Molecular Mediators/Regulators of Glucocorticoid Actions

糖皮质激素作用的分子介质/调节剂

• 批准号: 7209191
• 负责人: Tomoshige Kino
• 金额: --
• 依托单位: EUNICE KENNEDY SHRIVER NATIONAL INSTITUTE OF CHILD HEALTH & HUMAN DEVELOPMENT
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7209191
• 关键词: HeLa cells; acyltransferase; corticosteroid receptors; developmental genetics; fluorescence recovery after photobleaching; genetic disorder; glucocorticoids; guanine nucleotide binding protein; guanine nucleotide exchange factors; histones; hormone regulation /control mechanism; hormone sensitivity /resistance; human immunodeficiency virus 1; insulin; microarray technology; molecular biology; phosphorylation; protein protein interaction; transcription factor; virus protein; yeast two hybrid system

Glucocorticoids have a broad array of life-sustaining functions and play an important role in the therapy of several inflammatory/autoimmune/allergic and lymphoproliferative disorders. Thus, changes of tissue responsiveness to glucocorticoids may develop pathologic states and influence their disease course. We investigated pathophysiologic mechanism of one of such conditions, familial/sporadic glucocorticoid resistance syndrome, which is caused by mutations in the glucocorticoid receptor (GR) gene. We analyzed molecular defects of GRL773P, which is recently found as a heterozygotic mutation, replacing leucine at amino acid 773 of GR with proline. This mutant receptor demonstrated reduced transactivation activity due to its inability to form the ligand-dependent transactivation surface and behaved as a dominant negative mutant, thereby the patient developed glucocorticoid resistance even in the hetrozygotic condition. We recently found another two heterozygotic mutations that produce GRD401H and F737L. We will examine their molecular defects as well. To examine biologic defects of pathologic GR mutant receptors in living cells, we examined their motility inside the nucleus in living cells by using the fluorescence recovery after photobleaching (FRAP) method. Motility of the ligand-bound wild type GR is regulated inside the nucleus through its multiple interactions with the chromatin-associated molecules, transcriptional intermediate proteins, target DNAs and the ubiquitine/proteasomal pathway. We found that all of the examined mutant receptors demonstrated increased motility and some of them demonstrated altered responsiveness to a proteasomal inhibitor MG-132, possibly due to loss of one or some of the above-indicated important interactions. In order to find intracellular molecules, which potentially influence tissue sensitivity to glucocorticoids, we performed yeast two-hybrid screenings using several bait proteins. We found in the screening using the N-terminal domain of GR as a bait that the guanine nucleotide-binding protein (G) beta and the transforming growth factor beta/bone morphogenetic protein-downstream Smad6 specifically interacted with GR. Both proteins suppressed GR-induced transactivation of glucocorticoid-responsive promoters. The former protein, with its partner molecule Ggamma, co-migrated into the nucleus with GR in response to glucocorticoids, and was attracted to the glucocorticoid response elements (GREs) located in the promoter region of the glucocorticoid-responsive gene, suggesting that attracted Gbeta/gamma interferes with the activity of transcriptional machineries on the GR-bound promoters. Extracellularly administered somatostatin downregulated GR transactivation through the Gbeta/gamma complex. In contrast, Smad6 suppressed GR transcriptional activity both at cellular and animal levels via attracting histone deacetylases and antagonizing to histone acetylation induced by p160 type histone acetyltransferase coactivators. In another yeast two-hybrid screening using the GR DNA-binding domain, we found that SET/TAF-1beta and gas5 interact with this portion of the GR. The former molecule is known as a part of the SET-CAN oncogene product, as well as a component of the inhibitor of acetyltransferases (INHAT) complex that binds lysine residues of the histones and protects them from acetylation by the histone acetyltransferases. We found that SET/TAF-1beta acts as a negative regulator of GR transcriptional activity and ligand-activated GR stimulates transcription by displacing the INHAT complex from histones via physical interaction through the DBD. We speculate that the SET-CAN gene product may cause glucocorticoid insensitivity in leukemic cells, which harbors this translocation. In contrast, gas5 is none protein-coding mRNA, and is accumulated in growth-arrested cells. In our hands, overexpression of gas5 suppressed GR transcriptional activity, possibly by competing with GR for binding to target DNA sequences. We speculate that gas5 is a growth-related regulator of glucocorticoid action, attenuating the glucocorticoid-induced transcriptional activity in growth-arrested cells. Alternative splicing of the GR gene in exon 9 produces GRbeta in addition to the classic receptor GRalpha. This isoform receptor does not bind glucocorticoids and behaves as a dominant negative inhibitor of GRalpha-mediated transactivation. To explore whether GRbeta exerts distinct, intrinsic biologic effects on the human genome, we examined its impact on endogenous gene expression in human cervical carcinoma HeLa cells, by developing two HeLa cell lines, stably expressing enhanced green fluorescent protein (EGFP)-fused GRbeta or EGFP. We found that overexpression of GRbeta positively or negatively regulated mRNA expression of multiple glucocorticoid-unrelated genes in analyses using the 12K human cDNA microarray. Because most of the GRbeta-regulating genes play important roles in the embryonic development and the cell-to-cell contact, GRbeta might play an important physiologic role in early development, in contrast to GRalpha, the influence of which on gene expression are first evident in the late fetal stage. In addition to above proteins, we also worked on Brx, a Rho type guanine nucleotide exchange factor, which activates Rho family small G proteins by converting them from inactive GDP-bound to active GTP-bound form. This protein has a nuclear receptor-interacting domain in its C-terminal portion. We found that Brx enhanced GR transcriptional activity by activating and closely attracting small G proteins to GRE-bound GR. Brx mediated lysophosphatidic acid (LPA)-induced potentiation of GR transactivation. LPA is produced from activated platelets in patients with dysmetabolic syndrome, who frequently develop glucocorticoid hypersensitivity. Thus, Brx may contribute to increased glucocorticoid sensitivity seen in these patients, mediating LPA-induced signal to GR. We found that 14-3-3 proteins were physically associated with the human immunodeficiency virus type 1 accessory protein Vpr and contributed to its cell cycle-arresting activity. 14-3-3 proteins play a significant role in cell cycle progression at several different stages by regulating activities of their partner proteins, such as Cdc25C, Wee1 and Cdk2. They bind to these proteins through phosphorylated serine or threonine residues and modulate their activities by changing their subcellular localization and/or stability. Vpr binds 14-3-3 proteins at their C terminal region and alters the binding ability of the latter to their partner proteins. Thus, Vpr facilitates association of 14-3-3 and Cdc25C independently of the latter's phosphorylation status, promoting cell cycle arrest at the G2/M phase. In addition to the regulation of cell cycle progression, 14-3-3 proteins play a role in the intracellular signaling of insulin, by interacting with key molecules of its signaling cascade, such as insulin receptor substrate 1 (IRS1) and the FoxO subfamily of the forkhead transcription factors. We focused on FoxOs, which function as negative transcription factors of the insulin-signaling pathway. Unphosphorylated, nuclear FoxOs are transcriptionally active, while phosphorylated FoxOs generated through exposure of cells to insulin is segregated into the cytoplasm via binding to 14-3-3, and is thus inactive. We found that Vpr was associated with 14-3-3 and inhibited the latter's interaction with the FoxOs, even though 14-3-3-binding sites are created by insulin. Moreover, we found that Vpr antagonized insulin-induced suppression of the mRNA expression of several insulin/FoxO-target molecules. These results indicate that Vpr may participate in the development of lipodystrophy syndrome/metabolic disturbance frequently observed in AIDS patients through modification of FoxO activity.

糖皮质激素具有广泛的生命维持功能，在多种炎症/自身免疫/过敏和淋巴增殖性疾病的治疗中发挥着重要作用。因此，组织对糖皮质激素反应性的变化可能会形成病理状态并影响其病程。我们研究了其中一种疾病的病理生理机制，即家族性/散发性糖皮质激素抵抗综合征，该综合征是由糖皮质激素受体（GR）基因突变引起的。我们分析了 GRL773P 的分子缺陷，该缺陷是最近发现的一种杂合突变，将 GR 773 氨基酸处的亮氨酸替换为脯氨酸。该突变受体由于无法形成配体依赖性反式激活表面而表现出反式激活活性降低，并且表现为显性失活突变体，因此患者即使在杂合条件下也会出现糖皮质激素耐药性。我们最近发现了另外两个产生 GRD401H 和 F737L 的杂合突变。我们还将检查它们的分子缺陷。 为了检查活细胞中病理性GR突变受体的生物学缺陷，我们通过使用光漂白后荧光恢复（FRAP）方法检查了它们在活细胞细胞核内的运动性。配体结合的野生型 GR 的运动通过其与染色质相关分子、转录中间蛋白、靶 DNA 和泛素/蛋白酶体途径的多重相互作用在细胞核内受到调节。我们发现所有检查的突变受体都表现出运动性增加，其中一些表现出对蛋白酶体抑制剂 MG-132 的反应性改变，这可能是由于上述一种或一些重要相互作用的丧失。 为了找到可能影响组织对糖皮质激素敏感性的细胞内分子，我们使用几种诱饵蛋白进行了酵母双杂交筛选。我们在以GR的N端结构域为诱饵的筛选中发现，鸟嘌呤核苷酸结合蛋白（G）β和转化生长因子β/骨形态发生蛋白下游Smad6与GR特异性相互作用。两种蛋白均抑制 GR 诱导的糖皮质激素反应启动子的反式激活。前一种蛋白与其伴侣分子 Ggamma 响应糖皮质激素，与 GR 共同迁移到细胞核中，并被位于糖皮质激素反应基因启动子区域的糖皮质激素反应元件 (GRE) 吸引，这表明吸引的 Gbeta/gamma 会干扰 GR 结合启动子上转录机制的活性。细胞外施用生长抑素通过 Gbeta/gamma 复合物下调 GR 反式激活。相比之下，Smad6 通过吸引组蛋白脱乙酰酶并拮抗 p160 型组蛋白乙酰转移酶共激活剂诱导的组蛋白乙酰化，在细胞和动物水平上抑制 GR 转录活性。 在另一项使用GR DNA结合域的酵母双杂交筛选中，我们发现SET/TAF-1beta和gas5与GR的这一部分相互作用。前一种分子被认为是 SET-CAN 癌基因产物的一部分，也是乙酰转移酶抑制剂 (INHAT) 复合物的组成部分，该复合物结合组蛋白的赖氨酸残基并保护它们免受组蛋白乙酰转移酶的乙酰化。我们发现 SET/TAF-1beta 作为 GR 转录活性的负调节因子，配体激活的 GR 通过 DBD 的物理相互作用从组蛋白中取代 INHAT 复合物来刺激转录。我们推测，SET-CAN 基因产物可能会导致携带这种易位的白血病细胞对糖皮质激素不敏感。相比之下，gas5 不是蛋白质编码 mRNA，并且在生长停滞的细胞中积累。在我们看来，gas5 的过度表达可能通过与 GR 竞争与靶 DNA 序列的结合来抑制 GR 转录活性。我们推测gas5是糖皮质激素作用的生长相关调节剂，减弱生长停滞细胞中糖皮质激素诱导的转录活性。 GR 基因在外显子 9 中的选择性剪接除了产生经典受体 GRalpha 之外，还产生 GRbeta。该同种型受体不结合糖皮质激素，并且充当 GRalpha 介导的反式激活的显性负性抑制剂。为了探讨 GRbeta 是否对人类基因组发挥独特的内在生物学作用，我们通过开发两种稳定表达增强型绿色荧光蛋白 (EGFP) 融合的 GRbeta 或 EGFP 的 HeLa 细胞系，检查了其对人宫颈癌 HeLa 细胞内源基因表达的影响。在使用 12K 人类 cDNA 微阵列进行分析时，我们发现 GRbeta 的过度表达正向或负向调节多个与糖皮质激素无关的基因的 mRNA 表达。由于大多数 GRbeta 调节基因在胚胎发育和细胞间接触中发挥重要作用，因此 GRbeta 可能在早期发育中发挥重要的生理作用，与 GRalpha 不同，GRalpha 对基因表达的影响首先在胎儿晚期阶段显现出来。 除了上述蛋白质之外，我们还研究了 Brx，一种 Rho 型鸟嘌呤核苷酸交换因子，它通过将 Rho 家族小 G 蛋白从非活性 GDP 结合形式转化为活性 GTP 结合形式来激活它们。该蛋白的 C 端部分具有核受体相互作用结构域。我们发现 Brx 通过激活小 G 蛋白并将其紧密吸引到 GRE 结合的 GR 来增强 GR 转录活性。 Brx 介导溶血磷脂酸 (LPA) 诱导的 GR 反式激活增强。 LPA 是由代谢障碍综合征患者的活化血小板产生的，这些患者经常出现糖皮质激素过敏症。因此，Brx 可能有助于增加这些患者的糖皮质激素敏感性，介导 LPA 诱导的 GR 信号。 我们发现 14-3-3 蛋白与人类免疫缺陷病毒 1 型辅助蛋白 Vpr 存在物理关联，并有助于其细胞周期阻滞活性。 14-3-3 蛋白通过调节其伴侣蛋白（例如 Cdc25C、Wee1 和 Cdk2）的活性，在细胞周期进程的多个不同阶段发挥重要作用。它们通过磷酸化丝氨酸或苏氨酸残基与这些蛋白质结合，并通过改变其亚细胞定位和/或稳定性来调节其活性。 Vpr 在其 C 末端区域结合 14-3-3 蛋白，并改变后者与其伴侣蛋白的结合能力。因此，Vpr 促进 14-3-3 和 Cdc25C 的结合，而与后者的磷酸化状态无关，从而促进细胞周期停滞在 G2/M 期。 除了调节细胞周期进程外，14-3-3 蛋白还通过与信号级联的关键分子（例如胰岛素受体底物 1 (IRS1) 和叉头转录因子的 FoxO 亚家族）相互作用，在胰岛素的细胞内信号传导中发挥作用。我们关注 FoxO，它作为胰岛素信号通路的负转录因子。未磷酸化的核 FoxO 具有转录活性，而通过细胞暴露于胰岛素而产生的磷酸化 FoxO 通过与 14-3-3 结合而分离到细胞质中，因此不具有活性。我们发现 Vpr 与 14-3-3 相关，并抑制后者与 FoxO 的相互作用，尽管 14-3-3 结合位点是由胰岛素产生的。此外，我们发现 Vpr 可以拮抗胰岛素诱导的几种胰岛素/FoxO 靶分子 mRNA 表达的抑制。这些结果表明，Vpr 可能通过改变 FoxO 活性参与 AIDS 患者中常见的脂肪营养不良综合征/代谢紊乱的发生。