Differential expression of Prostaglandin-Endoperoxide Synthase 2 and Prostaglandins in the Epithelial and Mesenchymal cells of term amnion

Abstract

Objective: Prostaglandins (PGs) play an essential role in the initiation and maintenance of human labor. The primary source of intrauterine PGs is the amnion which is composed of two distinct cellular subsets: amnion epithelial cells (AECs) and amnion mesenchymal cells (AMCs). However, the expression and regulations patterns of PGs and Prostaglandin-endoperoxide synthase 2 (PTGS2), a key enzyme in the synthesis of PGs, in these cell types have not been clearly determined. This study was conducted to investigate PTGS2 expression and PGs production in AECs and AMCs at term pregnancy.

Materials and Methods: Primary AECs and AMCs obtained from cesarean deliveries at term (n=5) were studied for the changes in the expression of PTGS2, PGE2, and PGF2 following the treatment with pro-inflammatory cytokine IL-1. PTGS2 mRNA and protein expressions were analyzed by qRT-PCR and immunoblotting, and the concentrations of PGE2 and PGF2 were measured by specific immunoassays. The chorioamniotic membranes samples were obtained from women with singleton pregnancy: normal term labor and deliveries with (n=12) and without acute chorioamnionitis (ACA) (n=12). PTGS2 expression in the amnion was evaluated by immunohistochemistry and compared by grading of the immunoreactivity. Linear by linear association and generalized estimating equations regression models was used for comparisons. Results: 1) The amplitude of the increase in PTGS2 mRNA expression was significantly higher in AMCs than in AECs following IL-1 treatment [median fold change of 2^ddCT (range), 42.9 (14.9-171.3) vs. 11.7 (6.0-40.3); p=0.039]. 2) Basal PTGS2 protein expression was higher in AEC than in AMC. 3) PTGS2 protein expression in both AECs and AMCs increased after IL-1 treatment. 4) The increase of PTGS2 protein expression was more prominent in AMCs than in AECs [median fold change of PTGS2/-actin ratio (range), 2.53(1.17-2.75) vs. 1.48(1.02-1.93); p=0.007]. 5) Basal concentration (pg/ug of total protein) of PGE2 and PGF2 were significantly higher in AECs than in AMCs [PGE2: median (range), 427.5 (7.8-566.2) vs. 10.1 (8.0-12.3); p=0.002, PGF2: median (range), 1.10 (0.06-2.49) vs. 0.23 (0.15-0.35); p=0.024]. 6) Increase of PGE2 and PGF2 were more prominent in AMCs than in AECs [PGE2: median fold change (range), 42.5 (15.2-80.7) vs. 12.3 (3.5-37.5); p=0.018, PGF2: median fold change (range), 12.1 (4.4-14.5) vs. 6.3 (3.1-12.7); p=0.028] 7) There was no difference in the PTGS2 immunoreactivity of AECs between term labor cases with and without ACA (p=0.212). 8) There was a significant increase in PTGS2 immunoreactivity in AMCs in term labor cases with ACA compared to those without ACA (p<0.001).

Conclusions: The findings herein strongly suggest that AECs are key players of PG production at term pregnancy. In the presence of intra-amniotic inflammation, however, it is suggested that AMCs may play a more important role because their PTGS2 and PGs expressions are more inducible in nature.

연구 목적: 분만진통의 시작과 유지에 핵심 역할을 하는 프로스타글란딘(Prostaglandin, PG)은 상피세포와 기질세포로 이루어져 있는 양막(amnion)에서 주로 생성된다. 본 연구는 만삭 임신에서 양막 상피세포와 양막 기질세포에서 프로스타글란딘 생성과 프로스타글란딘 합성 효소인 PTGS2 (Prostaglandin-Endoperoxide Synthase 2) 의 발현을 알아보고자 시행되었다.

연구 방법: 제왕절개로 만삭 분만한 산모의 태반(n=5)에서 분리한 양막 상피세포와 양막 기질세포를 일차 배양한 후 인터류킨 1 베타 (IL-1)로 24시간 동안 처리하였다. PTGS2의 mRNA는 qRT- PCR, 단백질은 면역전기영동으로 확인하였다. PGE2 과 PGF2 농 도는 ELISA법으로 측정하였다. 분만 진통으로 만삭 질식 분만 한 단태아의 양막 중 급성 융모양막염이 있는 군(n=12)과 없는 군 (n=12)에서 PTGS2 발현은 면역조직화학법으로 평가하였다. 통계 기법은 Linear by linear association과 generalized estimating equations regression model를 이용하였다.

연구 결과: 1) IL-1 처리 전 PTGS2 단백질의 발현 및 PGE2 와 PGF2 의 농도는 양막 기질세포보다 양막 상피세포에서 더 높았다. 2) IL-1 처리 후 PTGS2 mRNA, PTGS2 단백질의 발현 및 PGE2 와 PGF2 농도는 IL-1 처리 전보다 양막 상피 세포 및 기질세포 모두에서 증가하였다. 3) IL-1 처리 후 양막 상피세포와 기질세포에서 PTGS2 mRNA, PTGS2 단백질의 발현 및 PGE2 와 PGF2 농도의 증가 폭은 양막 기질세포에서 더 뚜렷하였다 [median fold change of 2^ddCT (range), 42.9 (14.9-171.3) vs. 11.7 (6.0-40.3); p=0.039: median fold change of PTGS2/-actin ratio (range), 2.53(1.17-2.75) vs. 1.48(1.02-1.93); p=0.007: median fold change of PGE2(range), 42.5 (15.2-80.7) vs. 12.3(3.5-37.5); p=0.018: median fold change of PGF2 (range, 12.1 (4.4-14.5) vs. 6.3(3.1-12.7); p=0.028] 4) 실제 양막을 면역조직화학법을 염색하였을 때 양막 상피세포에서는 PTGS2의 발현이 융모양막염의 유무에 따라 차이가 없지만 (p=0.212) 양막 기질세포에서는 융모막양염이 있는 경우에는 PTGS2 발현이 증가하였다(p<0.001).

결론: 만삭에서 양막 상피세포가 프로스타글란딘의 생성에 핵심적인 역할을 함을 확인할 수 있었다. 하지만 IL-1의 자극에 PTGS2 와 프르스타글란딘의 발현의 증가 폭이 양막 기질세포에서 더 컸다. 이러한 소견은 자궁내 염증이 있는 경우에는 양막 기질세포가 프로스타글란딘 생성에 더 중요한 역할을 할 가능성이 있음을 시사한다.