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. 2016 Apr 12;7(15):20469-85.
doi: 10.18632/oncotarget.7814.

Overexpression of pig selenoprotein S blocks OTA-induced promotion of PCV2 replication by inhibiting oxidative stress and p38 phosphorylation in PK15 cells

Fang Gan  1   2 , Zhihua Hu  1   2 , Yu Huang  1   2 , Hongxia Xue  1   2 , Da Huang  1   2 , Gang Qian  1   2 , Junfa Hu  1   2 , Xingxiang Chen  1   2 , Tian Wang  3 , Kehe Huang  1   2
Affiliations

Overexpression of pig selenoprotein S blocks OTA-induced promotion of PCV2 replication by inhibiting oxidative stress and p38 phosphorylation in PK15 cells

Fang Gan et al. Oncotarget. .

Abstract

Porcine circovirus type 2 (PCV2) is the primary cause of porcine circovirus disease, and ochratoxin A (OTA)-induced oxidative stress promotes PCV2 replication. In humans, selenoprotein S (SelS) has antioxidant ability, but it is unclear whether SelS affects viral infection. Here, we stably transfected PK15 cells with pig pCDNA3.1-SelS to overexpress SelS. Selenium (Se) at 2 or 4 μM and SelS overexpression blocked the OTA-induced increases of PCV2 DNA copy number and infected cell numbers. SelS overexpression also increased glutathione (GSH), NF-E2-related factor 2 (Nrf2) mRNA, and γ-glutamyl-cysteine synthetase mRNA levels; decreased reactive oxygen species (ROS) levels; and inhibited p38 phosphorylation in PCV2-infected PK15 cells, regardless of OTA treatment. Buthionine sulfoximine reversed all of the above SelS-induced changes. siRNA-mediated SelS knockdown decreased Nrf2 mRNA and GSH levels, increased ROS levels, and promoted PCV2 replication in OTA-treated PK15 cells. These data indicate that pig SelS blocks OTA-induced promotion of PCV2 replication by inhibiting the oxidative stress and p38 phosphorylation in PK15 cells.

Keywords: ochratoxin A; overexpression of selenoprotein S; oxidative stress; p38 signaling pathway; porcine circovirus type 2.

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Conflict of interest statement

CONFLICTS OF INTEREST

All authors declare no financial conflict of interest.

Figures

Figure 1
Figure 1. Construction of the SelS overexpression plasmid, pc-SelS
SECISearch software was used to identify the SECIS sequence of porcine SelS A. A single target SelS gene, 1029 bp in length, was identified using PCR and electrophoresis B. pcDNA3.1-SelS was verified using colony PCR C. and restriction endonuclease digestion D.
Figure 2
Figure 2. Expression of the pc-SelS in PK 15 cells
SelS mRNA A. and protein B. levels after transfecting pc-SelS into PK15 cells were determined using real-time PCR and western blotting as described in Materials and Methods. Data are presented as means ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. control. #P < 0.05 and ##P < 0.01 vs. vector control.
Figure 3
Figure 3. SelS overexpression increased antioxidant ability in PK15 cells
SelS-overexpressing PK15 cells were incubated for 72 h in DMEM. The cell viability A. Nrf2 mRNA levels B. γ-GCS mRNA levels C, D. GSH levels E. and ROS levels F. were assayed as described in the Materials and Methods. Data are presented as means ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. control. #P < 0.05 and ##P < 0.01 vs. vector control.
Figure 4
Figure 4. Se supplementation blocked OTA-promoted PCV2 replication
PK15 cells were cultured for 12 h with 0, 1, 2 or 4 μM Se and then incubated for an additional 60 h with PCV2 in the presence or absence of 0.05 μg/ml OTA. Cells were assayed for PCV2 DNA copies A. using real-time PCR and the number of infected cells B. using IFA. Data are presented as means ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. control (without OTA or Se). Within the OTA treatment groups, #P < 0.05 and ##P < 0.01 vs. control cells without Se.
Figure 5
Figure 5. SelS overexpression blocked OTA-promoted PCV2 replication
PK15 cells were cultured for 12h with or without 4 μM Se and then incubated for an additional 60 h with PCV2 in the presence or absence of 0.05 μg/ml OTA. Cells were assayed for PCV2 viral DNA copies A. using real-time PCR and the number of infected cells B. using IFA. Data are presented as means ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. the respective Cont and Vcont groups.
Figure 6
Figure 6. Effects of OTA treatment and/or PCV2 infection on oxidative stress
Vector-PK15 cells were incubated for 24 h with or without PCV2 and then for 48 h in the presence or absence of 0.05 μg/ml OTA. Cells were harvested after an additional 48 h in the presence of OTA. Levels of Nrf2 A. and γ-GCS B, C. mRNA, GSH D. ROS E. and p38 phosphorylation F. were assayed as described in the Materials and Methods. Data are presented as means ± SE of three independent experiments. *p < 0.05 and **p < 0.01 vs. control.
Figure 7
Figure 7. SelS overexpression decreased OTA-induced oxidative stress in PK15 cells
PK15 cells overexpressing vector or SelS were cultured for 12h and then incubated for an additional 60 h with PCV2 in the presence or absence of 0.05 μg/ml OTA. Cellular levels of Nrf2 A. and γ-GCS B, C. mRNA, GSH D. and ROS E. were assayed as described in the Materials and Methods. Data are presented as means ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. control (without OTA and PCV2). Within the PCV2 infection group, #P < 0.05 and ##P < 0.01 vs. Vcont cells without OTA treatment and $P < 0.05 and $$P < 0.01 vs. Vcont cells with OTA treatment.
Figure 8
Figure 8. SelS overexpression inhibits OTA-induced p38 phosphorylation in PK15 cells
PK15 cells overexpressing vector or SelS were cultured for 12 h and then incubated for an additional 60 h without or with PCV2 in the presence or absence of 0.05 μg/ml OTA. Cells were assayed for p38 phosphorylation as described in the Materials and Methods. Data are presented as means ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. Vcont (without OTA and PCV2). Within the PCV2 infection group, #P < 0.05 and ##P < 0.01 vs. Vcont cells without OTA treatment and $P < 0.05 and $$P < 0.01 vs. Vcont cells with OTA treatment.
Figure 9
Figure 9. Effects of SelS overexpression and/or OTA and/or BSO on PCV2 replication, oxidative stress and p38 phosphorylation in PCV2-infected PK15 cells
PK15 cells overexpressing vector or SelS were cultured for 12 h and then incubated for an additional 60 h with PCV2 in the presence or absence of 0.05 μg/ml OTA and/or 50 μM BSO. Cells were harvested and assayed for PCV2 DNA copies A. the number of infected cells B. and levels of Nrf2 C. GSH D. ROS E. and p38 phosphorylation F. as described in the Materials and Methods. Data are presented as means ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. control. #P < 0.05 and ##P < 0.01 vs. OTA treatment. $P < 0.05 and $$P < 0.01 vs. OTA and SelS.
Figure 10
Figure 10. SelS gene expression after transfection of SelS-siRNA into PK15 cells
Vector-expressing PK15 cells were incubated with or without SelS-siRNA. Cell samples were assayed for levels of SelS mRNA A. actin and SelS protein B. Nrf2 and γ-GCS mRNA C. GSH D. and ROS E. Data are presented as means ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. control. #P < 0.05 and ##P < 0.01 vs. ssiRNA.
Figure 11
Figure 11. SelS knockdown enhanced OTA-induced oxidative stress and p38 phosphorylation
Vector-expressing PK15 cells were cultured for 12 h and then incubated for an additional 60 h with PCV2 in the presence or absence of 0.05 μg/ml OTA. Cells were assayed for levels of Nrf2 mRNA A. GSH B. ROS C. and p38 phosphorylation D. Data are presented as means ± SE of three independent experiments. *P < 0.05 and **P < 0.01 vs. sicont without OTA treatment. #P < 0.05 and ##P < 0.01 vs. sicont with OTA treatment.
Figure 12
Figure 12. SelS knockdown promoted OTA-induced PCV2 replication
Vector-expressing PK15 cells were cultured for 12 h and then incubated for an additional 60 h with PCV2 in the presence or absence of 0.05 μg/ml OTA. Cells were assayed for PCV2 viral DNA copies A. using real-time PCR and the number of infected cells B. using IFA. Data are presented as means ± SE of three independent experiments. *p < 0.05 and **p < 0.01 sicont without OTA treatment. #p < 0.05 and ## p < 0.01 sicont with OTA treatment.
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