Regulation of the sphingomyelin pathway in the CL

描述（由申请人提供）：黄体溶解是多方面的，涉及许多效应物，利用多种细胞信号传导机制。这些效应物及其引起反应的机制本身不足以解释整个黄体溶解过程。因此，必须有其他调解员参与。细胞因子，曾经被认为是次要的在黄体溶解过程中，被证明是活跃的参与者。最近的证据表明，细胞因子（FasL和TNFalpha）通过鞘磷脂途径的中间体，包括鞘磷脂酶（酸性或中性）和神经酰胺发出信号。鞘磷脂通路是调控细胞信号和细胞死亡的新途径。也有证据表明神经酰胺可以调节促性腺激素诱导的类固醇生成。这是直接作用、膜流动性改变的副产物还是由于促生存信号的破坏尚不清楚。也有可能这些事件对CL的结构演变至关重要。我们假设黄体细胞内多个位点产生的神经酰胺介导黄体溶解。更具体地说，在质膜的内部和/或外部小叶中，通过酸性鞘磷脂酶水解神经酰胺水平的增加改变了细胞膜中脂质排序的等级。因此，促性腺激素和PI(3)激酶信号被抑制，加速功能丧失。同时，通过神经酰胺合成酶在线粒体水平上增加神经酰胺，导致线粒体膜的扰动，导致功能丧失和细胞凋亡的激活。这一假设将通过以下具体目标进行检验：1)在体外和体内模型中确定酸性鞘磷脂酶是否在功能上需要小鼠鞘磷脂酶来破坏甾体生成和诱导细胞凋亡；2)确定质膜上产生的神经酰胺导致细胞因子诱导的功能丧失或黄体细胞死亡的机制；3)确定TNFa在什么细胞水平上抑制促性腺激素刺激的黄体细胞产生。4)通过隔离黄体细胞小窝部分的PI（3)-激酶，确定神经酰胺水平的升高是否会导致PI(3)-激酶的抑制；5）确定PGF2u或细胞因子介导的黄体细胞凋亡是否需要线粒体参与，以及神经酰胺是否会增强线粒体功能的失调。

DESCRIPTION (provided by applicant): Luteolysis is multi-faceted involving numerous effectors that utilize multiple cellular signaling mechanisms. These effectors and the mechanisms by which they elicit their responses are not by themselves sufficient to explain the process of luteolysis in its entirety. Thus, other mediators must be involved. Cytokines, once thought to be secondary in the luteolytic process, are proving to be active players. Recent evidence suggests that cytokines (FasL and TNFalpha) signal via intermediates of the sphingomyelin pathway including sphingomyelinases (acid or neutral) and ceramide. The sphingomyelin pathway is a novel pathway involved in modulating cell signaling and cell death. There is also evidence that ceramide can regulate gonadotropin-induced steroidogenesis. Whether this is a direct effect, a by-product of altered membrane fluidity or due to disruption of pro-survival signaling is unknown. It is also possible that these events are pivotal to the structural involution of the CL. We hypothesize that ceramide generated at multiple sites within luteal cells mediates luteolysis. More specifically, an increase in the levels of ceramide via acid sphingomyelinase hydrolysis in the inner and/or outer leaflet of the plasma membrane alters the hierarchy of lipid ordering in the cellular membranes. Consequently, gonadotropin and PI(3)kinase signaling is inhibited, accelerating loss of function. Concomitantly, an increase in ceramide at the level of the mitochondria via ceramide synthase results in perturbation of the mitochondrial membrane contributing to loss of function and activation of the apoptosome. This hypothesis will be tested by the following Specific Aims: 1) determine if acid sphingomyelinase is functionally required in mouse CL for the disruption of steroidogenesis and induction of apoptosis using in vitro and in vivo models, 2) determine the mechanism(s) by which ceramide generated at the plasma membrane contributes to cytokine-induced loss of function or luteal cell death 3) determine at what cellular level TNFa inhibits gonadotropin-stimulated progesterone production in the CL, 4) ascertain whether increased levels of ceramide result in inhibition of the PI(3)-kinase by sequestering PI(3)-kinase in the caveolae fraction of luteal cells, and 5) establish if PGF2u or cytokine-mediated luteal cell apoptosis require involvement of the mitochondria and whether or not ceramide potentiates the dysregulation of mitochondrial function.