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B형 간염바이러스 HBx 유전자에 의한 p53 조절기전 연구
Reports NRF is supported by Research Projects( B형 간염바이러스 HBx 유전자에 의한 p53 조절기전 연구 | 2004 Year 신청요강 다운로드 PDF다운로드 | 차만영(연세대학교) ) data is submitted to the NRF Project Results
Researcher who has been awarded a research grant by Humanities and Social Studies Support Program of NRF has to submit an end product within 6 months(* depend on the form of business)
사업별 신청요강보기
  • Researchers have entered the information directly to the NRF of Korea research support system
Project Number C00021
Year(selected) 2004 Year
the present condition of Project 종료
State of proposition 재단승인
Completion Date 2007년 10월 30일
Year type 결과보고
Year(final report) 2007년
Research Summary
  • Korean
  • Wnt/β-catenin pathway는 발생학에서 발견된 pathway이지만 이것이 대장암의 원인이 되는 주요 pathway라는 것이 밝혀짐에 따라 oncology측면에서 많은 연구가 진행이 되어왔다. 하지만 간암에서의 Wnt pathway의 역할은 아직 명확하게 밝혀지지 않음에 따라 본 연구에서는 간암에서의 Wnt signaling pathway에 대해서 연구를 진행하였다.
    우선 여러 가지 hepatoma cell lines을 이용하여 각각의 Wnt signaling activation을 reporter assay와 immunostaining으로 조사를 하였다. 그 결과 HBV가 감염되어 있는 cell lines-Hep3B, PLC/PRF/5-에서는 Wnt signaling activation이 관찰되었지만, HBV가 감염되어 있지 않은 cell lines-Huh7, Chang, SK-Hep1-에서는 그러한 activation이 관찰되지 않았다. 이에 hepatoma cell에서는 HBV 감염이 Wnt signaling activation에 관여할 것이라는 가설을 세우고 특히 HBV의 X protein이 그 역할을 수행할 것이라는 가정 하에 Wnt activation이 일어나지 않는 Huh7 cell에 HBx를 Wnt-1을 expression시켜 Wnt signaling activation을 관찰하였다. 그 결과 HBx와 Wnt-1에 의해서 Wnt signaling pathway가 activation되는 것을 reporter assay와 immunostaining으로 확인을 할 수 있었다.
    HBx에 의한 Src kinase activation이 HBx의 기능에 중요하기 때문에 HBx에 의한 Src kinase activation이 Wnt signaling pathway에 영향을 주는지를 조사하기 위하여 dominant-negative Src kinase (Src D.N)를 사용하여 HBx에 의한 Src activation이 Wnt/β-catenin에 중요한지를 조사하였다. 그 결과 Src D.N에 의해서 HBx에 의한 Wnt signaling activation이 감소되는 것을 관찰하였고, 특히 glycogen synthase kinase 3 (GSK-3) activity를 suppression함으로서 작용한다는 것을 관찰하였다. 이를 통하여 HBx가 Src kinase를 activation시키고 이렇게 activated Src kinase가 GSK-3를 suppression함으로서 Wnt signaling pathway를 activation시킨다는 결론을 내릴 수 있었다.
    HBx의 다른 중요한 기능인 virus replication의 molecular mechanism에 대해서 연구를 하였다. HBx는 17 kDa의 작은 size의 viral regulatory protein으로서 nucleus에서는 transcriptional transactivator로서, cytoplasm에서는 여러 signaling pathway를 induce시킨다는 것이 보고되었다. 이러한 HBx는 viral replication에 필수적인 것이라는 것이 알려져 있으나, 그 molecular pathway에 대해서는 아직 논란의 여지가 많이 남아있다. 최근, HBx는 cytoplasm에 존재하는 viral core protein의 C-terminal을 phosphorylation 시킴으로써 encapsidation을 증가시키고 이것이 viral replication에 collaboration할 것이라는 것이 보고되었다. 이에 반해 HBx가 viral transcription을 증가 시킴으로써 viral replication을 증가시킨다는 것이 보고된바 있다. 이에 본 연구에서는 HBx의 nuclear function과 cytoplasmic function 중에 어느 function이 virus replication에 중요한 지를 systemically 조사하였다. 먼저 HBx의 localization을 조사하여, HBx localization이 그 농도에 dependent하다는 것을 관찰하였다. HBx는 낮은 농도에서는 nucleus에, 그리고 높은 농도에서는 cytoplasm에 존재함을 알 수 있었으며, 이러한 농도에서 viral replication이 모두 stimulation됨을 관찰하였다. 또한, nucleus와 cytoplasm으로 retargeting시킨 mutants로 viral replication을 조사한 결과 cytoplasm에 존재하는 HBx보다 nucleus에 존재하는 HBx가 viral replication을 더 많이 support함을 관찰 하였다. 이를 통하여 HBx의 nuclear function이 cytoplasmic function보다 viral replication에 더 중요하다는 결론을 얻을 수 있었다.
  • English
  • Wnt/β-catenin signaling contributes to diverse cellular functions, such as Drosophila wing development and colon carcinogenesis. Recently, stabilizing mutations of β-catenin, a hallmark of Wnt signaling, were documented in significant numbers of primary hepatocellular carcinomas (HCC). However, it has not been established whether the β-catenin mutation leads to the activation of Wnt/β-catenin signaling in hepatoma cells. I found that Wnt/β-catenin signaling could be activated by ectopic expression of Wnt-1 in some hepatoma cells, such as Hep3B and PLC/PRF/5 cells, but not in others, such as Huh7 and Chang cells. Importantly, I noted that the former were derived from hepatitis B virus (HBV)-infected livers, whereas the latter were derived from HBV-negative livers. It was then speculated that HBx, a viral regulatory protein of HBV, is involved in activating Wnt/β-catenin signaling in hepatoma cells. In agreement with this notion, ectopic expression of HBx along with Wnt-1 activated Wnt/β-catenin signaling in Huh7 cells by stabilizing cytoplasmic β-catenin. Further, I showed that such stabilization of β-catenin by HBx was achieved by suppressing glycogen synthase kinase 3 activity via the activation of Src kinase. In conclusion, the data suggested that Wnt-1 is necessary but insufficient to activate Wnt/β-catenin signaling in hepatoma cells and the enhanced stabilization of β-catenin by HBx, in addition to Wnt-1, is essential for the activation of Wnt/β-catenin signaling in hepatoma cells.
    HBx, a small regulatory protein of hepatitis B virus, is essential for viral replication. HBx acts as a general transcriptional transactivator in nucleus, whereas it induces multiple signaling pathways in cytoplasm. Stimulation of the viral genome replication by HBx represents one of the most important functions of HBx during infection cycle. A recent study linked the stimulation by HBx to hyperphosphorylation of core protein that leads to the enhanced encapsidation and genome synthesis. In contrast, others attributed the stimulation of the viral genome replication by HBx to its nuclear function largely acting at transcription level. To clarify this discrepancy, I asked what extent nuclear or cytoplasmic HBx contributes to the genome replication. I first attempted to relate its expression level to its subcellular localization. Intriguingly, I found that its abundance primarily affects its subcellular localization in that it is confined to the nucleus at lower abundance, whereas it is predominantly detected in the cytoplasm when overexpressed. Further, I found that HBx, which expressed from endogenous viral promoter in a replicon context, was predominantly localized to nucleus. This finding is reinforced by the analysis of retargeting variants of HBx that harbor either nuclear localization signal (NLS) or nuclear export signal (NES) at its N-terminus. Importantly, NLS-HBx supported the viral genome replication, whereas cytoplasmically localized NES-HBx has impaired ability to support the viral genome replication. The data presented here led me to conclude that nuclear HBx, rather than cytoplasmic HBx, is linked to the stimulation of the viral genome replication.
Research result report
  • Abstract
  • Wnt/β-catenin signaling contributes to diverse cellular functions, such as Drosophila wing development and colon carcinogenesis. Recently, stabilizing mutations of β-catenin, a hallmark of Wnt signaling, were documented in significant numbers of primary hepatocellular carcinomas (HCC). However, it has not been established whether the β-catenin mutation leads to the activation of Wnt/β-catenin signaling in hepatoma cells. I found that Wnt/β-catenin signaling could be activated by ectopic expression of Wnt-1 in some hepatoma cells, such as Hep3B and PLC/PRF/5 cells, but not in others, such as Huh7 and Chang cells. Importantly, I noted that the former were derived from hepatitis B virus (HBV)-infected livers, whereas the latter were derived from HBV-negative livers. It was then speculated that HBx, a viral regulatory protein of HBV, is involved in activating Wnt/β-catenin signaling in hepatoma cells. In agreement with this notion, ectopic expression of HBx along with Wnt-1 activated Wnt/β-catenin signaling in Huh7 cells by stabilizing cytoplasmic β-catenin. Further, I showed that such stabilization of β-catenin by HBx was achieved by suppressing glycogen synthase kinase 3 activity via the activation of Src kinase. In conclusion, the data suggested that Wnt-1 is necessary but insufficient to activate Wnt/β-catenin signaling in hepatoma cells and the enhanced stabilization of β-catenin by HBx, in addition to Wnt-1, is essential for the activation of Wnt/β-catenin signaling in hepatoma cells.
    HBx, a small regulatory protein of hepatitis B virus, is essential for viral replication. HBx acts as a general transcriptional transactivator in nucleus, whereas it induces multiple signaling pathways in cytoplasm. Stimulation of the viral genome replication by HBx represents one of the most important functions of HBx during infection cycle. A recent study linked the stimulation by HBx to hyperphosphorylation of core protein that leads to the enhanced encapsidation and genome synthesis. In contrast, others attributed the stimulation of the viral genome replication by HBx to its nuclear function largely acting at transcription level. To clarify this discrepancy, I asked what extent nuclear or cytoplasmic HBx contributes to the genome replication. I first attempted to relate its expression level to its subcellular localization. Intriguingly, I found that its abundance primarily affects its subcellular localization in that it is confined to the nucleus at lower abundance, whereas it is predominantly detected in the cytoplasm when overexpressed. Further, I found that HBx, which expressed from endogenous viral promoter in a replicon context, was predominantly localized to nucleus. This finding is reinforced by the analysis of retargeting variants of HBx that harbor either nuclear localization signal (NLS) or nuclear export signal (NES) at its N-terminus. Importantly, NLS-HBx supported the viral genome replication, whereas cytoplasmically localized NES-HBx has impaired ability to support the viral genome replication. The data presented here led me to conclude that nuclear HBx, rather than cytoplasmic HBx, is linked to the stimulation of the viral genome replication.
  • Research result and Utilization method
  • 간암을 일으키는 가장 중요한 인자로서 HBV인 것이 밝혀진 것은 매우 오래전 일이지만, 그 molecular mechanism에 대해서는 알려진 바가 없었다. 본 논문에서는 hepatitis B virus의 X protein이 oncogenic signal로 잘 알려진 Wnt signaling pathway를 activation함으로써 간암을 유발시킨다는 것을 밝혔다. 이를 통하여 hepatitis B virus에 의한 간암 발생 mechanism을 명확히 함과 동시에 더 나아가 이를 이용한 치료법 개발에도 응용될 수 있을 것이라 예상한다.
    그 다음 주제로서 HBx에 의한 viral replication에의 증가 영향에 대한 연구를 진행하였다. HBx는 HBV의 중요한 viral regulatory protein으로써 HBV replication시에 HBx의 존재가 필수적인 것이 알려져 있으나, 이 HBx는 세포내에서 그 기능과 localization이 명확하게 규명되지 않은 상태이다. 본 논문에서는 HBx의 localization을 조사하고 tagging을 통해서 세포에서의 위치를 relocalization한 후에 replication에의 영향에 대해서 조사를 하였다. 그 결과 HBx는 세포내에서 핵과 cytoplasm에 그 농도에 따라서 다르게 분포 함을 알 수 있었으며, 그 mechanism은 다르겠지만 두 위치의 HBx 모두 viral replication에 영향을 준다는 것을 알 수 있었다. HBV는 위에서 언급한 바와 같이 간암을 일으키는 중요한 인자중에 하나이며, 따라서 HBV의 replication을 억제시키는 것은 이를 치료할 수 있는 중요한 clue가 될 수 있다. 이러한 viral replication에 그 viral protein인 HBx가 어떠한 mechanism으로 작용을 하는지를 파악하는 것은 단지 학술적인 의미뿐만이 아니라, 산업적으로도 응용될 가능성이 있을 것이라 예상한다.
  • Index terms
  • Hepatitis B virus, HBx, viral replication, Wnt/?]-catenin signaling pathway, Src kinase
  • List of digital content of this reports
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