The major goal of the study was the elucidation of the role of prostaglandin E2 (PGE2) receptors (EP) in chemically and UV induced mouse skin tumorigenesis. Epidemiological studies in humans have ? confirmed an important role of PGs, including PGE2, in a variety of cancers including skin cancer. PGE2 produced via the cyclooxygenases (COX-1/COX-2) act on four different cell surface receptors (EP 1, -2, -3, -4), which belong to the family of GPCRs.? ? In one series of studies, the mouse skin initiation/promotion model was used to investigate the contribution of the PGE2 receptors, EP2 and EP4, in papilloma development. Indomethacin was used to inhibit epidermal PG production and reduced papilloma formation about 60%. In indomethacin treated mice the EP2 agonist restored papilloma formation to TPA treatment levels, but the EP4 agonist was ineffective. In papillomas, the EP2 agonist increased Ras activation and levels as well as c-AMP, p-Src, p-EGFr, p-Erk and p-Akt levels. Treatment of papillomas with EGFr (Ag1478) or Src (PP2) inhibitors demonstrated that p-EGFr was downstream of p-Src. Ag1478 also inhibited the activation of Ras, Erk and AKT. EP2s role in papilloma development was confirmed by the observations that EP2 -/- mice showed a 60% reduction in papilloma numbers and reduced activation of Ras, Src, AKT and Erk. Immunoprecipitation of p-Src or EP2 indicated the presence of an EP2-b-arrestin1/2-p-Src complex in papillomas of wild type. B-arrestin can contribute to EP2 internalization as welll as serve as a docking protein and facilitate EP2 signaling. The data indicate that PGE2 acting via EP2 activates signal transduction pathways that can contribute to mouse skin papilloma development.? ? ? In a second serries of studies, the roles of EP2 and EP4 on UV induced skin damage was investigated. Wild type (WT) and COX-2-/- mice were acutely treated with UVB (5 kJ/m2) and apoptotic signaling pathways compared. Following exposure, apoptosis was 2.5-fold higher in COX-2-/- compared to WT mice. Because prostaglandin (PG) E2 is the major UV-induced PG and manifests its activity via four receptors, EP1 to 4, possible differences in EP signaling were investigated in WT and COX-2-/- mice. Following UVB exposure, protein levels of EP1, EP2, and EP4 were elevated in WT mice, but EP2 and EP4 levels were 50% lower in COX-2-/- mice. Activated cAMP-dependent protein kinase (PKA) and Akt are downstream in EP2 and EP4 signaling, and their levels were reduced in UVB-exposed COX-2-/- mice. Furthermore, p-Bad (Ser136 and Ser155), anti-apoptotic products of activated Akt and PKA, respectively, were significantly reduced in UVB-exposed COX-2-/- mice. To further study EP2 and EP4s roles, UVB-exposed CD-1 mice were topically treated with indomethacin to block endogenous PGE2 production, and PGE2, an EP2 agonist or an EP4 agonist applied. Indomethacin reduced PKA and Akt activation about 60%, but PGE2 and the agonists restored their activities. Furthermore, both agonists decreased apoptosis in COX-2-/- mice by 50%. The data suggest that COX-2 generated PGE2 has anti-apoptotic roles in UVB-exposed mouse skin that involves EP2 and EP4 mediated signaling that reduced keratinocyte apoptosis.
