Sufficiency of ErbB2 signaling in murine inner ear supporting cell proliferation

小鼠内耳中 ErbB2 信号传导的充分性支持细胞增殖

• 批准号: 9031098
• 负责人: Patricia M. White
• 金额: $ 38.38万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-06 至 2020-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9031098
• 关键词: Adenoviruses; Adolescent; Adult; Age; Alleles; Auditory; Biological Assay; Breast; Cell Cycle; Cell Death; Cell Differentiation process; Cell Proliferation; Cell division; Cells; Chemicals; Cochlea; Custom; Data; Development; Disease; Ductal Epithelium; Elderly; Equilibrium; Event; Family; Goals; Hair Cells; Health; Hearing; Impairment; In Vitro; Infection; Label; Labyrinth; Lead; Lesion; Ligands; Mammals; Mitogens; Mus; Mutate; Natural regeneration; Nature; Neonatal; Organ Culture Techniques; Pharmaceutical Preparations; Phosphotransferases; Physiologic pulse; Population; Proliferating; Proteins; Recovery of Function; Regulation; Reporter; Reporting; Role; Sensorineural Hearing Loss; Sensory Hair; Signal Transduction; Staging; Supporting Cell; System; Technology; Testing; Tetanus Helper Peptide; Time; Tissues; Transgenes; Transgenic Organisms; Utricle structure; Virus; age related; base; cell injury; erbB-2 Receptor; genetic approach; hair cell regeneration; in vivo; inhibitor/antagonist; injured; neonate; notch protein; overexpression; paracrine; prevent; receptor; receptor expression; receptor function; research study; response; small molecule; stem

 DESCRIPTION (provided by applicant): Sensorineural hearing loss and vestibular disorders are permanent impairments for many people. They often stem from the loss of sensory hair cells in the inner ear, a population of cells that cannot be replaced when they die. Recent reports have shown that inner ear supporting cells, which neighbor sensory hair cells, can be coaxed into directly transdifferentiating into sensory hair cells in injured tissue. However, to achieve tue regeneration, inner ear supporting cells must also be guided into proliferation. We hypothesize that signaling through the ErbB2 receptor is sufficient to drive inner ear supporting cells to proliferate. We will test this hypothesis by over-expressing a constitutively activated (CA) ErbB2 receptor in auditory and balance supporting cells in the inner ear, and assessing subsequent proliferation. We present preliminary data with two independent CA-ErbB2 expression systems. Moreover, we show that both neonatal cochlear supporting cells and adult utricular supporting cells proliferate in response to CA-ErbB2 signaling in organ culture. Our first system uses two custom-built adenoviruses to over-express two mutated versions of the ErbB2 protein: one that is constitutively active, and one that is inert. In Aim 1, we will infect neonatal cochlear supportng cells with these viruses in vitro. We show preliminary data demonstrating that only the CA-ErbB2 virus activates phosphoinositol 3-kinase's (PI3K) regulatory subunit. Moreover, we show that the CA-ErbB2 virus drives proliferation among neonatal cochlear supporting cells in vitro. Surprisingly, CA-ErbB2 exerts some of its effects indirectly, as cells neighboring infected cells are also stimulated to divide. This finding highlights the power of using overexpression to investigate receptor function. We will quantify these results and probe the downstream effectors of the CA-ErbB2 receptor with reporter assays and chemical inhibitors. These experiments will define ErbB2's role in proliferation in neonatal mouse cochlear supporting cells. Our second system uses transgenic Tet-On technology to over-express a mutated, constitutively active ErbB2 protein in mouse supporting cells at different stages. We show preliminary data demonstrating that this system also activates PI3K. We also show that transgenic CA-ErbB2 signaling drives proliferation in neonatal cochlear supporting cells in vitro. Because Tet-On technology allows us to control both the timing and duration of the CA-ErbB2 signal, we will deliver a pulse of signaling and assess first if supporting cells divide, and second, if they trans differentiate into hair cells. In Aim 2, we propose to use this system to activate CA-ErbB2 signaling in cochlear supporting cells at different stages, with and without hair cell injury, to determine if ErbB2 signaling is sufficient to promote proliferation in vivo. In Aim 3, we will use both the CA-ErbB2 virus and the transgenic system to drive proliferation in adult utricular supporting cells in vitro, after hair cell injury. These experiments will illuminate how proliferaton in mammalian cochlear supporting cells can be regulated and significantly advance our understanding of inner ear regeneration.

 描述（由申请人提供）：感音神经性听力损失和前庭障碍对许多人来说是永久性的损伤。它们通常源于内耳感觉毛细胞的丧失，这些细胞死亡后无法替代。最近的报告表明，与感觉毛细胞相邻的内耳支持细胞可以被诱导直接转分化为受损组织中的感觉毛细胞。然而，为了实现真正的再生，还必须引导内耳支持细胞增殖。我们假设通过 ErbB2 受体的信号传导足以驱动内耳支持细胞增殖。我们将通过在内耳听觉和平衡支持细胞中过度表达组成型激活 (CA) ErbB2 受体并评估随后的增殖来检验这一假设。我们提供了两个独立的 CA-ErbB2 表达系统的初步数据。此外，我们发现新生儿耳蜗支持细胞和成人椭圆囊支持细胞都会响应器官培养中的 CA-ErbB2 信号而增殖。我们的第一个系统使用两种定制的腺病毒来过度表达 ErbB2 蛋白的两种突变版本：一种是组成型活性的，另一种是惰性的。在目标 1 中，我们将在体外用这些病毒感染新生儿耳蜗支持细胞。我们展示的初步数据表明，只有 CA-ErbB2 病毒才能激活磷酸肌醇 3 激酶 (PI3K) 的调节亚基。此外，我们还发现 CA-ErbB2 病毒在体外可促进新生儿耳蜗支持细胞的增殖。令人惊讶的是，CA-ErbB2 间接发挥其一些作用，因为邻近受感染细胞的细胞也被刺激分裂。这一发现凸显了利用过度表达来研究受体功能的力量。我们将量化这些结果，并通过报告基因检测和化学抑制剂来探测 CA-ErbB2 受体的下游效应器。这些实验将确定 ErbB2 在新生小鼠耳蜗支持细胞增殖中的作用。我们的第二个系统使用转基因 Tet-On 技术在不同阶段的小鼠支持细胞中过度表达突变的、组成型活性的 ErbB2 蛋白。我们显示的初步数据表明该系统也激活 PI3K。我们还表明，转基因 CA-ErbB2 信号传导可在体外驱动新生儿耳蜗支持细胞的增殖。由于 Tet-On 技术使我们能够控制 CA-ErbB2 信号的时间和持续时间，因此我们将传递信号脉冲并首先评估是否支持细胞分裂，其次评估它们是否反式 分化为毛细胞。在目标 2 中，我们建议使用该系统激活不同阶段（有或没有毛细胞损伤）的耳蜗支持细胞中的 CA-ErbB2 信号传导，以确定 ErbB2 信号传导是否足以促进体内增殖。在目标 3 中，我们将使用 CA-ErbB2 病毒和转基因系统在毛细胞损伤后在体外驱动成年椭圆囊支持细胞的增殖。这些实验将阐明如何调节哺乳动物耳蜗支持细胞的增殖，并显着增进我们对内耳再生的理解。