Sufficiency of ErbB2 signaling in murine inner ear supporting cell proliferation

 描述（由申请人提供）：感音神经性听力损失和前庭障碍是许多人的永久性损伤。它们通常源于内耳感觉毛细胞的丢失，这是一群死亡后无法替代的细胞。最近的报道表明，内耳支持细胞，邻近的感觉毛细胞，可以被诱导直接转分化为受损组织中的感觉毛细胞。然而，为了实现内耳再生，内耳支持细胞也必须被引导增殖。我们假设通过ErbB 2受体的信号传导足以驱动内耳支持细胞增殖。我们将通过在内耳的听觉和平衡支持细胞中过度表达组成性激活（CA）ErbB 2受体并评估随后的增殖来验证这一假设。我们目前的初步数据与两个独立的CA-ErbB 2表达系统。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响应器官培养中的CA-ErbB 2信号传导而增殖。我们的第一个系统使用两种定制的腺病毒来过度表达ErbB 2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。目的一：用这些病毒体外感染新生儿耳蜗支持细胞。我们显示的初步数据表明，只有CA-ErbB 2病毒激活磷酸肌醇3-激酶（PI 3 K）的调节亚基。此外，我们表明，CA-ErbB 2病毒驱动增殖新生儿耳蜗支持细胞在体外。令人惊讶的是，CA-ErbB 2间接地发挥了一些作用，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，并探测CA-ErbB 2受体的下游效应与报告分析和化学抑制剂。这些实验将确定ErbB 2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表达突变的组成型活性ErbB 2蛋白。我们显示的初步数据表明该系统也激活了PI 3 K。我们还表明，转基因CA-ErbB 2信号驱动增殖新生儿耳蜗支持细胞在体外。由于Tet-On技术允许我们控制CA-ErbB 2信号的时间和持续时间，我们将提供一个信号脉冲，首先评估支持细胞是否分裂，其次评估它们是否转移。 分化成毛细胞。在目标2中，我们建议使用该系统激活不同阶段耳蜗支持细胞中的CA-ErbB 2信号，有和没有毛细胞损伤，以确定ErbB 2信号是否足以促进体内增殖。在目标3中，我们将使用CA-ErbB 2病毒和转基因系统在毛细胞损伤后，在体外驱动成人椭圆囊支持细胞的增殖。这些实验将阐明哺乳动物耳蜗支持细胞的增殖是如何受到调控的，并显着推进我们对内耳再生的理解。

 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.