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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信号转导途径。我们确定了一条保守的核外途径，通过该途径 17β-雌二醇 (E2) 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)，导致极其快速（<1 分钟）钙 (Ca2+) 从 EnR 内腔流入细胞体。细胞内 Ca2+ 升高为 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)，诱导重要的伴侣 BiP/GRP78（葡萄糖调节蛋白 78 kDa）。激活这个 E2-ERα 调节基因表达、诱导细胞增殖和保护细胞所需的途径对抗压力。我们通过具有医学前景的 ERα 生物调节剂 BHPI 来瞄准这一途径，它使用与 E2 相同的途径，但会诱导 UPR 的毒性过度激活。我们的假设是，产品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 快速启动该通路的传感器和信号（目标 3）。目标1。确定 E2-ERα 通路激活产物偶联的机制控制 E2-ERα 调节的基因表达。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，从而控制 E2-ERα 调节基因表达。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 对拮抗剂具有部分抗性。通过以下方式识别机制 UPR 激活有助于耐药性。检验以下假设：这些药物具有耐药性细胞的产生部分是因为 ERα 与孕酮-PR 一起协同激活 UPR，诱导 BiP 分子伴侣，可稳定 ERα，从而有助于耐药基因表达。目标 3. 识别 E2-ERα 启动该通路的多蛋白复合物的组成部分。使用无偏 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）

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.)
影响因子：
0
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.