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CaMKII Inhibits Hepatitis B Virus Replication via AMPK/AKT/mTOR Signaling Pathway

  • 김주미 (아주대학교 일반대학원)

초록/요약

- Abstract - CaMKII Inhibits Hepatitis B Virus Replication via AMPK/AKT/mTOR Signaling Pathway ` Ca2+/calmodulin‐dependent protein kinase II (CaMKII), which is involved in the calcium signaling pathway, is an important regulator of cancer cell proliferation, motility, growth, and metastasis. The effects of CaMKII on hepatitis B virus (HBV) replication have never been evaluated. Here, we found that phosphorylated, active CaMKII is reduced during HBV replication. Similar to other members of the AMPK/AKT/mTOR signaling pathway associated with HBV replication, CaMKII, which is associated with this pathway, was found to be a novel regulator of HBV replication. Overexpression of CaMKII reduced the expression of covalently closed circular DNA (cccDNA), HBV RNAs, and replicative intermediate (RI) DNAs while activating AMPK and inhibiting the AKT/mTOR signaling pathway. Findings in HBx‐deficient mutant‐transfected HepG2 cells showed that the CaMKII‐mediated AMPK/AKT/mTOR signaling pathway was independent of HBx. Moreover, AMPK overexpression reduced HBV cccDNA, RNAs, and RI DNAs through CaMKII activation. Although AMPK acts downstream of CaMKII, AMPK overexpression altered CaMKII phosphorylation, suggesting that CaMKII and AMPK form a positive feedback loop. These results demonstrate that HBV replication suppresses CaMKII activity, and that CaMKII upregulation suppresses HBV replication from cccDNA via AMPK and the AKT/mTOR signaling pathway. Thus, activation or overexpression of CaMKII may be a new therapeutic target against HBV infection. Keywords: hepatitis B virus, HBV replication, CaMKII, AMPK/AKT/mTOR, signaling pathway

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초록/요약

- 국문요약 - HBV 증식에 관여하는 CaMKII/AMPK/AKT/mTOR 신호전달 김 주 미 아주대학교 일반대학원 의생명과학과 (지도교수: 김 경 민) 칼슘 신호 전달 경로에 관여하는 Ca2+/calmodulin‐dependent protein kinase II (CaMKII)는 암세포의 증식, 운동성, 성장 및 전이의 중요한 조절제이다. CaMKII의 효과는 B형 간염 바이러스(HBV) 증식에 관해 평가된 적이 없다. 활성화 상태인 인산화 된 CaMKII가 HBV 증식하는 세포내에서 감소함을 밝혔다. HBV 증식에 관여하는AMPK/AKT/mTOR 신호 전달 경로와 연관된 CaMKII가 HBV 증식의 새로운 조절자임을 밝혔다. CaMKII의 과발현은 AMPK를 활성화하고 AKT/mTOR를을 억제하면서HBV의 cccDNA, HBV RNA, 그리고 RI DNA를 감소시켰다. CaMKII에 의한 AMPK/AKT/mTOR 신호 전달 경로는 HBx와 독립적이었다. 또한, AMPK의 과발현은 CaMKII를 활성화시켜 HBV cccDNA, RNA, 그리고 RI DNA를 감소시켰다. AMPK는 CaMKII의 downstream으로 작용하지만, AMPK의 과발현은 CaMKII의 인산화를 변화시켰다. 이를 통해 CaMKII와 AMPK가 positive한feedback loop를 형성함을 밝혔다. 이러한 결과는 HBV 증식이 CaMKII의 활성을 억제하고 CaMKII upregulation하면 AMPK/AKT/TOR의 신호 전달 경로를 통해 HBV cccDNA가 감소되어 HBV의 증식이 억제됨을 보여준다. 따라서 CaMKII의 활성화 또는 과발현은 HBV 감염에 대한 새로운 치료 target이 될 수 있다. 핵심어: B형 간염 바이러스, HBV 증식, CaMKII, AMPK/AKT/mTOR, 신호 전달 경로

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목차

Ⅰ. INTRODUCTION 1
Ⅱ. MATERIALS AND METHODS 4
A. DNA Constructs 4
B. Cell Culture 8
C. Transfection 8
D. Establishment of Stable Cells 9
E. SDS‐PAGE and Western Blotting 10
F. Core Particle Immunoblotting 13
G. Northern and Southern Blotting 13
H. HBV Preparation and Infection 14
I. cccDNA Extraction 15
J. Drug Treatment 16
K. Luciferase Reporter Assay 17
L. Quantitative Real-Time RT‐PCR and PCR 17
M. Statistical Analysis 18
Ⅲ. RESULTS 19
A. Active, Phosphorylated CaMKII Is Decreased in HBV Replicating Cells 19
B. CaMKII and AMPK Activities Tend to Be Lower in Tumor Tissues Than Adjacent Non-Tumor Tissues from Patients with HBV-Associated HCC 23
C. Effects of HBV Replication on CaMKII, AMPK, and AKT/mTOR Phosphorylation 27
D. CaMKII Overexpression Inhibits the HBV Replication through AKT/mTOR Signaling 32
E. AMPK Overexpression or Activation Inhibits HBV Replication 36
F. Inhibition of CaMKII Enhances HBV Replication 42
G. CaMKII and AMPK form a Feedback Loop 45
H. CaMKII Overexpression Reduces HBV RNAs Due to the Decreased HBV cccDNA 51
I. AMPK Overexpression Reduces HBV RNAs Due to the Decreased HBV cccDNA 55
J. Reduced HBV Replication by Overexpression of CaMKII or AMPK Is HBx Independent 57
Ⅳ. DISCUSSION 60
Ⅴ. CONCLUSION 65
REFERENCES 66

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