Regulation of the sphingomyelin pathway in the CL

描述（由申请人提供）：黄体分解是多方面的，涉及利用多种细胞信号传导机制的众多效应器。这些效应器及其引发反应的机制本身不足以解释整个黄体溶解过程。因此，必须有其他调解员的参与。细胞因子曾经被认为在黄体分解过程中是次要的，但事实证明它是活跃的参与者。最近的证据表明，细胞因子（FasL 和 TNFα）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。鞘磷脂途径是参与调节细胞信号传导和细胞死亡的新途径。还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。这究竟是直接影响、膜流动性改变的副产品还是由于促生存信号传导的破坏尚不清楚。这些事件也可能对 CL 的结构内卷至关重要。我们假设黄体细胞内多个部位产生的神经酰胺介导黄体溶解。更具体地，通过质膜的内部和/或外部小叶中的酸性鞘磷脂酶水解而增加的神经酰胺水平改变了细胞膜中的脂质排序的层次结构。因此，促性腺激素和 PI(3) 激酶信号传导受到抑制，加速功能丧失。与此同时，通过神经酰胺合酶增加线粒体水平的神经酰胺，导致线粒体膜扰动，导致凋亡体功能丧失和激活。该假设将通过以下具体目标进行检验：1) 使用体外和体内模型确定小鼠 CL 中是否需要酸性鞘磷脂酶来破坏类固醇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孕激素产生。 CL, 4) 确定神经酰胺水平的增加是否会通过将 PI(3)-激酶隔离在黄体细胞的小凹部分中来抑制 PI(3)-激酶，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的参与，以及神经酰胺是否会加剧线粒体功能的失调。

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.