ROLE OF PGE2 RECEPTORS EP2 AND EP3 ON SENESCENCE

EP2受体的生长刺激依赖于配体依赖性的腺苷酸环化酶的激活，伴随着环磷酸腺苷（cAMP）的升高。在角质形成细胞中，EP3受体偶联于二酰基甘油（DAG）的延迟增加。有证据表明，EP3受体刺激细胞衰老，EP2受体阻断细胞衰老，这些受体对细胞生长的调节是次要的。逃避衰老与体外细胞不朽和体内肿瘤生长高度相关。衰老与染色体复制能力下降有关，继发于端粒长度的丧失。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的丧失和细胞周期调节蛋白p21WAF1和p16INK4A的上调有关。反过来，p21WAF1的表达与端粒酶活性降低有关。p16INK4A和p21WAF1表达降低在皮肤恶性肿瘤中常见。利用生物化学、药理学和分子方法，本提案将研究一些问题：1。使用特异性受体激动剂和转染了EP3义和-反义结构的细胞，EP3受体利用的近似信号通路是什么？2. 通过检测EP2和EP3受体在正常、永生化和肿瘤角质细胞细胞系中的表达，EP3受体的缺失或EP2受体的获得与永生化和肿瘤相关吗？3. 利用正义和反义转染以及药物干预，EP2和EP3细胞内信号通路如何与关键细胞周期蛋白p21WAF1、端粒酶和p161NK4A的表达整合？4. EP2和EP3受体信号之间的相互抑制串扰是否解释了它们在细胞衰老中的相反活动？5. 受体表达的缺失/增加对移植到裸鼠体内的肿瘤人细胞系的致瘤性、侵袭性和转移潜力有什么影响？提出的研究为检验PGE2在正常和肿瘤角质细胞生长中的作用提供了一个模型。更一般地说，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改变如何在不同的细胞功能中定义类肾上腺素的作用。

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.