Bacteroides fragilis 장독소로 자극한 장상피세포에서의 matrix metallopeptidase 9 발현

Abstract

Bateroides fragilis는 장내 미생물무리로 알려져 있다. 이 중 장독소(B. fragilis enterotoxin, BFT)를 생산하는 enterotoxigenic B. fragilis는 장염과 염증성 장질환(inflammatory bowel disease, IBD)을 유발한다. IBD는 환경적 요인과 유전적 요인에 의해 발병된다고 알려져 있다. 장내 미생물무리의 조성변화에 의한 발병은 대표적인 환경적 요인에 포함되는데, 이로 인해 염증반응이 일어난 조직과 면역세포에서는 extracellular matrix (ECM) remodeling 효소가 생성된다. 이중에서 gelatinase가 IBD에 영향을 끼치는 대표적인 효소로 떠오르고 있다. 이 효소는 다양한 자극을 통해 장내 염증 조직에서 발현된다. 특히, IBD 환자의 염증 조직에서 matrix metallopeptidase 9 (MMP-9)이 많이 발현된다는 보고가 있다. MMP-9은 ECM의 구성요소 중 하나인 tight junction protein 1을 분해하여 장내에 존재하는 균이 점막 내로 쉽게 침투하도록 하여 tissue injury를 악화시킨다. 그러므로 BFT로 유도되는 장내 염증반응에서도 MMP-9의 역할과 이와 관련된 조절기전은 중요할 것으로 예상된다. 이번 연구는 BFT로 자극한 장상피세포에서의 MMP-9 발현경로와 신호전달을 규명하는 것을 목표로 하였다. 실험모델로 HCA7 장상피세포를 사용하였으며, MMP-9의 발현은 PCR을 이용하여 확인하였다. 그 결과 BFT자극 3시간부터 MMP-9 mRNA 발현증가가 관찰되었으며 16~ 24시간에서 최대치(대조군에 비해서 19배 증가)를 보였고 그 이후로 감소함을 확인하였다. 또한 NF-κB와 AP-1 전사인자의 활성과 MMP-9 발현의 상관관계를 규명하기 위해 dominant negative (dn)-IκBα DNA와 dn-TAM67 DNA를 도입한 lentiviral particle을 transduction 시켜 각 전사인자의 활성을 억제한 HCA7 세포를 제작하였다. 각각의 세포에 BFT를 자극하여 MMP-9의 발현을 관찰한 결과 MMP-9의 발현이 AP-1을 억제한 세포에서 감소하였다. 한편 BFT 처리된 HCA7 세포에서 mitogen-activated protein kinases (MAPKs)의 활성도 관찰하였다. MAPKs의 경우 HCA7 세포에서 BFT 자극에 의해 ERK, p38, JNK MAPK 모두 활성화되는 것을 확인하였으며, dn-ERK2 DNA, dn-p38 DNA, dn-JNK DNA가 전달된 세포에서 MMP-9의 발현을 관찰한 결과 JNK가 MMP-9 유도와 관련됨을 확인하였다. 따라서 AP-1과 JNK의 활성이 MMP-9의 발현과 관계가 있음을 확인하였다. 이 연구를 통해 BFT로 HCA7 장상피세포를 자극하였을 때 MMP-9의 발현이 유도될 수 있으며, 이와 관련된 유도기전에 전사인자 AP-1의 활성화와 JNK MAPK의 활성화가 포함된다는 것을 규명하였다. |Background and Purpose Enterotoxigenic bacteroides fragilis (ETBF) produces an approximately 20kDa heat-labile enterotoxin (BFT) that cause inflammation such as colitis and inflammatory bowel disease (IBD). IBD is known to be caused by genetic factors and environmental factors containing pathogenesis by change of microbial composition. The process of inflammation is occurred in inflammatory tissues and immune cells that produce extracellular matrix (ECM) remodeling enzyme. One of ECM enzymes, gelatinase B (also known as matrix metallopeptidase 9, MMP-9) play an important role in pathogenesis of IBD. This study has the investigation of the mechanisms of MMP-9 expression and the related signaling in BFT-stimulated human intestinal epithelial cells.

Methods HCA7 cells were cultured in presence or absence of BFT. MMP-9 expression was analyzed by reverse transcription-PCR. Activities of transcription factors such as nuclear factor-kappaB (NF-κB) and activator protein-1 (AP-1) were assessed by immunoblot after blocking IκBα and c-Jun and phosphorylation of signal molecules was measured by Immunoblot.

Results Stimulation of HCA7 cells with BFT increased MMP-9 expression from 3 h and peaked 16~24 h. BFT activated NF-κB and AP-1 signals in HCA7 cells. Suppression of NF-κB using lentiviruses containing dominant-negative (dn) DNA did not affect the increased MMP-9 mRNA expression in BFT-stimulated HCA7 cells, but suppression of AP-1 significantly decreased BFT-induced MMP-9 expression. Activated signals of mitogen-activated protein kinases (MAPKs) increased BFT-stimulated HCA7 cells. Infection with lentivirus containing dn-JNK significantly decreased MMP-9 expression compared with lentiviruses containing control DNA in BFT-stimulated cells. However, lentiviruses containing dn-ERK2 and dn-p38 did not inhibit the MMP-9 mRNA expression in HCA7 cells stimulated with BFT.

Conclusion These results suggest that BFT induces MMP-9 in BFT-stimulated intestinal epithelial cells through JNK and AP-1 signaling.; Background and Purpose Enterotoxigenic bacteroides fragilis (ETBF) produces an approximately 20kDa heat-labile enterotoxin (BFT) that cause inflammation such as colitis and inflammatory bowel disease (IBD). IBD is known to be caused by genetic factors and environmental factors containing pathogenesis by change of microbial composition. The process of inflammation is occurred in inflammatory tissues and immune cells that produce extracellular matrix (ECM) remodeling enzyme. One of ECM enzymes, gelatinase B (also known as matrix metallopeptidase 9, MMP-9) play an important role in pathogenesis of IBD. This study has the investigation of the mechanisms of MMP-9 expression and the related signaling in BFT-stimulated human intestinal epithelial cells.

Methods HCA7 cells were cultured in presence or absence of BFT. MMP-9 expression was analyzed by reverse transcription-PCR. Activities of transcription factors such as nuclear factor-kappaB (NF-κB) and activator protein-1 (AP-1) were assessed by immunoblot after blocking IκBα and c-Jun and phosphorylation of signal molecules was measured by Immunoblot.

Results Stimulation of HCA7 cells with BFT increased MMP-9 expression from 3 h and peaked 16~24 h. BFT activated NF-κB and AP-1 signals in HCA7 cells. Suppression of NF-κB using lentiviruses containing dominant-negative (dn) DNA did not affect the increased MMP-9 mRNA expression in BFT-stimulated HCA7 cells, but suppression of AP-1 significantly decreased BFT-induced MMP-9 expression. Activated signals of mitogen-activated protein kinases (MAPKs) increased BFT-stimulated HCA7 cells. Infection with lentivirus containing dn-JNK significantly decreased MMP-9 expression compared with lentiviruses containing control DNA in BFT-stimulated cells. However, lentiviruses containing dn-ERK2 and dn-p38 did not inhibit the MMP-9 mRNA expression in HCA7 cells stimulated with BFT.

Conclusion These results suggest that BFT induces MMP-9 in BFT-stimulated intestinal epithelial cells through JNK and AP-1 signaling.