The role of endocannabinoids receptors in regulating trophoblast cell function

背景 内源性大麻素是以脂质为基础的信号分子，调节疼痛、食欲和神经内分泌功能。最近有报道称，大麻素受体1和2(CB1和CB2)以及第三种亚型GPR55可能与生殖组织中的内源性大麻素信号有关。它们可能有助于胎盘的发育和滋养细胞(构成胎盘的干细胞)的功能。然而，这些受体调节母胎相互作用的初级界面的具体作用和机制还知之甚少。来自我的实验室的体外证据表明，刺激培养的滋养层细胞中的内源性大麻素受体(S)大大降低了它们入侵并形成融合的合胞体的能力，这是形成母胎界面的关键步骤。 假设 我们的假设是，滋养层细胞大麻素受体信号的中断将导致与胎盘发育、侵袭、血管重塑和内分泌功能相关的过程的改变。 研究计划 目的1.阐明大麻素受体信号对培养的人滋养层细胞功能的影响。由于没有普遍接受的啮齿动物滋养层细胞系，因此将使用人类滋养层细胞进行机制研究。我们将评估受体特异性激动剂和拮抗剂对滋养层细胞系中CB1、CB2和GPR55的激活和抑制作用。将监测滋养层细胞的功能结果，如侵袭、合体形成和内分泌信号(激素产生)。我们将使用分离的人类原代滋养层细胞来证实我们的观察结果。 目的2.线粒体在内源性大麻素信号转导中的作用。我们将通过用不同的药理学和分子方法扰乱线粒体自由基产生和细胞器形态变化对大麻素受体信号转导的贡献，并评估其对滋养细胞功能的影响(目标1)。 目的3.全身性干扰内源性大麻素受体信号对滋养层功能和胎盘形成的影响。我们将在妊娠期间的两个时间点评估亚型特异性大麻素受体信号通路的激活或抑制对胎盘形成进展的影响。我们将评估滋养层侵袭和血管重塑的变化，这两个过程对胎盘形成至关重要。我们将把这些结果与胎儿发育联系起来。 HQP的培训 我的项目通过技术培训和知识翻译的综合方法，促进研究生和本科生的培训。我对HQP培训的承诺超越了我的实验室，因为我已经开发了一个跨大学的推广计划，促进本科生和研究生在科学交流方面的培训。

BACKGROUND Endogenous cannabinoids are lipid-based signaling molecules which regulate pain, appetite, neuroendocrine function. Recently it has been reported that cannabinoid receptors 1 and 2 (CB1and CB2), along with a third subtype, GPR55, may be linked to endocannabinoid signaling in reproductive tissues. They may contribute to the development of the placenta and the function of trophoblast cells (the stem cells which comprise the placenta). However, the specific roles and the mechanisms by which these receptors regulate the primary interface that governs maternal/fetal interactions are poorly understood. In vitro evidence from my laboratory suggests that stimulation of the endocannabinoid receptor(s) in cultured trophoblast cells substantially reduce their ability to invade and form a fused syncytium; essential steps in forming the maternal-fetal interface. HYPOTHESIS Our hypothesis is that disruption of cannabinoid receptor signaling in trophoblast cells will result in altered progression of the processes associated with placental development; invasion, vascular remodeling and endocrine function. RESEARCH PROGRAM Aim 1. Elucidation of the functional pathways impacted by cannabinoid receptor signaling in cultured human trophoblasts. Mechanistic studies will be conducted using human trophoblast cells since there are no universally accepted rodent trophoblast cell lines. We will evaluate the impact of activating and inhibiting CB1, CB2 and GPR55 with receptor specific agonists and antagonists in trophoblast cell lines. Trophoblast functional outcomes such as invasion, syncytium formation and endocrine signaling (hormone production) will be monitored. We will confirm our observations using isolated human primary trophoblasts. Aim 2. The role of mitochondria in endocannabinoid signaling. We will determine the contribution of mitochondrial free radical production and changes in organelle morphology towards cannabinoid receptor signaling by disrupting these process with various pharmacological and molecular approaches and evaluate the consequences on trophoblast function (Aim 1). Aim 3. Consequences of systemic disruption of endocannabinoid receptor signaling on trophoblast function and placentation. We will evaluate the consequence of activating or inhibition of subtype-specific cannabinoid receptor signaling pathways on the progression of placentation at two time points during gestation. We will evaluate changes in trophoblast invasion and vascular remodeling; two processes which are critical to placentation. We will associate these outcomes with fetal growth. TRAINING OF HQP My program facilitates the training of graduate, undergraduate students using an integrated approach to technical training and knowledge translation. My commitment to training HQP extends beyond my laboratory as I have developed a pan-university outreach program which facilitates undergraduate and graduate student training in science communication.