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Induction of secreted type IIA phospholipase A₂ gene transcription by interleukin-1β -: Role of C/EBP factors

JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第30期275卷 22686-22694页

作者： Massaad, C Paradon, M Jacques, C Salvat, C Bereziat, G Berenbaum, F Olivier, JL Univ Paris 06 CNRS UPRESA 7079 UFR St Antoine F-75252 Paris 05 France

Secreted type IIA phospholipase A(2), which is involved in arachidonic acid release, is abundantly produced by chondrocytes and secreted in the synovial fluids of patients affected by rheumatoid arthritis. Transfection experiments showed that interleukin-1 beta stimulates the phospholipase A(2) [-1614;+20] promoter activity by 6-7-fold and that the [-210;-176] fragment is critical for this stimulation. CAAT enhancer-binding protein (C/EBP) beta and C/EBP delta transcription factors bind to this element as shown by bandshift experiments. Interleukin-1 beta increased the levels of C/EBP delta mRNA as soon as 2 h and up to 24 h without affecting those of C/EBP delta, Higher amounts of C/EBP delta proteins correlate with the stimulation of C/EBP delta mRNA Mutations or 5' deletions in the upstream [-247;-210] region reduced by 2-fold the basal and interleukin-1 beta-stimulated transcription activities. Two types of factors bind to overlapping sequences on this fragment: NF1-like proteins and the glucocorticoid receptor. The glucocorticoid receptor is responsible for a moderate stimulation of the promoter activity by dexamethasone and may interact with C/EBP factors to achieve a full transcription activity in basal conditions and in the presence of interleukin-1 beta. A [-114;-85] proximal regulatory element forms three complexes in bandshift experiments, the slowest mobility one involving the Sp1 zinc finger factor. Mutation of this sequence reduced to 2-fold the stimulation of the promoter activity by interleukin-1 beta or the C/EBP factors. Induction of the transcription of secreted type IIA phospholipase A(2) gene by interleukin-1 beta in chondrocytes absolutely requires C/EBP beta and C/EBP delta factors but does not involve NF-kappa B.

关键词：碱基序列 CCAAT增强子结合蛋白类细胞培养的软骨细胞/细胞学软骨细胞/酶学软骨细胞/代谢 DNA引物 DNA结合蛋白质类/遗传学 DNA结合蛋白质类/生理学基因表达调控酶学/生理学白细胞介素1/生理学核蛋白质类/遗传学核蛋白质类/代谢核蛋白质类/生理学磷脂酶A/遗传学启动区(遗传学) 蛋白质结合受体糖皮质激素/代谢调控序列核酸 Sp1转录因子/代谢转录遗传/生理学动物女(雌)性兔

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A role for the Hsp40 Ydj1 in repression of basal steroid receptor activity in yeast

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MOLECULAR AND CELLULAR BIOLOGY 2000年第9期20卷 3027-3036页

作者： Johnson, JL Craig, EA Univ Wisconsin Dept Biomol Chem Madison WI 53706 USA

In addition to its roles in translocation of preproteins across membranes, Ydj1 facilitates the maturation of Hsp90 substrates, including mammalian steroid receptors, which activate transcription in yeast in a hormone-dependent manner. To better understand Ydj1's function, we have constructed and analyzed an array of Ydj1 mutants in vivo. Both the glucocorticoid receptor and the estrogen receptor exhibited elevated activity in the absence of hormone in all ydj1 mutant strains, indicating a strict requirement for Ydj1 activity in hormonal control, Glucocorticoid receptor containing a mutation in the carboxy-terminal transcriptional activation domain, AF-2, retained elevated basal activity, while mutation of the amino-terminal transactivation domain, AF-1, eliminated the elevated basal activity observed in ydj1 mutant strains. This result indicates that the source of activity is AF-1, which is normally repressed by the carboxy-terminal hormone binding domain in the absence of hormone. Chimeric proteins containing the hormone binding domain of the estrogen or glucocorticoid receptor fused to heterologous activation and DNA binding domains also exhibited elevated activity in the absence of hormone. Thus, Ydj1 mutants appear to increase basal receptor activity by altering the ability of the hormone binding domain of the receptor to repress nearby activation domains. We propose that Ydj1 functions to present steroid receptors to the Hsp90 pathway for folding and hormonal control. In the presence of Ydj1 mutants that fail to bind substrate efficiently, some receptor escapes the Hsp90 pathway, resulting in constitutive activity.

关键词：电泳聚丙烯酰氨凝胶基因文库基因报告基因 src/遗传学 HSP40热休克蛋白质类 HSP70热休克蛋白质类/化学 HSP70热休克蛋白质类/遗传学 HSP70热休克蛋白质类/生理学 HSP90热休克蛋白质类/代谢热休克蛋白质类免疫印迹法诱变表型质粒蛋白质结构三级受体雌激素/代谢受体糖皮质激素/代谢受体类固醇/代谢重组融合蛋白质类/代谢酿酒酵母菌/代谢酿酒酵母蛋白质类温度双杂交系统技术 β半乳糖苷酶类/代谢

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EFFECTS OF CALCIUM AND MAGNESIUM-IONS ON INTERACTION OF CORTICOSTERONE WITH RAT-BRAIN CYTOSOL RECEPTOR(S)

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ENDOCRINE RESEARCH COMMUNICATIONS 1978年第2期5卷 81-90页

作者： NAKAI, T UEDA, M TAKEDA, R The Second Department of Internal Medicine School of Medicine Kanazawa University Kanazawa City 920 Takaramachi 13-1 Japan

The apparent maximum corticosterone binding (B max) with rat brain cytosol and the apparent dissociation constant of this steroid-receptor binding (Kd) estimated with a Scatchard plot was 2.9 × 10-13moles/mg cytosol protein and 4.0 × 10-9M, respectively. When increasing amounts of CaCl2or MgCl2up to 5.0 mM were added, a specific [3H] corticosterone binding increased 4-fold by CaCl2at concentrations of 1.0-2.0 mM and 1.5-fold by MgCl2at concentrations of 0.5-5.0 mM. The addition of MnCl2and KC1 did not affect this binding. Binding of corticosterone with rat brain cytosol receptor(s) were decreased by increasing amounts of EGTA and complete inhibition was observed at concentrations equal to and greater than 2.5 mM. Inhibition of this binding by EDTA was less than by EGTA. Either theophylline or dibutyryl cyclic AMP had no effect on this binding.

关键词：肾上腺切除术脑/代谢钙/药理学皮质酮/代谢胞质溶胶/代谢依地酸/药理学依他酸/药理学动力学镁/药理学受体糖皮质激素/药物作用受体糖皮质激素/代谢受体类固醇/代谢动物男(雄)性大鼠

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Structure and dynamic properties of a glucocorticoid receptor-induced chromatin transition

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MOLECULAR AND CELLULAR BIOLOGY 2000年第17期20卷 6466-6475页

作者： Fletcher, TM Ryu, BW Baumann, CT Warren, BS Fragoso, G John, S Hager, GL NCI Lab Receptor Biol & Gene Express NIH Bethesda MD 20892 USA

Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR), Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W, D, Pennie, S, John, and G, L, Eager, Mol. Cell. Biol, 18:3633-3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process;in the absence of ATP, GR binds to multiple sites on the chromatin array end prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which CR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a "hit-and-run" mechanism for GR action in living cells (J, G, McNally, W, G. Muller, D, Walker, and G, L, Hager, Science 287:1262-1264, 2000).

关键词： 3T3细胞腺苷三磷酸/代谢结合部位 CHO细胞细胞核/代谢染色质/遗传学染色质/代谢仓鼠亚科脱氧核糖核酸酶Ⅰ/代谢剂量效应关系药物电泳琼脂凝胶 HeLa细胞水解作用配体乳房瘤病毒鼠/遗传学诱变定点核小体/代谢质粒/代谢蛋白质结合受体糖皮质激素/遗传学受体糖皮质激素/代谢末端重复序列转染动物人类小鼠

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Creating a protein-based element of inheritance

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SCIENCE 2000年第5453期287卷 661-664页

作者： Li, LM Lindquist, S Univ Chicago Howard Hughes Med Inst Dept Mol Genet & Cell Biol Chicago IL 60637 USA

Proteins capable of self-perpetuating changes in conformation and function (known as prions) can serve as genetic elements. To test whether novel prions could be created by recombinant methods, a yeast prion determinant was fused to the rat glucocorticoid receptor. The fusion protein existed in different heritable functional states, switched between states at a Low spontaneous rate, and could be induced to switch by experimental manipulations. The complete change in phenotype achieved by transferring a prion determinant from one protein to another confirms the protein-only nature of prion inheritance and establishes a mechanism for engineering heritable changes in phenotype that should be broadly applicable.

关键词：真菌蛋白质类/化学真菌蛋白质类/遗传学真菌蛋白质类/代谢基因报告胍/药理学热休克蛋白质类/药理学肽终止因子表型朊病毒/化学朊病毒/遗传学朊病毒/代谢受体糖皮质激素/化学受体糖皮质激素/遗传学受体糖皮质激素/代谢重组融合蛋白质类/化学重组融合蛋白质类/代谢酿酒酵母菌/化学酿酒酵母菌/遗传学酿酒酵母蛋白质类转录遗传转化遗传动物大鼠

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Differential interaction of steroid hormone receptors with LXXLL motifs in SRC-1a depends on residues flanking the motif

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JOURNAL OF STEROID BIOCHEMISTRY AND MOLECULAR BIOLOGY 2000年第1-2期72卷 35-46页

作者： Needham, M Raines, S McPheat, J Stacey, C Ellston, J Hoare, S Parker, M AstraZeneca Pharmaceut Macclesfield SK10 4TG Cheshire England Imperial Canc Res Fund Mol Endocrinol Lab London WC2A 3PX England

Steroid hormones induce the transcriptional activity of their cognate receptors by recruiting a variety of cofactors. One of these, steroid receptor co-activator-1 (SRC-1) interacts with the ligand binding domains of a number of different receptors by means of LXXLL motifs. We have investigated the relative interaction of four such motifs in SR%-1a using a yeast two-hybrid assay. We demonstrate that ER alpha, ER beta and ER beta 2 preferentially interact with motif 2 while GR, AR, PPAR alpha and PPAR gamma preferentially interact with motif 4. We show that the interactions depend not only on the LXXLL motif itself but also on residues flanking the motif. (C) 2000 Elsevier Science Ltd. All rights reserved.

关键词：氨基酸特征结构氨基酸序列氨基酸取代/遗传学结合部位地塞米松/代谢双氢睾酮/代谢雌激素类/代谢组蛋白酰基转移酶亮氨酸/遗传学亮氨酸/代谢配体核受体共激活因子1 肽类/化学肽类/代谢蛋白质结合受体雄激素/代谢受体胞质和核/代谢受体雌激素/代谢受体糖皮质激素/代谢受体类固醇/遗传学受体类固醇/代谢重组融合蛋白质类/化学重组融合蛋白质类/遗传学重组融合蛋白质类/代谢底物特异性热力学转录因子/化学转录因子/遗传学转录因子/代谢双杂交系统技术动物人类大鼠

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EVIDENCE THAT NATURAL VS SYNTHETIC STEROID-HORMONES BIND TO PHYSICOCHEMICALLY DISTINCT CELLULAR RECEPTORS

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 1976年第3期73卷 767-772页

作者： AGARWAL, MK INSERM 36 UNIT F-75005 PARIS FRANCE

Natural (corticosterone) vs. synthetic (dexamethasone) bound rat liver cytosols were fractionated on 3 chromatographic systems. Dexamethasone bound radioactivity eluted in positions different from those that were labe... 详细信息

关键词：结合竞争性皮质酮/代谢胞质溶胶/代谢地塞米松/代谢肝/代谢受体糖皮质激素/分离和提纯受体糖皮质激素/代谢受体类固醇/代谢动物男(雄)性大鼠

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Glucocorticoid receptor interaction with 14-3-3 and Raf-1, a proposed mechanism for cross-talk of two signal transduction pathways

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JOURNAL OF BIOLOGICAL CHEMISTRY 2000年第50期275卷 39296-39301页

作者： Widén, C Zilliacus, J Gustafsson, JÅ Wikström, AC Karolinska Inst Novum Dept Med Nutr S-14186 Huddinge Sweden

The glucocorticoid receptor (GR) functions as a ligand-dependant transcription factor. In the present study we describe a specific immunoaffinity chromatography purification of GR from liver cytosol from adrenalectomized rats that may be used to identify hitherto unknown cytosolic GR interacting proteins. me have identified the ubiquitously expressed 14-3-3 as well as Raf-1, a downstream effector of Ras, as GR co-purifying proteins. In our semi-quantitative analysis liganded/activated GR showed the strongest interaction with 14-3-3 and Raf-l, but 14-3-3 was also found to co-purify with GR in a nonliganded/nonactivated state. By extensive salt washes we were also able to demonstrate that the glucocorticoid induced interaction between GR, 14-3-3, and Raf-1, respectively, is remarkably stable and withstood 2.4 M salt. The interaction between GR and 14-3-3 was also verified by 14-3-3 co-immunoprecipitation studies. Our observations that GR and Raf-1 are found within the same protein complex ("receptosome") in the cytoplasm of rat liver cells could provide a mechanistic explanation for glucocorticoid effects on the Raf-1-Ras signaling pathway.

关键词： 14-3-3蛋白质类印迹法蛋白质色谱法亲和细胞质/代谢胞质溶胶/代谢电泳聚丙烯酰氨凝胶配体肝/代谢沉淀素试验蛋白质结合原癌基因蛋白质c-raf/分离和提纯原癌基因蛋白质c-raf/代谢受体糖皮质激素/分离和提纯受体糖皮质激素/代谢信号传导氯化钠/药理学酪氨酸单氧化酶/分离和提纯酪氨酸单氧化酶/代谢动物女(雌)性大鼠

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The phosphoenolpyruvate carboxykinase gene glucocorticoid response unit:: Identification of the functional domains of accessory factors HNF3β (hepatic nuclear factor-3β) and HNF4 and the necessity of proper alignment of their cognate binding sites

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MOLECULAR ENDOCRINOLOGY 1999年第4期13卷 604-618页

作者： Wang, JC Strömstedt, PE Sugiyama, T Granner, DK Vanderbilt Univ Sch Med Dept Physiol & Mol Biophys Nashville TN 37232 USA

Complete induction of hepatic phosphoenolpyruvate carboxykinase (PEPCK) gene transcription by glucocorticoids requires a complex glucocorticoid response unit (GRU). The GRU is comprised of two glucocorticoid receptor (GR)-binding sites (GR1 and GR2) and four accessory factor-binding sites [AF1, AF2, AF3, and cAMP response element (CRE)] that bind distinct transcription factors. Hepatic nuclear factor 4 (HNF4) and chicken ovalbumin upstream promoter transcription factor (COUP-TF) bind to the AF1 element and account for AF1 activity. Members of the hepatic nuclear factor 3 (HNF3) family bind to the AF2 element and provide AF2 activity. In this report, we show that the functions of AF1 and AF2 are dependent on their positions in the promoter, since they cannot substitute for each other nor can they be exchanged without a reduction in the response to glucocorticoids. We also identified the domains of HNF4 and HNF3 beta that are required for the AF1 and AF2 activities, respectively. The carboxy-terminal transactivation domain of HNF4 (amino acids 128-374) confers most of the AF1 activity, while the carboxyterminal transactivation domain of HNF3 beta (amino acids 361-458) mediates AF2 activity. These domains of HNF4 and HNF3 beta appear to have distinct roles in the response to glucocorticoids, as there are unique structural requirements for each, as judged by the failure of most other classes of transactivation domains to serve as accessory factors. These results suggest that the regulation of the PEPCK gene by glucocorticoids requires specific interactions between GR, accessory factors, and coactivators, and that the transactivation domains of AF1 and AF2 are of fundamental importance in the assembly of this multiprotein complex.

关键词：碱性螺旋-环-螺旋亮氨酸拉链转录因子类结合部位 DNA结合蛋白质类/代谢糖皮质激素类/代谢肝细胞核因子3β 肝细胞核因子4 核蛋白质类/代谢磷酸烯醇丙酮酸羧激酶(GTP)/药物作用磷酸烯醇丙酮酸羧激酶(GTP)/遗传学磷酸烯醇丙酮酸羧激酶(GTP)/代谢磷蛋白类/代谢受体糖皮质激素/代谢重组蛋白质类/遗传学重组蛋白质类/代谢反应元件/药物作用反应元件/生理学反式激活(遗传学) 转录因子/代谢人类

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Inhibition of heat shock factor activity prevents heat shock potentiation of glucocorticoid receptor-mediated gene expression

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CELL STRESS & CHAPERONES 1999年第4期4卷 223-234页

作者： Li, DP Calzi, SL Sánchez, ER Med Coll Ohio Dept Pharmacol Toledo OH 43614 USA

Using mouse L929 cells stably transfected with a glucocorticoid receptor (GR)-responsive murine mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) reporter gene (LMCAT2 cells), we have shown that cellular stress (heat or chemical shock) can cause a dramatic increase in the levels of dexamethasone (Dex)-induced CAT gene expression. We refer to this response as the heat shock potentiation effect, or HSPE. As the cellular heat shock response also involves the activation of heat shock transcription factor (HSF), we have, in the present study, examined the role of HSF in the stress potentiation of GR by use of a flavonoid compound, quercetin, recently shown to selectively inhibit the stress response in a variety of human and murine cell lines. Analysis of the HSPE, as well as heat shock protein synthesis and activation of HSF during time-courses of recovery following heat shock, revealed a similar pattern for each response, with peak activities occurring about 16 h after stress. These data suggest a correlation between the activation of both GR and HSF in stressed cells. In L929 cells stably transfected with a CAT reporter plasmid under the control of the HSF-responsive hsp70 promoter (LHSECAT cells), pretreatment with quercetin was found to cause a dose- and time-dependent inactivation of HSF activity following heat shock, but only when added before the stress event. In LMCAT2 cells, quercetin similarly inhibited both heat and chemical shock potentiation of Dex-induced GR activity. This activity of quercetin was not the result of post-transcriptional or general cytotoxic properties, as quercetin (1) did not significantly affect GR or HSF activities when added after the stress event, (2) did not reduce CAT gene expression as controlled by the constitutive SV40 early promoter, and (3) did not alter normal (non-stress), Dex-induced MMTV-CAT expression. Thus, quercetin appears to be an effective and selective inhibitor of HSF stress-induced activation and its

关键词：亚砷酸盐类/药理学细胞系氯霉素O-乙酰转移酶 DNA结合蛋白质类/拮抗剂和抑制剂地塞米松/药理学剂量效应关系药物基因表达调控 HSP70热休克蛋白质类/代谢 HSP90热休克蛋白质类/代谢热休克蛋白质类/药物作用热休克蛋白质类/代谢模型生物学槲皮素/药理学受体糖皮质激素/药物作用受体糖皮质激素/代谢信号传导温度转录因子转染动物人类小鼠

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