ROLE OF PGE2 RECEPTORS EP2 AND EP3 ON SENESCENCE

EP2 受体的生长刺激依赖于腺苷酸环化酶的配体依赖性激活，以及环磷酸腺苷 (cAMP) 的升高。 在角质形成细胞中，EP3 受体与二酰甘油 (DAG) 的延迟增加相结合。 有证据表明，这些受体的生长调节是对细胞衰老的相反作用的继发性作用，其中 EP3 受体刺激细胞衰老，EP2 受体阻止衰老。 逃避衰老与体外细胞永生和体内肿瘤生长高度相关。 衰老与端粒长度丧失继发的染色体复制能力下降有关。 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相关。 在皮肤恶性肿瘤中经常观察到 p16INK4A 和 p21WAF1 表达减少。 该提案将使用生化、药理学和分子方法来研究许多问题： 1. 使用特定受体激动剂和用 EP3 正义和反义构建体转染的细胞，EP3 受体利用的邻近信号传导途径是什么？ 2. 通过检查正常、永生化和肿瘤性角质细胞系中 EP2 和 EP3 受体的表达，EP3 受体的丧失或 EP2 受体的增加是否与永生化和肿瘤相关？ 3. 使用正义和反义转染子以及药理学干预，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 受体信号传导之间的相互抑制串扰是否解释了它们对细胞衰老的相反活性？ 5. 受体表达的缺失/增加对人肿瘤细胞系异种移植到裸鼠体内的致瘤性、侵袭性和转移潜力有何影响？ 拟议的研究提供了一个模型来检查 PGE2 在正常和肿瘤性角质形成细胞生长中的作用。 更一般地说，这些研究将定义 cAMP- 和 DAG- 细胞内信号传导途径在调节细胞死亡率中的作用。 这些研究将强调受体表达的改变如何定义类胡萝卜素在不同细胞功能中的作用。

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.