Studies On Signal Transduction And Effect On Metastasis Of Tumor Cells Of Prostaglandin E Receptor 3 Subtype

Prostaglandins are generated from arachidonic acid(AA) via enzymatic reaction catalyzed by cyclooxygenase(COX) and have autocrine and paracrine activities in regulating the homeostasis of microenvironment in vivo. Prostaglandin E2(PGE2)is an important member with the most diverse biological activity and has multiple regulating functions in inflammation, pain response, and bone metabolism process. Prostaglandin E2 receptor(EP) has four subtypes EP1, EP2, EP3 and EP4, which all belong to G-protein coupled receptors(GPCR) family. EP receptors have different physiological functions and are differently expressed in our tissues. EP3 is unique because of the alternative splicing of its C-terminal, which reveals the existence of several subtypes. These EP3 subtypes behave differently both in their G protein-coupling preference and downstream signaling characteristics, which indicates it is important to understand their subtle signal transduction pathways.First, we tried to take a glance at the binding sites of EP3 and PGE2. We used homology modeling to generate the 3D structure model of the EP3 receptor truncated at amino acid 359(EPtr359) and found a hole around 300 A3 which we believed was the potential binding sites of EP3 and PGE2. We also found two amino acids residues that may be involved in ligand binding through molecular docking of PGE2 and the model described above, respectively, Met137 and Lys323. Subsequently, we concluded these two residues involved in the binding of EP3 and PGE2 through the Met137 and Lys323 mutant and the competitive binding ligands(3H-PGE2, PGE2), which laid the foundation for the exploration EP3 structure in the future.Secondly, we have preliminarily explored the heterologous dimerization of EP3 subtypes and their effects on direct downstream of the second messenger signaling. We transiently transfected different EP3 subtypes fusion with different fluorescent proteins(YFP/CFP)in HEK 293, and confirmed the heterologous dimerization of EP3 subtypes by fluorescence resonance energy transfer(FRET). We further detected changes in real time intracellular calcium ion concentration with the calcium probe Fluo 4 by LS-55 spectrofluorophotometer after adding PGE2. The result indicated that the calcium signaling property of the heterodimer was significantly altered.In addition, this study explores whether the presence of EP3 will influence the metastasis of tumor cells and its pathway. It is reported that f PGE2 was significantly higher in tumor tissues than normal tissue. And researchers also has explored which EP receptors participates in the effects of PGE2 on development and metastasis associated with tumor. but there are some controversy regarding this. We transiently transfected EP3 in tumor cells and found that metastatic ability of tumor cells are affected. Then we used gelatinase spectrum and fluorescence quantitative PCR(Realtime PCR) to study the effects of EP3 receptors on matrix metalloproteinase activities, as well as the influence of expression of MMP-9, MMP-2, ERK 1/2 and PKC. We found EP3 receptors regulates MMP-9 and MMP-2 expression through Ca2+-ERK1 / 2 pathway. This process will reduce the capacity of tumor cells in breaking down extracellular matrix thereby affecting transfer of tumor cells. We also found that this effect of EP3 resulting from different types of tumor cell and tissue, and these studies also provide certain evidences for the future development of a more accurate treatment.