Steroid Responsive Mechanisms in the Ear

描述（由申请人提供）：糖皮质激素（强的松、甲基强的松、地塞米松）用于控制听力损失已有几十年的历史。然而，在它们的控制下，耳朵的细胞和分子过程知之甚少。这些类固醇反应机制的知识对于我们理解正常耳蜗功能以及设计适当的临床治疗至关重要。因此，本研究的长期目标是充分表征类固醇驱动的耳部细胞和分子机制。这项研究的进展表明，MRL/MpJ-Faslpr自身免疫小鼠的听力损失对糖皮质激素强的松龙和矿皮质激素醛固酮均有反应。此外，研究表明糖皮质激素与糖皮质激素受体和矿糖皮质激素受体的结合具有相同的亲和力，从而分别通过免疫抑制和离子稳态调节耳蜗基因表达。因此，我们的工作假设是，耳朵中存在两种类固醇反应机制：一种是矿皮质激素受体介导的钠钾转运（离子稳态）基因的直接表达，另一种是糖皮质激素受体介导的间接炎症基因抑制机制。计划中的研究将通过类固醇治疗来描述这些类固醇驱动的细胞和分子过程，这些过程将在功能上分离受体，并测量它们控制的离子稳态和炎症基因表达。研究这些类固醇驱动耳部机制的具体目的有：目的1：确定矿化皮质激素醛固酮和糖皮质激素强的松龙对内耳离子稳态和炎症基因表达的剂量依赖性控制。目的2：确定两种类固醇联合剂量对内耳离子稳态和炎症基因表达过程的最有效控制。目的3：确定每种类固醇受体介导的耳蜗细胞和分子功能。目的4：确定中耳类固醇注射是否能诱导内耳稳态和抗炎基因的有效表达。在所有的研究中，1)内耳功能将通过听觉脑干反应测听和耳蜗内电位来评估；2)光镜、电镜检查内耳形态；3)全身性自身免疫性疾病将通过血清免疫复合物、血细胞比容和抗核抗体进行评估；4)采用ELISA检测耳蜗特异性自身抗体，5)采用ELISA、Western blot、细胞因子RNA表达和定量RT-PCR检测类固醇介导的耳蜗基因产物。这些研究结果将为类固醇控制下的耳细胞和分子机制提供新的发现。这也将为开发比目前使用的更有效的类固醇疗法奠定重要的基础。一项研究提出了评估的机制，类固醇控制内耳稳态的维持。这些研究旨在更好地了解糖皮质激素和矿物皮质激素在正常内耳基因表达中的作用，以及类固醇治疗如何逆转听力损失。自身免疫性内耳疾病（一种类固醇反应性疾病）小鼠模型（MRL/lpr）将被用来区分类固醇的离子稳态和免疫抑制功能以及两类类固醇联合使用的疗效。最后，将比较中耳注射与全身给药的功能影响，以确定每种方法控制耳蜗中糖皮质激素和矿皮质激素受体介导的基因表达的能力。特别感兴趣的是炎症细胞因子和相关转录因子的免疫抑制，而不是控制流体稳态的离子转运途径的调节。

DESCRIPTION (provided by applicant): Glucocorticoids (prednisone, methlyprednisolone, dexamethasone) have been employed for decades for control of hearing loss. However, little is known of the cellular and molecular processes of the ear that are under their control. Knowledge of these steroid-responsive mechanisms is critical for our understanding of normal cochlear function, as well as the design of appropriate clinical therapies. Therefore, the long term goal of this research is to fully characterize the steroid-driven cellular and molecular mechanisms of the ear. Progress on this study has shown that hearing loss in the MRL/MpJ-Faslpr autoimmune mouse responds to both the glucocorticoid prednisolone and the mineralocorticoid aldosterone. Furthermore, studies have shown glucocorticoids bind with equal affinity to both the glucocorticoid receptor and the mineralocorticoid receptor, thus regulating cochlear gene expression via immune suppression and ion homeostasis, respectively. Therefore, our working hypothesis is that two steroid-responsive mechanisms exist in the ear: a direct sodium and potassium transport (ion homeostatic) gene expression mediated by the mineralocorticoid receptor, and an indirect inflammatory gene suppression mechanism mediated by the glucocorticoid receptor. The planned studies will characterize these steroid driven cellular and molecular processes in the ear with steroid treatments that will functionally isolate the receptors and measure the ion homeostaticand inflammatory gene expression they control. The specific aims to investigate these steroid driven mechanisms of the ear are: Aim 1: Determine the dose-dependent control of inner ear ion homeostatic and inflammatory gene expression by the mineralocorticoid aldosterone and the glucocorticoid prednisolone. Aim 2: Determine the most effective control of both inner ear ion homeostatic and inflammatory gene expression processes by combination doses of the two steroids. Aim 3: Determine which cochlear cellular and molecular functions are mediated by each steroid receptor. Aim 4: Determine if effective inner ear homeostatic and anti-inflammatory gene expression can be induced by middle ear steroid delivery. In all studies, 1) inner ear function will be assessed by auditory brainstem response audiometry and endocochlear potential; 2) inner ear morphology will be assessed by light and electron microscopy; 3) systemic autoimmune disease will be assessed by serum immune complexes, hematocrits, and antinuclear antibodies; 4) cochlear specific autoantibodies will be assessed with ELISA, and 5) steroid-mediated cochlear gene products will be assessed with ELISA, Western blot, cytokine RNA expression, and quantitative RT-PCR. The results from these studies will provide new findings regarding the cellular and molecular mechanisms of the ear that are under the control of steroids. This also will lay important groundwork for the potential development of steroid therapies more effective than those currently employed. A study is proposed to evaluate the mechanisms by which steroids control the maintenance of inner ear homeostasis. Studies are designed to better understand the role of glucocorticoids and mineralocorticoids in normal inner ear gene expression, and how steroid treatments may be operating to reverse hearing loss. Mouse models (MRL/lpr) with autoimmune inner ear disease, a steroid responsive disorder, will be employed to differentiate the ion homeostasis and immune suppressive functions of steroids and the efficacy of the two steroid classes combined. Finally, the functional impact of middle ear injections versus systemic delivery will be compared to determine the ability of each method to control glucocorticoid and mineralocorticoid receptor-mediated gene expression in the cochlea. Of particular interest are immune suppression of inflammatory cytokines and related transcription factors compared to the regulation of ion transport pathways that control fluid homeostasis.