The korean journal of orthodontics (대한치과교정학회지)

The Korean Association Of Orthodontists (대한치과교정학회)
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A study on the regulatory effect of p-38 MAP kinase on nitric oxide and interleukin-6 in osteoblasts

조골세포에시 p-38 MAP kinase의 nitric oxide 및 interleukin-6 생성조절에 관한 연구

  • Lee, Kyung-Won (Department of Orthodontics, School of Dentistry, Wonkwang University) ;
  • Lee, Doe-Hoon (Department of Orthodontics, School of Dentistry, Wonkwang University) ;
  • Kang, Kyung-Hwa (Department of Orthodontics, School of Dentistry, Wonkwang University) ;
  • Kim, Sang-Cheol (Department of Orthodontics, School of Dentistry, Wonkwang University)
  • 이경원 (원광대학교 치과대학 교정학교실) ;
  • 이도훈 (원광대학교 치과대학 교정학교실) ;
  • 강경화 (원광대학교 치과대학 교정학교실) ;
  • 김상철 (원광대학교 치과대학 교정학교실)
  • Published : 2003.06.01
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Abstract

Tooth movement is the result of bone metabolism in the periodontium, where various cytokines take important roles. Interleukin-6(II-6) and nitrous oxide (NO) were reported to be secreted from osteoblasts in the process of bone resorption. The mechanism of the process has not been clearly understood, but the activation of mitogen-activated protein kinase (MAPK) was known to be an important process in the release of the inflammatory cytotines in macrophages. In this regard, to prove the role of MAPK in the release of IL-6 and NO in MC3T3E-1 osteoblasts, Northern blot analysis, Western blot analysis and immune complex kinase assay were used. As a result, the treatment of MC3T3E-1 osteoblast cultures with combined $interferon-\gamma(IFN-\gamma)$, lipopolysaccharide (LPS) and tumor necrosis $factor-\alpha(TNF-\alpha)$ induces expressions of inducible nitric oxide synthase (iNOS) and IL-6, resulting in sustained releases of large amounts of NO and IL-6. However, $IFN-\gamma,\;LPS,\;and\;TNF-\alpha$ individually induce a non-detectable or small amount of NO and IL-6 in MC3T3E-1 osteoblasts. The role of MAPK activation in the early intracellular signal transduction involved in iNOS and IL-6 transcription in the combined agents-stimulated osteoblasts has been investigated. The p38 MAPK pathway is specifically involved in the combined agents-induced NO and IL-6 release, since NO and IL-6 release in the presence of a specific inhibitor of p38 MAPK, 4-(4-fluorophenyl)-2-(4-metylsulfinylphenyl)-5-(4-metylsulfinylphenyl)-5-(4-pyridyl)imidazole) (SB203580), were significantly diminished. In contrast, PD98059, a specific inhibitor of MEK1, had no effect on NO and IL-6 release. Northern blot analysis showed that the p3a MAPK pathway controlled the iNOS and IL-6 transcription level. These data suggest that p38 MAPK play an important role in the secretion of NO and IL-6 in $LPS/IFN{\gamma}-or\;TNF-\gamma-treated\;MC3T3E-1$ osteoblasts.

치아이동 시 발생하는 골흡수에서 이미 여러 cytokine의 중요성이 강조된 바 있으며 이 가운데 interleukin-6는 구강 및 연골조직 등에서 많은 연구의 초점이 되어 왔으나 확실한 기전은 아직까지 정확히 확립되어 있지 못하다 골흡수 시 조골세포에서 유리되는 interleukin-6 (IL-6)와 nitric oxide (NO) 등이 골흡수의 조절자로 최근 대두되고 있으며 Mitogen-activated Protein kinase (MAPK)의 활성화로 인해 염증성 cytokine등이 유리될 수 있음이 최근 macrophage 등에서 증명된 바 있다. 그러므로 치아이동을 비롯한 구강 내 여러 염증의 조건에서 골흡수의 대표인자인 IL-6및 NO유리가 MAPK등의 활성 등을 통해 조절될 수 있는 가능성을 시사하고 있다. 본 연구에서 조골세포 특징을 대부분 가지고 있는 조골세포주 MC3T3El에서 p-38 MAP kinase을 매개로 NO와 IL-6가 유리됨을 확인하고자 하였다. $10\%$ Fetal Bovine Serum이 첨가된 -MEM 배양액으로 배양한 조골세포주인 MC3T3El 세포에 tumor necrosis $factor-\alpha(TNF-\alpha)$, $interferon-\gamma(IFN-\gamma)$ 및 lipopolysacchalide(LPS) 등의 단독처리 시 NO와 IL-6의 증가는 확인되지 않았으나 $TNF-\alpha/IFN-\gamma$ 혹은 $LPS/IFN-\gamma$ 등의 처치시 NO와 IL-6의 유의한 증가를 보였으며, NO발현에 직접 관여하는 inducible nitric oxide synthase (iNOS)와 IL-6 단백질 및 mRNA의 발현을 관찰하였다. 또한 specific p-38 MAP kinase inhibitor인 SB203580의 NO와 IL-6의 생성 억제를 관찰하고 단백질과 mRNA발현억제를 통해서도 확인함으로써 SB203580은 transcription 단계에서 NO와 IL-6의 생성을 조절하고 있음을 시사하여 주고 있다. $TNF-\alpha/IFN-\gamma$ 혹은 $LPS/IFN-\gamma$ 처치 시 p-38 MAP Kinase의 활성을 관찰하였으나 단독 처치 시 역시 P-38 MAP Kinase의 활성을 확인함으로써 NO와 IL-6생성기전에는 p-38 MAP Kinase이외에 다른 인자 역시 관여하고 있음을 보여주고 있다. 본 연구에서는 치아 등의 골조직의 구성 세포인 조골세포에서 NO와 IL-6유리를 확인하였으며, 또한 이들의 생성기전중의 하나로 p-38 MAP Kinase가 transcription 단계에서 관여하고 있음을 확인하였다.

Keywords

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