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: 9915884
• 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 2022-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9915884
• 关键词: 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; Regulatory Pathway; Resistance; Role; Scaffolding Protein; Signal Transduction; Signal Transduction Pathway; Steroid Receptors; Stress; System; Testing; Therapeutic; Time; Work; activation product; arm; cell growth; endoplasmic reticulum stress; glucose-regulated proteins; human data; human disease; mutant; pre-clinical; programs; receptor; recruit; 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）发挥作用，调节基因表达，激活 信号转导途径我们确定了一个保守的17β-雌二醇（E2）， 通过ERα作用，迅速激活磷脂酶C γ（PLCγ），导致三磷酸肌醇的产生 （IP3）。IP 3结合并打开内质网（EnR）IP 3受体（IP 3R），导致非常快速的细胞凋亡。 （<1分钟）钙（Ca 2+）从EnR腔流出进入细胞体。细胞内Ca 2+升高 为E2-ERα的后续作用准备细胞; EnR Ca 2+耗竭激活未折叠蛋白反应 (UPR)诱导重要的分子伴侣BiP/GRP 78（葡萄糖调节蛋白78 kDa）。激活此 E2-ERα通路是E2-ERα调节基因表达、诱导细胞增殖和保护细胞所必需的 对抗压力我们用我们在医学上有前途的ERα生物调节剂BHPI靶向这一通路， 与E2相同的途径，但诱导UPR的毒性过度活化。我们的假设是 激活这一新发现的途径，升高细胞内钙（目的1），并在稍后的时间，BiP 伴侣蛋白（Aim 2），连接并调节随后E2-ERα调节的基因表达，稳定ERα， 影响ERα的耐药性和基因组作用。我们的目标是确定 这些产物偶联并控制基因表达（目的1）、ERα稳定性和对药物的反应（目的2）， 并鉴定使E2-ERα快速启动通路的传感器和信号（目的3）。目标1. 确定E2-ERα激活途径的产物与 控制E2-ERα调节的基因表达。测试数据驱动的假设，即Ca 2+由 E2-ER α：CaM通路的激活通过Ca 2+感受器钙调素（CaM）调节细胞核E2-ERα：CaM 相互作用、E2-ERα二聚化和核定位，从而控制E2-ERα调节基因 表情目标2.背景：在CRISPR/Cas9产生的表达组成型活性ERα的细胞系中， 突变体中，UPR被激活，ERα对拮抗剂部分耐药。通过以下方式确定机制： UPR的激活导致了抗药性。测试这些细菌的抗药性 细胞的产生部分是因为ERα与孕酮-PR协同激活UPR， BiP分子伴侣，稳定ERα，从而促进耐药基因表达。目标3.识别 E2-ERα启动通路的多蛋白复合物组分。用无偏差 CRISPR/Cas9致死性筛选，然后验证和分析多蛋白复合物，我们将确定 活化激酶、支架蛋白、复合物的其它组分、影响细胞增殖的基因、以及它们的结合。 途径和探针ERα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识别新的 类固醇受体调控的转录与细胞核信号的偶联机制。

项目成果

Fluorescence anisotropy microplate assay to investigate the interaction of full-length steroid receptor coactivator-1a with steroid receptors.

荧光各向异性微孔板测定法研究全长类固醇受体辅激活剂-1a 与类固醇受体的相互作用。

• DOI: 10.1007/978-1-62703-284-1_27
• 发表时间: 2013
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Zhang,Chen;Nordeen,StevenK;Shapiro,DavidJ
• 通讯作者: Shapiro,DavidJ

Antiestrogen Resistant Cell Lines Expressing Estrogen Receptor α Mutations Upregulate the Unfolded Protein Response and are Killed by BHPI.

• DOI: 10.1038/srep34753
• 发表时间: 2016-10-07
• 期刊: Scientific reports
• 影响因子: 4.6
• 作者: Mao C;Livezey M;Kim JE;Shapiro DJ
• 通讯作者: Shapiro DJ

A New Role for Estrogen Receptor α in Cell Proliferation and Cancer: Activating the Anticipatory Unfolded Protein Response.

雌激素受体α在细胞增殖和癌症中的新作用：激活预期的展开的蛋白质反应。

• DOI: 10.3389/fendo.2018.00325
• 发表时间: 2018
• 期刊: Frontiers in endocrinology
• 影响因子: 5.2
• 作者: Livezey M;Kim JE;Shapiro DJ
• 通讯作者: Shapiro DJ

RUNX3 acts as a tumor suppressor in breast cancer by targeting estrogen receptor α.

RUNX3 通过靶向雌激素受体 α 作为乳腺癌肿瘤抑制因子。

• DOI: 10.1038/onc.2011.252
• 发表时间: 2012-01-26
• 期刊: ONCOGENE
• 影响因子: 8
• 作者: Huang, B.;Qu, Z.;Ong, C. W.;Tsang, Y-H N.;Xiao, G.;Shapiro, D.;Salto-Tellez, M.;Ito, K.;Ito, Y.;Chen, L-F
• 通讯作者: Chen, L-F

Anticipatory estrogen activation of the unfolded protein response is linked to cell proliferation and poor survival in estrogen receptor α-positive breast cancer.

• DOI: 10.1038/onc.2014.292
• 发表时间: 2015-07
• 期刊: Oncogene
• 影响因子: 8