Bacteroides fragilis enterotoxin으로 자극한 장상피세포에서 chemokine CCL20 발현

Abstract

Enterotoxigenic B. fragilis (ETBF)와 같은 특정균은 비침입성 설사질환 및 염증성 질병과 관련이 있으며, 이로부터 발생되는 B. fragilis toxin (BFT)는 ETBF의 유일한 독성인자로 상피 형태의 붕괴와 친염증성 사이토카인의 분비 등을 통해 염증반응을 발생시키는 것으로 보고되고 있다. 장상피세포는 미생물 감염이나 염증성 물질들에 대하여 cytokine이나 chemokine을 분비하여 neutrophil, lymphocyte 등의 유입을 발생시켜 염증반응을 매개하며, 이와 같은 반응은 다양한 염증성 질환의 병인론과도 관련되어 있다. 장상피세포는 BFT에 의해서도 다양한 chemokine을 분비함으로 leukocyte의 유입을 증가시키는 것으로 알려져 있지만, 이와 관련된 분자생물학적 기전에 대해서는 밝혀진 바가 거의 없다. CCL20는 상피조직으로부터 발현이 되어 CCL20-CCR6 결합을 통해 immature dendritic cells, effector memory T cell, B cell과 같은 세포의 이동을 발생시키며, 상피세포와 관련된 다양한 염증성 질환에서 그 발현이 증가되는 것으로 보고되어 왔다. BFT로 유도되는 장내 염증반응에서도 CCL20의 역할과 이와 관련된 조절기전은 중요할 것으로 예상된다. 이번 연구는 장상피세포에서 BFT로 인한 CCL20의 유도와 CCL20 유도기전에 있어 염증성 신호경로의 구성원인 Nuclear factor-kappaB (NF-κB), activator protein-1 (AP-1), mitogen-activated protein kinase (MAPK)의 역할을 함께 확인하기 위하여 실행되었다. BFT 자극은 HT-29 및 COLO 205 인간 장상피세포주에서 CCL20의 발현을 상당히 증가시켰다. 또한 전사인자 NF-κB와 AP-1은 두 장상피세포 모델에서 BFT에 의해 각각 활성화 되었다. 각 전사인자의 활성을 감소시킨 세포 모델을 제작하여 BFT를 자극한 결과 BFT에 의한 CCL20의 발현 유도가 전적으로 NF-κB에 의해 조절되는 것으로 AP-1과는 그 관련성이 다소 부족한 것으로 나타났다. MAPK의 경우 HT-29 및 COLO 205 세포에서 BFT 자극에 의해 ERK, p38, JNK MAPK 세 가지 구성원이 모두 활성화되는 것으로 나타났으며, 각각에 대한 inhibitor를 적용한 결과 이 중 ERK와 p38 MAPK가 CCL20 유도와 관련되는 것으로 확인되었다. 그리고 각 MAPK 활성을 억제한 모델에서 p38 MAPK의 활성 억제가 NF-κB 활성과 CCL20의 발현을 함께 감소시키는 것으로 관찰되었다. 이 연구는 결과적으로 BFT로 자극된 장상피세포에서 CCL20의 발현유도가 발생될 수 있으며, 이와 관련된 유도기전에 전사인자 NF-κB의 활성화와 상위 신호분자인 p38 MAPK의 활성화가 포함된다는 것을 규명하였다. |Background and Purpose: Bacteroides fragilis entrotoxin (BFT) causes inflammation in intestinal epithelial cells. Although BFT is known to induce chemokine response which could attract leukocyte to the site of inflammation, there has been little understanding on its exact molecular mechanisms. CC Chemokine ligand 20 (CCL20) is expressed by surface-lining epithelium, and is related in the link between innate and acquired immunity by attracting immature dendritic cells, effector memory T cells and B cells via CCR6. This study has the investigation of the mechanisms of CCL20 expression and the role of Nuclear factor-kappaB (NF-κB), activator protein-1 (AP-1) and mitogen-activated protein kinase (MAPK) signals in BFT-stimulated human intestinal epithelial cells. Methods: HT-29 and COLO 205 cells were cultured in the presence or absence of purified BFT. CCL20 expression was analyzed by Reverse transcription-PCR. Activities of transcription factors such as NF-κB and AP-1 were assessed by electrophoretic mobility shift assays (EMSA), and Phosphorylation of signal molecules was measured by Immunoblot. Results: BFT stimulation increased CCL20 expression in HT-29 and COLO 205 cells. BFT activated NF-κB and AP-1 signals in both HT-29 and COLO 205 cells. Suppression of NF-κB using lentiviruses containing dominant negative (dn) DNA significantly decreased the expression of CCL20 in HT-29 and COLO 205 cells stimulated by BFT, but suppression of AP-1 did not affect BFT-induced CCL20 expression. Activated MAPKs increased in HT-29 and COLO 205 cells stimulated by BFT. Infection with lentiviruses containing dn-p38 and dn-ERK2 decreased CCL20 expression compared with that with lentiviruses containing control DNA and that dn-JNK1. However, NF-κB activity was decreased in BFT-stimulated HT-29 cells transfected with lentiviruses containing dn-p38 only. Conclusion: These results suggest that p38 MAPK may act as the upstream signaling molecule for the activation of NF-κB, which induces CCL20 expression in BFT-stimulated human intestinal epithelial cells.; Background and Purpose: Bacteroides fragilis entrotoxin (BFT) causes inflammation in intestinal epithelial cells. Although BFT is known to induce chemokine response which could attract leukocyte to the site of inflammation, there has been little understanding on its exact molecular mechanisms. CC Chemokine ligand 20 (CCL20) is expressed by surface-lining epithelium, and is related in the link between innate and acquired immunity by attracting immature dendritic cells, effector memory T cells and B cells via CCR6. This study has the investigation of the mechanisms of CCL20 expression and the role of Nuclear factor-kappaB (NF-κB), activator protein-1 (AP-1) and mitogen-activated protein kinase (MAPK) signals in BFT-stimulated human intestinal epithelial cells. Methods: HT-29 and COLO 205 cells were cultured in the presence or absence of purified BFT. CCL20 expression was analyzed by Reverse transcription-PCR. Activities of transcription factors such as NF-κB and AP-1 were assessed by electrophoretic mobility shift assays (EMSA), and Phosphorylation of signal molecules was measured by Immunoblot. Results: BFT stimulation increased CCL20 expression in HT-29 and COLO 205 cells. BFT activated NF-κB and AP-1 signals in both HT-29 and COLO 205 cells. Suppression of NF-κB using lentiviruses containing dominant negative (dn) DNA significantly decreased the expression of CCL20 in HT-29 and COLO 205 cells stimulated by BFT, but suppression of AP-1 did not affect BFT-induced CCL20 expression. Activated MAPKs increased in HT-29 and COLO 205 cells stimulated by BFT. Infection with lentiviruses containing dn-p38 and dn-ERK2 decreased CCL20 expression compared with that with lentiviruses containing control DNA and that dn-JNK1. However, NF-κB activity was decreased in BFT-stimulated HT-29 cells transfected with lentiviruses containing dn-p38 only. Conclusion: These results suggest that p38 MAPK may act as the upstream signaling molecule for the activation of NF-κB, which induces CCL20 expression in BFT-stimulated human intestinal epithelial cells.