Discussion and Conclusion GPR30/Src kinase- mediated EGFR signalling is involved in regulation of cyclooxygenase-2 expression in rat oviductal epithelial cells Pooja Popli, Vijay Kumar Sirohi, Vinay Shukla, Jyoti Bala Kaushal, Kanchan Gupta , Anila Dwivedi Division of Endocrinology, CSIR-Central Drug Research Institute, Lucknow-226031, U.P., India. Email: popli.pooja@rediffmail.com Results Introduction E2-BSA induced GPR30 mediated COX-2 expression in rat OECs The oviduct plays a crucial role in female reproduction by regulating gamete transport, providing a specific microenvironment for fertilization and early embryonic development to take place Cyclooxygenase (COX) -derived prostaglandins play essential role in carrying out these oviduct- specific functions Previous reports suggest that steroid hormones regulate COX-2 synthesis in various cell types including endothelial, endometrial cells and rat oviduct However, non-genomic mechanisms responsible for regulation of COX-2 expression & subsequent prostaglandin release in rat oviductal epithelial cells (OECs) remain unclear Demonstration of GPR 30- mediated COX-2 expression in primary rat oviductal epithelial cells (OECs). Results are expressed as mean ± S.E., n = 3. p values are a-p<0.001, b-p<0.01, c-p<0.05 and d-p>.05 vs. control. Immunofluorescence to show effect of E2-BSA or ICI 182,780 or G1 or G-15 on sub-cellular localization of COX-2 protein Measurement of cAMP level Measurement of PGE2 and PGF2a by ELISA Objective To demonstrate and explore the GPR30/Src kinase- mediated EGFR signalling involved in COX-2 expression in primary rat oviductal epithelial cells (OECs) Methodology Rat oviductal epithelial cells (OECs) primary culture was established Expression of GPR-30, COX-2, NF-kB, phosphorylation of PI-3 kinase and Akt was demonstrated by immunoblotting Detection of E2-BSA binding to GPR30 receptors in oviductal epithelial cells was done by immunofluorescence imaging Measurement of PGE2 and PGF2α levels was done by ELISA E2-BSA induced transcriptional activation of NF-kB was measured by luciferase assay Effect of E2-BSA, ICI 182,780, G-1 or G-15 on cAMP level in primary rat oviductal epithelial cells (OECs). Effect of E2-BSA, ICI 182,780, G-1 or G-15 on PGE2 & PGF2a release in in primary rat oviductal epithelial cells (OECs). Co-localization of E2-BSA-FITC and GPR30 in rat primary OECs COX-2 expression in GPR30 siRNA transfected cells (B) Immunofluoresence to demonstrate co-localization of E2-BSA-FITC and GPR30 in primary rat oviductal epithelial cells cultured for 24 h Discussion and Conclusion Effect of GPR 30 silencing on E2-BSA induced COX-2 expression in in primary rat oviductal epithelial cells (OECs) E2-BSA induced COX-2 expression in rat OECs through membrane estrogen receptor GPR30. G-15, a selective pharmacological antagonist of GPR30, completely suppressed E2-BSA-induced increase in COX-2 protein expression while ICI 182,780 and GPR30-agonist (G-1) upregulated COX-2 expression and subsequent prostaglandin release in rat OECs Co-localization experiments revealed GPR30 to be primarily located in the perinuclear space, which was also the site of E2-BSA-FITC binding. However, knockdown of GPR-30 with siRNA prevents E2-BSA binding to GPR-30 receptors in peri-nuclear area of OECs. Furthermore, treatment with chloroquine (clathrin-dependent endocytosis inhibitor) inhibited the internalization of cell-surface-bound GPR30 and enhanced co-localization and accumulation of E2-BSA FITC and GPR-30 at the plasma membrane. E2-BSA induced- COX-2 was found to be regulated by both EGFR and PI3K signaling as inhibitors of c-Src kinase (PP2), EGFR (EGFR inhibitor) and PI-3 kinase (LY294002) attenuated E2-BSA mediated effect In addition, E2-BSA induced the nuclear translocation of p65-NF-κB and up-regulated the NF-κB promoter activity in rat OECs. However, E2-BSA induced COX-2 protein expression and subsequent prostaglandin release was prevented by NF-κB inhibitor In conclusion, E2-BSA binding to GPR30 receptors results in activation of PI-3 kinase, Akt and NF-κB through transactivation of EGFR pathway via Src-dependent activation of MMPs/HB-EGF. These signaling pathways might enforce each other and contribute to sustained activation of transcription factors required for COX-2 gene expression. Effect of c-src kinase inhibitor PP2 on E2-BSA induced COX-2 expression Demonstration of co-localization of E2-BSA-FITC and GPR30 in scram siRNA treated cells or GPR30 siRNA treated cells E2-BSA induced c-Src like tyr kinase mediated COX-2 expression occurs via EGFR activation in primary rat oviductal cells (OECs). Treatment with chloroquine (endocytosis inhibitor) causes enhanced E2-BSA binding at the plasma membrane as compared to peri-nuclear area of OECs E2-BSA induced COX-2 expression involves transcription factor, NF-kB E2-BSA induced COX-2 expression involves EGFR- mediated PI-3 kinase and Akt activation Pre-treatment of OECs with QNZ (NF-kB activation inhibitor) significantly inhibited E2-BSA iduced COX-2 protein expression E2-BSA incresaed nuclear translocation of NF-kB p65 subunit. Pre-treatment of OECs with EGFR and PI-3 kinase inhibitor significantly inhibited E2-BSA iduced COX-2 protein expression E2-BSA induces NF-kB translocation into nucleus and regulates transcriptional activation of NF-kB References Pérez et al., Prostaglandins Other Lipid Mediat. 80 (2006) 155-164 Kleuser et al., Mol Pharmacol. 74 (2008) 1533-1543 Wang et al., Endocrinology 149 (9) (2008) 4452-4461 Saxena et al., PLoS One 8 (6) ( 2013) e66246 Confocal microscopy to show the effect of E2-BSA on localization pattern of NF-κB (p65) in OECs E2-BSA Effect of E2-BSA on transcriptional activation of NF-kB by luciferase assay Schematic representation of signaling pathways involved in E2-BSA induced COX-2 expression and PGE2 and PGF2α release in OECs