Applied Microscopy

Korean Society of Microscopy (한국현미경학회)
권호 표지

Increased Protein of the Secretory Leukocyte Pretense Inhibitor (SLPI) and the Expression of Growth Factors in NIH3T3 Cells by LPS Stimulation

NIH3T3 세포주에서 LPS자극에 의한 분비백혈구단백분해효소억제제 (SLPI)의 단백질증가와 성장인자들의 발현

  • Lee, Sang-Hwa (Department of Periodontology, College of Dentistry, Chosun University) ;
  • Choi, Baik-Dong (Department of Oral Histology, College of Dentistry, Chosun University) ;
  • Jeong, Soon-Jeong (Department of Oral Histology, College of Dentistry, Chosun University) ;
  • Jang, Hyun-Seon (Department of Periodontology, College of Dentistry, Chosun University) ;
  • Kim, Byung-Ock (Department of Periodontology, College of Dentistry, Chosun University) ;
  • Lim, Do-Seon (Department of Dental Hygiene, Seoul Health College) ;
  • Park, Joo-Cheol (Department of Oral Histology, College of Dentistry, Chosun University) ;
  • Wang, Guan-Lin (Department of Oral Histology, College of Dentistry, Chosun University) ;
  • Jeong, Moon-Jin (Department of Oral Histology, College of Dentistry, Chosun University)
  • 이상화 (조선대학교 치과대학 치주과학교실) ;
  • 최백동 (조선대학교 치과대학 구강조직학교실) ;
  • 정순정 (조선대학교 치과대학 구강조직학교실) ;
  • 장현선 (조선대학교 치과대학 치주과학교실) ;
  • 김병옥 (조선대학교 치과대학 치주과학교실) ;
  • 임도선 (서울보건대학 치위생과) ;
  • 박주철 (조선대학교 치과대학 구강조직학교실) ;
  • 왕관림 (조선대학교 치과대학 구강조직학교실) ;
  • 정문진 (조선대학교 치과대학 구강조직학교실)
  • Published : 2006.09.30
Download PDF
⟨ Previous Next ⟩

Abstract

Secretory leukocyte protease inhibitor (SLPI) involves tissue protection against the destructive action of neutrophil elastase at the site of inflammation. Several studies on new functions of SLPI have demonstrated that SLPI may play a primary role in innate immunity than protease inhibitor, To identify the function of SLPI by lipopolysaccharide (LPS) stimulation in the embryonic fibroblast (NIH3T3) cells. we studied the expression of SLPI compared to other growth factors involving the LPS treatment. To address this, we performed the reverse transcriptase polymerase chain reaction (RT-PCR) and Western blots for the detection of mRNA and protein expression of the SLPI and some growth factors such as VEGF. bFGF, and PDGF-BB after LPS stimulation. NIH3T3 cells were exposed 100 ng/mL Escherichia coli LPS for 30min, 60min, 90min, 24h, and 48h, respectively. The result of RT-PCR showed that SLPI and VEGF mRNA was expressed strongly in NIH3T3 without related to LPS stimulation. mRNA of bFGF was weakly expressed such as the expression of the control. PDGF mRNA expression gradually increased follows at time course. However, SLPI protein level was increased in lysates and culture medium by LPS stimulation. Phase contrast microscopic and scanning electron microscopic observation showed that the increased cell number and cytoplasmic enlargement of the NIH3T3 cells. Therefore, it suggests that the LPS upregulates SLPI expression in NIH3T3 cells. Moreover, secreted SLPI may stimulate cell proliferation and migration.

분비백혈구단백분해효소억제제 (SLPI)는 염증 부위에서 중성구 엘라스타제에 의한 손상 작용에 대해 조직을 보호하는 역할을 한다. SLPI의 새로운 기능에 대한 연구들은 단백질분해효소억제제 역할보다는 선천적 면역반응 작용에 주로 관여 할 것임을 보고하였다. 따라서 본 연구는 섬유모세포 세포주(NIH3T3)에서 박테리아성지질다당류(LPS)자극에 의한 SLPI의 기능을 확인하기 위하여 LPS처리에 따른 여러 성장 인자들과 비교하여 SLPI의 발현을 알아보았다. 역전사효소 중합반응(RT-PCR)과 면역학적 단백질 검출법 (Western blot)은 LPS 처리 후 SLPI와 몇몇 성장 인자들(VEGF, bFGF, PDGF)의 mRNA와 단백질의 검출을 위해 수행하였다. NIH3T3 세포주를 mL당 100ng의 LPS에 각각 30, 60, 90분, 24, 48시간 동안 노출시켰다. RT-PCR 결과 SLPI와 VEGF mRNA는 LPS처리와는 상관없이 강한 발현 양상을 보였다. bFGF mRNA는 대조군과 같이 약하게 발현하였고, PDGF mRNA는 LPS 노출 시간에 따라 점진적으로 증가하는 양상을 나타냈다. 그러나 세포질 용해액과 세포 배양액에서 SLPI단백질의 수준은 LPS처리에 의해 증가하였다. 또한 광학 현미경 관찰과 전자 현미경 관찰은 LPS가 NIH3T3세포주의 형태학적인 변화를 유발시킴을 증명하였다. 따라서 LPS는 NIH3T3세포에서 SLPI의 발현 증가를 조절하며, 분비된 SLPI는 세포분열과 이동을 자극할 것이라는 결론을 얻었다. SLPI가 세포분열과 세포이동에 어떻게 관여하는지는 아직까지 규명되지 않은 실정이므로-추후에 SLPI단백질이나 유전자 도입을 통하여 세포 이동에 관련된 실험이 진행되어야 할 것이다.

Keywords

References

  1. Abe T, Kobayashi N, Yoshimura K, Trapnell BC, Kim H, Hubbard RC, Brewer MT, Thompson RC, Crystal RG: Expression of the secretory leukoprotease inhibitor gene in epithelial cells. J Clin Invest 87 : 2207-2215, 1991 https://doi.org/10.1172/JCI115255
  2. Albert B, Johnson A, Lewis J, Martin R, Roberts K, Walter P : The molecular biology of The cell 4rd edn pp. 1300-1301, 2002
  3. Bartold PM, Narayanan AS, Page RC: Platelet-derived growth factor reduces the inhibitory effects of lipopolysaccharide on gingival fibroblast proliferation. J Periodontal Res 27 : 499-505, 1992 https://doi.org/10.1111/j.1600-0765.1992.tb01823.x
  4. Botero TM, Mantellini MG, Song W, Hanks CT, Nor JE: Effect of lipopolysaccharides on vascular endothelial growth factor expression in mouse pulp cells and macrophage. Eur J Oral Sci 111 : 228-234, 2003 https://doi.org/10.1034/j.1600-0722.2003.00041.x
  5. Coin PG, Lindroos PM, Bird GS, Osornio-Vargas AR, Roggli VL, Bonner JC: Lipopolysaccharide up-regulates plateletderived growth factor (PDGF) alpha-receptor expression in rat lung myofibroblasts and enhances response to all PDGF isoforms. J Immunol 156 : 4797-4806, 1996
  6. Fritz H: Human mucus proteinase inhibitor (human MPI). Human seminal inhibitor I (HUSI-I), antileukoprotease (ALP), secretory leukocyte protease inhibitor (SLPI). Biol Chem Hoppe-Seyler 369 : 79-82, 1988
  7. Jeffrey RJ, Seed MP, Kircher CH, Willoughby DA, Winkler JD: The codependence of angiogenesis and chronic inflammation. FASEB J 11 : 457-465, 1997 https://doi.org/10.1096/fasebj.11.6.9194526
  8. Jin F, Carl N, Danuta R, Aihao D: Secretory leukocyte protease inhibitor: A macrophage product induced by and antagonistic to bacterial lipopolysaccharide. Cell 48 : 417-426, 1997 https://doi.org/10.1016/0092-8674(87)90193-0
  9. Kaoru T, Teruo K, Masayasu N: Lipopolysaccharide (LPS)-induced IL-6 production by embryonic fibroblasts isolated and cloned from LPS-responsive and LPS-hyporesponsive mice. Molecular Immunology 34 : 16-17, 1997
  10. Kumagai Y, Yagishita H, Yajima A, Okamoto T, Konishi K: Molecular mechanism for connective tissue destruction by dipeptidyl aminopeptidase IV produced by the periodontal pathogen Porphyromonas gingivalis. Infect Immun 73 : 2655-2664, 2005 https://doi.org/10.1128/IAI.73.5.2655-2664.2005
  11. Laurie RG, Audrey LA, Diane CS: Construction, non-denaturing affinity purification, and characterization of baculovirally expressed human secretory leukocyte protease inhibitor. Protein Expression and Purification 26 : 179-186, 2002 https://doi.org/10.1016/S1046-5928(02)00529-6
  12. Lavnikova N, Laskin DL: Unique patterns of regulation of nitric oxide production in fibroblasts. J Leukoc Biol 58 : 451-458, 1995 https://doi.org/10.1002/jlb.58.4.451
  13. Leung DW, Cachianes G., Kuang WJ, Goeddel DV, Ferrara N: Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 246 : 1306-1309, 1989 https://doi.org/10.1126/science.2479986
  14. Love RM, Jenkinson HF: Invasion of dental tubules by oral bacteria. Crit Rev Oral Biol Med 13(2) : 171-183, 2002 https://doi.org/10.1177/154411130201300207
  15. Nick D, Gholamreza HG, Jing Z, Lea B, Patrick de B, Hilde R: Secretory leukocyte protease inhibitor promotes the tumorigenic and metastatic potential of cancer cells. PNAS 100 : 5778-5782, 2003 https://doi.org/10.1073/pnas.1037154100
  16. Ohlsson K, Bergenfeldt M, Bjork P: Functional studies of human secretory leukocyte protease inhibitor. Adv Exp Med Biol 240 : 123-131, 1988
  17. Pinheiro ML, Feres-Filho EJ, Graves DT, Takiya CM, Elsas MI, Elsas PP, Luz RA: Quantification and localization of platelet-derived growth factor in gingiva of periodontitis patients. J Periodontol 74 : 323-328, 2003 https://doi.org/10.1902/jop.2003.74.3.323
  18. Prochnau D, Rodel J, Hartmann M, Straube E, Figulla HR: Growth factor production in human endothelial cells after Chlamydia pneumoniae infection. Int J Med Microbiol 294 : 53-57, 2004 https://doi.org/10.1016/j.ijmm.2003.11.001
  19. Ross HM, Romrell LJ, Kaye GI: Histology: a text and atlas, 3rd edn, pp. 107-110, 1995
  20. Slavin, J: Fibroblast growth factors; At the heart of angiogenesis. Cell Biol Int 19 : 431-444, 1995 https://doi.org/10.1006/cbir.1995.1087
  21. Stetler G, Brewer MT, Thompson RC: Isolation and sequence of a human gene encoding a potent inhibitor of leukocyte proteases. Nucleic Acids Res 14 : 7883-7896, 1986 https://doi.org/10.1093/nar/14.20.7883
  22. Sunderkotter C, Steinbrink K, Goebeler M, Bhardwaj R, Sorg C: Macrophages and angiogenesis. J Leuk Biol 55 : 410-422, 1994 https://doi.org/10.1002/jlb.55.3.410
  23. Thompson RC, Ohlsson K: Isolation, properties, and complete amino acid sequence of human secretory leukocyte protease inhibitor, a potent inhibitor of leukocyte elastase. Proc Natl Acad Sci U. S. A. 83 : 6692-6696, 1986
  24. Zhang D, Simmen RC, Michel FJ, Zhao G, Vale-Cruz D, Simmen FA: Secretory leukocyte protease inhibitor mediates proliferation of human endometrial epithelial cells by positive and negative regulation of growth-associated genes. J Biol Chem 277 : 29999-30009, 2002 https://doi.org/10.1074/jbc.M203503200