Transcription profiling of mouse 26-day-old K-ras conditional mutant mice and 3-month-old K-ras conditional mutant mice reveals cell-specific Kras and Pten mutations document proliferation arrest in granulosa cells vs. oncogenic insult to OSE cells

The small G-protein KRAS is crucial for mediating gonadotropin-induced events associated with ovulation. However, constitutive expression of KrasG12D in granulosa cells disrupted normal follicle development leading to the persistence of abnormal follicle-like structures containing non-mitotic cells. To determine what factors mediate this potent effect of KrasG12D, gene profiling analyses were done. We also analyzed KrasG12D;Cyp19-Cre and KrasG12D;Pgr-Cre mutant mouse models that express Cre prior to or after the initiation of granulosa cell differentiation, respectively. KrasG12D induced cell cycle arrest in granulosa cells of the KrasG12D;Cyp19-Cre mice but not in the KrasG12D;Pgr-Cre mice, documenting the cell context specific effect of KrasG12D. Expression of KrasG12D silenced the Kras gene, reduced cell cycle activator genes and impaired expression of granulosa cell and oocyte specific genes. Conversely, levels of PTEN and phosphorylated p38MAPK increased markedly in the mutant granulosa cells. Because disrupting Pten in granulosa cells leads to increased proliferation and survival, Pten was disrupted in the KrasG12D mutant mice. The Pten/Kras mutant mice were infertile but lacked GCTs. By contrast, the Ptenfl/fl;KrasG12D;Amhr2-Cre mice developed aggressive ovarian surface epithelial (OSE) cell tumors that did not occur in the Ptenfl/fl;KrasG12D;Cyp19-Cre or Ptenfl/fl;KrasG12D;Pgr-Cre mouse strains. These data document unequivocally that Amhr2-Cre is expressed in and mediates allelic recombination of oncogenic genes in OSE cells. That KrasG12D/Pten mutant granulosa cells do not transform but rather undergo cell cycle arrest indicates that they resist the oncogenic insults of Kras/Pten by robust self-protecting mechanisms that silence the Kras gene and elevate PTEN and phospho-p38MAPK. Experiment Overall Design: Whole ovaries were collected from 26-day-old wild type mice, 26-day-old K-ras conditional mutant mice and 3-month-old K-ras conditional mutant mice. The gene expression profiles of these samples were compared using microarray method.小G蛋白KRAS（small G-protein KRAS）在介导促性腺激素诱导的排卵相关事件中发挥关键调控作用。然而，在颗粒细胞中组成型表达KrasG12D会破坏正常卵泡发育，导致含有非增殖细胞的异常类卵泡结构持续存在。为明确介导KrasG12D这一强效效应的因子，本研究开展了基因表达谱分析。此外，我们还分析了KrasG12D;Cyp19-Cre与KrasG12D;Pgr-Cre两种突变小鼠模型，二者分别在颗粒细胞分化启动前及启动后表达Cre重组酶。 KrasG12D可诱导KrasG12D;Cyp19-Cre小鼠的颗粒细胞发生细胞周期阻滞，但对KrasG12D;Pgr-Cre小鼠无此作用，这证实了KrasG12D的细胞情境特异性效应。KrasG12D的表达会沉默Kras基因、下调细胞周期激活基因的表达，并损害颗粒细胞与卵母细胞特异性基因的表达。与之相反，突变颗粒细胞中PTEN（磷酸酶和张力蛋白同源物，PTEN）与磷酸化p38丝裂原活化蛋白激酶（p38MAPK）的水平显著升高。 鉴于在颗粒细胞中敲除Pten可促进细胞增殖与存活，我们在KrasG12D突变小鼠中敲除了Pten基因。Pten/Kras双突变小鼠不孕，但未出现生殖细胞肿瘤（GCTs）。与之形成鲜明对比的是，Ptenfl/fl;KrasG12D;Amhr2-Cre小鼠会形成侵袭性卵巢表面上皮（OSE）细胞肿瘤，而该肿瘤在Ptenfl/fl;KrasG12D;Cyp19-Cre或Ptenfl/fl;KrasG12D;Pgr-Cre小鼠品系中并未发生。上述数据明确证实，Amhr2-Cre可在卵巢表面上皮细胞中表达并介导致癌基因的等位基因重组。KrasG12D/Pten双突变颗粒细胞未发生恶性转化，反而出现细胞周期阻滞，这表明其可通过沉默Kras基因、升高PTEN与磷酸化p38MAPK水平的强效自我保护机制，抵御Kras/Pten介导的致癌应激。 实验总体设计：从26日龄野生型小鼠、26日龄K-ras条件性突变小鼠以及3月龄K-ras条件性突变小鼠中收集完整卵巢，采用微阵列（microarray）技术比较各组样本的基因表达谱。