Uterine bitter taste receptors in pregnancy and preterm labor management

摘要 子宫收缩力由复杂的系统调节和控制，从而维持妊娠， 分娩发生在足月。妊娠37周前发生协调的子宫收缩，即， 早产（PTL）可能导致早产（PTB），影响1500万新生儿， 全球每年的新生儿死亡率。考虑到宫缩是PTL的中心特征， 在PTB管理中，目前的宫缩抑制剂还不够有效。我们最近发现子宫光滑 来自小鼠和人的肌肉（USM）细胞表达苦味受体（TAS 2 R）， 信令组件（即，G蛋白味蛋白和PLCβ2）。还有苦味促味剂（例如，菲咯啉（PHEN）， 和氯喹，一种FDA批准的抗疟疾药物）使子宫收缩剂预收缩的子宫条松弛（例如， 催产素和前列腺素F2α）比目前常用的宫缩抑制剂（即，硝苯地平， 吲哚美辛和MgSO 4）。此外，苦味促味剂（例如，氯喹）可预防小鼠PTB诱导的 细菌内毒素脂多糖（LPS）和核孕酮受体拮抗剂RU 486更经常 比通常使用的宫缩抑制剂更强，并且是以依赖于gustducin的方式。在初步研究中，我们进一步 发现（1）PHEN能阻断5-羟色胺、内皮素-1诱导的孕鼠子宫收缩， （2）Tas 2 r在USM中的表达与子宫内膜的病理生理介质neuromedin S和U-46619有关， 当妊娠小鼠接近分娩时降低，并在分娩后恢复，以及（3）同时 在USM中表达的三种主要Tas 2 rs的缺失增加了小鼠中PTB的可能性。因此我们 提出（1）TAS 2 R是一类调节子宫收缩和妊娠持续时间的蛋白质，以及（2） TAS 2 R激动剂是可能适用于PTB管理的广谱宫缩抑制剂。测试这些 假设，我们将直接研究苦味促味剂如何激活TAS 2 R信号通路来放松人体。 用药理学方法和siRNA慢病毒表达技术对子宫肌层进行研究（Aim 1）。然后我们将 使用Tas 2 r缺失小鼠来确定促苦味剂是否激活TAS 2 R以引起USM松弛，以及 TAS 2 R是否对维持妊娠期间子宫静止至关重要， 持续时间，在小鼠中（目标2）。最后，我们将研究促苦味剂预防小鼠肺结核的有效性 TAS 2 R在促苦味剂预防LPS-或RU 486-诱导的苦味中的作用 诱导PTB（目的3）。这项研究将揭示苦味促味剂放松小鼠的分子机制， 和人类子宫，确定TAS 2 R家族是否在妊娠期间子宫静止中起主要作用 和分娩，确定TAS 2 R家族是否是治疗人类PTL的有吸引力的治疗靶点。 并帮助确定是否可以开发苦味促味剂作为新的和更有效的宫缩抑制剂 用于PTB管理。

Abstract Uterine contractility is regulated and controlled by a complex system such that pregnancy is maintained and parturition occurs at full term. Coordinated uterine contractions occurring prior to 37 weeks gestation, i.e., preterm labor (PTL), could lead to preterm birth (PTB) which affects 15 million newborns and causes 1 million neonatal deaths worldwide annually. Given that contractions are a central feature of PTL, tocolytics are used in PTB management, yet current tocolytics are not sufficiently effective. We recently found that uterine smooth muscle (USM) cells from mouse and human express bitter taste receptors (TAS2Rs) and their canonical signaling components (i.e., G-protein gustducin and PLCβ2). Also bitter tastants (e.g., phenanthroline (PHEN), and chloroquine, a FDA-approved antimalarial drug) relax uterine strips pre-contracted by uterotonics (e.g., oxytocin and prostaglandin F2α) more completely than current commonly used tocolytics (i.e., nifedipine, indomethacin and MgSO4). Moreover, bitter tastants (e.g., chloroquine) can prevent mouse PTB induced by bacterial endotoxin lipopolysaccharide (LPS) and nuclear progesterone receptor antagonist RU486 more often than commonly used tocolytics and in a gustducin dependent manner. In our preliminary studies, we further found that (1) PHEN can stop pregnant mouse uterine contractions induced by serotonin, endothelin-1, neuromedin S and U-46619, all pathophysiological mediators in the uterus, (2) Tas2r expression in USM is decreased when pregnant mice approach parturition and is restored after labor, and (3) the simultaneous deletion of the three main Tas2rs expressed in USM increases the probability of PTB in mice. We therefore propose that (1) TAS2Rs are a class of proteins regulating uterine contraction and gestational duration, and (2) TAS2R agonists are broad spectrum tocolytics potentially suitable for PTB management. To test these hypotheses, we will directly study how bitter tastants activate the TAS2R signaling pathway to relax human myometrium with pharmacological approaches and siRNA lentiviral expression technology (Aim 1). We will then use Tas2r deletion mice to determine whether bitter tastants activate TAS2Rs to cause USM relaxation, and whether the TAS2Rs are critical for maintaining uterine quiescence during pregnancy, setting gestational duration, in mice (Aim 2). Finally, we will study the effectiveness of bitter tastants in preventing mouse PTB induced by LPS and RU486, and determine the role of TAS2Rs in bitter tastants' prevention of LPS- or RU486- induced PTB (Aim 3). This study will uncover the molecular mechanisms by which bitter tastants relax mouse and human uteri, determine whether the TAS2R family plays a major role in uterine quiescence during pregnancy and parturition, establish whether the TAS2R family is an attractive therapeutic target for treating PTL in human pregnancy, and help determine whether bitter tastants can be developed as new and more effective tocolytics for PTB management.