Stem cell niche and Wnt in liver injury and regeneration.

描述（由申请人提供）：卵圆细胞是成体肝脏祖细胞，在肝脏再生中发挥生理作用并具有有吸引力的治疗潜力。据信它们居住在门静脉周围区域的一个小生境中。然而，损伤后激活卵圆细胞的信号尚不清楚。 Wnt 蛋白家族在多种哺乳动物组织中成体干细胞的维持中发挥着重要作用。 Wnt 信号传导也参与肝脏发育和修复。有证据表明 Wnt 信号传导对于激活卵圆细胞的损伤反应至关重要。组织修复中一个尚未解答的主要问题是损伤如何导致重建组织所需的信号激活。 Nusse 实验室进行了一系列新的研究，发现了一种转录控制元件（称为 CAIRE，顺式作用损伤反应元件），它可以控制 Wnt 在损伤反应中的表达。通过使用与该增强子相关的报告基因，CAIRE 功能已在各种组织中进行了检查。在初步工作中，实验室已表明它位于肝脏的门静脉周围区域并对损伤做出反应。假设是 CAIRE 因肝损伤而被激活，从而激活卵圆细胞区室。它通过驱动局部 Wnt 表达来诱导卵圆细胞增殖，这是再生反应所必需的。 CAIRE 上游存在一条特定的信号通路，控制 CAIRE 活性和 Wnt 表达。拟议的研究将解决这些假设。首先，将检查 CAIRE 在三种不同形式的肝损伤中的作用，这三种肝损伤表现出不同程度的卵圆细胞活化。 CAIRE 报告基因的表达预计会出现在激活卵圆细胞群的损伤中。接下来，将产生 CAIRE 有条件激活 Wnt 抑制剂 Dkk 的转基因小鼠，以确定卵圆细胞再生反应是否需要 CAIRE 驱动的局部 Wnt 信号传导。 Wnt 抑制预计会导致损伤后卵圆细胞再生减少。最后，将鉴定结合并激活 CAIRE 的转录因子。生物信息学将用于识别与 CAIRE 结合的潜在转录因子。将检查这些因素是否与 CAIRE 直接结合。基于这些结果，CAIRE 内的转录因子结合位点将发生突变。转基因小鼠将被用来评估功能丧失。预计这些小鼠的卵圆细胞再生反应会降低。这些研究将提供 Wnt 表达上游激活剂的第一个证据。他们将为 Wnt 信号在肝损伤和随后涉及卵圆细胞的再生反应中发挥关键作用提供更多证据。最后，结果将深入了解生态位在将损伤反应与组织再生联系起来方面的作用。 公共卫生相关性：肝硬化是美国发病和死亡的最重要原因之一。卵圆细胞在肝硬化的细胞替代疗法中具有巨大的潜力。更好地了解来自微环境的信号如何调节卵圆细胞介导的再生将是实现基于细胞的肝病治疗潜力的重要一步。

DESCRIPTION (provided by applicant): Oval cells are adult liver progenitor cells that play a physiologic role in liver regeneration and have attractive therapeutic potential. They are believed to reside in a niche within the periportal region. However, the signals that activate oval cells after injury are not known. The Wnt family of proteins plays important roles in adult stem cell maintenance in a variety of mammalian tissues. Wnt signaling is also involved in liver development and repair. Evidence suggests that Wnt signaling is central to the injury response that activates oval cells. A main unanswered question in tissue repair is how injury leads to activation of the signals necessary for rebuilding the tissue. Pursuing a novel line of research, the Nusse lab has identified a transcriptional control element (called CAIRE, for Cis Acting Injury Responsive Element) that governs the expression of Wnt in response to injury. Through the use of reporter genes linked to this enhancer, CAIRE function has been examined in various tissues. In preliminary work, the lab has shown that it is in the periportal region of the liver and responds to injury. The hypothesis is that CAIRE undergoes activation in response to liver injury that activates the oval cell compartment. It induces oval cell proliferation by driving local Wnt expression, which is required for the regenerative response. A specific signaling pathway exists upstream of CAIRE that controls CAIRE activity and Wnt expression. The proposed studies will address these hypotheses. First, the role of CAIRE in three distinct forms of liver injury that exhibit different degrees of oval cell activation will be examined. CAIRE reporter expression is expected in injury that activates the oval cell population. Next, transgenic mice in which CAIRE conditionally activates the Wnt inhibitor Dkk will be generated to determine if local Wnt signaling driven by CAIRE is required for the oval cell regenerative response. Wnt inhibition is expected to result in decreased oval cell regeneration after injury. Lastly, transcription factors that bind to and activate CAIRE will be identified. Bioinformatics will be used to identify potential transcription factors that bind to CAIRE. These factors will be examined for direct binding to CAIRE. Based on these results, mutations will be made in transcription factor binding sites within CAIRE. Transgenic mice will be made to assess for loss-of- function. These mice are expected to have decreased oval cell regenerative response. These studies will provide the first evidence of an upstream activator of Wnt expression. They will add to the evidence for a key role for Wnt signaling in liver injury and the subsequent regenerative response involving oval cells. Lastly, the results will give insight into the role of the niche in linking the injury response to tissue regeneration. PUBLIC HEALTH RELEVANCE: Cirrhosis is one of the most significant causes of morbidity and mortality in the United States. Oval cells have tremendous potential in cell replacement therapy for cirrhosis. Improved understanding of how signals from the niche regulate oval cell mediated regeneration will be an important step towards realizing the potential of cell based therapy for liver disease.