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.

Exosomes maintain cellular homeostasis by excreting harmful DNA from cells

“…MISSION siRNA targeting for Slp4/Sytl4 (5′-CUGUUAAUCCACUAUAUGA-3′) was purchased by Sigma Genosys. ON-TARGETplus siRNAs (Dharmacon) and non-targeting control siRNA were used to target Sting 65 66 67 and Alix 34 . Knockdown efficiency was confirmed by quantitative real-time PCR.…”

Phosphatidylserine Exposure Controls Viral Innate Immune Responses by Microglia

“…Viral particle sensing results in an initial burst of pro-inflammatory cytokines and modulates PLSCR1 activity. The latter may be induced downstream of interferon regulatory factor 3 (IRF3), activated by calcium release and/or through an independent mechanism triggered upon viral uptake (Anand and Tikoo, 2013; Dong et al, 2004; Kodigepalli and Nanjundan, 2015; Lu et al, 2007). Activated PLSCR1 can translocate into the nucleus, for example, to enhance IP3R expression, thereby influencing intracellular calcium homeostasis (Ben-Efraim et al, 2004; Zhou et al, 2005).…”

Resveratrol significantly inhibits the occurrence and development of cervical cancer by regulating phospholipid scramblase 1

“…Phospholipid scramblase 1 is a calcium‐dependent endofacial plasma membrane protein. The first function ascribed to PLSCR1 was to catalyze rapid, bidirectional, and nonspecific distribution of phospholipids (lipid scrambling) between the inner and outer leaflets of the plasma membrane, resulting in collapse of the phospholipid asymmetry . However, little is known about the role of PLSCR1 in the occurrence and development of CC.…”