REGULATING GLI FUNCTION IN HAIR FOLLICLE PROGENITORS

描述(申请人提供)：成体干细胞是多能细胞，具有程序性器官替换的能力，并具有诱导器官修复的承诺，以应对损伤或损害。小鼠毛囊经历了重复的、由基因控制的增殖(生长期)、退化期(退化期)和静止期(休止期)，因此提供了一种宝贵的方法来模拟基本的再生过程。最近的焦点集中在阐明干细胞静止的控制和重新进入增殖生长期阶段的时机。以前的研究已经显示了几个关键的信号通路，如Wnt和Sonic hedgehog(Shh)刺激生长期，由父母拨款5ARO54780资助的研究集中在通过Shh信号控制生长期的上皮信号输入。相比之下，尽管最近的数据支持钙调神经磷酸酶/NFAT通路在维持静止中的作用，但对周期计时的高阶机制仍知之甚少。钙调神经磷酸酶/NFAT的参与使我们研究了钙调节蛋白在毛发周期中的作用。对局部调节毛发周期的小分子的筛选显示，硝苯地平是一种L类型的电压门控钙通道拮抗剂，可导致小鼠早熟进入生长期。与这一发现一致的是，有条件地消融CACNA1C(CaV1.2)，这是主要的皮肤通道，在小鼠中会导致类似的毛发循环缺陷和早熟进入生长期。此外，含有CaV1.2功能获得突变的小鼠会推迟进入生长期，这种突变模仿蒂莫西综合征(TS)，这是一种人类多器官系统疾病。这些数据支持这一假说，即通过电压门控钙通道的钙瞬变通过NFATc1调节调节毛发干细胞的静止。为了进一步剖析钙信号和干细胞静止的机制，我们建议：解剖CaV1.2突变小鼠体内的分子缺陷，检测需要CaV1.2功能的细胞谱系和已知信号通路在突变小鼠中的变化；利用比率钙染料和基因编码的钙传感器测量野生型和Cav1.2突变小鼠干细胞在体外和体内的钙含量差异。这些研究将为器官发生过程中使用的计时机制提供新的见解。 公共卫生研究进展：成体干细胞是一种多能细胞，具有程序性器官替换的能力，并具有诱导器官修复的前景，以应对损伤或损害。我们利用小分子拮抗剂和小鼠突变体发现，L型电压门控钙通道调节毛发再生的时间。我们认为，通过电压门控钙通道的钙瞬变通过NFATc1调节调节毛发干细胞的静止。我们建议解剖CaV1.2突变小鼠的分子缺陷，并测量其在 野生型和Cav1.2突变体的干细胞中的钙含量。这些研究将 对器官发生过程中使用的计时机制提供了新的见解。

DESCRIPTION (provided by applicant): Adult stem cells are multipotent cells that possess the capacity for programmed organ replacement and carry the promise of induced organ repair in response to injury or damage. Murine hair follicles provide an invaluable approach to modeling fundamental processes of regeneration because they undergo repeated, genetically controlled cycles of proliferation (anagen), regression (catagen) and quiescence (telogen). Intense recent focus has come to elucidate the controls of stem cell quiescence and the timing of re-entering the proliferative anagen stage. Previous studies have shown several key signaling pathways such as the Wnt and Sonic hedgehog (Shh) stimulate anagen, and studies funded through the parent grant 5ARO54780 have focused on the epithelial signaling inputs that control anagen via Shh signaling. By contrast, while recent data supports the calcineurin/NFAT pathways in maintaining quiescence, the higher order mechanisms of cycle timing remain poorly understood. The involvement of calcineurin/NFAT led us to investigate the role of calcium regulatory proteins in hair cycling. A screen for small molecules that topically regulate hair cycling revealed that nifedipine (DHP), an L-type voltage gated calcium channel antagonist, causes precocious entry to anagen in mice. Consistent with this finding, conditional ablation of CACNA1C (CaV1.2), the dominant cutaneous channel, causes similar hair cycling defects and precocious entry to anagen in mice. Further, mice containing a CaV1.2 gain-of-function mutation that mimics the Timothy syndrome (TS), a human multi-organ system disorder, delays entry into anagen. These data support the hypothesis that calcium transients via the voltage gated calcium channels regulate hair stem cell quiescence through Nfatc1 regulation. In order to further dissect the mechanism of calcium signaling and stem cell quiescence, we propose to: Dissect the molecular defects in of CaV1.2 mutant mice, examining the cell lineage requiring CaV1.2 function and the alterations in known signaling pathways in mutant mice; Measure the differences in amount of calcium in stem cells from wild type and Cav1.2 mutant mice in vitro and in vivo by using ratiometric calcium dyes and genetically-encoded calcium sensors. These studies will provide new insights into the timing mechanisms used during organogenesis. PUBLIC HEALTH RELEVENCE: Adult stem cells are multipotent cells that possess the capacity for programmed organ replacement and carry the promise of induced organ repair in response to injury or damage. We have found using small molecule antagonists and mouse mutants that L-type voltage gated calcium channels regulate the timing of hair regeneration. We believe that calcium transients via the voltage gated calcium channels regulate hair stem cell quiescence through Nfatc1 regulation. We propose to dissect the molecular defects in of CaV1.2 mutant mice, and measure the differences in amount of calcium in stem cells from wild type and Cav1.2 mutants. These studies will provide new insights into the timing mechanisms used during organogenesis.