Estrogen and TMJ Pain

描述（由申请人提供）：女性报告更大的颞下颌关节（TMJ）疼痛时，雌激素的浓度正在减少。与这些结果一致，雌激素降低TMJ敏感性在发情前期大鼠，当雌激素浓度最高。在我们的实验室中，一项大型基因阵列研究（> 10，000个基因）表明，大鼠三叉神经节（TG）中的基因表达受到雌激素增加的显著影响。高动情前期与低动情间期水平 17 β-雌二醇导致GABAA受体亚基α6（Gabrα6）、甘氨酸受体亚基α2（Glrα2）和β-肾上腺素能受体亚基β 1（Adr β 1）增加16 - 34倍。这种显著增加发生在未处理大鼠和咬肌肌腱结扎大鼠中，这是一种慢性（>6个月）肌源性TMJ超敏反应模型。由于GABA、甘氨酸和拟交感神经胺可以抑制超敏反应，因此预期Gabrα6、Glrα2和Adr β 1表达的敲低会增加超敏反应。在初步研究中，siRNA敲低TG中Gabrα6的表达增加了超敏性，增加了TG神经元中磷酸化ERK（p-ERK）的水平，并增加了神经元的电活动。对这三个基因的雌激素受体α和雌激素受体β（ER α和ER β）结合位点的同源性搜索表明，它们至少存在一个潜在的结合位点，但其调节雌激素反应的机制尚不清楚。基于这些信息，我们假设17 β-雌二醇通过增加TG中Gabrα6、Glrα2和Adr β 1的表达来降低动情前期TMJ超敏性，并且这种雌激素效应是由于与雌激素受体和转录起始位点近端序列的相互作用。为了解决这一假设，我们提出了两个具体的目标，在目标#1中，我们将在TG中确定Gabrα6、Glrα2和Adr β 1在调节男性和女性TMJ超敏性/细胞活性中的作用。为了完成目标#1，将在咬肌肌腱上放置结扎线，并通过拮抗剂或siRNA降低Gabrα6、Glrα2和Adr β 1的TG表达。将在急性阶段（结扎后7天）和慢性阶段（结扎后6个月）使用von Frey细丝、膳食持续时间、电生理记录和定量p-ERK水平来评估超敏反应/细胞活性。目标#2的目标是使用染色质免疫沉淀、电泳迁移率变动分析和荧光素酶报告基因构建体表征ERα和ER β在控制Gabrα6、Glrα2和Adr β 1表达中的作用。我们希望表明，这三个基因的表达改变可以影响TMJ伤害性反应，这些基因是负责的，在一定程度上，减少在发情前期大鼠观察到的超敏反应。我们还希望确定雌激素调节TG细胞中这些基因表达的机制。

DESCRIPTION (provided by applicant): Women report greater temporomandibular joint (TMJ) pain when the concentration of estrogen is diminishing. Consistent with these results, estrogen reduces TMJ hypersensitivity in proestrus rats, when estrogen concentrations are highest. In our lab, a large gene array study (>10,000 genes) indicated that gene expression in the rat trigeminal ganglia (TG) was significantly affected by increased estrogen. High proestrus versus low diestrus levels of 17ß-estradiol resulted in a 16 to 34 fold increase in the GABAA receptor subunit α6 (Gabrα6), the glycine receptor subunit α2 (Glrα2) and the beta-adrenergic receptor subunit ß1 (Adrß1). This significant increase occurred in the TG of naïve rats and in rats with ligature of the masseter tendon, a model of chronic (>6 months) myogenic TMJ hypersensitivity. Since GABA, glycine and sympathomimetic amines can inhibit hypersensitivity the knock-down of Gabrα6, Glrα2 and Adrß1 expression would be expected to increase hypersensitivity. In preliminary studies siRNA knock-down of Gabrα6 expression in the TG increased hypersensitivity, increased the level of phosphorylated-ERK (p-ERK) in TG neurons and increased neuronal electrical activity. A homology search for estrogen receptor α and ß (ER α and ERß) binding sites 10kb of the transcriptional start site for these three genes showed that at least one potential binding site was present but the mechanism regulating the estrogen response of these genes is unknown. Based on this information we hypothesize that 17 ß-estradiol decreased TMJ hypersensitivity at proestrus by increasing Gabrα6, Glrα2 and Adrß1 expression in the TG and that this estrogen effect was due to interaction with the estrogen receptor and sequence proximal of the transcriptional start site. To address this hypothesis we propose two specific aims, in Aim #1 we will determine in the TG the role of Gabrα6, Glrα2 and Adrß1 in modulating TMJ hypersensitivity/cellular activity in both males and females. To complete aim #1 a ligature will be placed on the masseter tendon and TG expression of Gabrα6, Glrα2, and Adrß1 will be reduced through antagonists or siRNA. Hypersensitivity/cellular activity will be assessed at an acute stage (7 days post-ligature) and at a chronic stage (6 months post-ligature) using von Frey filaments, meal duration, electrophysiological recordings and by quantitating p-ERK levels. The goal of Aim #2 will be to characterize the role of ERα and ERß in controlling Gabrα6, Glrα2, and Adrß1 expression using chromatin immunoprecipitation, electrophoretic mobility shift assays and luciferase reporter constructs. We expect to show that an altered expression of these three genes can affect the TMJ nociceptive response and that these genes are responsible, in part, for the decrease in hypersensitivity observed in proestrus rats. We also expect to determine the mechanism by which estrogen modulates expression of these genes in TG cells.