ROLE OF PGE2 RECEPTORS EP2 AND EP3 ON SENESCENCE

EP 2受体的生长刺激依赖于腺苷酸环化酶的配体依赖性激活，伴随环磷酸腺苷（cAMP）的升高。 在角质形成细胞中，EP 3受体与二酰基甘油（DAG）的延迟增加偶联。 有证据表明，这些受体的生长调节是次要的细胞衰老的反作用，与EP 3受体刺激细胞衰老和EP 2受体阻断衰老。 逃避衰老与体外细胞永生和体内肿瘤生长高度相关。 衰老与染色体复制能力降低有关，而染色体复制能力降低是端粒长度丧失的结果。 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p21 WAF 1和p16 INK 4A的上调有关。 p21 WAF 1的表达反过来又与端粒酶活性降低相关。 p16 INK 4A和p21 WAF 1表达的降低在皮肤恶性肿瘤中是常见的。 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用EP 3正义和反义构建体转染的细胞，EP 3受体利用的最接近的信号通路是什么？2.通过检测正常、永生化和肿瘤性角膜细胞系中EP 2和EP 3受体的表达，EP 3受体的缺失或EP 2受体的获得是否与永生化和肿瘤形成相关？3.使用正义和反义转染子，以及药物干预，EP 2和EP 3细胞内信号通路如何与关键细胞周期蛋白，p21 WAF 1，端粒酶和p161 NK 4A的表达整合？4. EP 2和EP 3受体信号之间的相互抑制串扰是否解释了它们对细胞衰老的相反作用？5.受体表达的缺失/获得对移植到裸鼠体内的人肿瘤细胞系的致瘤性、侵袭性和转移潜力有什么影响？ 该研究提供了一个模型，用于检查PGE 2在正常和肿瘤性角质形成细胞生长中的作用。 更一般地，这些研究将确定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钙激活蛋白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.