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

描述（由申请人提供）：糖皮质激素是小鼠皮肤癌发生的有效抑制剂。糖皮质激素的活性是由已知的转录因子糖皮质激素受体（GR）介导的。GR调控基因表达的两种主要机制是依赖dna结合的转抑制和通过与其他转录因子负相互作用的非依赖dna结合的转抑制。我们发现角化蛋白。GR （K5.GR）转基因动物中，GR在基底角质形成细胞和毛囊中过表达并自发激活，在肿瘤多样性和肿瘤大小方面都对ras诱导的皮肤癌具有抗性。人们普遍认为，小鼠皮肤肿瘤起源于位于毛囊凸起区域的上皮干细胞（SC）。我们也知道，肿瘤促进剂可以激活位于凸起处的静止表皮SC的增殖。我们的实验表明，在K5的凸起中假定SC的数量。转基因小鼠的死亡率几乎是对照组的两倍。我们还发现从K5皮肤中分离的富含sc的角化细胞具有克隆活性。与从w.t.动物中分离的sc富集的角质形成细胞相比，GR小鼠的角质形成细胞明显减少。我们转录分析了从K5中收获的富含sc的角质形成细胞和小鼠皮肤肿瘤。使用基因阵列的GR和wt小鼠。出乎意料的是，在SC中受GR影响的基因中有70%的表达被GR抑制，在皮肤肿瘤中受GR影响的基因中有86%的表达被GR抑制。这些新结果表明，基因负调控（转抑制）在GR对表皮SC维持和皮肤中GR肿瘤抑制作用中起着关键作用。我们拟证明以下假设：(i) SC状态的改变，包括SC生长潜能和SC对促瘤刺激的敏感性是GR在皮肤中抑瘤作用的重要机制，以及（ii） GR的转抑制活性是其抑瘤作用的关键。与这些研究目标相关的具体目标是：1)。用遗传学和药理学方法确定GR对体内促瘤刺激诱导的SC增殖的影响；2）. 利用遗传学和药理学方法确定GR对体外SC生长潜力和分化敏感性的影响；3)。利用KS鉴定gr诱导的基因转抑制在皮肤SC维持和肿瘤抑制作用中的作用。转基因小鼠。这项工作是高度创新的，因为GR和糖皮质激素介导的信号在表皮SC维持中的作用以前从未被解决过。我们希望这些研究将为糖皮质激素维持SC提供新的见解，糖皮质激素是表皮稳态的重要生理和药理调节剂。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.