ROLE OF PGE2 RECEPTORS EP2 AND EP3 ON SENESCENCE

EP2受体的生长刺激依赖于配体依赖的腺苷环化酶的激活，并伴随环腺苷一磷酸(CAMP)的升高。在角质形成细胞中，EP3受体与二酰甘油(DAG)的延迟增加有关。有证据表明，这些受体的生长调节作用次于对细胞衰老的相反作用，EP3受体刺激细胞衰老，EP2受体阻止衰老。逃脱衰老与体外细胞永生和体内肿瘤生长密切相关。衰老与继发于端粒长度丧失的染色体复制能力降低有关。端粒长度由端粒酶维持。衰老诱导的生长停滞与端粒酶活性丧失和细胞周期调控蛋白p21WAF1和p16INK4A上调有关。P21WAF1的表达反过来又与端粒酶活性降低有关。P16INK4A和p21WAF1的表达降低在皮肤恶性肿瘤中是常见的。本研究将利用生化、药理学和分子生物学的方法，研究一些问题：1.使用特定的受体激动剂和EP3-正义和反义结构的细胞，EP3受体使用的最接近的信号通路是什么？2.通过检测EP2和EP3受体在正常、永生化和肿瘤角质形成细胞系中的表达，EP3受体的缺失或获得是否与永生化和肿瘤有关？3.使用正义和反义基因转染剂以及药物干预，EP2和EP3细胞内信号通路如何与关键的细胞周期蛋白p21WAF1、端粒酶和表达结合和p161NK4A？4.EP2和EP3受体信号之间相互抑制的串扰是否是它们对抗细胞衰老活性的原因？5.受体表达的丧失/获得对移植到裸鼠体内的人肿瘤细胞系的致瘤性、侵袭性和转移潜能有什么影响？建议的研究为检验PGE2在正常和肿瘤角质形成细胞生长中的作用提供了一个模型。更广泛地说，这些研究将确定cAMP和DAG细胞内信号通路在调节细胞死亡中的作用。这些研究将强调受体表达的变化如何在不同的细胞功能中定义eicasonid的作用。

Growth stimulation by EP2 receptors is dependent on ligand- dependent activation of adenylate cyclase, with elevation of cyclic adenosine monophosphate (cAMP). In keratinocytes, EP3 receptors are coupled to a delayed increase in diacylglycerol (DAG). Evidence suggests that growth regulation by these receptors is secondary to an opposing action on cellular senescence, with EP3 receptors stimulating cellular senescence and EP2 receptors blocking senescence. Escape from senescence is highly associated with cellular immortality in vitro and neoplastic growth in vivo. Senescence has been linked to decreased chromosomal replicative capacity secondary to loss of telomere length. Telomere length is maintained by the enzyme telomerase. Senescence-induced growth arrest is associated with loss of telomerase activity and upregulation of the cell cycle regulatory proteins, p21WAF1 and p16INK4A. Expression of p21WAF1, in turn, is associated with decreased telomerase activity. Decreased p16INK4A and p21WAF1 expression is routinely observed in cutaneous malignancy. Using biochemical, pharmacological, and molecular approaches, this proposal will examine a number of questions: 1. Using specific receptor agonists and cells transfected with EP3-sense and -antisense constructs, what are the proximate signaling pathways utilized by EP3 receptors? 2. By examining the expression of EP2 and EP3 receptors in normal, immortalized, and neoplastic kertinocyte cell lines, is loss of EP3, or gain of EP2, receptors associated with immortalization and neoplasia? 3. Using sense and antisense transfectants, as well as pharmacological interventions, how do EP2 and EP3 intracellular signaling pathways integrate with the expression of the key cell cycle proteins, p21WAF1, telomerase, and p161NK4A? 4. Does mutually inhibitory crosstalk between EP2 and EP3 receptor signaling account for their opposing activities on cellular senescence? 5. What affect does loss/gain of receptor expression have on the tumorigenicity, invasiveness, and metastatic potential of neoplastic human cell lines xenografted into nude mice? The proposed studies provide a model for examining the role of PGE2 in normal and neoplastic keratinocyte growth. More generally, these studies will define the role of cAMP- and DAG- intracellular signaling pathways in regulating cellular mortality. These studies will underscore how alterations of receptor expression serve to define the role of eicasonoids in diverse cellular functions.