PERK-eIF2a-ATF4 경로에 의한 ferroptosis 조절 기전 규명 = Regulation of ferroptosis via the PERK-eIF2a-ATF4 pathway

다국어 초록 (Multilingual Abstract)

Ferroptosis, a regulated form of cell death characterized by lipid peroxidation and iron accumulation, is increasingly recognized for its role in disease pathogenesis. The unfolded protein response (UPR) has been implicated in both endoplasmic reticulum (ER) stress and ferroptosis-mediated cell fate decisions; yet, the specific mechanism remains poorly understood. In this study, we demonstrated that ER stress induced by tunicamycin and ferroptosis triggered by erastin both activate the UPR, leading to the induction of ferroptotic cell death. This cell death was mitigated by the application of chemical chaperones and a ferroptosis inhibitor. Among the three arms of the UPR, the PERK-eIF2α-ATF4 signaling axis was identified as a crucial mediator in this process. Mechanistically, the ATF4-driven induction of DDIT4 plays a pivotal role, facilitating ferroptosis via the inhibition of the mTORC1 pathway. Furthermore, acetaminophen (APAP)-induced hepatotoxicity was investigated as a model of eIF2α-ATF4-mediated ferroptosis. Our findings reveal that the inhibition of eIF2α-ATF4 or ferroptosis protects against APAP-induced liver damage, underscoring the therapeutic potential of targeting these pathways. Overall, this study not only clarifies the intricate role of the PERK-eIF2α-ATF4 axis in ER-stress-and erastin-induced ferroptosis but also extends these findings to a clinically relevant model, providing a foundation for potential therapeutic interventions in conditions characterized by dysregulated ferroptosis and ER stress.

국문 초록 (Abstract)

지질 과산화와 철 축적을 특징으로 하는 조절된 세포 사멸 형태인 **페롭토시스(ferroptosis)**는 질병 병태생리에 관여하는 중요한 기전으로 점차 주목받고 있다. **미접힘 단백질 반응(unfolded protein response, UPR)**은 소포체(endoplasmic reticulum, ER) 스트레스뿐만 아니라 페롭토시스 매개 세포 운명 결정에도 관여하는 것으로 보고되어 왔으나, 그 구체적인 기전은 아직 충분히 규명되지 않았다.
본 연구에서는 tunicamycin에 의해 유도된 ER 스트레스와 erastin에 의해 유도된 페롭토시스가 모두 UPR를 활성화시키며, 이로 인해 페롭토시스성 세포 사멸이 유도됨을 입증하였다. 이러한 세포 사멸은 화학적 샤페론 및 페롭토시스 억제제의 처리에 의해 완화되었다.
UPR의 세 가지 신호 경로 중 PERK–eIF2α–ATF4 신호축이 이 과정의 핵심 매개 인자로 확인되었다. 기전적으로, ATF4에 의해 유도되는 DDIT4의 발현 증가가 mTORC1 경로의 억제를 통해 페롭토시스를 촉진하는 데 중요한 역할을 수행함을 규명하였다.
또한 eIF2α–ATF4 매개 페롭토시스의 모델로서 아세트아미노펜(APAP) 유발 간독성을 분석하였다. 그 결과, eIF2α–ATF4 신호 또는 페롭토시스의 억제가 APAP에 의한 간 손상을 효과적으로 보호함을 확인하였으며, 이는 해당 경로를 표적으로 하는 치료 전략의 가능성을 시사한다.
종합하면, 본 연구는 ER 스트레스 및 erastin 유도 페롭토시스에서 PERK–eIF2α–ATF4 축의 정교한 역할을 명확히 규명함과 동시에, 이를 임상적으로 중요한 질환 모델로 확장함으로써, 페롭토시스와 ER 스트레스의 조절 이상이 관여하는 질환에서의 잠재적 치료 전략을 위한 기초를 제공한다.

목차 (Table of Contents)

• PART I The PERK-eIF2α-ATF4 Axis Is Involved in Mediating ER-Stress-Induced Ferroptosis via DDIT4-mTORC1 Inhibition and Acetaminophen-Induced Hepatotoxicity 1
• I.I ABSTRACT 2
• I.II INTRODUCTION 3
• I.III MATERIALS AND METHODS 5
• 1. Animal 5
• 2. Hepatic damage assay 5
• 3. Chemicals and reagents 6
• 4. Cell Culture 6
• 5. Real-time quantitative RT-PCR 7
• 6. Western blot analysis 9
• 7. Small interfering RNA transfection 10
• 8. Adenovirus infection 10
• 9. Cell viability 11
• 10. Measurement of Lipid ROS 11
• 11. Iron measurement 11
• 12. Transmission Electron Microscopy (TEM) 12
• 13. Histology and immunohistochemistry 12
• 14. RNA Sequencing 14
• 15. Statistical analysis 14
• I.IV RESULTS 15
• 1. Ferroptosis pathway is enriched under ER stress condition 15
• 2. ER stress triggers either apoptosis or ferroptosis 17
• 3.The branch of PERK among UPR is activated in response to ferroptosis inducers 19
• 4. Inhibition of ER stress effectively abrogates erastin-induced ferroptosis 21
• 5. PERK signaling pathway is involved in mediating ferroptotic cell death under ER stress condition 23
• 6. PERK deficiency abolishes ferroptosis induced by erastin or cystine deprivation 25
• 7.PERK inhibition abolishes erastin-induced ferroptosis 28
• 8.PERK activator by SB202190 promotes either ferroptotic or apoptotic cell death 29
• 9. Mitochondrial dysfunction is accompanied by PERK activation during ferroptosis 31
• 10. PERK-eIF2a phosphorylation signaling pathway is a key mediator of erastin-induced ferroptosis 33
• 11. ATF4 deficiency protects against erasin-induced ferroptotic cell death 35
• 12. ATF4 plays an indispensable for ferroptosis induction triggered by erastin 37
• 13. Inhibition of mTOR pathway enhances ferroptosis through reducing GPX4 expression 39
• 14. DDIT4 as a hub gene under erastin-induced ATF4 activation 41
• 15. ATF4-mediated upregulation of DDIT4 suppress mTOR signaling 44
• 16. Knockdown of DDIT4 protects against erastin-induced ferroptosis 46
• 17. ATF4-induced DDIT4 expression and subsequent mTORC1 inhibition trigger ferroptotic signaling through reducing GPX4 expression 48
• 18. eIF2α-ATF4 signaling activated by APAP triggers ferroptosis in mouse hepatocytes 50
• 19. eIF2α-ATF4 signaling activated by APAP triggers ferroptosis in human hepatocytes 52
• 20. Ferroptosis pathway and response to unfolded protein is enriched under APAP and erastin treatment 54
• 21. Inhibition of eIF2α-ATF4 Protects the Liver from Acetaminophen-Induced Ferroptotic Damage 56
• I-V DISCUSSION 59
• I-VI CONCLUSION 64
• I-VII REFERENCES 65
• PART II PERK activation contributes to carbon monoxide-enhanced sensitivity of cancer cells to erastin-induced ferroptosis through mitochondrial ROS production 75
• II.I ABSTRACT 76
• II.II INTRODUCTION 77
• II.III MATERIALS AND METHODS 80
• 1. Reagents 80
• 2. Cell cullture 80
• 3. Western blot 81
• 4. Cell viability 82
• 5. Cell death 82
• 6. Measurement of Lipid ROS 82
• 7. Measurement of mitochondrial ROS 83
• 8. Statistic analysis 83
• II.IV RESULTS 84
• 1.Carbon monoxide sensitizes B16F10 melanoma to ferroptosis 84
• 2. Carbon monoxide sensitizes human cancer cells to ferroptosis 86
• 3. PERK activation plays an essential role for the carbon monoxide-enhanced sensitivity of cancer cells to ferroptosis 88
• 4.Loss of PERK attenuates sensitivity to carbon monoxide-induced ferroptosis 91
• 5. Mitochondrial ROS production is induced by erastin, but not RSL3, and is further amplified by carbon monoxide to enhance ferroptosis sensitivity 93
• 6. PERK activation is essential for carbon monoxide-mediated enhancement of mitochondrial ROS production in response to erastin 96
• II.V DISCUSSION 98
• II.VI CONCLUSION 101
• II.VII REFERENCES 102