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

描述(申请人提供)：糖皮质激素是抑制小鼠皮肤癌发生的有效药物。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于DNA结合的反式激活和通过与其他转录因子的负相互作用而非依赖于DNA结合的反式抑制。我们发现，角蛋白5.GR(K5.GR)转基因动物在基底角质形成细胞和毛囊中过表达并自发激活GR，在肿瘤多样性和肿瘤大小方面都对ras诱导的皮肤癌具有抵抗力。众所周知，小鼠皮肤肿瘤起源于毛囊隆起区的上皮干细胞(SC)。众所周知，肿瘤促进剂可以激活位于隆起中的静止的表皮干细胞的增殖。我们的实验表明，K5.GR转基因小鼠的突起中可能存在的SC数量几乎是产仔对照小鼠的两倍。我们还发现，用流式细胞仪从K5、GR小鼠皮肤分离的富含SC的角质形成细胞的克隆形成活性明显低于从W.T分离的富含SC的角质形成细胞。动物。我们从K5.GR和W.T获得了富含SC的角质形成细胞和小鼠皮肤肿瘤的转录图谱。使用基因阵列的小鼠。意外的是，70%受GR影响的基因在SC和86%受GR影响的基因在皮肤肿瘤中的表达受到GR的抑制。这些新的结果表明，负基因调控(转录抑制)在GR对皮肤SC的维持和GR对皮肤的肿瘤抑制作用中起关键作用。我们提出的假设是：(I)SC状态的改变，包括SC的生长潜能和SC对促肿瘤刺激的敏感性是GR在皮肤中抑制肿瘤的重要机制，以及(Ii)GR的反转录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研究GR在体内对促肿瘤刺激诱导的SC增殖的影响；用遗传学和药理学方法研究GR对SC体外生长潜能和分化敏感性的影响；利用KS.GRdim转基因小鼠，确定GR诱导的基因反式表达在干细胞维持和皮肤肿瘤抑制效应中的作用。这项工作具有很高的创新性，因为GR和糖皮质激素介导的信号在维持表皮SC中的作用以前从未被解决过。我们期待所提出的研究将为糖皮质激素维持SC提供新的见解，糖皮质激素是表皮动态平衡的重要生理和药理学调节因素。这些研究还将显著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.