Glucocorticoid ligands specify different interactions with NF-kappaB by allosteric effects on the glucocorticoid receptor DNA binding domain

Glucocorticoids inhibit inflammation by acting through the glucocorticoid receptor (GR) and powerfully repressing NF-kappaB function. Ligand binding to the C-terminal of GR promotes the nuclear translocation of the receptor and binding to NF-kappaB through the GR DNA binding domain. We sought how ligand recognition influences the interaction between NF-kappaB and GR. Both dexamethasone (agonist) and RU486 (antagonist) promote efficient nuclear translocation, and we show occupancy of the same intranuclear compartment as NF-kappaB with both ligands. However, unlike dexamethasone, RU486 had negligible activity to inhibit NF-kappaB transactivation. This failure may stem from altered co-factor recruitment or altered interaction with NF-kappaB. Using both glutathione S-transferase pull-down and bioluminescence resonance energy transfer approaches, we identified a major glucocorticoid ligand effect on interaction between the GR and the p65 component of NF-kappaB, with RU486 inhibiting recruitment compared with dexamethasone. Using the bioluminescence resonance energy transfer assay, we found that RU486 efficiently recruited NCoR to the GR, unlike dexamethasone, which recruited SRC1. Therefore, RU486 promotes differential protein recruitment to both the C-terminal and DNA binding domain of the receptor. Importantly, using chromatin immunoprecipitation, we show that impaired interaction between GR and p65 with RU486 leads to reduced recruitment of the GR to the NF-kappaB-responsive region of the interleukin-8 promoter, again in contrast to dexamethasone that significantly increased GR binding. We demonstrate that ligand-induced conformation of the GR C-terminal has profound effects on the functional surface generated by the DNA binding domain of the GR. This has implications for understanding ligand-dependent interdomain communication.     

肺内调节肽对支气管上皮细胞ICAM-1表达及NF-κB活性的调控

细胞间粘附分子—1(ICAM—1)是介导细胞与细胞之间粘附的重要生物分子;核因子—κB(NF—κB)是体内普遍存在、能迅速对刺激产生反应的重要核转录因子。越来越多的证据显示,ICAM—1表达与NF—κB激活是炎症反应的重要步骤。我们应用免疫组化、RT—PCR、凝胶阻滞电泳(EMSA)等多种实验方法,观察了肺内调节肽对支气管上皮细胞ICAM—1表达及NF—κB活性的影响,以及NF—κB抑制剂MG—132对ICAM—1表达的影响。实验结果发现,VIP、EGF可使臭氧应激BECS的ICAM—1表达降低;ET—1、CGRP可使未受应激BECs的ICAM—1表达增加。NF—κB抑制剂MG—132可阻断O3、ET—1、CGRP引起的ICAM—1表达,提示NF—κB在调控ICAM—1表达中起重要作用。EMSA结果显示,BECs中NF—κB在臭氧应激下反复激活,CGRP与ET—1可促进NF—κB的激活;VIP与EGF可抑制臭氧应激的BECs中NF—κB的激活。以上结果说明,VIP、EGF可通过下调ICAM—1转录及NF—κB激活减轻炎症反应,而ET—1、CGRP可通过上调ICAM—1转录及NF—κB激活、加大炎症反应。ICAM—1与NF—κB的持续表达和反复激活是炎症持续加重发展的重要因素。     

Glucocorticoid binding in the hen oviduct

[³H]Triamcinolone acetonide (15nm) was incubated with cytosol (150000g fraction) prepared from oviducts of egg-laying hens. The extent of steroid binding, as determined by charcoal assays, was greatest between 2–4h at 4°C. A similar time curve was obtained when cytosol preparations were first fractionated with (NH₄)₂SO₄ before labelling. The addition of 10mm-Na₂MoO₄ or 10mm-ATP during the incubation of hen oviduct cytosol with [³H]triamcinolone acetonide lowered the extent of steroid binding. The presence of glycerol (20%), however, increased the extent of [³H]triamcinolone acetonide binding in cytosol fractions from chick (330%) and hen (160%) oviducts. The [³H]triamcinolone acetonide–receptor complex was stable for over 4h at 4°C, but dissociated rapidly at 37°C, exhibiting a half-life of about 10min. The presence of 10mm-Na₂MoO₄ and 10mm-ATP or both had a small protective effect on the dissociation of [³H]triamcinolone acetonide–receptor complex. The receptor from hen oviduct showed significant affinity for unlabelled triamcinolone acetonide, cortisol, compound R5020 and dihydrotestosterone and, to a lesser extent, for oestradiol, oestrone and progesterone. Diethylstilboestrol treatment of immature chicks appeared to induce a more specific binder, which showed affinity for unlabelled triamcinolone acetonide, cortisol and compound R5020 only. Scatchard analysis of [³H]triamcinolone acetonide binding in hen oviduct cytosol revealed a K_d value of 6.4nm. The steroid–receptor complex sedimented as a 7–8S and a 4S entity on low-salt (0.01m-KCl)- and high-salt (0.3m-KCl)-containing sucrose gradients respectively. The cytosol [³H]triamcinolone acetonide–receptor complex showed no affinity for ATP–Sepharose or DNA–cellulose, but acquired this ability on heat activation (23°C, 40min). The data indicate the avian oviduct possesses a high-affinity binding molecule that fulfils the criteria of a glucocorticoid receptor.