Estrogen and TMJ Pain

描述(由申请人提供)：当雌激素浓度降低时，女性报告更大的颞颌关节(TMJ)疼痛。与这些结果一致的是，当雌激素浓度最高时，雌激素可以降低发情前期大鼠的TMJ过敏症。在我们的实验室，一项大型基因阵列研究(&gt；10,000个基因)表明，雌激素增加显著影响了大鼠三叉神经节(TG)的基因表达。高发情前期与低发情间期水平的比较 17？-雌二醇使GABAA受体亚单位α6(GABRα6)、甘氨酸受体亚单位α2(GLRα2)和β-肾上腺素能受体亚单位？1(Adr？1)增加16~34倍。这种显著的升高发生在幼稚的大鼠和结扎咬肌肌腱的大鼠，这是一种慢性(6个月)肌源性TMJ超敏反应的模型。由于GABA、甘氨酸和拟交感神经胺可抑制超敏反应，下调GABRα6、GLRα2和Adr？1的表达有望增强超敏反应。在初步研究中，siRNA下调TG中GABRERK6的表达，增加了TG神经元的超敏反应，增加了磷酸化α的水平，并增加了神经元的电活动。对这三个基因转录起始点10kb的雌激素受体α和？(ER、α和ER？)结合部位的同源性搜索表明，至少存在一个潜在的结合部位，但调控这些基因的雌激素反应的机制尚不清楚。在此基础上，我们推测17？雌二醇通过增加GABR-α-6、GLR-α-2和Adr？1在三叉神经节中的表达而降低动情前期的TMJ超敏感度，并且这种雌激素效应是通过与雌激素受体和转录起始点序列近端的相互作用而实现的。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？1在调节男性和女性TMJ过敏性/细胞活动中的作用。为了完成目标1，将在咬肌腱上结扎，并通过拮抗剂或小干扰RNA降低GABRα6、GLRα2和Adr？1的TG表达。在急性阶段(结扎后7天)和慢性阶段(结扎后6个月)，将使用von Frey细丝、进餐时间、电生理记录和通过定量p-ERK水平来评估超敏反应/细胞活性。目标#2的目标将是通过染色质免疫沉淀、凝胶迁移率改变分析和荧光素酶报告构建来表征ERα和ER？在控制GABRα6、GLRα2和Adr？1表达中的作用。我们希望证明这三个基因的表达改变可以影响TMJ的伤害性反应，并且这些基因在一定程度上是动情前期大鼠过敏性降低的原因。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.