The crucial role of LKB1 in the regulation of hepatic glucose metabolism

고유진 성균관대학교 일반대학원 2014 국내석사

Liver kinase B 1 (LKB1), as a tumor suppressor, is initially identified as the product of the gene mutated in the autosomal dominantly inherited Peutz-Jeghers syndrome. Recently, it has been reported that LKB1 is a key regulator of AMPK activity in glucose metabolic pathway. LKB1 phosphorylates AMPK to inhibit glucose production and lipid synthesis in the liver. However, it is not clear how LKB1 is regulated by upstream signal. First, I wanted to confirm it the functional significance of LKB1 in vivo. So, I generated Cre adenovirus mediated in liver-specific LKB1 knockout mice to determine the effect of LKB1 deletion in glucose metabolism. Next, to discover the potential modification site of LKB1 that is involved in the regulation of AMPK, I generated expression vectors for 4 LKB1 mutants by site-directed mutagenesis ; phosphorylation defective forms (T363A and S428A) and phosphorylation mimic form (T363E and S428E). To confirm whether the DNA constructs work well before experiment, I performed expression test in HepG2 cells. All of DNA constructs were expressed well and showed similar expression levels. Last, to test whether there are functional differences between LKB1 wild type and its mutants, I observed phospho-AMPK levels in HeLa cells, and measured CRE luc activity in HepG2 cells. I found that T363A mutant failed to function properly compared to wild type. This data suggested that Thr 363 might be one of the important regulatory sites for LKB1 functions. Based on this data, I will further identify upstream signals of LKB1 that are involved in hepatic glucose metabolism. LKB1은 상염색체 우성 유전질환인 Peutz-Jeghers syndrome에서 돌연변이가 일어나 있는 것을 처음으로 발견하면서 tumor 억제 유전자로 알려졌다. 최근에 들어서는 LKB1이 포도당 대사과정에 있어서 AMPK의 활성을 조절하는 중요한 요소임이 보고가 되었다. 즉, LKB1은 AMPK를 인산화 하여 간에서의 포도당의 생산과 지방합성을 방지시킨다. 그러나 LKB1이 그 과정 중 상위 단계의 어떤 신호를 통해서 조절 받는지에 대해서는 확실하게 밝혀지지 않았다. 먼저 mice 내에서의 LKB1의 기능적 중요성에 대해 알아보기 위해서 LKB1 flox/ flox mice에 Cre recombinase를 발현하는 adenovirus를 미정맥 주사하여 Cre recombinase에 의한 LKB1 knockout 효과를 확인해보았다. 다음으로 LKB1의 AMPK 활성화와 관련된 modification site를 찾기 위하여 우리는 site-directed mutagenesis를 이용해서 4개의 LKB1의 mutant를 만들어내었다. 인산화 비활성화 형태인 T363A와 S428A, 인산화의 모방 형태인 T363E와 S428E이 그 것이다. 실험에 앞서서 만들어낸 DNA construct들이 비슷한 정도로 발현을 하는지 살펴보았고, 마지막으로 LKB1 wild type과 mutant construct들이 HeLa cell, HepG2 cell에서 차이를 갖고 다르게 기능하는지를 확인해 보았다. 이 실험결과들을 비교해보았을 때 363번 Threonine기가 LKB1이 기능하는데 있어서 중요한 위치 중에 하나일거라고 생각하였다. 이러한 실험결과를 토대로 우리는 간에서의 포도당 대사과정과 연관된 LKB1의 상위단계의 신호전달 경로에 대해 밝혀낼 수 있을 것이다.

Identification of regulatory mechanisms of CYP24A1 expression and VDR activity in Lung cancer

박건석 아주대학교 2021 국내석사

LKB1 (STK11) is a tumor suppressor gene as a serine/threonine kinase that regulates various biological functions such as transcriptional control of cells, cell growth, cell polarity, and cell mobility. LKB1 is mutated about 17 to 30% in lung cancer cells, the effect of treatment with the LKB1 mutation is still insufficient. Therefore, we tried to discover new signal transduction regulated by LKB1. We found in a preliminary research that the tumor suppressor gene LKB1 regulate CYP24A1 expression. CYP24A1 (Cytochrome P450 family 24 subfamily A member 1) is an enzyme that catabolizes vitamin D, 1,25-dihydroxyvitamin D3 (calcitrol). which has various biological functions in the body, and is essential for vitamin D signaling. But recently, CYP24A1 overexpression was reported on many lung cancer cancers as oncogene. Increased CYP24A1 offsets the anti-cancer mechanism of VDR, but it is speculated that the mechanism of regulation of CYP24A1 expression is not yet clear. In this paper, we identified increase in aerobic glycolysis in LKB1 mutant type cells compared to LKB1 wild type. The resulting evidence suggests that lactate by aerobic glycolysis contributes to the increase of CYP24A1 expression. Conversely, the glucose starvation capable of inhibiting glycolysis reduced CYP24A1 expression, consistently NaHCO3 and NaOH treatments that induce alkalization also reduced expression of CYP24A1. In addition, low of cytosolic pH by glycolysis plays an important role in acidification of lysosome through V-ATPase, that essential for increasing CYP24A1 expression. Moreover, we suggest that calcium-dependent transcription factors TFEB regulates CYP24A1 expression in lysosomes. Thapsigargin (TG), Tunicamycin (TM), which is well known as an ER stressor, inhibits calcium release to lysosomes along with calcium depletion in ER, decreases function through disruption of lysosome, and increases pH in lysosome, significantly reducing the expression of CYP24A1. Our data suggest that a novel tumor suppressor mechanism of LKB1, the vitamin D metabolic pathway, and an anti-cancer strategy to neutralize acidic tumor microenvironment caused by overexpression of CYP24A1 through elevated lactate in LKB1 mutant cells. LKB1(STK11)은 종양 억제유전자로써, 세포의 전사적 제어, 세포의 성장, 세포 극성, 세포 이동성 등 다양한 생물학적 기능을 조절하는 세린/트레오닌 키나아제 이다. LKB1은 폐암세포에서 약 17~30%정도 돌연변이 되어있으며, 아직까지LKB1돌연변이에 의한 치료제의 발굴은 미흡한 상황이다. 따라서 우리는 LKB1에 의해 조절되는 새로운 신호전달을 발굴하고자 하였다. 우리는 예비 연구에서 종양 억제 유전자 LKB1이 CYP24A1 발현에 영향을 미치는 것을 발견했다. CYP24A1(사이토크롬 P450 패밀리 24 하위 패밀리 A 멤버 1)는 비타민 D,1,25-디하이드록시비타민 D3(칼시트리올)을 분해시키는 효소로써 몸에 다양한 생물학적 기능을 가지는 비타민 D 신호 전달에 필수적이다. 그러나 최근 폐암세포에서 높은 CYP24A1 발현이 종양 유전자로 보고되었다. 게다가 CYP24A1의 과 발현은 VDR의 항암 효과를 상쇄한다고 보고되었지만, 아직까지 폐암에서 CYP24A1발현조절 기전은 아직 명확하지 않다. 이 연구를 통해 우리는 LKB1 야생 형과 비교하여 돌연변이 세포에서 호기성 해당과정의 증가를 확인하였고, 그 결과 해당과정으로 인한 젖산의 생성 증가는 CYP24A1 발현의 증가에 기여함을 제시하였다. 반대로 호기성 해당과정을 억제할 수 있는 저포도당 상태는 젖산을 감소시켰으며, 그로 인해 CYP24A1 발현이 감소되었으며, NaHCO3 처리 및 NaOH 에 의한 세포 외 알칼리화 는 CYP24A1 발현을 감소시킴을 확인하였다. 또한 해당과정 증가에 의한 세포질 pH의 감소는 CYP24A1 발현 증가에 필수적인 V-ATPase를 통한 리소좀의 산성화에 중요한 역할을 하며, 우리는 칼슘 의존적 전사 인자인 TFEB가 리소좀 에서 CYP24A1 발현을 부분적으로 조절 함을 밝혀냈다. ER 스트레스 유발 물질로 잘 알려진 Thapsigargin (TG), Tunicamycin (TM)은 ER의 칼슘 고갈과 함께 리소좀으로의 칼슘 방출을 억제하고, 리소좀 붕괴를 통해 기능을 저하시키고, 리소좀의 pH를 증가시켜 CYP24A1의 발현을 현저하게 감소시킨다. 우리는 LKB1의 새로운 종양 억제 메커니즘인 비타민 D 대사 경로에서 비타민 D분해 효소인 CYP24A1의 과발현으로 인한 내성 폐암 세포에서 산성 종양 미세 환경을 중화시키는 항암 전략을 제시한다.

Development and application of aryl hydrocarvon receptor(AhR) activity assay systems in cancer

장희진 아주대학교 2023 국내석사

Aryl hydrocarbon receptor (AhR) is one of the basic hex-loop-helx families and is activated by ligand. Activated AhR mainly plays an important role in toxic substance metabolism and inflammation. Recent studies suggest that AhR can be a target for cancer treatment because AhR is abnormally activated in various carcinomas and affects the prognosis of cancer patients. In a previous study, we confirmed a mutually exclusive tendency between AhR and LKB1 through TCGA data of cancer patients. In addition, positive correlations were confirmed when AhR, LKB1, and AMPK expressions were examined in LKB1 wild-type and mutant-type lung cancer patients. Therefore, we studied to confirm the correlation and causal relationship between the LKB1/AMPK pathway and AhR in lung cancer cells. It was confirmed that when the LKB1/AMPK pathway is activated, AhR expression decreases, and when AhR expression increases, the LKB1/AMPK pathway decreases. In addition, we confirmed the anticancer effect of inhibiting the growth of cancer cells when AhR activity was inhibited using doxycyclin. Therefore, we constructed a Lucifrease assay system to measure the activity of AhR, a transcription factor, and confirmed whether it acts specifically on AhR. However, when screening AhR inhibitors using the Luciferase assay, it is impossible to confirm whether DNA binding is directly inhibited by the AhR inhibitor. There is a need to build a transcription factor direct binding assay system in order to secure the disadvantages. This study presents an AhR inhibitor screening system that shows anticancer effects by establishing an AhR activity measurement system. Aryl hydrocarvon receptor(AhR)은 기본 helx-loop-helx 계열 중 하나로 Ligand에 의해 활성화된다. 활성화된 AhR은 주로 독성물질 대사와 염증에 중요한 역할을 한다. 최근 연구를 통해 다양한 암종에서 AhR이 비정상적으로 활성화되어 있으며 암 환자의 예후에도 영향을 주기 때문에 AhR은 암 치료의 표적이 될 수 있음을 시사합니다. 우리는 이전연구에서 암환자의 TCGA data를 통해 AhR과 LKB1 사이의 상호 배타적 경향을 확인했습니다. 또한, LKB1 wild type과 mutant type 폐암환자에서 AhR, LKB1, AMPK 발현을 조사했을 때 서로 positive correlation관계를 확인했습니다. 이에 이번 연구에서 우리는 폐암세포에서도 LKB1/AMPK pathway와 AhR 사이의 상관관계와 인과관계를 확인하기 위해서 연구했다. LKB1/AMPK pathway가 활성화되면 AhR 발현이 감소되고 AhR 발현이 증가되면 LKB1/AMPK pathway가 감소되는 것을 확인했습니다. 또한 우리는 Doxycyclin을 이용해 AhR 활성을 억제했을 때 암세포의 성장이 억제되는 항암 효과를 확인했다. 따라서 우리는 전사인자인 AhR의 활성을 측정하기 위해 Lucifrease assay system을 구축했으며 이것이 AhR 특이적으로 작용하는지 확인했습니다. 그러나 Luciferase assay를 이용하여 AhR inhibitor를 스크리닝할 경우 AhR inhibitor에 의해 DNA 결합이 직접적으로 억제되는지 여부를 확인하는 것은 불가능합니다. 이러한 단점을 보완하기 위해서는 전사인자 직접 결합 분석 시스템 구축이 필요성이 있습니다. 이번 연구는 AhR 활성 측정 시스템을 구축하여 항암효과를 보이는 AhR inhibitor 스크리닝 system을 제시합니다.

Development and application of transcription factors TFEB/TFE3 activity assay system in cancer

김도연 아주대학교 2023 국내석사

Cancer cells survive in various metabolic and oxidative stress condition. In previous study AMPK/LKB1 activated in nutrient depletion condition, and NRF2 activated by toxin accumulation condition. In previous study between AMPK/LKB1 and NRF2 interaction, NRF2 was activated by regulating TFEB/TFE3 expression by AMPK/LKB1. Therefore, we found TFEB/TFE3 is an important regulator, but there is no drugs targeting TFEB/TFE3. So we established various TFEB/TFE3 assay system to measure activity to use in the drugs screening. Transcriptional factor EB/E3 (TFEB/TFE3) is a master regulator of lysosome biogenesis. At first, we checked TFEB/TFE3 affected in lung cancer, and confirmed that there was an anticancer effect during TFEB/TFE3 double knock down. CLEAR motif Luciferase system was established to measure activity of TFEB/TFE3, and it might be used for inhibitor screening in future. Based on previous studies, TFEB/TFE3 activity was affected by lysosomal stress, lysosomal acidity system was established to measure lysosomal stress in real time. This can be used to elucidate the role of TFEB/TFE3 in lysosome stress in future. Then, when the correlation between LKB1 and TFEB/TFE3 was checked, it was found that LKB1 activates TFEB/TFE3 via PP2A. Also, it was confirmed that LKB1 regulates the activation of TFEB/TFE3 by pH. By confirming with the lysosome and cytosol pH system, LKB1 was required for maintaining lysosome and cytosol pH. Based on this, we thought that LKB1 affects v-ATPase, a type of lysosome pump that delivers hydrogen ions through the regulation of TFEB/TFE3 expression. 암의 다양한 대사스트레스와, 환경스트레스에서 암세포는 살아 남게 되는데, 선행연구에 의하면 이때 영양소가 결핍 되어 있을 경우에는 AMPK/LKB1 이 활성화 되고, 독성물질이 축적되어 있을 경우 NRF2 가 활성화된다. 이들의 상호작용에 관해 연구 진행 결과 AMPK/LKB1 에 의해 TFEB/TFE3 발현을 조절하여 NRF2 가 활성화 됨을 알게 되었다. 따라서 우리는 TFEB/TFE3 가 중요한 조절자 임을 알았지만, 현재까지 TFEB/TFE3 를 억제하는 약물이 존재 하지 않다. 이에 TFEB/TFE3 에 대한 약물을 스크리닝 할 때 사용할 수 있는 다양한 TFEB/TFE3 활성도를 측정할 수 있는 시스템을 확립 했다. TFEB/TFE3(Transcriptional factor EB/E3)는 전사인자로서 리소좀 생합성을 조절하는 대표적인 조절 전사 인자 이다. 우선 TFEB/TFE3 에 의해 폐암 세포에 영향이 있는지 확인을 하였고, TFEB/TFE3 동시 발현억제시 항암 효과가 있음을 확인하였다. 그후 CLEAR motif luciferase system 을 확립하여 TFEB/TFE3 의 활성을 측정할 수 있게 하였고, 이러한 시스템을 통해 추후에 억제제 스크리닝에 사용될 수 있을 것이라 생각 한다. 리소좀 스트레스에 의해 TFEB/TFE3 가 영향을 받기 때문에, 리소좀 스트레스를 실시간으로 측정 할 수 있는 리소좀 산성도 시스템을 구축 하였다. 이 또한 추후에 리소좀 스트레스 에서 TFEB/TFE3 의 상관관계를 확인할 수 있을 것이다. 이러한 시스템을 사용하여 AMPK/LKB1 과 TFEB/TFE3 의 상관관계를 확인 하였을 때, LKB1 이 PP2A 통해서 TFEB/TFE3 를 활성화 시킨다는 사실을 알았다. 또한 세포의 산성도에 의해서도 TFEB/TFE3 가 조절됨을 확인했다. 실시간 리소좀과 세포질 산성도를 측정하는 시스템에 의해 확인을 한 결과, LKB1 이 있을 때 리소좀과 세포질 산성이 유지 되는 반면 LKB1 이 돌연변이 되어있을 경우 리소좀과 세포질 산성이 유지 되지 않음을 확인 했다. 따라서 LKB1 이 TFEB/TFE3 발현 조절을 통해 수소이온을 전달하는 수소이온 이동 펌프에 작용하여 리소좀과 세포질 산성도 유지에 필요로 함을 확인 했다.

Novel connection between tumor suppressor LKB1 and vitamin D signaling in lung cancer

신은애 아주대학교 아주대학교 일반대학원 2018 국내석사

Liver kinase B1 (LKB1) is mutated in certain types of cancers including ~30 % of lung cancer cases and in Peutz-Jeghers syndrome (PJS) which is characterized by development of benign intestinal hamartomatous polys. Although its role in tumor suppression has been extensively studied, related mechanisms have not been fully understood. Here, we show that LKB1 regulates the vitamin D signaling pathway via a novel tumor suppressor mechanism. Vitamin D has been consistently known as one of the essential nutrients for basic human health as well in cancer patient health. It is well-known that vitamin D deficiency is commonly found in patients with cancer. We found that LKB1 deficiency increased expression of the cytochrome P450 family 24 subfamily A member 1 (CYP24A1) that induces degradation of calcitriol, the active metabolite of vitamin D. LKB1 reconstitution in LKB1-deficient tumor cells suppressed CYP24A1 expression and provoked vitamin D receptor (VDR) activity. Silencing CYP24A1 expression in LKB1-deficient tumor cells promoted vitamin D receptor (VDR) activity and enhanced calcitriol-induced VDR activation, suggesting that the LKB1-CYP24A1 axis regulates VDR activity. In summary, we investigated mechanisms of LKB1-induced CYP24A1 expression and its role in tumor development. Our data suggest that regulation of CYP24A1 mRNA expression is mediated by LKB1 via miRNA. 1983년 이래로 국내 사망원인 1위는 현재까지 암이 차지하고 있는 실정이다. 그 중에서도 폐암으로 인한 사망률은 2015년 기준 10만명당 34.1명으로 간암에 비해 10명 이상 높은 수치로 1위를 독점하였다. 폐암은 재발률이 높고 화학적치료요법에 대한 내성이 강해 아직까지 현대 과학기술로 치료는 물론 진단의 한계를 겪고 있어 치명적인 암 종류 중 하나로 알려져 있다. 폐암에서는 종양억제인자 LKB1이 ~30% 정도 돌연변이 되어있다고 잘 알려져 있으며 우리는 흥미롭게도 예비연구에서 microarray 분석을 통해 LBK1 돌연변이와 CYP24A1 유전자의 상호연관성을 발견하게 되었다. LKB1의 돌연변이는 체내에서 다양하게 생물학적 작용을 가지는 활성 형태의 vitamin D를 분해하는 효소인 CYP24A1의 발현을 높이며, CYP24A1의 높은 발현은 많은 암에서 발견되어 있는 특징 중 하나로 잘 알려져 있다. 그러나 그 기전은 아직 명확하지 못한 상황이다. LKB1 또한 여러가지 하위 유전자가 밝혀져 있으나 암을 조절하는 구체적 신호전달 경로에 대한 연구가 많이 부족하다. 따라서 이 연구에서는 종양억제 유전자 LKB1이 CYP24A1대사효소를 promoter activity에 의한 조절이 아닌 후성 유전적 조절을 통해 간접적으로 mRNA 발현을 억제함으로써 암을 억제할 수 있는 새로운 기전을 밝혔다.

LKB1 is regulated by p53 in response to glucose deprivation

신하연 경희대학교 대학원 2011 국내석사

The tumor suppressor LKB1 is an evolutionarily conserved serine/theronine kinase. In humans, LKB1 can be inactivated either by germ line mutations resulting in Peutz-Jeghers syndrome or by somatic mutations causing predisposition to multiple sporadic cancers. LKB1 has wide-ranging functions involved in tumor suppression and cell homeostasis, including establishing cell polarity, setting energy metabolic balance (via phosphorylation of AMP-dependent kinase), regulating the cell cycle, and promoting apoptosis. LKB1 function was previously linked to the tumor suppressor p53 and shown to activate the p53 target gene p21/WAF1. To investigate correlation between p53 and LKB1, we used HEK293, HCT116 p53 WT and Null cell line. In this study, we show that protein levels of LKB1 in HCT116 p53 null cells are more down-regulated than them of HCT116 p53 WT Cells by glucose deprivation. Here, the protein levels of LKB1were increased over-expression of p53 WT. Also, the LKB1 mRNA levels were increased by over-expression of p53 WT but not p53 DN(dominant negative). Therefore, these results indicate that the p53 is positive regulator of LKB1 in glucose deprivation.

Effects of Ursolic Acid and Loquat Leaf Extract on AGEs Formation and Pro-inflammatory Signaling in db/db Mouse Liver

현민경 부산대학교 대학원 2019 국내석사

Hyperglycemia induces oxidative stress and leads to formation and accumulation of advanced glycation endproducts (AGEs) in diabetes mellitus (DM), in which proteins produced from non-enzymic glycation reactions. Bioactivities of loquat leaf extract (LE) are known for liver function improvement such as anti-diabetic effects and anti-glycative effects. Ursolic acid (UA) is a major compound of LE, which has anti-hyperglycemic, antioxidant and anti-inflammatory properties. Although UA and LE were shown to suppress AGE/RAGE signaling in Type 1 diabetes mellitus (T1DM), its roles in T2D liver is not fully defined. Here, the study showed inhibitory effects of UA and LE on ROS generation, AGE formation, and pro-inflammatory mediators in db/db mouse model and HepG2 cells. The UA and LE administrated orally at 50 mg/kg/day for 15 days to db/db mice. Results showed that UA and LE significantly suppressed hepatic reactive oxygen species (ROS). Additionally, UA and LE attenuated AGEs formation, mitogen-activated protein kinase (MAPK), and pro-inflammatory gene expressions in liver. Furthermore, UA reduced AGE formation and nuclear factor kappa B (NF-κB) activity through inhibition of glucose-induced ROS in HepG2 cells. The in silico molecular docking simulation results predicted that UA binds to liver kinase B1 (LKB1) with significant affinity. Moreover, UA phosphorylated AMP- activated protein kinase (AMPK) through LKB1 activation, and regulated transcription factor forkhead Box O3 (FoxO3), which initiates transcription of antioxidant genes such as superoxide dismutase 2 and catalase. Therefore, UA may suppress ROS via LKB1/AMPK/FoxO3 pathway. Together, these data suggest that UA and LE inhibited ROS-induced AGEs and pro-inflammatory mediators through LKB1/AMPK/FoxO3 pathway in diabetic liver, and this study indicates the potential therapeutic role of UA and LE, which modulates hepatic injury by a mechanism of LKB1/AMPK/FoxO3 in DM.

The Mechanism of dual activation and interaction of AMPK and NRF2 during metabolic stress

유환식 아주대학교 아주대학교 일반대학원 2018 국내석사

영양소의 부족은 종양 미세 환경의 특징 중 하나이기 때문에 종양 세포는 생존을 위해 대사 스트레스에 대한 효율적인 적응 기전을 나타내야 한다. LKB1-AMPK 경로가 NADPH 및 산화 환원 균형의 유지를 통해 암의 대사 적응 기전에 중요한 역할을 하는 것으로 나타났지만 그에 대한 보완 기전으로 여러 기전이 있을 수 있다. 더욱이, p62 의존성 Autophagy 경로에 대해서는 알려진 바가 없다. 여기에서 우리는 LKB1-AMPK 경로 이외에도 항산화 유전자 발현의 주요 조절 인자인 NRF2의 활성화와 p62 의존성 Autophagy 경로를 통해 또 다른 항산화 기전을 활성화 시킬 수 있음을 보여준다. 우리는 대사 스트레스 동안 NRF2 의 활성화가 ROS 의 증가에 의존한다는 것을 발견했다. ROS 는 두가지 기작을 통해 NRF2 를 활성화 시킨다 : 1) 대사 스트레스가 KEAP1 의 ROS- 및 p62- 의존성 Autophagic Degradation 을 통해 NRF2를 활성화 시킨다. 2) 대사 스트레스 동안 AMPK 및 NRF2의 이중 활성화는 항산화 방어기작을 상승 시켜 세포 생존을 촉진시킨다. 또한 대사 스트레스는 p62 의 증가를 통해 대사 적응 기전을 가진다 : 1) 대사 스트레스 동안 활성화된 p62 는 AMPK 와 상호 보완적 관계를 갖는다. 2) 대사 스트레스 동안 LKB1-AMPK 는 p62 와 함께 AXIN 과 결합하여 상호 보완적인 활성화를 일으킨다. 3) 대사 스트레스에 의해 조절되는 AMPK 는 p62 mRNA 안정성의 증가를 촉진하여 p62 의 발현 수준을 높인다.. As nutrient deficiency is one of the hallmarks of tumor microenvironment, tumor cells should develop efficient adaptive responses to metabolic stress for their survival. Although the LKB1-AMPK pathway has been shown to play a key role in metabolic adaptation in cancer through the maintenance of NADPH and redox balance (1, 2), multiple mechanisms could be involved in as complementary mechanisms. Moreover, there is no known about the p62-dependent autophagy pathway. Here, we show that in addition to the LKB1-AMPK pathway, metabolic st-ress can also activate another antioxidant defense mechanisms through ac-tivation of NRF2, which is the master regulator of antioxidant gene expres-sion, and p62-dependent autophagy pathway. We found that NRF2 activation during metabolic stress isdependent on the increase of ROS. ROS activate NRF2 through two mechanisms: 1) Metabolic stressactivates NRF2 via ROS- and p62-dependent autophagic degradation of KEAP1 and 2) Dual activation of AMPK and NRF2 during metabolic stress synergizes antioxidant defense to promote cell survival. In addition, metabolic stress has a metabolic adaptation mechanism through an increase of p62: 1) Activated p62 during metabolic stress has a complementary relationship with AMPK. 2) AMPK, which is regulated by metabolic stress, promotes the increase of p62 mRNA stability and thus enhances the expression level of p62.

세포노화 및 TPA?유도 역노화에서 protein kinase C 동종효소의 역할규명

이윤영 아주대학교 2016 국내박사

Cellular senescence plays an important role in biological processes such as development, aging and tumorigenesis, and it is a process of permanent growth arrest when cells lose ability to reactivate cell division cycle. One of the common molecular features of senescent cells is the failure of phospho-extracellular signal-regulated protein kinase 1/2 (pErk1/2) translocation to nuclei in response to growth factor stimulation. However, when senescent cells are treated with 12-O-tetradecanoylphorbol-13-acetate (TPA), old cell morphology starts to change young cell like in addition to molecular markers of cellular senescence, such as increases of DNA synthesis, pRB hyperphosphorylation, reductions of p53, p21Sdi1/WAF1/CIP1 and SA-β-galactosidase activity. Mechanism of how 12-O-tetradecanoylphorbol-13-acetate (TPA) bypasses cellular senescence was investigated using replicative senescence of HDF cells and DMBA-TPA induced carcinogenesis in CD-1 mice. Upon TPA treatment, PKCα and PKCβ1 played differentially in the nuclear translocation of senescence-associated pErk1/2, which regulates reversal of senescence; PKCα carried pErk1/2 to nuclei after freed from PEA-15pS104 by PKCβ1, and then rapidly degraded by ubiquitination. MAPK docking motif and kinase activity in PKCα were required to carry pErk1/2. Moreover, repetitive application of TPA on mouse skin revealed significant loss of PKCα expression along with acanthosis in epidermis and hair follicle, indicating that downregulation of PKCα was accompanied with epidermal proliferation. The above observations were further supported by the RNA-seq analyses in HDF old cells: TPA-mediated PKCα degradation allows pErk1/2 to be free to promote cell proliferation in both senescence and carcinogenesis, emphasizing the role of PKC isozymes and cytoplasmic pErk1/2 in the regulation of cellular senescence. Mitochondrial dysfunction is linked between age-related accumulation of oxidative damage and alterations of physiological function associated with senescence. Recent studies suggest that mitochondrial metabolism is upregulated in oncogene induced senescent cells to meet with metabolic demand of cytokine production. In addition, a partial uncoupling of oxidative phosphorylation in mitochondria has been reported in the senescence fibroblast cells; thus ATP production is insufficient despite increased oxygen consumption. We report herein increased mass and DNA contents of mitochondria in the replicative HDF cells along with mitochondrial hyperfusion, and elevated expression of OXPHOS complex 4 and 5 proteins than those of the young cells. Furthermore, increased 5-bromo-2'-deoxyuridine (BrdU) incorporation at the mitochondrial nucleoid along with mitochondrial transcription factors A (TFAM). Nevertheless, we observed that mitochondria dysfunction was increased via alteration of ROS level, integrity of membrane potential, mitochondria cristae structure and ATP content. To explore signaling pathways regulating the phenotypes, we analyzed the mitochondrial TFAM protein expression which was found to be significantly increased in old cells. Up regulation of TFAM was accompanied with increased PGC-1α and NRF1 expressions through the activations of LKB1 and AMPK due to increased activity of PKCζ in old cells. These signaling pathways are important in regulating the mitochondrial encoding OXPHOS complex subunit genes and respiration as well as ATP generation, evidenced by employing siRNA against PKCζ. All of the findings were further confirmed in the doxorubicin-induced premature senescence of young HDF cells. These datas indicate that continuous increase of BrdU positive cells and mitochondrial biogenesis is regulated by LKB1, AMPK and PKCζ, despite higher ROS accumulation in stress induced senescence model. In summary, the activation of mitochondrial biogenesis pathway via PKCζ-LKB1-AMPK in senescent cells might be due to the compensation of mitochondrial dysfunction, which stimulated maintaining of nucleoid structure and TFAM activity via PGC-1α and NRF1 increased by LKB1-AMPK-PKCζ signal pathway. Our present study suggests a new concept about mitochondrial function in old cells and offers a plausible explanation on the role of mitochondrial hyperfunction in senescence, being a survival reaction when exposed to lower energy condition and cell stress.

(The) effects of cocaine on the AMPK system in the rat brain

Xu, Shijie 서울대학교 대학원 2013 국내석사

Introduction: Cocaine is a potent psychostimulant that inhibits the dopamine transporter and increases the dopamine (DA) content in the frontal cortex and striatum. Acute treatments with cocaine increases psychological and physical activities in the animal model and thus can serve as a model of mania. In the striatum, where dopamine content is related to the motivated action and psychological energy, it is expected that the energy metabolism of brain would be increased by the cocaine treatment. The AMP-activated protein kinase (AMPK) not only plays a key role in sensing intracellular ATP levels, but also acts as a crucial component in maintaining the energy balance within cells. AMPK can be activated directly by three kinases, LKB1, TAK1 and CaMKKβ and can affect the downstream ACC. Members of EGR family genes known to play a prominent role in neuronal plasticity and may therefore participate in mediating long-term adaptations produced by cocaine in the brain. The expression of these genes reflects short-term neural activity. So it is important to detect whether the systemic administration of cocaine affected the activity of AMPK system proteins and the expression of EGR in the rat brain. Methods: Male Sprague-Dawley rats (n=24) were randomly assigned to either the cocaine (15mg/kg) group or the control (saline, 0.9% NaCl) group. The animals were decapitated at the 0.5 and 2 hours after injection. The locomotor activity of rats was recorded, and their frontal cortex and striatum were dissected and immunoblot analysis was performed. Results: The locomotor activity in the cocaine group was significantly higher compared to the control group and it was normalized at 1 h. The phosphorylation level of AMPKα (Thr172) was significantly higher after cocaine injection at 30 min (p=0.04) in the frontal cortex. But no significant differences were found between the two groups at 2 h. The phosphorylation level of LKB1 (Ser431) and CaMK4 (Thr196) was significantly increased after cocaine injection at 30min compared to control (p<0.05, respectively) in the frontal cortex. The differences disappeared at 2 h. The phosphorylation level of ACC (Ser79) did not change significantly after cocaine injection in the frontal cortex. However, the administration of cocaine significantly decreased p-AMPKα (Thr172) and LKB1 (Ser431) level (p<0.05, respectively) at 30 min in the striatum. The differences disappeared at 2 h. The other proteins were not significantly affected by cocaine in the striatum. The expression of EGR1 was significantly higher after cocaine injection at 30min comparing with saline injection group in the frontal cortex, but no significant differences were found after 2h. However, the expressions of EGR1 were significantly higher at both 30min and 2h in the striatum. Conclusion: After an acute treatment with cocaine, the phosphorylation level of AMPK was increased in the frontal cortex, while it decreased in the striatum. It was suggested that cocaine activated AMPK via LKB1 and CaMK4 in the frontal cortex, but LKB1 only affected AMPK phosphorylation in the striatum. The level of p-AMPK and LKB1 showed opposite directions between striatum and cortex. Although EGR1 was increased in both tissues, suggesting the neuronal activation, AMPK signaling was different between the two tissues. Moreover, the downstream ACC phosphorylation was not affected despite the alterations in the phosphrylation of AMPK, which suggests that the role of AMPK system in the brain after cocaine treatment needs a further investigation.