Estrogen Receptor Activation of p38 Kinase and Apoptosis

描述：（扫描自申请人摘要）在稳定转染的ER+中 在HeLa-ER细胞中，我们发现4-羟基他莫昔芬（OHT）-ER和17 β-雌二醇（E2） （E2）-ER，引起p38和Jun N-末端的极其快速的活化， 激酶（JNK）途径。OHT-ER或E2-ER对p38或iNK的激活作用强烈， 与诱导凋亡相关。在MCF-7细胞中，OHT，而不是E2， 诱导p38和细胞凋亡，p38抑制剂阻断p38的诱导， 凋亡p38的激活和凋亡的诱导是新的研究方向。 雌激素受体的作用。 具体目的是：（1）鉴定OHT-ER的通路（和 En-ER）对p38和JNK的磷酸化和活化的影响。OHT-ER诱导 p38和JNK在2分钟内磷酸化，表明非基因组效应。(a)我们 将确定OHT-ER所需的ER结构域（如核定位） （B）评估p38和JNK的活化在p38活化中的可能作用， 膜相关ER使用富集在推定的膜ER中的细胞。通过 特异性靶向ER细胞膜，并通过研究潜在的 ER-小窝相互作用：（c）使用显性负性MKK来确定ER-小窝相互作用的作用。 MKKs在响应OHT-ER和E2-ER磷酸化p38和JNK中的表达。(2)到 研究OHT-ER激活p38与 E2-ER与细胞凋亡的诱导。我们将确定 在我们的初步研究中看到的p38和JNK激活的第二个晚期阶段， 确定凋亡是否需要p38和JNK的晚期激活，和 无论晚期激活是基因组的还是非基因组的。我们要写一部小说， 与乳腺癌化学预防和治疗相关的可检验的假设 他莫昔芬（Tamoxifen，Tam），或许还有其他SERMs。在乳腺上皮细胞中， 由DNA损伤产生的细胞死亡途径的激活水平通常 负责转化的人有时可能不足以诱导 所有受损细胞的凋亡。在ER水平较低的细胞中， OHT-ER对p38和JNK的弱诱导增强DNA诱导的细胞凋亡 损伤，从而刺激癌前和早期阶段的细胞凋亡 乳腺癌细胞(3)我们会轻微损伤乳腺上皮细胞的DNA 细胞和ER+细胞系与低水平的UV光，并确定是否Tam 激活p38或JNK并增强细胞凋亡。(4)以确定 乳腺癌细胞系对Tam耐药的进展是否涉及 p38或JNK的Tam诱导丧失，或p38或JNK诱导 我们将使用现有的Tam抗性乳腺癌细胞系和细胞凋亡， 我们分离的品系，抑制剂和显性阴性突变体。 这些研究有助于确定ER作用的新途径，并测试一种新的 关于SERMs如何发挥作用的假设。

DESCRIPTION: (Scanned from the applicant's abstract) In stably transfected ER+ HeLa-ER cells, we find that 4-hydroxytamoxifen (OHT)-ER and l7b-estradiol (E2)-ER, elicit an extremely rapid activation of the p38 and Jun N-terminal kinase (JNK) pathways. Activation of p38 or iNK by OHT-ER or E2-ER is strongly correlated with the induction of apoptosis. In MCF-7 cells, OHT, but not E2, induces p38 and apoptosis and a p38 inhibitor blocks induction of p38 and apoptosis. Activation of p38 and induction of' apoptosis represent newly identified actions of estrogen receptor. The Specific Aims are: (1) To identify the pathway leading from OHT-ER (and En-ER) to the phosphorylation and activation of p38 and JNK. OHT-ER induces phosphorylation of p38 and JNK in 2 min. suggesting a nongenomic effect. (a) We will identify the ER domains (such as nuclear localization) required for OHT-ER activation of p38 and JNK; (b) evaluate the possible role in p38 activation of a membrane-associated ER using cells enriched in a putative membrane ER. by specifically targeting ER to cell membranes, and through studies of potential ER-caveolae interactions: (c) use dominant negative MKKs to determine the role of MKKs in phosphorylating p38 and JNK in response to OHT-ER and E2-ER. (2) To investigate the relationship between the activation of p38 by OHT-ER and by E2-ER and the induction of apoptosis. We will determine the kinetics of the second late stage of activation of p38 and JNK seen in our preliminary studies, determine whether apoptosis requires the late activation of p38 and JNK, and whether the late activation is genomic or non-genomic. We propose a novel, testable, hypothesis relevant to both breast cancer chemoprevention and therapy by tamoxifen (Tam), and perhaps by other SERMs. In mammary epithelial cells, the level of activation of cell death pathways produced by the DNA damage often responsible for transformation may sometimes be insufficient to induce apoptosis of all damaged cells. In cells with low levels of ER, the additional weak induction of p38 and JNK by OHT-ER potentiates apoptosis induced by DNA damage, and thereby stimulates apoptosis of pre-malignant and early stage breast cancer cells. (3) We will slightly damage the DNA of mammary epithelial cells and ER+ cell lines with a low level of UV light and determine whether Tam activates p38 or JNK and potentiates apoptosis of the cells. (4) To determine whether the progression of breast cancer cell lines to Tam resistance involves loss of Tam induction of p38 or JNK, or loss of ability of p38 or JNK to induce apoptosis, we will use existing Tam resistant breast cancer cell lines and cell lines we isolate, inhibitors and dominate negative mutants. These studies help define a new pathway for ER action and test a novel hypothesis for how SERMs function.