Journal of the Korea Academia-Industrial cooperation Society (한국산학기술학회논문지)

The Korea Academia-Industrial cooperation Society (한국산학기술학회)
권호 표지
DOI QR코드

DOI QR Code

Expression Study of The Mouse Collectin-Placenta 1 Gene

마우스 Collectin-Placenta 1 유전자의 발현 연구

  • Kim, Geun Ho (Division of Life Science and Biochemical Engineering, Sun Moon University) ;
  • Kim, Youn Uck (Division of Life Science and Biochemical Engineering, Sun Moon University)
  • 김근호 (선문대학교 대학원 생명공학과) ;
  • 김연욱 (선문대학교 대학원 생명공학과)
  • Received : 2019.07.02
  • Accepted : 2019.08.02
  • Published : 2019.08.31
Download PDF
⟨ Previous Next ⟩

Abstract

Several types of scavenger receptors, including the Collectin-Placenta 1 (CL-P1) receptorthat is present in mammals, are molecules that are expressed on the surfaces of endothelial cells, macrophages and smooth muscle cells. These molecules are cell surface glycoproteins that can be conjugated to oxidized low density lipoprotein (oxLDL). Among these molecules, the effect of quercetin on CL-P1 activation has been confirmed. Quercetin is known as an antioxidant that stops oxidation because it acts to remove free radicals that are responsible for the oxidation reaction. In this study, fragments from the transcription start site of the mouse CL-P1 gene promoter to the -500th base were cloned using DNA polymerase. These fragments were then introduced into macrophage like RAW264.7 cells and fibroblast-like NIH3T3 cells to study the effect of quercetin on the CL-P1 gene expression. As a result, we found that bases ranging from -250 to -350 in the anterior part where gene expression starts are important for producing CL-P1 protein. Among them, the DNA mutation experiments we performed confirmed that the E2F binding sites are critical for producing the CL-P1 protein? In addition, when quercetin was added to the RAW264.7 culture medium, which was a culture of adherent cells, observedthe phenomenon of the cells falling off from the surface of the culture container.

포유류에 존재하는 Collectin-Placenta 1 (CL-P1)을 포함한 여러 종류의 scavenger 수용체는 주로 내피 세포, 대식 세포 및 평활근 세포 표면에 발현되는 분자이다. 이들 분자는 산화 된 저밀도 지질 단백질 (oxLDL)에 결합하여 처리 할 수 있는 세포 표면 당 단백질이다. 이들 분자 중 케르세틴이 CL-P1 활성화에 어떤 영향을 미치는가를 확인하였다. 케르세틴은 산화 반응을 담당하는 자유 라디칼의 제거제 역할을 하여 산화를 중지시키는 항산화제로 알려져 있다. 본 논문에서는 마우스 CL-P1 유전자 promoter 부분의 전사 시작 점부터 -500 번째 염기까지의 단편을 DNA 중합효소를 이용하여 클로닝 하였다. 그 후에 대식세포 계열인 RAW264.7 및 섬유아세포계열의 NIH3T3 세포에 도입하여 케르세틴이 CL-P1 유전자 발현에 어떠한 영향을 미치는지에 대한 연구를 하였다. 이 부위에는 세포주기 조절 인자인 E2F 결합부위를 비롯해서 여러 종류의 전사 인자가 결합하는 염기서열이 다수 위치하고 있다. 이러한 500염기 단편을 pGL4.10 기본 벡터 및 프로모터에 연결시킨 후 세포에 도입시켰다. 그리고 배양 중에 케르세틴을 처리하여 유전자 발현양을 형광 색소 발현기법으로 측정하였다. 그 결과 유전자 발현이 시작되는 앞쪽 부분의 -250에서 -350사이의 염기들이 CL-P1 단백질을 만드는데 중요하다는 것을 확인하였다. 그 중에서도 E2F결합 부위가 결정적인 것 이라는 것을 DNA 돌연변이 실험을 통해 확인 하였다. 또한 부착 세포인 RAW264.7 배양액에 케르세틴을 첨가 한 결과, 배양용기 표면에서 탈락하는 현상을 확인하였다. 즉 발현된 CL-P1단백질이 케르세틴에 의해 세표 표면의 부착 분자에도 영향을 주는 것을 확인하였다.

Keywords

References

  1. M. S. Brown, S. K. Basu, J. R. Falck, Y. K. Ho, J. L. Goldstein, "The scavenger cell pathway for lipoprotein degradation: Specificity of the binding site that mediates the uptake of negatively- charged LDL by macrophages" J. Supramol. Struct, Vol.13, No.1, pp.67-81, 1980. DOI: http://doi.org/10.1002/jss.400130107
  2. J. L. Goldstein, Y. K. Ho, S. K. Basu, M. S. Brown, "Binding site on macrophages that mediates uptake and degradation of acetylated low density lipoprotein, producing massive cholesterol deposition" Proc. Natl. Acad .Sci. USA, Vol.76, No.1, pp.333-337, Jan. 1979. DOI: http://doi.org/10.1073/pnas.76.1.333
  3. T. Kodama, P. Reddy, C. Kishimoto, M. Krieger, "Purification and characterization of a bovine acetyl low density lipoprotein receptor", Proc. Natl. Acad. Sci. USA, Vol.85, No.23, pp.9238-9242, Dec. 1988. DOI: 10.1073/pnas.85.23.9238
  4. C. Santiago-Fernandez, L. M. Perez-Belmonte, M. Millan-Gomez, I. Moreno-Santos, F. Carrasco-Chinchilla, "Overexpression of scavenger receptor and infiltration of macrophage in epicardial adipose tissue of patients with ischemic heart disease and diabetes", J Transl Med, Vol.20, No.17, pp.95-99, Mar. 2019. DOI: http://doi.org/10.1186/s12967-019-1842-2
  5. K. Ohtani, Y. Suzuki, S. Eda, T. Kawai, T. Kase, H, "The membrane-type collectin CL-P1 is a scavenger receptor on vascular endothelial cells", J. Biol. Chem, Vol.262, No.47, pp.44222-44228, Nov. 2001. DOI: http://doi.org/10.1074/jbc.M103942200
  6. F. Bogie, J. Mailleux, E. Wouters, W. Jorissen, E. Grajchen, "Scavenger receptor collectin placenta 1 is a novel receptor involved in the uptake of myelin by phagocytes", Sci Rep, Vol.20, No.7, pp.44794-44799, Mar. 2017. DOI: http://doi.org/10.1038/srep44794
  7. K. Drickamer, "Two distinct classes of carbohydrate-recognition domains in animal lectins", J. Biol. Chem, Vol.263, No.20, pp.9557-9560, Jul. 1988. https://www.ncbi.nlm.nih.gov/pubmed/3290208/ https://doi.org/10.1016/S0021-9258(19)81549-1
  8. K. Nakamura, H. Funakoshi, K. Miyamoto, F. Tokunaga, T. Nakamura, "Molecular cloning and functional characterization of a human scavenger receptor with C-type lectin (SRCL), a novel member of a scavenger receptor family", Biochem. Biophys. Res. Commun, Vol.280, No.4, pp.1028-1035, Feb. 2001. DOI: http://doi.org/10.1006/bbrc.2000.4210
  9. R. T. White, D. Damm, J. Miller, K. Spratt, J. Schilling, "Isolation and characterization of the human pulmonary surfactant apoprotein gene" Nature, Vol.317, No.6035, pp.361-363, Sep. 1985. DOI: http://doi.org/10.1038/317361a0
  10. A. Persson, D. Chang, K. Rust, M. Moxley, W. Longmore, "Purification and biochemical characterization of CP4 (SP-D), a collagenous surfactant-associated protein", Biochemistry, Vol.28, No.15, pp.6361-6367, Jul. 1989. DOI: http://doi.org/10.1021/bi00441a031
  11. K. Ohtani, Y. Suzuki, S. Eda, T. Kawai, T. Kase, "Molecular cloning of a novel human collectin from liver (CL-L1)", J. Biol. Chem, Vol.274, No.19, pp.1368113689, May 1999. DOI: http://doi.org/10.1074/jbc.274.19.13681
  12. H. Keshi, T. Sakamoto, T. Kawai, K. Ohtani, T. Katoh, "Identification and characterization of a novel human collectin CL-K1", Microbiol Immunol, Vol.50, No.12, pp.1001-1013, 2006. DOI:https://doi.org/10.1111/j.1348-0421.2006.tb03868.x
  13. S. Jang, K. Ohtani, A. Fukuoh, T. Yoshizaki, M. Fukuda, "Scavenger receptor collectin placenta 1 (CL-P1) predominantly mediates zymosan phagocytosis by human vascular endothelial cells", J. Biol. Chem, Vol.284, No.6, pp.3956-3965, Feb. 2009. DOI: http://doi.org/10.1074/jbc.M807477200
  14. N. Roy, K. Ohtani, Y. Matsuda, K. Mori, I. Hwang, "Collectin CL-P1 utilizes C-reactive protein for complement activation", Biochim Biophys Acta, Vol.1860, No6, pp.1118-1128. Jun. 2016. DOI: http://doi.org/10.1016/j.bbagen.2016.02.012
  15. Y. U. Kim, K. Ohtani, K. Mori, S. J. Jang, Y. Suzuki, "Gene Regulation Function of the Three Specificity Protein-1 (Sp1) within the Human Collectin Placenta-1 Proximal Promoter", Genes & Genomics, Vol.33, No.3, pp.275-283, Jun 2011. DOI: http://doi.org/10.1007/s13258-011-0001-9
  16. R. J. Williams, J. P. Spencer, C. Rice-Evans, "Flavonoids: antioxidants or signalling molecules?", Free Radical Biology & Medicine, Vol.36, No.7, pp.838-849, Apr. 2004. DOI: http://doi.org/10.1016/j.freeradbiomed.2004.01.001
  17. G. L. Russo, M. Russo, C. Spagnuolo, I. Tedesco, S. Bilotto, "Quercetin: a pleiotropic kinase inhibitor against cancer", Cancer Treatment and Research, Vol.159, pp.185-205, 2014. DOI: http://doi.org/10.1007/978-3-642-38007-5_11
  18. G. Juergenliemk, K. Boje, S. Huewel, C. Lohmann, H. J. Galla, "In vitro studies indicate that miquelianin (quercetin 3-O-beta-D-glucuronopyranoside) is able to reach the CNS from the small intestine", Planta Medica, Vol.69 No.11, pp.1013-1017, Nov. 2003. DOI: http://doi.org/10.1055/s-2003-45148
  19. A. Massi, O. Bortolini, D. Ragno, T. Bernardi, G. Sacchetti, "Research Progress in the Modification of Quercetin Leading to Anticancer Agents", Molecules. Vol.29, No.22, pii: E1270, Aug. 2017. DOI: http://doi.org/10.3390/molecules22081270, Review.
  20. M. Russo, A. Milito, C. Spagnuolo, V. Carbone, A. Rosen, "CK2 and PI3K are direct molecular targets of quercetin in chronic lymphocytic leukaemia", Format: AbstractSend to Oncotarget. Vol.8, No.26, pp.42571-42587, Jun. 2017. DOI: http://doi.org/10.18632/oncotarget.17246
  21. W. W. Lorenz, R. O. McCann, M. Longiaru, M. J. Cormier, "Isolation and expression of a cDNA encoding Renilla reniformis luciferase", Proc Natl Acad Sci USA, Vol.88, No.10, pp.4438-4442, May 1991. DOI: http://doi.org/10.1073/pnas.88.10.4438
  22. A. Farr, A. Roman, "A pitfall of using a second plasmid to determine transfection efficiency", Nucleic Acids Res, Vol.20, No.4, pp.920-930, Feb. 1992. DOI: http://doi.org/10.1093/nar/20.4.920