after addition of DTT. The presence of peroxide during heating of the cytosol also prevents dissociation of the receptor complex as assayed both by reduction in sedimentation value of the receptor and by dissociation of the 90-kDa heat shock protein from the steroid-binding protein. These results strongly suggest that critical sulfur moieties in the receptor complex must be in a reduced form for the temperature-mediated dissociation of the receptor to occur.     

Aluminum ions are required for stabilization and inhibition of hepatic microsomal glucose-6-phosphatase by sodium fluoride

Stabilization and inhibition of hepatic microsomal glucose-6-P phosphohydrolase (EC 3.1.3.9) by F- requires the presence of Al3+ ions. At millimolar concentrations, reagent grade NaF inhibited glucose-6-P hydrolysis and protected the enzyme against inactivation induced by heat in the presence of 0.025% (w/v) Triton X-100 or by reaction of the catalytic site with the histidine-specific reagent, diethyl pyrocarbonate. The presence of millimolar EDTA in all test systems abolished the effectiveness of NaF, yet EDTA by itself was without significant influence on the kinetics of phosphohydrolase reaction, the thermal stability of the enzyme or its reactivity with diethyl pyrocarbonate. Although ultrapure NaF was ineffectual in all test systems, its potency as a competitive inhibitor or protective agent was markedly increased by micromolar AlCl3 or when assays were carried out in flint glass test tubes. The latter response is explained by the well documented ability of fluoride solutions to extract Al3+ from glass at neutral pH. Our analysis indicates that the effectiveness of fluoride in all test systems derives from the formation of a specific complex with Al3+, most likely Al(F)4-. The apparent dissociation constant for interaction of the enzyme and Al(F)4- is 0.1 microM. The combination of NaF and AlCl3 holds promise as an unusually effective and versatile means to stabilize this notoriously labile enzyme during efforts to purify it.     

Intracellular inhibition of chromatin binding and transformation of androgen receptor by 3'-deoxyadenosine

Incubation of minced rat ventral prostate with 3'-deoxyadenosine (3'-dA) prior to labeling with the androgen, tritiated 7 alpha, 17 alpha-dimethyl-19-nortestosterone, reduced the level of androgen receptor bound to chromatin and increased the level of cytosolic androgen receptor and the fraction of cytosolic androgen receptor that did not bind to DNA. This effect was specific for 3'-dA and not mimicked by adenosine, 2'-deoxy-adenosine, cytidine, guanosine, or uridine. Adenosine was a competitive inhibitor of the 3'-dA effect. Labeled cytosolic androgen receptor from 3'-dA-treated prostate had properties that were similar to those exhibited by untransformed androgen receptor from prostate cytosol prepared in the presence of Na2MoO4, an inhibitor of receptor transformation in cell-free systems. Both androgen receptors had sedimentation coefficients of 8-9 S in low-salt gradients, did not bind to DNA tightly, and had a high affinity for DEAE-cellulose. The 3'-dA effect on these properties was not observed if androgen receptor from 3'-dA-treated prostate was isolated on high-salt gradients. These findings show that androgen receptor transformation does take place in intact prostate cells and suggest that 3'-dA inhibits chromatin binding of androgen receptor by interfering with androgen receptor transformation. The transformation process appears to involve removal of components from androgen receptor. Since 3'-dA is a potent inhibitor of the synthesis, polyadenylation, and nucleocytoplasmic transport of RNA, the 3'-dA effect may indicate a role for RNA in the mechanism of receptor transformation in intact target cells.     

Evidence that removal of an endogenous metal that stabilizes the untransformed glucocorticoid receptor in cytosol allows ligand-independent receptor transformation

Cytosol preparations contain an endogenous heat-stable factor which stabilizes the glucocorticoid receptor in its untransformed, non DNA-binding form. Elution of a partially purified preparation of this stabilizing factor through a metal chelating resin (Chelex-100) leads to the loss of its ability to inhibit temperature-mediated transformation of the receptor. Sodium molybdate mimicks the ability of this endogenous metal to stabilize the untransformed receptor, and it too is adsorbed by Chelex resin. When an L-cell cytosol preparation containing the glucocorticoid receptor is passed through a column of Chelex-100 resin and then incubated at 15 degrees C, the receptor is rapidly transformed to the DNA-binding state, regardless of whether it is steroid-bound or not. In contrast, whole cytosol containing endogenous metals is transformed to the DNA-binding state only when the receptor is both steroid-bound and exposed to elevated temperature. these data suggest that a metal (or metals) may be involved in conferring the property of ligand-dependency to the transformation process.     

Enzyme inactivation and inhibition by Gossypol

Summary Three lactate dehydrogenase isozymes and malate dehydrogenase purified from mouse tissues were inactivated with time by low concentration of gossypol. The degree of enzyme inactivation is both gossypoland enzyme-concentration-dependent. Under the same experimental conditions, lactate dehydrogenase-X and lactate dehydrogenase-5 were inactivated faster than lactate dehydrogenase-1. NADH was shown to partially protect the enzymes against inactivation by gossypol. The results of this study suggest that the enzymes are inactivated by the minor components in gossypol preparations. Isozymes of glutathione S-transferases were reversibly inhibited by gossypol. The inhibition of transferases by gossypol was shown to be competitive with respect to the 1-chloro-2,4-dinitrobenzene. It is proposed that the male antifertility effect of gossypol may be related to the selective inactivation of sperm-specific lactate dehydrogenase-X.     

Interaction of glucocorticoid analogues with the human glucocorticoid receptor

Transient co-transfection of receptor cDNA and suitable reporter genes was used to study human glucocorticoid receptor (hGR) function in a neutral mammalian cell background. A variety of natural and synthetic steroids were analyzed for their ability to activate gene expression through the hGR and to bind to extracts of cells expressing the hGR cDNA. There was very good correlation between these two in vitro parameters for these compounds. Furthermore, correlation of these data with reported in vivo anti-inflammatory potencies was surprisingly close, with two exceptions. The in vitro data suggest an explanation for the discrepant compounds, consistent with published data on their metabolic fate in vivo. The co-transfection assay has utility as a quantitative predictor of in vivo glucocorticoid pharmacology.