트립토판에 의한 폐섬유화 촉진: TPH-1 (Tryptophan Hydroxylase-1) 증가와 AKT-mTORC1 및 TGF-β1 신호 활성화 = Tryptophan-Induced Aggravation of Pulmonary Fibrosis: Increased TPH-1 (Tryptophan Hydroxylase-1) Expression and Activation of AKT-mTORC1 and TGF-β1 Signaling

다국어 초록 (Multilingual Abstract)

Background
Idiopathic pulmonary fibrosis (IPF) is characterized by aberrant lung remodeling and excessive extracellular matrix (ECM) accumulation. Previously, we observed elevated tryptophan (Trp) levels in the lungs of patients with IPF; however, the contribution of Trp to fibrogenesis remained unclear. Therefore, this study aimed to determine whether Trp directly promotes fibroblast activation and contributes to the progression of lung fibrosis.
Methods
To determine whether Trp modulates the severity of lung fibrosis, ECM component expression was measured in Trp-treated primary lung fibroblasts. The expression of key Trp-metabolizing enzymes-tryptophan hydroxylase-1 (TPH-1) and indoleamine 2,3-dioxygenase (IDO/TDO)-was evaluated in IPF lung tissues. Additionally, the effects of exogenous Trp administration were assessed in a bleomycin-induced lung fibrosis mouse model.
Results
Exogenous Trp significantly exacerbated lung injury and fibrosis in bleomycin-treated mice. TPH-1 levels were elevated in IPF lung tissues and primary IPF fibroblasts compared to controls, whereas IDO/TDO expression showed no significant differences. In vitro, Trp treatment increased fibroblast proliferation and upregulated collagen, fibronectin, and α-smooth muscle actin, whereas Trp deprivation reduced these ECM components. Trp also enhanced activation of the AKT-mTORC1 and TGF-β1 signaling pathways in IPF fibroblasts.
Conclusion
These findings indicate that elevated Trp levels contribute to IPF progression through increased TPH-1 expression and activation of fibrogenic signaling pathways. Targeting Trp metabolism may therefore offer a potential therapeutic strategy for lung fibrosis, including IPF.

국문 초록 (Abstract)

특발성 폐섬유증(Idiopathic Pulmonary Fibrosis, IPF)은 비정상적인 폐 재형성과 과도한 세포외기질(extracellular matrix, ECM)축적을 특징으로 하는 만성 진행성 질환이다. 이전 연구에서 IPF 환자 폐 조직에서 트립토판(Tryptophan, Trp) 수치가 상승되어 있음을 확인하였으나, Trp가 섬유화 반응에 어떻게 기여하는지는 명확히 규명되지 않았다.
본 연구에서는 Trp가 폐섬유화의 진행에 미치는 영향을 규명하기 위해, 폐섬유아세포에서 ECM 단백질 발현 변화를 분석하였다. 또한 Trp 대사 관련 효소인 트립토판 하이드록실라아제-1(Tryptophan hydroxylase-1, TPH-1)과 인돌아민 2,3-디옥시게나제(indoleamine 2,3-dioxygenase, IDO/TDO)의 발현을 IPF 폐조직에서 확인하였으며, 블레오마이신 유도 폐섬유화 마우스 모델에서 외인성 Trp 투여 효과를 추가적으로 분석하였다.
연구 결과, Trp 투여는 블레오마이신 처리 마우스에서 폐손상과 섬유화를 유의하게 악화시켰다. TPH-1 발현은 IPF 폐조직과 일차 IPF 섬유아세포에서 증가하였으나, IDO/TDO 발현에는 유의한 차이가 없었다. 시험관 내 실험에서 Trp는 섬유아세포 증식을 증가시키고 콜라겐, 피브로넥틴, α-평활근액틴 발현을 상승시켰으며, Trp 결핍 환경에서는 이러한 ECM 성분이 감소하였다. 또한 Trp는 섬유아세포에서 AKT-mTORC1 및 TGF-β1 신호경로 활성화를 촉진하였다.
이러한 결과는 폐 내 Trp 증가가 대사 및 신호전달 경로를 통해 섬유아세포의 섬유화 반응을 강화함으로써 IPF의 병태 생리에 기여함을 보여준다. 더불어 Trp 대사 조절이 폐섬유증, 특히 IPF의 새로운 치료 전략으로 활용될 가능성을 시사한다.

목차 (Table of Contents)

• Contents of Figures i
• ABSTRACT ii
• I. INTRODUCTION 1
• II. MATERIALS AND METHODS 3
• 1. Reagents and Antibodies 3
• 2. Human Samples 4
• 3. Fibroblast isolation and culture 4
• 4. Proliferation assays 5
• 5. Immunoblotting 5
• 6. Enzyme-linked immunosorbent assays (ELISAs) for TPH-1 and IDO 6
• 7. Immunohistochemical staining 6
• 8. Animal model of BLM-induced lung fibrosis and treatment with L-tryptophan 7
• 9. Histological assays 8
• 10. Masson’s trichrome staining 8
• 11. Measurement of hydroxyproline in the lungs of mice 9
• 12. Compliance with Ethical Standards 9
• 13. Measurement of Active TGF-β1 9
• 14. Statistical analyses 10
• III. RESULTS 11
• 1. Treatment with TRP promotes the proliferation of IPF fibroblasts 11
• 2. Tryptophan regualtes the production of collagen, fibronectin, and α-SMA in IPF fibroblasts 11
• 3. Tryptophan upregulates AKT-mTOR signaling pathway proteins 12
• 4. Effect of tryptophan on the TGF-β signaling pathway 12
• 5. Tryptophan hydroxylase-1 is upregulated in the IPF lung and in IPF fibroblasts 13
• 6. Effect of TRP administration on BLM-induced lung injury/fibrosis 13
• IV. DISCUSSIONS 14
• V. CONCLUSION 20
• REFERENCES 21
• 국문요약 32
• 감사의 글 33
• Contents of Figures
• Fig. 1. Tryptophan (Trp) levels in the lung are elevated in idiopathic pulmonary fibrosis (IPF), and the proliferation of IPF fibroblasts is significantly increased by Trp 25
• Fig. 2. L-Trp regulates the expression of extracellular matrix (ECM) components in IPF fibroblasts 26
• Fig. 3. Trp regulates AKT-mTOR signaling and the SMAD pathway in IPF fibroblasts 27
• Fig. 4. TPH-1 levels are increased in the lungs of patients with IPF and in those of bleomycin (BLM)-treated mice 28
• Fig. 5. L-Trp exacerbates lung inflammation and fibrosis in BLM-treated mice 29