Department of Pediatrics, National Jewish Health, Denver, Colorado 80206, USA.
Am J Respir Cell Mol Biol. 2012 Jan;46(1):115-24. doi: 10.1165/rcmb.2011-0099OC.
The expression of β-catenin-dependent genes can be increased through the Cre recombinase (Cre)-mediated elimination of the exon 3-encoded sequence. This mutant β-catenin is termed DE3, and promotes the expression of β-catenin-dependent genes. Our previous study used the DE3 model to demonstrate that persistent β-catenin activity inhibited bronchiolar Clara-to-ciliated cell differentiation. The present study was designed to evaluate the roles of β-catenin in regulating the tracheal progenitor cell hierarchy. However, initial experiments demonstrated that the tetracycline-responsive element-Cre transgene (TRE-Cre) was active in the absence of a reverse tetracycline transactivator driver or inducer, doxycycline (Dox). This spurious TRE-Cre transgene activity was not detected using the ROSA26-floxed STOP-LacZ reporter. To determine if the phenotype was a consequence of genotype or treatment with Dox, tracheal and lung specimens were evaluated using quantitative histomorphometric techniques. Analyses of uninduced mice demonstrated a significant effect of genotype on tracheal epithelial cell mass, involving basal, Clara-like cell types. The bronchial and bronchiolar Clara cell mass was also decreased. Paradoxically, an effect on ciliated cell mass was not detected. Activation of the β-catenin reporter transgene TOPGal demonstrated that β-catenin-dependent gene expression led to the genotype-dependent tracheal and bronchiolar phenotype. Comparative analyses of wild-type or keratin 14-rtTA(+/0)/TRE-cre(+/0)/DE3(+/+) mice receiving standard or Dox chow demonstrated an effect of treatment with Dox on basal, Clara-like, and Clara cell masses. We discuss these results in terms of cautionary notes and with regard to alterations of progenitor cell hierarchies in response to low-level injury.
β-连环蛋白依赖性基因的表达可以通过 Cre 重组酶（Cre）介导的外显子 3 编码序列的消除而增加。这种突变的β-连环蛋白被称为 DE3，并促进β-连环蛋白依赖性基因的表达。我们之前的研究使用 DE3 模型表明，持续的β-连环蛋白活性抑制了细支气管 Clara 细胞向纤毛细胞的分化。本研究旨在评估β-连环蛋白在调节气管祖细胞谱系中的作用。然而，最初的实验表明，四环素反应元件-Cre 转基因（TRE-Cre）在没有反向四环素转录激活剂驱动或诱导剂（多西环素，Dox）的情况下是活跃的。这种非特异性的 TRE-Cre 转基因活性在 ROSA26 基因敲除 STOP-LacZ 报告基因中没有被检测到。为了确定表型是基因型的结果还是 Dox 的处理结果，使用定量组织形态计量学技术评估了气管和肺标本。对未诱导的小鼠进行分析表明，基因型对气管上皮细胞质量有显著影响，涉及基底、Clara 样细胞类型。支气管和细支气管 Clara 细胞质量也减少了。矛盾的是，没有检测到纤毛细胞质量的影响。β-连环蛋白报告基因 TOPGal 的激活表明，β-连环蛋白依赖性基因表达导致了基因型依赖性的气管和细支气管表型。对野生型或角蛋白 14-rtTA(+/0)/TRE-cre(+/0)/DE3(+/+)小鼠进行标准或 Dox 饲料的比较分析表明，Dox 的处理对基底、Clara 样和 Clara 细胞质量有影响。我们根据警示说明和低水平损伤对祖细胞谱系的改变来讨论这些结果。
