초파리 장조직에서 Caudal 전사조절인자에 의한 matrix metalloproteinase-1 발현 조절 Regulation of Matrix Metalloproteinase-1 Expression by the Homeodomain Transcription Factor Caudal in Drosophila Intestine원문보기
Matrix metalloproteinase (MMP)는 세포외골격의 주요 조절효소로, 배아발생, 혈관생성, 상처치료 및 조직 재생과정에 중요한 인자로 알려져 있다. MMP의 조절 이상은 비정상적 세포외골격 분해로 인해 암 전이와 같은 질병을 일으킨다. 따라서, MMP의 발현과 활성은 엄격하게 조절되고 있다. 최근, 초파리 Mmp1이 소화기관에서 강하게 발현되며, 장줄기세포의 비정상적인 활성을 억제하여 장의 항상성 유지에 중요함을 밝혔다. 하지만, 장조직에서 Mmp1의 발현 조절 기전은 아직 밝혀지지 않았다. 본 연구에서는, 장조직에서 Mmp1의 발현이 장 발생과 항상성 유지에 중요한 Caudal homeobox 유전자에 의해 조절되는지를 연구하였다. GAL4/UAS 조절계를 이용하여 장조직 특이적으로 Caudal의 발현을 감소시켰을 때, Mmp1의 발현이 감소함을 확인하였으며, Caudal을 과발현 시켰을 때, Mmp1의 발현이 증가함을 in vitro와 in vivo 실험 모두에서 확인하였다. 또한, Mmp1 promoter에 Caudal 전사인자 결합 부위가 존재하며, 이 부위가 Mmp1 발현에 중요한 역할을 함을 확인하였다. 이상의 본 연구는, 정상적 혹은 암화 과정에서 Mmp1이 Caudal의 표적 유전자일 수 있음을 의미한다.
Matrix metalloproteinase (MMP)는 세포외골격의 주요 조절효소로, 배아발생, 혈관생성, 상처치료 및 조직 재생과정에 중요한 인자로 알려져 있다. MMP의 조절 이상은 비정상적 세포외골격 분해로 인해 암 전이와 같은 질병을 일으킨다. 따라서, MMP의 발현과 활성은 엄격하게 조절되고 있다. 최근, 초파리 Mmp1이 소화기관에서 강하게 발현되며, 장줄기세포의 비정상적인 활성을 억제하여 장의 항상성 유지에 중요함을 밝혔다. 하지만, 장조직에서 Mmp1의 발현 조절 기전은 아직 밝혀지지 않았다. 본 연구에서는, 장조직에서 Mmp1의 발현이 장 발생과 항상성 유지에 중요한 Caudal homeobox 유전자에 의해 조절되는지를 연구하였다. GAL4/UAS 조절계를 이용하여 장조직 특이적으로 Caudal의 발현을 감소시켰을 때, Mmp1의 발현이 감소함을 확인하였으며, Caudal을 과발현 시켰을 때, Mmp1의 발현이 증가함을 in vitro와 in vivo 실험 모두에서 확인하였다. 또한, Mmp1 promoter에 Caudal 전사인자 결합 부위가 존재하며, 이 부위가 Mmp1 발현에 중요한 역할을 함을 확인하였다. 이상의 본 연구는, 정상적 혹은 암화 과정에서 Mmp1이 Caudal의 표적 유전자일 수 있음을 의미한다.
The matrix metalloproteinase (MMP) family plays essential roles in physiological processes such as embryonic development, angiogenesis, wound healing, and tissue homeostasis as a consequence of MMPr capacity for breaking down many types of extracellular matrix proteins. Imbalanced regulation of MMP ...
The matrix metalloproteinase (MMP) family plays essential roles in physiological processes such as embryonic development, angiogenesis, wound healing, and tissue homeostasis as a consequence of MMPr capacity for breaking down many types of extracellular matrix proteins. Imbalanced regulation of MMP expression can also lead to pathological conditions such as tumor progression. We recently reported that the Drosophila Mmp1 gene is highly expressed in the digestive tract and is required for the maintenance of intestinal homeostasis such as by restriction of uncontrolled intestinal stem cell proliferation. However, the regulatory mechanisms of MMP gene expression in the intestine remain unclear. In this study, we determined that the expression of Mmp1 is regulated by the homeodomain transcription factor Caudal. Experiments using the targeted expression of Caudal under the regulation of Gal4-UAS system indicated that endogenous Caudal is required for the Mmp1 gene expression in the adult Drosophila intestine and that exogenous Caudal induces Mmp1 expression. Transient transfection experiments indicated that Caudal can activate the promoter activity of Mmp1 and that several putative Caudal binding sites in the 5'-flanking region of the Mmp1 gene may be critical to the upregulation by Caudal. Our data suggest that Mmp1 is one of the target genes of Caudal in physiological normal condition and in tumorigenesis.
The matrix metalloproteinase (MMP) family plays essential roles in physiological processes such as embryonic development, angiogenesis, wound healing, and tissue homeostasis as a consequence of MMPr capacity for breaking down many types of extracellular matrix proteins. Imbalanced regulation of MMP expression can also lead to pathological conditions such as tumor progression. We recently reported that the Drosophila Mmp1 gene is highly expressed in the digestive tract and is required for the maintenance of intestinal homeostasis such as by restriction of uncontrolled intestinal stem cell proliferation. However, the regulatory mechanisms of MMP gene expression in the intestine remain unclear. In this study, we determined that the expression of Mmp1 is regulated by the homeodomain transcription factor Caudal. Experiments using the targeted expression of Caudal under the regulation of Gal4-UAS system indicated that endogenous Caudal is required for the Mmp1 gene expression in the adult Drosophila intestine and that exogenous Caudal induces Mmp1 expression. Transient transfection experiments indicated that Caudal can activate the promoter activity of Mmp1 and that several putative Caudal binding sites in the 5'-flanking region of the Mmp1 gene may be critical to the upregulation by Caudal. Our data suggest that Mmp1 is one of the target genes of Caudal in physiological normal condition and in tumorigenesis.
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가설 설정
Average values obtained from 3 independent experiments with ±SE values are given. B. Effect of Caudal on the activity of Mmp1 deletion reporter plasmids. Wild type pMmp1-luc or deletion reporter plasmids (100 ng) were co-transfected with the pAc-cad (600 ng) into S2 cells and the luciferase activities were normalized with co-transfected β-galactosidase activity.
The third instar larvae expressing cad under heat shock-inducible hs-Gal4 driver (hs>cad) and driver alone (hs>+) were incubated at 25℃ for 24 hr after heat shock and the epidermis (A and B) and guts (C and D) were stained with anti-Mmp1 (red), and DAPI (blue). E. Mmp1 expression level was dependent with cad expression in larvae. The third instar larvae expressing cadRNAi or cad under heat shock-inducible hs-Gal4 driver (hs>cadRNAi or hs>cad) and driver alone (hs>+) were incubated at 25℃ for 24 hr after heat shock and the whole larvae extracts were blotted with anti-Mmp1 antibody.
In this study, we investigated whether the expression of Mmp1 is regulated by Caudal transcription factor in Drosophila. We found that endogenous Caudal is required for the Mmp1 gene expression in the adult Drosophila intestine and exogenous Caudal expression can activate Mmp1 promoter in vitro and in vivo.
제안 방법
4A). To investigate the potential of Caudal binding sites in Mmp1 gene, deletion constructs of pMmp1-luc were constructed as described in the Materials and Methods and named from their sites deleted (Fig. 4A).
대상 데이터
Drosophila stocks were consistently maintained at 25℃ on standard cornmeal-sugar-yeast medium under a 12 hr/12 hr light/dark cycle. The food consisted of 79.2% water, 1% agar, 7% cornmeal, 2% yeast, 10% sucrose, 0.3% bokinin, and 0.5% propionic acid. To avoid larval overpopulation in culture vials, 25-30 adult flies were cultured in a vial and transferred to new vials containing fresh food every 2-3 days.
The samples were then washed in PBST, incubated with secondary antibodies for 45 min at 25℃, washed and mounted with Vectashield (Vector Labs, CA, USA). The images were analyzed using a Zeiss AxioSkop 2Plus microscope (Carl Zeiss Inc., Germany). Primary antibodies were diluted as follows: mouse anti-Mmp1 1:50 (Developmental Studies Hybridoma Bank, IA, USA); rabbit anti-GFP 1:500 (Molecular Probes, OR, USA).
이론/모형
After centrifugation for 5 min, these supernatant were subjected to β-galactosidase and luciferase assays. Luciferease assay was performed with a luminometer (TD-20/20, Turner Designs, CA, USA). Normalized luciferase activity was calculated by determining the luciferase/β-galactosidase activity ratio and by averaging the values from 3-8 experiments and the average values, and standard errors were calculated.
성능/효과
In addition, Cdx2 has been well known to function as tumor suppressor gene in the intestine [2,14]. From these facts, our results suggest that the regulation of MMP expression by endogenous CDX in physiological normal condition may be associated with the role of CDX as tumor suppressor.
Normalized luciferase activity was calculated by determining the luciferase/β-galactosidase activity ratio and by averaging the values from 3-8 experiments and the average values, and standard errors were calculated.
후속연구
Ectopic expression of human CDX1 has been known to be associated with intestinal metaplasia in gastric and esophageal tumors [27], suggesting that Cdx1 has an oncogenic potential. From these studies, it will be interesting to further investigate the specific functions of Caudal/CDX and MMPs in regulating ISC proliferation and tumorigenesis.
Caudal was also reported to be accumulated in the midguts with age and oxidative stress, which is mediated with NFκB [4]. Thus, the increase of MMPs expression with age and exposure to oxidative stress may also be regulated by the Caudal transcription factor, although further investigations are required. The overexpression of Mmp1 also induces intestinal stem cell proliferation [15] as consistent with a great deal of reports to show the tumorigenic function of MMP in mammal.
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