Growth differentiation control in primary keratinocytes

描述(由申请人提供)：Noch信号是细胞间通信的一种重要形式，在细胞命运的决定和分化中发挥关键作用(Artavanis-Tsakonas等人，1999)。激活或抑制这一途径的生物学结果高度依赖于上下文。在许多哺乳动物系统中，Notch信号增强干细胞潜能并抑制分化，而在其他系统中，尤其是角质形成细胞，它发挥相反的作用。我们的主要工作假设是，Notch在角质形成细胞中的这种促分化功能依赖于与其他关键调控途径的特定细胞类型的整合。参与控制人和小鼠角质形成细胞生长/分化的信号机制，包括它们的Notch反应，只是部分重叠。因此，在我们今后的工作中，我们将优先关注人类细胞，在必要时利用鼠标系统作为参照点。Notch信号通过一条涉及CBF-1/Maml_1复合体转录激活的“规范”途径和一条不依赖于CBF-1/Maml_1功能的较不明确的“非规范”途径进行。我们将致力于以下目标：1)我们将测试“规范”的Notch途径通过与小的Rho GTP酶信号的相互作用促进角质形成细胞分化的假说。特别是，我们发现Notch/CBF1通路参与了小Rho GTP酶的三个关键效应因子：ROCK1、2和MRCKa激酶的细胞类型特异性负调控。因此，我们将评估这些分子的下调是否是角质形成细胞Notch反应中的一个关键事件，以及这些激酶是否反过来控制这些细胞中的Notch信号2)我们将通过与干扰素反应因子，特别是IRF6的相互作用，检验“非规范的”Notch途径在角质形成细胞的生长/分化控制中发挥平行重要作用的假设。我们将评估与另一种角质形成细胞分化选择性调节因子Ikka有关的Notch信号在控制IRF6表达中的作用。与此同时，我们将以角质形成细胞的关键转录因子p63为重要终点，评估IRF6和其他IRFs对角质形成细胞Notch反应的影响。3)我们将检验这一假设，即上述机制的整合在自我更新干细胞群体的长期控制和肿瘤发生中发挥关键作用。通过体外和体内试验，我们将评估自我更新和可逆与不可逆承诺的种群之间的平衡。同时，我们将通过转基因角质形成细胞的皮肤重建/移植实验来确定上述途径在控制角质形成细胞肿瘤发展中的作用。

DESCRIPTION (provided by applicant): Notch signaling is an important form of inter-cellular communication with a key role in cell-fate determination and differentiation (Artavanis-Tsakonas et al., 1999). The biological outcome of activation or suppression of this pathway is highly context-dependent. In many mammalian systems, Notch signaling enhances stem cell potential and suppresses differentiation, while in others, notably keratinocytes, it exerts an opposite role. Our main working hypothesis is that this pro-differentiation function of Notch in keratinocytes depends on a cell-type specific integration with other key regulatory pathways. Signaling mechanisms involved in growth/differentiation control of human and mouse keratinocytes, including their Notch response, are only partially overlapping. Therefore, in our future work, we will focus preferentially on human cells, utilizing the mouse system whenever necessary and as a point of reference. Notch signaling proceeds through a "canonical" pathway involving transcriptional activation of the CBF-1/Maml_1 complex and a less defined "non-canonical" pathway independent of CBF-1/Maml_1 function. We will address the following aims : 1) We will test the hypothesis that the "canonical" Notch pathway promotes keratinocyte commitment to differentiation through a cross-talk with signaling by small Rho GTPases. In particular, we have found that the Notch/CBF1 pathway is involved in cell type specific negative regulation of three key effectors of small Rho GTPases : the ROCK1, 2 and MRCKa kinases. Accordingly, we will assess whether down-modulation of these molecules is a key event in the Notch response of keratinocytes, and whether these kinases in turn control Notch signaling in these cells 2) We will test the hypothesis that the "non-canonical" Notch pathway plays a parallel important function in keratinocyte growth/differentiation control through an interplay with Interferon Response Factors and in particular IRF6, a transcription factor with an essential specific role in keratinocytes. We will evaluate the involvement of Notch signaling, in connection with IKKa, another selective regulator of keratinocyte differentiation, in control of IRF6 expression. Concomitantly, we will assess the impact of IRF6 and other IRFs on the Notch-response of keratinocytes, with p63, a key transcription factor for these cells, as an important endpoint. 3) We will test the hypothesis that integration of the above mechanisms plays a key role in long term control of self-renewing stem cell populations, and tumorigenesis. By both in vitro and in vivo assays, we will assess the balance between self-renewing and reversibly versus irreversibly committed populations. Concomitantly, we will determine the role of the above pathways in control of keratinocyte tumor development, by skin reconstitution / grafting experiments with genetically modified keratinocytes.