High mobility group box-1 promotes proliferation and invasion in endometriotic stromal cells through toll-like receptor-4/nuclear factor-kappa B

Abstract

The objective of this study was to evaluate whether high mobility group box (HMGB)-1 induces cell proliferation, invasion, and mediates inflammation in ectopic human endometrial stromal cells via toll-like receptor (TLR) 4 and the nuclear factor (NF)-κB pathway. An experimental study was performed in a tertiary university hospital and laboratory, in which ten women with ovarian endometriomas participated. Ectopic endometrial tissue from the endometriomas was collected. Human endometrial stromal cells (HESCs) were treated with recombinant HMGB-1 in a dose-dependent manner. Using real-time reverse transcription polymerase chain reaction (qRT-PCR) and western blotting, cell proliferation and invasion, TLR4 mRNA and proteins, receptors for advanced glycation end products (RAGE), and vessel endothelial growth factor (VEGF) were examined. The expression of mRNA for the adhesion molecules intracellular adhesion molecule-1 (ICAM-1) and E-cadherin were measured. Inflammatory cytokines were measured from the supernatants of HESCs in accordance with rHMGB-1 treatment. During rHMGB-1 treatment, expression of the TLR4 and RAGE genes and proteins increased in HESCs. VEGF synthesis also increased under these circumstances. Gene expression of ICAM-1 was upregulated, whereas that of E-cadherin was downregulated with rHMGB-1 treatment. Inflammatory cytokine secretion increased significantly during rHMGB-1 treatment. After blocking TLR4 by siTLR4 transfection during rHMGB treatment, cell proliferation and invasion showed a marked decrease. HMGB-1 activates the NF-κB pathway via TLR4 to increase cell proliferation, invasion, and the production of various inflammatory markers.

본 연구는 인간 이소성 자궁내막기질세포에서 high mobility group box(HMGB)-1이 세포 증식 및 침윤 증가, 염증성 변화를 toll-like receptor(TLR)-nuclear factor(NF)-kB 경로를 통해 매개하는지 확인하고자 하였다. 3차 대학병원과 연구실에서 실험연구로 진행하였고, 10명의 난소 자궁내막종 환자가 참여하였다. 이소성 자궁내막세포를 자궁내막종 조직에서 채취하였고, 이를 이용한 원발성 세포 배양으로 세포를 얻었다. 인간 자궁내막기질세포(HESCs)는 recombinant HMGB-1으로 농도를 다르게 처리하였고, 이에 따른 세포 증식과 침윤, TLR4, receptor for advanced glycation end products (RAGE), vessel endothelial growth factor (VEGF) 의 mRNA와 단백질 발현을 정량하였다. 또한 adhesion molecules-intracellular adhesion molecule-1 (ICAM-1)과 E-cadherin의 mRNA 발현과, 세포배양 부유액에서 염증성 사이토카인을 ELISA로 측정하였다. rHMGB-1의 처리에 따라, 이소성 HESCs에서 TLR4와 RAGE의 발현은 증가하였고, VEGF의 합성도 증가하였다.rHMGB-1의 처리 농도 증가에 따라 ICAM-1의 유전자 발현은 상승하였고, E-Cadherin의 발현은 감소하였다. 염증성 사이토카인의 분비 또한 rHMGB-1의 농도 증가에 따라 유의하게 증가하였다. TLR4를 siTLR4 transfection을 통해 차단하였을 때, HESCs 세포 증식과 침윤은 rHMGB-1을 처리함에 따라 유의한 감소를 보였다. 본 결과들을 종합할 때, HMGB-1은 TLR4를 통해 NF-kB 경로를 활성화해 자궁내막기질세포의 증식과 침윤 및 염증성 변화를 유발하여 자궁내막증의 발생에 기여하는 것으로 생각해볼 수 있다.