Growth differentiation control in primary keratinocytes

描述（由申请人提供）：Notch信号是细胞间通讯的一种重要形式，在细胞命运决定和分化中起关键作用（Artavanis-Tsakonas et al., 1999）。激活或抑制该途径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，尤其是角化细胞，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。人类和小鼠角质形成细胞生长/分化控制的信号机制，包括它们的Notch反应，只是部分重叠。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作为参考点。Notch信号通过一个涉及CBF-1/ maml1复合物转录激活的“规范”途径和一个不太明确的独立于CBF-1/ maml1功能的“非规范”途径进行。我们将解决以下目标：1)我们将验证“典型”Notch通路通过与小Rho gtpase信号传导的串导促进角质形成细胞分化的假设。特别是，我们发现Notch/CBF1通路参与了小Rho gtpase的三个关键效应物的细胞类型特异性负调控：ROCK1, 2和MRCKa激酶。因此，我们将评估这些分子的下调是否是角化细胞Notch反应的关键事件，以及这些激酶是否反过来控制这些细胞中的Notch信号。2)我们将验证“非规范”Notch通路通过与干扰素反应因子，特别是IRF6的相互作用，在角化细胞生长/分化控制中发挥平行重要作用的假设。在角质形成细胞中起重要特殊作用的转录因子。我们将评估Notch信号与IKKa（另一种角化细胞分化的选择性调节因子）在控制IRF6表达中的作用。同时，我们将评估IRF6和其他irf对角化细胞notch反应的影响，p63是这些细胞的关键转录因子，是一个重要的终点。3)我们将验证上述机制的整合在干细胞群体自我更新和肿瘤发生的长期控制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，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.