PARK7 regulates inflammation-induced pro-labour mediators in myometrial and amnion cells

in Reproduction
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Preterm birth is a prevalent cause of neonatal deaths worldwide. Inflammation has been implicated in spontaneous preterm birth involved in the processes of uterine contractility and membrane rupture. Parkinson protein 7 (PARK7) has been found to play an inflammatory role in non-gestational tissues. The aims of this study were to determine the expression of PARK7 in myometrium and fetal membranes with respect to term labour onset and to elucidate the effect of PARK7 silencing in primary myometrium and amnion cells on pro-inflammatory and pro-labour mediators. PARK7 mRNA expression was higher in term myometrium and fetal membranes from women in labour compared to non-labouring samples and in amnion from preterm deliveries with chorioamnionitis. In human primary myometrial cells transfected with PARK7 siRNA (siPARK7), there was a significant decrease in IL1B, TNF, fsl-1 and poly(I:C)-induced expression of pro-inflammatory cytokine IL6, chemokines (CXCL8, CCL2), adhesion molecule ICAM1, prostaglandin PGF and its receptor PTGFR. Similarly, amnion cells transfected with siPARK7 displayed a decrease in IL1B-induced expression of IL6, CXCL8 and ICAM1. In myometrial cells transfected with siPARK7, there was a significant reduction of NF-κB RELA transcriptional activity when stimulated with fsl-1, flagellin and poly(I:C), but not with IL1B or TNF. Collectively, our novel data describe a role for PARK7 in regulating inflammation-induced pro-inflammatory and pro-labour mediators in human myometrial and amnion cells.

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    PARK7 expression in human myometrium and fetal membranes. (A) Human myometrium was obtained from non-labouring and labouring women at term Caesarean section (n = 8 patients per group). (B) Fetal membranes were obtained from women not in labour at term Caesarean section and women after term spontaneous labour onset and delivery (n = 9 patients per group). (C) Fetal membranes were obtained from women not in labour at preterm Caesarean section and women after preterm spontaneous labour onset and delivery (n = 9 patients per group). (D) Amnion was obtained from women at preterm Caesarean section with or without histological chorioamnionitis (n = 8 patients per group). PARK7 mRNA abundance was analysed by qRT-PCR. All data are displayed as mean ± s.e.m. *P ≤ 0.05 vs term no labour (Student’s t-test); **P ≤ 0.05 vs preterm no chorioamnionitis (Student’s t-test).

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    Effect of siPARK7 on pro-inflammatory cytokines and chemokines induced by IL1B or TNF in primary myometrial cells. Human primary myometrial cells were transfected with 50 nM siCONT or 50 nM siPARK7 for 48 h and then treated with (A, B, E, F, I and J) 1 ng/mL IL1B or (C, D, G, H, K and L) 10 ng/mL TNF for an additional 20 h (n = 5 patients). (A, C, E, G, I and K) IL6, CXCL8 and CCL2 mRNA expression was analysed by qRT-PCR. (B, D, F, H, J and L) The concentration of IL6, CXCL8 and CCL2 in the incubation medium was assayed by ELISA. For all data, the fold change was calculated relative to IL1B- or TNF-stimulated siCONT-transfected cells and displayed as mean ± s.e.m. *P ≤ 0.05 vs IL1B-stimulated siCONT-transfected cells; **P ≤ 0.05 vs TNF-stimulated siCONT-transfected cells. All data were analysed by one-way repeated-measures ANOVA.

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    Effect of siPARK7 on pro-inflammatory cytokines and chemokines induced by TLR ligands in primary myometrial cells. Human primary myometrial cells were transfected with 50 nM siCONT or 50 nM siPARK7 for 48 h and then treated with (A, B, G, H, M and N) 250 ng/mL fsl-1, (C, D, I, J, O and P) 1 μg/mL flagellin (flag) or (E, F, K, L, Q and R) 5 µg/mL poly(I:C) for an additional 20 h (n = 5 patients). (A, C, E, G, I, K, M, O and Q) IL6, CXCL8 and CCL2 mRNA expression was analysed by qRT-PCR. (B, D, F, H, J, L, N, P and R) The concentration of IL6, CXCL8 and CCL2 in the incubation medium was assayed by ELISA. For all data, the fold change was calculated relative to fsl-1-, flag- or poly(I:C)-stimulated siCONT-transfected cells and displayed as mean ± s.e.m. ***P ≤ 0.05 vs fsl-1-stimulated siCONT-transfected cells; §P ≤ 0.05 vs flag-stimulated siCONT-transfected cells; #P ≤ 0.05 vs poly(I:C)-stimulated siCONT-transfected cells. All data were analysed by one-way repeated-measures ANOVA.

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    Effect of siPARK7 on pro-inflammatory cytokines and chemokines induced by IL1B in primary amnion cells. Human primary amnion cells were transfected with 50 nM siCONT or 50 nM siPARK7 for 48 h and then treated with 1 ng/mL IL1B for an additional 20 h (n = 5 patients). (A, C and E) IL6, CXCL8 and CCL2 mRNA expression was analysed by qRT-PCR. (B, D and F) The concentration of IL6, CXCL8 and CCL2 in the incubation medium was assayed by ELISA. For all data, the fold change was calculated relative to IL1B-stimulated siCONT-transfected cells, and displayed as mean ± s.e.m. *P ≤ 0.05 vs IL1B-stimulated siCONT-transfected cells (one-way repeated-measures ANOVA).

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    Effect of siPARK7 on ICAM1 in primary myometrial and amnion cells. Human primary (A, B, E, F, G, H, I, J, K and L) myometrial and (C and D) amnion cells were transfected with 50 nM siCONT or 50 nM siPARK7 for 48 h and then treated with (A, B, C and D) 1 ng/mL IL1B, (E and F) 10 ng/mL TNF, (G and H) 250 ng/mL fsl-1, (I and J) 1 μg/mL flagellin (flag) or (K and L) 5 µg/mL poly(I:C) for an additional 20 h (n = 5 patients). (A, C, E, G, I and K) ICAM1 mRNA expression was analysed by qRT-PCR. (B, D, F, H, J and L) The concentration of sICAM1 in the incubation medium was assayed by ELISA. For all data, the fold change was calculated relative to IL1B-, TNF-, fsl-1- or flag-stimulated siCONT-transfected cells, and displayed as mean ± s.e.m. *P ≤ 0.05 vs IL1B-stimulated siCONT-transfected cells; **P ≤ 0.05 vs TNF-stimulated siCONT-transfected cells; ***P ≤ 0.05 vs fsl-1-stimulated siCONT-transfected cells; §P ≤ 0.05 vs flag-stimulated siCONT-transfected cells; #P ≤ 0.05 vs poly(I:C)-stimulated siCONT-transfected cells. All data were analysed by one-way repeated-measures ANOVA.

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    Effect of siPARK7 on PTGS2-prostaglandin pathway induced by IL1B or TNF in primary myometrial cells. Human primary myometrial cells were transfected with 50 nM siCONT or 50 nM siPARK7 for 48 h and then treated with (A, B and C) 1 ng/mL IL1B or (D, E and F) 10 ng/mL TNF for an additional 20 h (n = 5 patients). (A, B, D and E) PTGS2 and PTGFR mRNA expression was analysed by qRT-PCR. (C and F) The concentration of PGF in the incubation medium was assayed by ELISA. For all data, the fold change was calculated relative to TNF-stimulated siCONT-transfected cells, and displayed as mean ± s.e.m. *P ≤ 0.05 vs IL1B-stimulated siCONT-transfected cells; **P ≤ 0.05 vs TNF-stimulated siCONT-transfected cells. All data were analysed by one-way repeated-measures ANOVA.

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    Effect of siPARK7 on RELA transcriptional activity in primary myometrial cells. Human myometrial cells were transfected with 300 ng/mL RELA reporter construct. After 6 h, cells were transfected with 50 nM siCONT or 50 nM siPARK7 for 48 h, then treated with (A) 1 ng/mL IL1B, (B) 10 ng/mL TNF, (C) 250 ng/mL fsl-1, (D) 1 µg/mL flagellin (flag) or (E) 5 μg/mL poly(I:C) for an additional 20 h (n = 5 patients). RELA promoter activity is expressed as a ratio of luciferase activity of IL1B-, TNF-, fsl-1-, flag- or poly(I:C)-stimulated siCONT transfected cells. All data displayed as mean ± s.e.m. *P ≤ 0.05 vs IL1B-stimulated siCONT-transfected cells; **P ≤ 0.05 vs TNF-stimulated siCONT-transfected cells; ***P ≤ 0.05 vs fsl-1-stimulated siCONT-transfected cells; §P ≤ 0.05 vs flag-stimulated siCONT-transfected cells; #P ≤ 0.05 vs poly(I:C)-stimulated siCONT-transfected cells. All data were analysed by one-way repeated-measures ANOVA.

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