title: Induction of PLSCR1 in a STING/IRF3-dependent manner upon vector transfection in ovarian epithelial cells.
abstract: Toll-like receptors (TLRs) are the primary sensors of the innate immune system that recognize pathogenic nucleic acids including double-stranded plasmid DNA (dsDNA). TLR signaling activates multiple pathways including IRF3 which is involved in transcriptional induction of inflammatory cytokines (i.e. interferons (IFNs)). Phospholipid scramblase 1, PLSCR1, is a highly inducible IFN-regulated gene mediating anti-viral properties of IFNs. Herein, we report a novel finding that dsDNA transfection in T80 immortalized normal ovarian surface epithelial cell line leads to a marked increase in PLSCR1 mRNA and protein. We also noted a comparable response in primary mammary epithelial cells (HMECs). Similar to IFN-2α treated cells, de novo synthesized PLSCR1 was localized predominantly to the plasma membrane. dsDNA transfection, in T80 and HMEC cells, led to activation of MAPK and IRF3. Although inhibition of MAPK (using U0126) did not modulate PLSCR1 mRNA and protein, IRF3 knockdown (using siRNA) significantly ablated the PLSCR1 induction. In prior studies, the activation of IRF3 was shown to be mediated by cGAS-STING pathway. To investigate the contribution of STING to PLSCR1 induction, we utilized siRNA to reduce STING expression and observed that PLSCR1 protein was markedly reduced. In contrast to normal T80/HMECs, the phosphorylation of IRF3 as well as induction of STING and PLSCR1 were absent in ovarian cancer cells (serous, clear cell, and endometrioid) suggesting that the STING/IRF3 pathway may be dysregulated in these cancer cells. However, we also noted induction of different TLR and IFN mRNAs between the T80 and HEY (serous epithelial ovarian carcinoma) cell lines upon dsDNA transfection. Collectively, these results indicate that the STING/IRF3 pathway, activated following dsDNA transfection, contributes to upregulation of PLSCR1 in ovarian epithelial cells.
authors: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, 33620, United States of America.
authors: Karthik M
authors: KM
authors: Kodigepalli
authors: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, 33620, United States of America.
authors: Meera
authors: M
authors: Nanjundan
mesh_terms: D002460:Cell Line; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D005260:Female; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D050838:Interferon Regulatory Factor-3 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007372:Interferons / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D010053:Ovary / Q000166:cytology; D049294:Phospholipid Transfer Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D050794:STAT1 Transcription Factor / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051193:Toll-Like Receptors / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D000099197:STING Protein
chemical_list: D050838:Interferon Regulatory Factor-3; D007372:Interferons; D008565:Membrane Proteins; D020928:Mitogen-Activated Protein Kinases; D049294:Phospholipid Transfer Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D050794:STAT1 Transcription Factor; D051193:Toll-Like Receptors; C494232:IRF3 protein, human; C493422:PLSCR1 protein, human; C532857:STING1 protein, human; D000099197:STING Protein