p38 MAP Kinase and Akt Interaction in HUVEC

描述（由申请人提供）：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，重点研究PI-3激酶/Akt和p38 MAP激酶途径之间的相互作用。研究发现，男婴的死亡率高于女婴。所观察到的男女胎儿死亡率差异的分子基础尚不清楚。磷脂酰肌醇3-激酶（PI-3激酶）/ Akt通路是内皮细胞活力的关键调控因子，决定内皮细胞的活力。停用生长因子或暴露于细胞因子可抑制内皮细胞的PI-3激酶/Akt信号传导并诱导细胞凋亡。PI-3激酶/Akt的下调可诱导应激诱导的p38 MAP激酶上调。有丝分裂原活化蛋白激酶（MAP激酶）与PI-3激酶/Akt通路之间的串扰以及活化的p38 MAP激酶在细胞凋亡中的作用尚不清楚。在这个新颖的建议中，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，而HUVEC是由PI-3激酶/Akt信号传导维持的。虽然缺乏文本数据，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wortmannin （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HUVECs对各种压力的敏感性来巩固这一初步发现。本研究也将确定男性和女性HUVEC对应激易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p38 MAP激酶的小抑制RNA （si-RNA）将被用于抑制p38 MAP激酶的活性。p38 MAP激酶上游激酶MKK6的过表达也将用于阐明p38 MAP激酶与PI-3激酶/ Akt通路之间潜在的串扰。凋亡诱导将通过TUNEL试验、Annexin V染色和caspase-3和-9活性来确定。我们还将研究17-¿-雌二醇（E2）是否调节男性和女性HUVEC中p38 MAP激酶和PI-3激酶/Akt信号之间的串扰。研究人员将利用人脐静脉内皮细胞对应激诱导的细胞凋亡反应的性别差异。这一调查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.