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; 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（由适用提供）：感官听力损失和前庭疾病是许多人的永久损害。它们通常源于内耳内部的感觉毛细胞的丧失，这些细胞群无法替代。最近的报道表明，邻居感觉毛细胞的内耳支撑细胞可以直接转变为受伤组织中的感觉毛细胞。但是，为了实现TU再生，还必须引导内耳支撑细胞增殖。我们将通过在听觉中过度表达组成型激活（CA）ERBB2受体来检验这一假设，并在内耳中平衡支撑细胞，并评估随后的增殖。我们使用两个独立的CA-ERBB2表达系统介绍了初步数据。此外，我们表明，新生儿人工耳蜗辅助细胞和成年尿路支撑细胞响应器官培养中的CA-ERBB2信号而增殖。我们的第一个系统使用两个自定义的腺病毒过表达ERBB2蛋白的两个突变版本：一种始终是活跃的，一种是惰性的。在AIM 1中，我们将在体外用这些病毒感染新生儿耳蜗支撑细胞。我们显示的初步数据表明，只有CA-ERBB2病毒激活磷酸肌醇3-激酶（PI3K）调节亚基。此外，我们表明CA-ERBB2病毒在体外驱动了新生儿耳蜗支撑细胞中的增殖。令人惊讶的是，CA-ERBB2间接地执行其某些效果，因为还刺激了邻近受感染细胞的细胞分裂。这一发现突出了使用过表达来研究受体功能的力量。我们将量化这些结果，并使用报告基因分析和化学抑制剂探测CA-ERBB2受体的下游效应。这些实验将定义ERBB2在新生小鼠人工耳蜗支撑细胞中的增殖中的作用。我们的第二个系统使用转基因Tet-On技术过表达在不同阶段的小鼠支持细胞中突变的，组成型活性的ERBB2蛋白。我们显示的初步数据表明该系统还激活了PI3K。我们还表明，转基因CA-ERBB2信号传导在体外促进新生儿耳蜗支撑细胞的增殖。由于Tet-On技术使我们能够控制CA-ERBB2信号的时机和持续时间，因此，如果支撑单元划分，我们将首先提供信号传导和评估的脉冲 分化为毛细胞。在AIM 2中，我们建议使用该系统在不同阶段的人工耳蜗支撑细胞中激活CA-ERBB2信号传导，而有或没有毛细胞损伤，以确定ERBB2信号传导是否足以促进体内增殖。在AIM 3中，我们将同时使用CA-ERBB2病毒和转基因系统来驱动毛细胞损伤后体外成年uttricular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撑细胞中的增殖，并显着提高我们对内耳再生的理解。