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.

Fig 1. IFN and empty plasmid transfection induce PLSCR1 mRNA and protein.

(A) T80 cells were treated with 3000 IU/ml IFN-2α from 15 minutes up to 24 hours. Cell lysates were analyzed by western blotting with the indicated antibodies (n = 3). (B) Total RNA was isolated from cells treated as described in (A). PLSCR1 mRNA levels, detected via real-time PCR, are presented (n = 3). (C) T80 cells were transfected with empty pcDNA3 plasmid (“pcDNA3”) or transfection reagent only (“mock”). Cell lysates were harvested from 6 up to 48 hours post-transfection and analyzed via western blotting with the indicated antibodies (n = 3). (D) Total RNA was isolated from cells treated as described in (C). PLSCR1 mRNA levels, detected via real-time PCR, are presented (n = 4). (E) HMEC cells were treated similarly as described for T80 cells (C) and cell lysates were then analyzed by western blotting with the indicated antibodies (n = 2). (F) Annexin V-PI staining was performed in 48 hours mock or pcDNA3 transfected cells (n = 3).

Fig 2. Subcellular localization of PLSCR1 following IFN treatment and dsDNA transfection.

(A) T80 cells, transfected with empty pGL3-basic, pBABE-puro, pcDNA3, pQCXIN vectors, or transfection reagent only (“Mock”) were analyzed via western blotting with the specified antibodies (n = 3). (B) T80 cells (grown on coverslips) were treated with 3000 IU/ml IFN-2α (left panel) for 18 hours, transfected with empty pcDNA3, or mock transfected. Cells were immunostained for PLSCR1 (4D2) and DAPI. Alexafluor-488 refers to control cells stained only with secondary antibody (no primary antibody was applied). Images were then captured using an inverted fluorescence microscope (n = 3). (C) T80 cells were treated with 3000 IU/ml IFN-2α for 24 hours or transfected with empty pcDNA3 for 48 hours. Cell lysates were analyzed via western blotting with the specified antibodies (n = 3) (left panel). Densitometric analysis of PLSCR1 western was performed using Image J (n = 3) (right panel)

Fig 3. dsDNA transfection leads to induction of TLR and IFN mRNA.

T80 cells were mock or empty pcDNA3 transfected. RNA was isolated following 6 to 48 hours transfection. TLR4/TLR9 (A) and IFN-α/IFN-β (B) mRNA levels were detected by real-time PCR (n = 4).

Fig 4. Knockdown of STING, STAT1, or IRF3 leads to reduced PLSCR1 protein.

(A) T80 cells were pre-treated with 10 μM U0126 or DMSO for at least 2 hours prior to and 6 hours after transfection with pcDNA3. PLSCR1 mRNA levels were quantified by real-time PCR (n = 3). (B) Empty plasmid or mock transfection was performed in T80 cells that were treated with PLSCR1 siRNA. Cell lysates were isolated at the indicated time points following transfection and analyzed via western blotting with the indicated antibodies (n = 2). (C) T80 cells were transfected with control, STAT1, or STING siRNA followed by mock or pcDNA3 transfection. Cell lysates were isolated 48 hours post-transfection and analyzed via western blotting with the indicated antibodies (n = 3). (D) Empty plasmid or mock transfection was performed in T80 cells that were treated with IRF3 siRNA. Cell lysates were isolated at 48 hours following transfection and analyzed via western blotting with the indicated antibodies (n = 2).

Fig 5. Lack of PLSCR1 induction upon plasmid transfection in ovarian cancer cells.

(A) T80, HEY, TOV112D, and TOV21G cells were “mock” or empty pcDNA3 transfected. Cell lysates were collected 48 hours post-transfection followed by western analysis with the indicated antibodies (n = 2). (B) Densitometric analyses of westerns shown in (A) are displayed. (C) Transfection efficiency was assessed in T80, HEY, TOV112D, and TOV21G cells (grown on coverslips) using pEGFP-C1 vector. Representative images are presented (top panel). Percentages of GFP positive cells in each of these cell lines is graphically presented (lower panel) (n = 2). (D) Transfection efficiency was assessed in T80, HEY, TOV112D, and TOV21G cells using pEGFP-C1 vector by flow cytometry. The percentage of GFP positive cells in each of these cell lines is graphically presented.

Fig 6. Induction of TLR9 and IFN-α mRNA and a lack of PLSCR1 or TLR4 mRNA in ovarian cancer cells.

(A) HEY cells were “mock” or pcDNA3 transfected. Cell lysates were collected from 6 up to 48 hours post-transfection and analyzed by western blotting with the indicated antibodies (n = 2). (B) Total RNA was isolated from HEY cells that were either mock (48 hours) or pcDNA3 transfected (from 6 up to 48 hours post-transfection). PLSCR1, TLR4, TLR9, and IFN-α mRNA levels were quantified by real-time PCR (n = 2).

Fig 7. dsDNA transfection in normal epithelial cells induces PLSCR1 expression mediated by IRF3 activation.

Plasmid dsDNA transfection in T80 immortalized normal ovarian surface epithelial cells leads to induction of PLSCR1 through the STING/IRF3 pathway. dsDNA transfection also results in activation of MAPK (ERK1/2) and induction of TLR4/9 and IFN-α/β mRNA.