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.
