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Induction of PLSCR1 in a STING/IRF3-dependent manner upon vector transfection in ovarian epithelial cells.

Kodigepalli KM, Nanjundan M - PLoS ONE (2015)

Bottom Line: Toll-like receptors (TLRs) are the primary sensors of the innate immune system that recognize pathogenic nucleic acids including double-stranded plasmid DNA (dsDNA).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.In prior studies, the activation of IRF3 was shown to be mediated by cGAS-STING pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, 33620, United States of America.

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.

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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).
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pone.0117464.g003: 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).

Mentions: Toll-like receptors (TLRs) are the major component of the innate immune system and are responsible for recognition of pathogen-associated molecular patterns (PAMPs) including pathogenic foreign nucleic acids (viral and bacterial DNA). Upon recognition of PAMPs, TLRs stimulate the downstream signaling cascade which activates transcription factors, NFκB, and IRF3/7, resulting in induction of inflammatory cytokines including Type 1 IFNs [3]. In addition, the cGAS-STING pathway can upregulate IFNs transcription via activation of IRF3 [12,13]. Since we have observed that IRF3 is phosphorylated following dsDNA transfection (Fig. 1C) and that PLSCR1 can regulate TLR9 signaling in plasmacytoid dendritic cells (pDCs) [21], we next assessed whether TLR4 as well as TLR9 were altered at their transcript level following dsDNA transfection in T80 cells. Both TLR4 and TLR9 have been previously shown to function as nucleic acid sensing TLRs [6]. As expected, there was a significant induction of both TLR4 and TLR9 upon transfection of dsDNA (Fig. 3A, upper and lower panels). Similarly, as shown in Fig. 3B (upper and lower panels), we also detected significant increases in the mRNA levels of IFN-α and IFN-β (IFN-γ was undetectable). Collectively, these results indicate that dsDNA transfection leads to induction of TLR4 and TLR9 as well as IFN-α and IFN-β.


Induction of PLSCR1 in a STING/IRF3-dependent manner upon vector transfection in ovarian epithelial cells.

Kodigepalli KM, Nanjundan M - PLoS ONE (2015)

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).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4320088&req=5

pone.0117464.g003: 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).
Mentions: Toll-like receptors (TLRs) are the major component of the innate immune system and are responsible for recognition of pathogen-associated molecular patterns (PAMPs) including pathogenic foreign nucleic acids (viral and bacterial DNA). Upon recognition of PAMPs, TLRs stimulate the downstream signaling cascade which activates transcription factors, NFκB, and IRF3/7, resulting in induction of inflammatory cytokines including Type 1 IFNs [3]. In addition, the cGAS-STING pathway can upregulate IFNs transcription via activation of IRF3 [12,13]. Since we have observed that IRF3 is phosphorylated following dsDNA transfection (Fig. 1C) and that PLSCR1 can regulate TLR9 signaling in plasmacytoid dendritic cells (pDCs) [21], we next assessed whether TLR4 as well as TLR9 were altered at their transcript level following dsDNA transfection in T80 cells. Both TLR4 and TLR9 have been previously shown to function as nucleic acid sensing TLRs [6]. As expected, there was a significant induction of both TLR4 and TLR9 upon transfection of dsDNA (Fig. 3A, upper and lower panels). Similarly, as shown in Fig. 3B (upper and lower panels), we also detected significant increases in the mRNA levels of IFN-α and IFN-β (IFN-γ was undetectable). Collectively, these results indicate that dsDNA transfection leads to induction of TLR4 and TLR9 as well as IFN-α and IFN-β.

Bottom Line: Toll-like receptors (TLRs) are the primary sensors of the innate immune system that recognize pathogenic nucleic acids including double-stranded plasmid DNA (dsDNA).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.In prior studies, the activation of IRF3 was shown to be mediated by cGAS-STING pathway.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, Florida, 33620, United States of America.

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.

Show MeSH
Related in: MedlinePlus