p38 MAP Kinase and Akt Interaction in HUVEC

描述（由申请方提供）：本研究的总体目的是了解男性和女性人脐静脉内皮细胞（HUVEC）对应激反应的分子机制和性别差异，重点是PI-3激酶/Akt和p38 MAP激酶通路之间的串扰。结果发现，男性胎儿的死亡率高于女性。对所观察到的男性和女性胎儿死亡率性别差异的分子基础知之甚少。磷脂酰肌醇3-激酶（PI-3 Kinase）/ Akt通路是决定内皮细胞活力的关键调控因子。撤除生长因子或暴露于细胞因子抑制内皮细胞中的PI-3激酶/Akt信号传导并诱导凋亡。PI-3激酶/Akt的下降诱导应激诱导的p38 MAP激酶的上调。有丝分裂原活化蛋白激酶（MAP激酶）和PI-3激酶/Akt通路之间的相互作用以及活化的p38 MAP激酶在细胞凋亡中的作用知之甚少。在这个新的提议中，我们将胎儿的生存能力与HUVEC的功能完整性和存活联系起来，HUVEC的功能完整性和存活是由PI-3激酶/Akt信号转导维持的。尽管缺乏文本数据，但我们已经纳入了初步数据，其中男性HUVEC比女性更容易受到渥曼青霉素（PI-3激酶抑制剂）诱导的应激，导致pAkt急剧下降和细胞凋亡的急剧发生。我们将通过评估男性和女性HUVEC对各种压力的耐受性来巩固这一初步发现。本研究还将确定男性和女性HUVEC对应激敏感性的性别差异是否受p38 MAP激酶的调节。SB 203580（p38 MAP激酶的特异性抑制剂）或针对p38 MAP激酶的小抑制RNA（si-RNA）将用于抑制p38 MAP激酶活性。P38 MAP激酶的上游激酶MKK 6的过表达也将用于阐明P38 MAP激酶和PI-3激酶/ Akt通路之间的潜在串扰。将通过TUNEL测定、膜联蛋白V染色和半胱天冬酶-3和半胱天冬酶-9活性来确定细胞凋亡诱导。我们还将研究17-<$-雌二醇（E2）是否调节雄性和雌性HUVEC中p38 MAP激酶和PI-3激酶/Akt信号之间的串扰。将来自男性和女性脐带的人脐静脉内皮细胞用于研究对应激诱导的细胞凋亡的反应的性别差异。本研究有助于我们了解男性胎儿和新生儿死亡率高于女性的机制。

DESCRIPTION (provided by applicant): Over all objective of this study is to understand the molecular mechanisms and gender differences in response of male and female human umbilical vein endothelial cells (HUVEC) to stress, with emphasis on cross talk between PI-3 Kinase/Akt and p38 MAP Kinase pathways. It is found that male fetus has greater mortality rate than female. The molecular basis of the observed gender difference in mortality of male and female fetus is poorly understood. Phosphatidylinositol 3-kinase (PI-3 Kinase)/ Akt pathway is the key controller of endothelial cell viability determining endothelial cell viability. Withdrawal of growth factor or exposure to cytokines inhibits PI-3 Kinase /Akt signaling in endothelial cells and induces apoptosis. Decline of PI-3 Kinase/Akt induces an upregulation of stress induced p38 MAP Kinase. The cross-talk between mitogen -activated protein kinase (MAP Kinase) and PI-3 Kinase/Akt pathway and the role of activated p38 MAP Kinase in apoptosis is poorly understood. In this novel proposal we link the viability of fetus to the functional integrity and survival of HUVEC which is maintained by PI-3 Kinase /Akt signaling. Although there is lack of textual data, we have included preliminary data where male HUVEC is more susceptible than female to wortmannin (an inhibitor of PI-3 Kinase) induced stress, leading to sharper decline in pAkt and dramatic onset of apoptosis. We will consolidate this preliminary finding by assessing the susceptibilities of male and female HUVECs to a variety of stress. This proposal will also determine whether gender difference in susceptibility of male and female HUVEC to stress is regulated by p38 MAP Kinase. SB 203580 (a specific inhibitor of p38 MAP Kinase) or small-inhibitory RNA (si-RNA) against p38 MAP Kinase will be used to inhibit p38 MAP Kinase activity. Overexpression of MKK6, an upstream kinase of p38 MAP Kinase will also be utilized to elucidate the potential cross-talk between p38 MAP Kinase and PI-3 Kinase/ Akt pathway. Apoptosis induction will be determined by TUNEL assay, Annexin V staining and caspase-3 and -9 activities. We will also examine whether 17-¿-estradiol (E2) modulates the cross talk between p38 MAP Kinase and PI-3 Kinase /Akt signaling in male and female HUVEC. Human umbilical vein endothelial cells from male and female umbilical cord will be used to study the gender difference in the response to stress induced apoptosis. This investigation could help us understand the mechanisms by which the mortality rate of male fetus and neonates is higher than female.