HDAC1-Sp1-PTEN-PIP3信号通路在噪声性听力损伤的分子机制研究

Inhibition of Akt survival signal plays an important role in noise-induced outer hair cells (OHCs) apoptosis. However, the up-stream regulation of the inhibition of Akt survival signal in OHCs remains an open question. Our preliminary results show that: 1) in the noise-induced permanent threshold shift (PTS) mice model (Broad band noise, 2-20 KHz, 106 dB SPL, 120 min), Histone deacetylase 1 (HDAC1) in OHCs increases significantly; 2) in the mean time, the acetylation of transcription factor specificity factor 1 (Sp1) in OHCs decreases significantly; 3) the level of phosphatidylinosital-3,4,5-triphosphate (PIP3) in OHCs decreases significantly; 4) HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) significantly attenuates noise-induced OHCs apoptosis and noise-induced hearing loss (NIHL) as determined by hair cell count and auditory brainstem response (ABR) test. Acetylation of lysine is a reversible post-translational modification that play a key role in gene transcriptional regulation which is controlled by Histone acetyltransferases (HAT) and HDAC. HDAC is actually lysine deacetylase (KDAC) because it catalyses not only Histone protein deacetylation, but also non-Histone protein (e.g.: Sp1) deacetylation. It’s reported that HDAC1 can bind and cause Sp1 deacetylation, decrease the binding abilility of Sp1 to core promoter of phosphatase and tensin homologue deleted on chromosome ten (PTEN) gene DNA and loss the ability in PTEN gene transcription inhibition. The increased level of PTEN can inhibit phosphoinositide 3-kinase (PI3K), decrease PIP3 level and finally inhibit Akt survival signal. Based on our preliminary results and literatures, we therefor propose that following noise exposure, increased HDAC1 causes Sp1 deacetylation and increases the expression of PTEN gene, which leads to down-regulation of PIP3 and inhibition of Akt, results in the apoptosis of OHCs. To examine our hypothesis we propose three specific aims by using mouse model of NIHL, siRNA and type specific inhibitors. In specific aim 1, we will examine how increased HDAC1 afftects Sp1 acetylation in OHCs. In specific aim 2, we will examine how Sp1 deacetylation affects PTEN gene expression in OHCs. In specific aim 3, we will exaine how increased level of PTEN affects PIP3 and Akt in OHCs. Results from these experiments promise to significantly contribute to our understanding of how noise exposure induces apoptosis of OHCs via HDAC1-Sp1-PTEN-PIP3 signal pathway, and will aid in research of new and effective prophylactic intervention in NIHL.

Akt存活信号抑制是噪声引起耳蜗外毛细胞凋亡的重要机制，但其上游调控机制尚不明确。我们前期研究发现，噪声暴露后耳蜗外毛细胞内组蛋白去乙酰化酶1（HDAC1）上升、转录因子特异性因子1（Sp1）的乙酰化和磷酯酰肌醇-3,4,5-三磷酸（PIP3）水平显著降低；HDAC抑制剂可以显著减轻噪声性听力损伤。文献报道HDAC1可以与Sp1结合并使之去乙酰化，增加PTEN基因的转录，而PTEN水平升高可下调PIP3水平，抑制Akt活性。我们根据前期结果和已知文献提出假说：噪声性听力损伤过程中存在HDAC1上升-Sp1乙酰化水平下降-PTEN上调-PIP3下调-Akt抑制这一诱导耳蜗外毛细胞凋亡的信号转导通路。本课题拟通过噪声性听力损伤动物模型，采用siRNA和特异性抑制剂，阐明HDAC1-Sp1-PTEN-PIP3这一噪声性听力损伤的分子机制，为噪声性耳聋临床防治新策略的建立奠定理论基础。

噪声是引起听力损伤的主要外界因素。耳蜗外毛细胞凋亡是噪声性听力损伤的典型性病理改变，是导致听力障碍的主要原因。本课题组既往发现噪声暴露后耳蜗外毛细胞内组蛋白去乙酰化酶1（HDAC1）水平上调。然而，HDAC1介导耳蜗外毛细胞凋亡的确切机制尚不清楚。本课题提出噪声暴露后耳蜗外毛细胞内HDAC1上调-转录因子特异性因子1（Sp1）乙酰化水平下降-第10 号染色体上缺失与张力蛋白同源的磷酸酶（PTEN）上调-PIP3下调-磷酯酰肌醇-3,4,5-三磷酸（PIP3）-Akt存活信号下降这一噪声性耳聋发病新机制，使用噪声性听力损伤模型，采用特异性抑制剂和siRNA证实上述机制的存在。结果发现：1. 噪声暴露后耳蜗组织HDAC1，HDAC3，HDAC4和HDAC8的蛋白表达水平上升，而HDAC抑制剂辛二酰苯胺异羟肟酸（SAHA）可下调这些HDAC蛋白表达水平。噪声暴露后耳蜗外毛细胞内HDAC1、HDAC3、HDAC6和HDAC8蛋白表达水平上升，组蛋白乙酰化水平下降，抑制HDACs功能可减轻噪声暴露引起的听力损伤。2. 噪声暴露后耳蜗外毛细胞内转录因子Sp1蛋白水平不变，乙酰化水平降低。噪声暴露后内耳外毛细胞PTEN蛋白表达水平上升，但PI3K-p85α，PI3K-p110α和磷酸化Akt ser473水平下降。Sp1抑制剂光辉霉素A（MitA）预处理上调PTEN表达水平。Sp1抑制剂MitA在噪声暴露后第1天，3天，7天降低噪声暴露后听力阈移，而在噪声暴露后第14天对听力阈移没有影响。3. PTEN抑制剂双过氧化钒（bpV）不但上调耳蜗外毛细胞内PI3K-p85α，PI3K-p110α和磷酸化Akt ser473信号水平，而且可预防噪声暴露后听力损伤并减少耳蜗毛细胞损失。4. PTEN siRNA不但可以下调PTEN蛋白表达水平，而且可以上调PI3K-p85α水平，预防噪声暴露后听力损伤。5. 噪声暴露后耳蜗外毛细胞内PIP3和PIP2水平下调，耳蜗组织SGK磷酸化水平降低。本项目的研究结果具有潜在的预防噪声性听力损伤的临床应用前景，使用食物添加补充HDAC1抑制剂或使用药物HDAC1抑制剂或PTEN抑制剂可能预防军事噪声性听力损伤或强噪声环境下工作人员的听力损伤，包括耳鸣和听力下降。临床应用潜能的转化研究是本项目进一步计划的重要内容。