Altiratinib inhibits osteoarthritis by regulating the c-MET/β-catenin pathway = c-MET/β-catenin 경로 조절을 통한 알티라티닙의 골관절염 치료에 관한 연구

다국어 초록 (Multilingual Abstract)

Osteoarthritis (OA) is a prevalent degenerative joint disease with no effective disease-modifying therapies. The canonical Wnt/β-catenin signaling pathway is a key catabolic regulator in OA pathogenesis and a promising therapeutic target. This study aimed to investigate the therapeutic potential of the multi-kinase inhibitor altiratinib for OA and elucidate its mechanism of action on the Wnt/β-catenin pathway. The effects of altiratinib were evaluated in HEK293, MH7A, and SW1353 cell lines using Western blotting, immunofluorescence, and siRNA-mediated gene knockdown. In vivo therapeutic efficacy was evaluated in a monoiodoacetate (MIA)-induced OA rat model using gross evaluation, qRT-PCR, histological examination, and micro-computed tomography (μCT). We demonstrated for the first time that the inhibitory effect of altiratinib on Wnt/β-catenin signaling is primarily mediated by the c-MET in HEK293 cells. Specifically, altiratinib blocked c-MET-mediated phosphorylation of β-catenin at tyrosine 654 (Y654), thereby inhibiting its nuclear translocation in a dose-dependent manner. Consequently, altiratinib broadly suppressed the expression of the upstream inflammatory regulator NF-κB, inflammatory cytokines (TNF-α, IL-1β, IL-6), catabolic matrix metalloproteinases (MMP-1, MMP-3, MMP-13), and fibrosis-related proteins (Vimentin, α-SMA, Fibronectin) in MH7A cells. In SW1353 cells, altiratinib significantly inhibited the expression of inflammatory cytokines and MMP-13. In the MIA-induced OA rat model, subcutaneous administration of altiratinib mitigated joint swelling and cartilage damage, preserved subchondral bone structure, and downregulated mRNA expression of inflammation- and catabolism-related genes in synovial tissue. In conclusion, altiratinib mitigates OA progression by downregulating inflammatory and catabolic responses through inhibition of c-MET-mediated phosphorylation of β-catenin at Y654. These findings suggest that altiratinib is a novel, potent disease-modifying therapeutic candidate for osteoarthritis.

국문 초록 (Abstract)

골관절염(OA)은 효과적인 질병 조절 치료제가 부족한 만연한 퇴행성 관절 질환이다. 표준 Wnt/β-catenin 신호 전달 경로는 OA 발병 기전의 핵심 이화작용 조절자로서 유망한 치료 표적으로 주목받고 있다. 본 연구는 다중 키나아제 억제제인 알티라티닙의 OA 치료 잠재력을 평가하고, Wnt/β-catenin 경로에 대한 작용 기전을 규명하고자 하였다.
알티라티닙의 효과는 HEK293, MH7A, SW1353 세포주에서 Western blotting, 면역형광법, siRNA 매개 유전자 침묵 기법을 이용하여 분석하였으며, in vivo 치료 효능은 monoiodoacetate(MIA) 유도 OA 랫드 모델에서 육안 평가, qRT-PCR, 조직학적 분석 및 마이크로 컴퓨터 단층촬영(μCT)을 통해 평가하였다.
본 연구는 알티라티닙의 Wnt/β-catenin 신호 전달 억제 효과가 HEK293 세포에서 주로 c-MET 수용체를 통해 매개됨을 최초로 규명하였다. 알티라티닙은 c-MET 매개 β-catenin의 Tyrosine 654(Y654) 인산화를 차단하여 β-catenin의 핵 내 이동을 억제하였으며, 이를 통해 MH7A 세포에서 염증 반응, 이화작용 및 섬유화 관련 인자의 발현을 유의하게 감소시켰다. 또한 SW1353 세포에서도 염증성 인자 및 이화작용 효소의 발현을 억제하였다.
MIA 유도 OA 랫드 모델에서 알티라티닙의 피하 투여는 관절 부종과 연골 손상을 완화하고 연골하골 구조를 보존하였으며, 활막 조직에서 염증 및 이화작용 관련 유전자 발현을 감소시켰다. 결론적으로, 알티라티닙은 c-MET 매개 β-catenin의 Y654 인산화를 억제함으로써 OA의 염증 및 이화 반응을 조절하며, 골관절염에 대한 새로운 질병 조절 치료 후보 물질로서의 가능성을 시사한다.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Methods and Materials 4
• 2.1 Cell culture, reporter assay and ethanol extraction method 4
• 2.2 siRNA-mediated gene silencing 4
• 2.3 Western Blot analysis and cellular fractionation 5
• 2.4 Immunofluorescence staining 6
• 2.5 MIA-induced osteoarthritis animal model and drug administration 6
• 2.6 Macroscopic and histological analysis 7
• 2.7 Quantitative real-time PCR (qRT-PCR) 7
• 2.8 Micro-computed tomography (μCT) analysis 7
• 2.9 Statistical analysis 8
• 3. Results 10
• 3.1 Altiratinib's β-catenin regulatory effect is mediated through the MET pathway in HEK293 cells. 10
• 3.2 Altiratinib inhibits Wnt3a-Induced β-catenin phosphorylation and nuclear translocation in MH7A Cells 13
• 3.3 Altiratinib comprehensively inhibits key osteoarthritis pathological factors—inflammation, catabolism, and fibrosis—in MH7A cells 16
• 3.4 Altiratinib inhibits β-catenin nuclear translocation and pathological factor expression in the chondrosarcoma cell line SW1353. 19
• 3.5 Altiratinib alleviates joint swelling in a MIA-induced OA rat model 22
• 3.6 Altiratinib inhibits inflammatory and catabolic gene expression in a MIA-induced OA rat model 25
• 3.7 Altiratinib alleviates histological damage in articular cartilage 28
• 3.8 Altiratinib preserves subchondral bone structural integrity (μCT Analysis) 31
• 4. Discussion 34
• 5. Conclusion 37
• 6. References 38
• 7. 국문초록 40