Growth differentiation control in primary keratinocytes

描述（由申请人提供）：Notch信号传导是细胞间通讯的重要形式，在细胞命运决定和分化中具有关键作用（Artavanis-Tsakonas等人，1999年）。该通路的激活或抑制的生物学结果是高度依赖于环境的。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，特别是角质形成细胞中，它发挥相反的作用。我们的主要工作假设是，角质形成细胞中Notch的这种促分化功能取决于与其他关键调控途径的细胞类型特异性整合。参与人和小鼠角质形成细胞的生长/分化控制的信号传导机制，包括它们的Notch反应，仅部分重叠。因此，在我们未来的工作中，我们将优先关注人类细胞，在必要时利用小鼠系统并作为参考点。Notch信号传导通过涉及CBF-1/Maml_1复合物的转录激活的“经典”途径和不太确定的不依赖于CBF-1/Maml_1功能的“非经典”途径进行。1）我们将检验“经典”Notch途径通过与小Rho GTP酶的信号传导的串扰促进角质形成细胞定向分化的假设。特别是，我们已经发现Notch/CBF 1通路参与小Rho GTP酶的三个关键效应子的细胞类型特异性负调控：ROCK 1，2和MRCK α激酶。因此，我们将评估这些分子的下调是否是角质形成细胞的Notch应答中的关键事件，以及这些激酶是否反过来控制这些细胞中的Notch信号传导。2）我们将检验“非经典”Notch途径通过与干扰素应答因子特别是IRF 6的相互作用在角质形成细胞生长/分化控制中发挥平行的重要功能的假设，一种在角质形成细胞中具有重要特异性作用的转录因子。我们将评估Notch信号传导与IKKa（角质形成细胞分化的另一种选择性调节因子）在控制IRF 6表达中的参与。同时，我们将评估IRF 6和其他IRFs对角质形成细胞Notch反应的影响，并将这些细胞的关键转录因子p63作为重要终点。3)我们将检验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起关键作用的假设。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群体之间的平衡。同时，我们将确定上述途径在控制角质形成细胞肿瘤发展中的作用，通过皮肤重建/移植实验与遗传修饰的角质形成细胞。

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.