The Bradykinin System Contributes to the Regulation of Prostaglandin-Endoperoxide Synthase 2 Expression in Human Amnion Fibroblasts: Implications for Term and Preterm Birth

Abstract

Background: Bradykinin (BK) and its biologically active metabolite des-Arg9 bradykinin (DABK) play a pivotal role in inflammation. Since chorioamnionitis is the leading cause of preterm birth and prostaglandin E2 (PGE2) derived from the amnion is key to labor initiation, we investigated if bradykinin peptides are part of the regulatory network of PGE2 synthesis in human amnion at parturition.

Methods: Human amnion tissue was obtained from term and preterm birth for the study of the changes of the bradykinin system at parturition. Cultured primary human amnion fibroblasts, the major source of PGE2, were used to study the effects of bradykinin peptides on PTGS2 expression and PGE2 production as well as the effects of infection mediators on bradykinin receptors.

Results: Bradykinin peptides and their receptors BDKRB1 and BDKRB2 were present in human amnion, and their abundance increased in term and preterm labor. However, transcripts of the genes encoding the bradykinin precursor and its proteolytic cleavage enzymes were hardly detectable in human amnion despite the increased abundance of bradykinin peptides in term and preterm labor, suggesting that there is an alternative source of bradykinin peptides for human amnion and their actions are enhanced in human amnion at parturition. In-vitro studies in cultured human amnion fibroblasts showed that both BK and DABK increased the expression of prostaglandin-endoperoxide synthase 2 (PTGS2), the rate-limiting enzyme in prostaglandin synthesis, and subsequent PGE2 production. These effects of BK and DABK were mediated through BDKRB2 and BDKRB1 receptors, respectively, with subsequent activation of the p38 and ERK1/2 pathways. Moreover, lipopolysaccharide (LPS) and serum amyloid A1 (SAA1), the important mediators of infectious inflammation, induced the expression of both BDKRB1 and BDKRB2 through toll-like receptor 4 (TLR4). Induction of BDKRB1 and BDKRB2 expression by LPS and SAA1 enhanced BK- or DABK-induced PTGS2 expression and PGE2 production in human amnion fibroblasts.

Conclusions: This study demonstrated for the first time that the human amnion is a target tissue of bradykinin peptides and the bradykinin system may be part of the regulatory network of PTGS2 expression and PGE2 production in human amnion fibroblasts at both term and preterm birth, which may be enhanced by infection.

Keywords: PGE2; bradykinin; chorioamnionitis; cyclooxygenase-2; fetal membranes; inflammation; parturition; preterm birth.

Figure 1

Pathway of bradykinin synthesis and the expression of the bradykinin system in human amnion. (A) Pathway of bradykinin synthesis. Italic word in brackets is the gene name of the corresponding protein. UD, undetected. (B) FPKM of gene transcripts related to bradykinin synthesis in the amnion at term with labor (TL, n = 3) and without labor (TNL, n = 3) as revealed by transcriptomic sequencing. (C) Immunohistochemical staining of bradykinin B1 receptor (BDKRB1) and bradykinin B2 receptor (BDKRB2) in human amnion, and comparison of BDKRB1 and BDKRB2 mRNA expression in human amnion fibroblasts and epithelial cells (n = 3). ae, amnion epithelial cells; af, amnion fibroblasts; scale bar, 50 µm. Statistical analysis was performed with unpaired Student’s t-test. **p < 0.01 vs. ae.

Figure 2

Bradykinin and its receptor abundance in human amnion at term and preterm birth with labor. (A, B) BDKRB1 and BDKRB2 mRNA abundance in the amnion of term labor (TL, n = 12) and term non-labor (TNL, n = 11) groups. (C, D) BDKRB1 and BDKRB2 mRNA abundance in the amnion of preterm labor (PL, n = 11) and preterm non-labor (PNL, n = 7). (E) Bradykinin abundance in the amnion of TL (n = 14) and TNL (n = 13) groups. (F) Bradykinin abundance in the amnion of PL (n = 11) and PNL (n = 7) groups. Statistical analysis was performed with the Mann–Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. TNL or PNL.

Figure 3

Effects of bradykinin (BK) and des-Arg9 bradykinin (DABK) on PTGS2 expression and PGE2 production in human amnion fibroblasts. (A) Time-dependent induction of PTGS2 mRNA (n = 3) and protein (n = 5) expression by BK (0.1 μM). (B) Time-dependent induction of PTGS2 mRNA (n = 4) and protein (n = 4) expression by DABK (0.1 μM). (C) Concentration-dependent induction of PTGS2 mRNA (n = 4) and protein (n = 4) expression by BK (0, 0.01, 0.1, 1 μM, 4 h). (D) Concentration-dependent induction of PTGS2 mRNA (n = 3), protein (n = 4) expression by DABK (0, 0.01, 0.1, 1 μM, 4 h). Top panels are the representative immunoblots. Statistical analysis was performed with one-way ANOVA test followed by Dunnett’s test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. 0 h or 0 μM. (E, F) Induction of PGE2 production by BK (0.1 μM, 4 h) (n = 9) and DABK (0.1 μM, 4 h) (n = 10). Statistical analysis was performed with Wilcoxon signed-rank test. **p < 0.01 vs. control (CTR).

Figure 4

Induction of PTGS2 expression by BK and DABK was mediated by BDKRB2 and BDKRB1, respectively, in human amnion fibroblasts. (A) BDKRB1 inhibitor ELN-441958 failed to block BK (0.1 μM, 4 h)-induced PTGS2 mRNA (n = 4) and protein (n = 3) expression. (B) The BDKRB2 inhibitor icatibant blocked BK (0.1 μM, 4 h)-induced PTGS2 mRNA (n = 4) and protein (n = 3) expression. (C) The BDKRB1 inhibitor ELN-441958 blocked DABK (0.1 μM, 4 h)-induced PTGS2 mRNA (n = 4) and protein (n = 5) expression. (D) The BDKRB2 inhibitor icatibant failed to block DABK (0.1 μM, 4 h)-induced PTGS2 mRNA (n = 5) and protein (n = 6) expression. Statistical analysis was performed with one-way ANOVA test followed by Tukey test. Top panels are the representative immunoblots. *p < 0.05, **p < 0.01 vs. group without BK/DABK and antagonist treatment. ^# p < 0.05, ^## p < 0.01 vs. BK- or DABK-treated groups.

Figure 5

Phosphorylation of p38, ERK1/2, and JNK by BK and DABK in human amnion fibroblasts. (A–C) Time-dependent effects of BK (0.1 μM) on p38, ERK1/2, and JNK phosphorylation. (D–F) Time-dependent effects of DABK (0.1 μM) on p38, ERK1/2, and JNK phosphorylation. n = 3–4. Statistical analysis was performed with one-way ANOVA test followed by Dunnett’s test. Top panels are the representative immunoblots. *p < 0.05, **p < 0.01 vs. 0 min.

Figure 6

Role of ERK1/2, p38, and JNK in BK- and DABK-induced PTGS2 expression in human amnion fibroblasts. (A, B) BK (0.1 μM, 4 h)-induced PTGS2 expression was blocked by the ERK1/2 inhibitor PD98059 (20 µM) (mRNA: n = 4; protein: n = 3) or the p38MAPK inhibitor SB203580 (10 µM) (mRNA: n = 4; protein: n = 3). (C, D) DABK (0.1 μM, 4 h)-induced PTGS2 expression was blocked by the ERK1/2 inhibitor PD98059 (20 µM) (mRNA: n = 3; protein: n = 4) or the p38MAPK inhibitor SB203580 (10 µM) (mRNA: n = 3; protein: n = 4). (E, F) The JNK inhibitor SP600125 (10 µM) failed to block the induction of PTGS2 expression by BK (0.1 μM, 4 h) (mRNA: n = 4; protein: n = 3) or by DABK (0.1 μM, 4 h) (mRNA: n = 5; protein: n = 3). Statistical analysis was performed with one-way ANOVA test followed by Tukey test. Top panels are the representative immunoblots. *p < 0.05, **p < 0.01, ***p < 0.001 vs. the group without BK/DABK and antagonist. ^# p < 0.05, ^## p < 0.01 vs. BK- or DABK-treated groups.

Figure 7

Induction of BDKRB1 and BDKRB2 expression by lipopolysaccharide (LPS) and serum amyloid A1 (SAA1) in human amnion fibroblasts. (A, B) Concentration-dependent induction of BDKRB1 and BDKRB2 mRNA expression by LPS (0, 1, 10, 50 ng/ml, 24 h) (n = 4) and SAA1 (0, 10, 50, 100 ng/ml, 24 h) (n = 5). (C, D) Upregulation of BDKRB1 and BDKRB2 protein levels by LPS (50 ng/ml, 24 h) and SAA1 (50 ng/ml, 24 h) (n = 5). (E, F) The TRL4 inhibitor CLI-095 (5 µM) blocked LPS (n = 3)- and SAA1 (n = 4)-induced BDKRB1 and BDKRB2 mRNA expression. Data are the means ± SEM. Statistical analysis was performed with one-way ANOVA test followed by Tukey test. Top panels are the representative immunoblots. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. 0 ng/ml for (A) and (B); vs. control (CTR) for (C) and (D); vs. group without LPS/SAA1 and CLI-095 treatment for (E) and (F) ^# p < 0.05, ^## p < 0.01 vs. LPS- or SAA1-treated groups.

Figure 8

Pretreatment with LPS and SAA1 enhanced the subsequent induction of PTGS2 expression and PGE2 production by BK and DABK in human amnion fibroblasts. (A) Time line illustrating pretreatment of amnion fibroblast with LPS (50 ng/ml) or SAA1 (50 ng/ml) for 24 h, followed by BK (0.1 μM) or DABK (0.1 μM) treatment without LPS or SAA1 for another 4 h. (B–E) Enhancement of BK (0.1 μM)- and DABK (0.1 μM)-induced PTGS2 mRNA expression by LPS (50 ng/ml) or SAA1 (50 ng/ml) (n = 6). (F–I) Enhancement of BK (0.1 μM)- and DABK (0.1 μM)-induced PTGS2 protein expression by LPS (50 ng/ml) or SAA1 (50 ng/ml). n = 6 (F); n = 5 (G); n = 6 (H); n = 6 (I). (J–M) Enhancement of BK- and DABK (0.1 μM)-induced PGE2 production by LPS (50 ng/ml) or SAA1 (50 ng/ml). n = 4 (J); n = 4 (K); n = 7 (L); n = 6 (M). Statistical analysis was performed with two-way ANOVA test. Top panels are the representative immunoblots. *p < 0.05, **p < 0.01 indicate that the interaction factor was significant between groups with and without LPS/SAA1.