Tumor suppressor effects of GR in skin: implication of stem cells

性状（由申请方提供）：糖皮质激素是小鼠皮肤致癌作用的强效抑制剂。糖皮质激素的活性由已知的转录因子糖皮质激素受体（GR）介导。GR调节基因表达的两种主要机制是DNA结合依赖性反式激活和通过与其他转录因子的负相互作用的DNA结合非依赖性反式阻遏。我们发现，角蛋白5.GR（K5.GR）的转基因动物中，GR过表达和自发激活的基底角质形成细胞和毛囊，是抵抗ras诱导的皮肤癌发生的肿瘤多样性和肿瘤大小。众所周知，小鼠皮肤肿瘤来源于位于毛囊隆突区的上皮干细胞（SC）。还已知肿瘤促进剂激活位于隆突中的静止表皮SC的增殖。我们的实验表明，在K5.GR转基因小鼠的隆起的推定SC的数量几乎是在同窝对照的两倍少。我们还发现，与从w.t.动物我们对从K5.GR和w.t.使用基因阵列。出乎意料的是，70%的基因的表达，其表达受GR在SC和86%的基因，其表达受GR在皮肤肿瘤中的影响被抑制GR。这些新的结果表明，负基因调控（transrepression）发挥了关键作用，在GR对表皮SC的维护和GR肿瘤抑制剂在皮肤中的作用。我们提出的假设证明：（i）SC状态的改变，包括SC的生长潜力和敏感性的肿瘤促进刺激是重要的机制，GR在皮肤中的肿瘤抑制作用，和（ii）GR的反式阻遏活性是其肿瘤抑制作用的关键。与这些研究目标相关的具体目标是：1）。采用遗传学和药理学方法研究GR对促肿瘤刺激物诱导的SC增殖的影响; 2）.使用遗传学和药理学方法确定GR对SC生长潜力和体外分化敏感性的影响; 3）。使用KS.GRdim转基因小鼠鉴定GR诱导的基因反式阻遏在SC维持和皮肤肿瘤抑制效应中的作用。这项工作是高度创新的，因为GR和糖皮质激素介导的信号传导在维持表皮SC中的作用以前从未被提及。我们期望，拟议的研究将提供新的见解维持SC的糖皮质激素是重要的生理和药理调节表皮稳态。这些研究也将大大提高我们对干细胞在皮肤癌抵抗中的作用的理解。

DESCRIPTION (provided by applicant): Glucocorticoid hormones are potent inhibitors of skin carcinogenesis in mice. The activity of glucocorticoids is mediated by the known transcription factor, the glucocorticoid receptor (GR). Two major mechanisms of the regulation of gene expression by GR are DNA-binding dependent transactivation, and DNA-binding independent transrepression through negative interaction with other transcription factors. We found that keratin5.GR (K5.GR) transgenic animals in which GR was overexpressed and spontaneously activated in basal keratinocytes and in hair follicles, are resistant to ras-induced skin carcinogenesis both in terms of tumor multiplicity and tumor size. It is well accepted that mouse skin tumors derive from epithelial stem cells (SC) located in the bulge region of the hair follicle. It is also known that tumor promoters activate proliferation of quiescent epidermal SC located in the bulge. Our experiments showed that the number of putative SC in the bulge of K5.GR transgenic mice was almost two-fold less than in littermate controls. We also found that the clonogenic activity of SC-enriched keratinocyties isolated by FACS from skin of K5.GR mice was strongly decreased in comparison to SC-enriched keratinocytes isolated from w.t. animals. We transcriptionally profiled SC-enriched keratinocyties and mouse skin tumors harvested from K5.GR and w.t. mice using gene arrays. Unexpectedly, the expression of 70% of genes whose expression was affected by GR in SC and 86% of genes whose expression was affected by GR in skin tumors was inhibited by GR. These novel results suggest that negative gene regulation (transrepression) plays a key role in GR effects on epidermal SC maintenance and in GR tumor suppressor effects in skin. We propose to prove the hypotheses that (i) alteration of SC status including SC growth potential and sensitivity of SC to tumor promoting stimuli are important mechanisms of the tumor suppressor effect of GR in skin, and that (ii) transrepression activity of GR is critical for its tumor suppressor role. Pertinent to these research goals the Specific Aims are: 1). Define the effect of GR on proliferation of SC induced by tumor promoting stimuli in vivo using genetic and pharmacological approaches; 2). Determine the effect of GR on SC growth potential and sensitivity to differentiation in vitro using genetic and pharmacological approaches; 3). Identify the role of GR-induced gene transrepression in SC maintenance and in tumor suppressor effect in skin using KS.GRdim transgenic mice. This work is highly innovative because the role of GR and glucocorticoid-mediated signaling in the maintenance of epidermal SC has never been previously addressed. We expect that proposed studies will provide new insights into the maintenance of SC by glucocorticoids which are important physiological and pharmacological regulators of epidermal homeostasis. These studies will also significantly enhance our understanding of the role of stem cells in skin cancer resistance.