How Rapid Anticipatory Estrogen Activation of the Unfolded Protein Response Acts as an Authorizing Signal for Estrogen Receptor Action

未折叠蛋白反应的快速预期雌激素激活如何作为雌激素受体作用的授权信号

• 批准号: 9294047
• 负责人: DAVID J SHAPIRO
• 金额: $ 37.38万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-15 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9294047
• 关键词: Alpha Cell; Binding; Biological Response Modifiers; CRISPR/Cas technology; Calcium; Calmodulin; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell physiology; Cells; Complex; Couples; Coupling; Cytosol; DNA Polymerase II; Data; Development; Dimerization; Drug Targeting; Drug resistance; Elements; Endoplasmic Reticulum; Epidermal Growth Factor Receptor; Estradiol; Estrogen Receptor alpha; Estrogen Receptors; Estrogens; Event; Fulvestrant; GRP78 gene; Gene Expression; Genes; Genetic Complementation Test; Genetic Transcription; Genomics; Goals; Hormones; Inositol; Investigation; Knowledge; Link; Mediating; Medical; Molecular; Molecular Chaperones; Multiprotein Complexes; Mus; Mutation; Nuclear; Pathway interactions; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Play; Process; Production; Progesterone; Proteins; RNA; Recruitment Activity; Regulatory Pathway; Resistance; Role; Scaffolding Protein; Signal Transduction; Signal Transduction Pathway; Steroid Receptors; Stress; System; Testing; Therapeutic; Time; Work; activation product; arm; cell growth; dimer; endoplasmic reticulum stress; glucose-regulated proteins; human data; human disease; killings; mutant; pre-clinical; programs; receptor; response; sensor; steroid hormone; therapeutic candidate; therapeutic target; tripolyphosphate

Estrogens, acting via estrogen receptor α (ERα), were known to regulate gene expression and to activate signal transduction pathways. We identified a conserved extranuclear pathway by which 17β-estradiol (E2), acting through ERα, rapidly activates phosphoplipase C γ (PLCγ) leading to production of inositol triphosphate (IP3). The IP3 binds to and opens endoplasmic reticulum (EnR) IP3 receptors (IP3R) leading to extremely rapid (<1 min.) efflux of calcium (Ca2+) from the lumen of the EnR into the cell body. Elevated intracellular Ca2+ primes cells for subsequent actions of E2-ERα; depletion of EnR Ca2+ activates the unfolded protein response (UPR), inducing the important chaperone BiP/GRP78 (glucose regulated protein 78 kDa). Activation of this pathway is required for E2-ERα-regulated gene expression, induction of cell proliferation and protects cells against stress. We target this pathway with our medically promising ERα biomodulator, BHPI, which uses the same pathway as E2, but induces toxic hyperactivation of the UPR. Our hypothesis is that the products of activation of this newly unveiled pathway, elevated intracellular calcium (Aim 1), and at later times, BiP chaperone (Aim 2), link to and regulate subsequent E2-ERα-regulated gene expression and stabilize ERα, influencing drug resistance and genomic actions of ERα. Our goals are to identify the mechanism(s) by which these products couple to, and control, gene expression (Aim 1), ERα stability and response to drugs (Aim 2), and to identify the sensors and signals that allow E2-ERα to rapidly initiate the pathway (Aim 3). Aim 1. Identify the mechanism(s) by which the product of E2-ERα activation of the pathway couples to and controls E2-ERα-regulated gene expression. Test the data-driven hypothesis that Ca2+ produced by pathway activation acts through the Ca2+ sensor calmodulin (CaM) to regulate nuclear E2-ERα:CaM interaction, E2-ERα dimerization and nuclear localization and thereby controls E2-ERα-regulated gene expression. Aim 2. Background: In CRISPR/Cas9 generated cell lines expressing constitutively active ERα mutants, the UPR is activated and ERα is partially resistant to antagonists. Identify the mechanism by which UPR activation contributes to drug resistance. Test the hypothesis that drug resistance in these cells arises in part because ERα, together with progesterone-PR, synergistically activate the UPR, inducing BiP chaperone, which stabilizes ERα, thereby contributing to drug resistant gene expression. Aim 3. Identify components of the multiprotein complex by which E2-ERα initiates the pathway. Using an unbiased CRISPR/Cas9 lethality screen, followed by verification and analysis of multiprotein complexes, we will identify the activating kinase(s), scaffolding proteins, other components of the complex(es), genes that impact the pathway and probe ERα interactions in the complex. These studies will establish the initial events that occur when estrogen contacts a cell and identify new mechanisms coupling steroid receptor regulated transcription to extranuclear signals.

雌激素通过雌激素受体α(ERα)作用，调节基因表达和激活 信号转导通路。我们发现了一条保守的核外途径，17β-雌二醇(E_2)， 通过内质网α，迅速激活磷脂酶Cγ(PLCγ)，导致肌醇三磷酸的产生 (IP3)。IP3与内质网(ENR)IP3受体(IP3R)结合并开放，导致极快的 (&lt；1分钟。)钙离子从ENR的管腔流出到细胞体。细胞内钙离子升高 为E2-ERα的后续作用启动细胞；ENR钙离子的耗竭激活未折叠的蛋白质反应 (UPR)，诱导重要的伴侣蛋白Bip/GRP78(葡萄糖调节蛋白78 kDa)。激活此选项 E2-ERα调控基因表达、诱导细胞增殖和保护细胞所需的途径 对抗压力。我们用我们医学上很有前途的ERα生物调节剂BHPI来靶向这一途径，它使用 与E2途径相同，但可诱导UPR的毒性过度激活。我们的假设是， 激活这一新揭示的通路，升高细胞内钙(Aim 1)，然后再激活Bip 伴侣(Aim 2)，连接并调节随后的E2-ERα调节的基因表达，稳定ERα， 影响ER-α的耐药性和基因组作用。我们的目标是确定(S)通过什么机制 这些产物偶联并控制基因表达(目标1)、ERα稳定性和对药物的反应(目标2)、 并确定允许E2-ERα快速启动该途径的传感器和信号(目标3)。目标1。 确定该途径的E2-ERα激活产物偶联到和的机制(S) 控制E2-ERα调控的基因表达。测试数据驱动的假设，即 钙通道激活通过钙调素(CaM)调节核内E2-ERα：CaM 相互作用、E2-ERα二聚化和核定位从而调控E2-ERα调控基因 表情。目的2.背景：在CRISPR/CAS9中建立表达活性ERα的细胞系 突变体中，UPR被激活，ERα对拮抗剂产生部分抗性。通过以下方式确定机制 其中UPR的激活与耐药有关。验证这样一种假设，即在这些细胞中存在耐药性 细胞的产生部分是因为ER-α和孕激素-PR协同激活UPR，诱导 BIP分子伴侣，稳定ERα，从而促进耐药基因表达。目标3.确定 多蛋白复合体的组成部分，E2-ERα通过它启动该途径。使用不带偏见的 CRISPR/Cas9致死性筛选，随后对多蛋白复合体进行验证和分析，我们将鉴定 激活蛋白(S)，支架蛋白，复合体(ES)的其他成分，影响 途径和探针ERα在复合体中的相互作用。 这些研究将确定雌激素接触细胞时发生的初始事件，并确定新的 类固醇受体调节转录与核外信号耦合的机制。