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

基本信息

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
项目类别：
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

项目摘要

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，已知可调节基因表达并激活我们确定了 17β-雌二醇 (E2) 的保守核外途径，通过 ERα 作用，快速激活磷脂酶 C γ (PLCγ)，从而产生三磷酸肌醇 (IP3) IP3 结合并打开内质网 (EnR) IP3 受体 (IP3R)，从而导致极其快速的反应。（<1 分钟）钙 (Ca2+) 从 EnR 内腔流入细胞体内。为 E2-ERα 的后续作用做好准备；EnR Ca2+ 的消耗激活未折叠的蛋白质反应； (UPR)，诱导重要伴侣 BiP/GRP78（葡萄糖调节蛋白 78 kDa）的激活。 E2-ERα 调节基因表达、诱导细胞增殖和保护细胞所需的途径我们利用具有医学前景的 ERα 生物调节剂 BHPI 来针对这一途径，它使用与 E2 相同的途径，但会诱导 UPR 的毒性过度激活。激活这一新发现的途径，提高细胞内钙（目标 1），并在随后的时间，BiP 分子伴侣（目标 2），连接并调节随后的 E2-ERα 调节基因表达并稳定 ERα，影响 ERα 的耐药性和基因组作用我们的目标是确定其机制。这些产品耦合并控制基因表达（目标 1）、ERα 稳定性和对药物的反应（目标 2），并识别允许 E2-ERα 快速启动通路的传感器和信号（目标 3）。确定 E2-ERα 通路激活产物偶联的机制检验 Ca2+ 产生的数据驱动假设。通路激活通过 Ca2+ 传感器钙调蛋白 (CaM) 调节核 E2-ERα:CaM 相互作用、E2-ERα二聚化和核定位并由此控制E2-ERα调节基因目标 2. 背景：在 CRISPR/Cas9 生成的细胞系中表达组成型活性 ERα。突变体中，UPR 被激活，ERα 对拮抗剂具有部分抵抗力。 UPR 激活有助于耐药性的假设。细胞的出现部分是因为 ERα 与孕酮-PR 一起协同激活 UPR，诱导 BiP 分子伴侣，可稳定 ERα，从而促进耐药基因表达。目标 3：识别。 E2-ERα 启动该途径的多蛋白复合物的组成部分。 CRISPR/Cas9致死率筛选，随后对多蛋白复合物进行验证和分析，我们将鉴定激活激酶、支架蛋白、复合物的其他成分、影响复合物中途径和探针 ERα 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节转录与核外信号的耦合机制。

项目成果

期刊论文数量（19）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Differential requirements of Hsp90 and DNA for the formation of estrogen receptor homodimers and heterodimers.

Hsp90 和 DNA 对雌激素受体同二聚体和异二聚体形成的不同需求。

DOI：
发表时间：
2010-05-21
期刊：
影响因子：
0
作者：
Powell, Emily;Wang, Yidan;Shapiro, David J;Xu, Wei
通讯作者：
Xu, Wei

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

表达雌激素受体α突变的抗雌激素抗性细胞系上调未折叠蛋白反应并被BHPI杀死。

DOI：
发表时间：
2016
期刊：
影响因子：
4.6
作者：
Mao, Chengjian;Livezey, Mara;Kim, Ji Eun;Shapiro, David J
通讯作者：
Shapiro, David J

8-alkylthio-6-thio-substituted theophylline analogues as selective noncompetitive progesterone receptor antagonists.

8-烷硫基-6-硫代取代的茶碱类似物作为选择性非竞争性黄体酮受体拮抗剂。

DOI：
发表时间：
2012-05
期刊：
影响因子：
2.7
作者：
Aninye, Irene O;Berg, Kenneth C;Mollo, Andy R;Nordeen, Steven K;Wilson, Elizabeth M;Shapiro, David J
通讯作者：
Shapiro, David J

Evaluation of a luciferase-based reporter assay as a screen for inhibitors of estrogen-ERα-induced proliferation of breast cancer cells.

评估基于荧光素酶的报告测定作为雌激素-ERα 诱导的乳腺癌细胞增殖抑制剂的筛选。