Chondrocyte oxiapoptophagy mediated by 7α,25-DHC-EBI2 axis in IL-1β-induced arthritis

Abstract

7α,25-dihydroxycholesterol(7α,25-DHC)은 염증 조건 하에서 cholesterol by cytochrome P450 family 7 subfamily B member 1(CYP7B1)이라는 효소에 의해 25-hydroxycholesterol(25-HC)로부터 합성되는 옥시스테롤이며, 이는 다양한 유형의 세포에서 세포사멸과 관련되어 있다. 본 연구에서는 interleukin-1β(IL-1β)와 7α,25-DHC가 유도하는 연골세포 oxiapoptophagy에서 7α,25-DHC 수용체 Epstein-Barr virus-induced G-protein-coupled receptor 2(EBI2)의 길항작용과 그 기전을 분석하였다. IL-1β는 흰쥐 관절 연골에서 세포외 기질의 주요 구성요소인 aggrecan과 Type II collagen의 발현을 감소시켜 연골세포의 점진적인 퇴행을 증가시켰으며 세포에서 관절염의 주요 인자인 matrix metalloproteinase(MMP)-1, -3, -13과 염증 매개체인 inducible nitric oxide synthase, cycloxygenase-2, 산화질소 및 prostaglandin E2의 발현을 증가시켰다. 또한, 흰쥐 관절 연골세포에서 IL-1β는 외인성 세포사멸 인자인 caspase-8과 내인성 세포사멸 인자인 caspase-9를 활성화시켜 caspase-3과 Poly ADP-ribose polymerase의 활성화를 유도하였으며, 이와 더불어 자가포식작용 바이오마커인 beclin-1과 microtubule-associated proteins 1A/1B light chain 3B의 발현 증가를 유도하여 연골세포의 생존력을 감소시켰다. 이러한 결과는 흰쥐 연골세포에서 IL-1β가 산화적 스트레스를 동반한 연속적인 caspase 활성화에 의한 세포사멸과 자가포식작용을 동시에 유도할 수 있음을 시사한다. 또한 IL-1β에 의한 연골세포 사멸 시 25-HC와 7α,25-DHC 등과 같은 옥시스테롤 합성이 동반됨을 제시하고 있다. 이에 따라 본 연구에서는 염증 상태에서 콜레스테롤로부터 합성되는 25-HC와 그의 하위 옥시스테롤인 7α,25-DHC가 흰쥐 관절연골의 프로테오글리칸 소실의 가속화와 더불어 산화적 스트레스와 자가포식작용을 동반한 세포사멸인 oxiapoptophagy를 통해 관절연골 퇴행성을 유도한다는 것을 입증하였다. 반면 7α,25-DHC 수용체 EBI2 길항제인 NIBR189는 7α,25-DHC에 의해 유도되는 연골세포 산화작용, 세포사멸 및 자가포식작용을 억제하였다. NIBR189는 7α,25-DHC에 의해 증가된 pro-apoptotic factor 및 autophagy 바이오마커의 발현 억제를 통하여 연골세포사멸을 감소시켰으며, 생체 외 장기 배양 관절 연골에서 7α,25-DHC에 의해 매개되는 프로테오글리칸 소실을 억제하였다. 이러한 결과는 7α,25-DHC 수용체 억제 시 IL-1β에 의해 유도되는 관절연골퇴행성이 지연 또는 예방될 수 있음을 제시한다. 본 연구에서는 NIBR189는 흰쥐 관절연골에서 IL-1β가 유도하는 프로테오글리칸 소실 및 세포외 기질 성분 감소를 억제하였으며, 연골세포에서 IL-1β에 의한 연골 분해 효소 및 염증 매개체의 발현증가를 억제하였다. 또한 NIBR189는 연골세포에서 IL-1β로 유도된 세포독성과 세포사멸 및 자가포식작용을 억제하였다. 본 연구의 결과는 7α,25-DHC 수용체 EBI2의 길항작용이 IL-1β로 유도되는 연골세포 oxiapoptophagy를 억제시킬 수 있음을 시사한다. 따라서 본 연구의 결과로, EBI2 억제를 이용한 관절염 예방 및 치료전략의 방향을 제시할 수 있을 것으로 사료된다.| Oxysterol 7α,25-dihydroxycholesterol (7α,25-DHC) is synthesized by cytochrome P450 family 7 subfamily B member 1 (CYP7B1) from 25-hydroxycholesterol (25-HC) under inflammatory conditions, associated with the apoptosis of various cell types. In this study, the antagonism of the 7α,25-DHC receptor Epstein-Barr virus-induced G-protein-coupled receptor 2 (EBI2) and its mechanism were analyzed in interleukin-1β (IL-1β)- or 7α,25-DHC-induced chondrocyte oxiapoptophagy. This study showed that IL-1β induced the progressive degeneration of articular cartilage though the acceleration of proteoglycan loss in the ex vivo organ cultured explant of articular cartilage dissected from rat knee joint. Sequentially, IL-1β-induced arthritis was mediated by the suppression of extracellular matrix (ECM) component such as aggrecan and type II collagen, the expression and activation of cartilage degrading enzymes such as matrix metalloproteinase -1, -3, and -13, and even the increase of inflammatory mediators including inducible nitric oxide synthase, cyclooxygenase-2, nitric oxide, and prostaglandin E2 in primary rat chondrocytes. Furthermore, IL-1β decreased the viability of chondrocytes through apoptosis mediated by the cascade activation of pro-apoptotic caspases, such as caspase-8, -9, and -3. In addition, the expression of autophagy-related biomarkers, such as beclin-1 and Microtubule-associated protein 1A/1B-light chain 3 (LC3), was significantly increased in both chondrocytes and articular cartilage under IL-1β-induced inflammatory condition. Additionally, IL-1β did not only increase the expression of oxysterol syntheses such as CH25H and CYP7B1, but also up-regulate the synthesis of 25-HC in chondrocytes. Hence, these data indicate that IL-1β-induced arthritis is not only mediated by apoptosis of chodnrocytes, but also is accompanied with the synthesis of oxysterols such as 25-HC and 7α,25-DHC under inflammatory condition. Next, the present study demonstrated that both 25-HC and 7α,25-DHC induced the caspase dependent apoptosis, the upregulation of ROS production, and the expression of autophagy biomarkers such as beclin-1 and LC3. In addition, both oxysterols induced the progressive degeneration of articular cartilage through the acceleration of proteoglycan loss. Hence, these data indicate that both oxysterols induce the degeneration of articular cartilage through the oxiapoptophagy accompanied with oxidative stress, apoptosis, and autophagy in chondrocytes. Furthermore, the antagonism of EBI2, a receptor activated by 7α,25-DHC, did not only counteract the 7α,25-DHC-induced cytotoxicity and apoptosis, but also suppress both the production of ROS and the expression of autophagy biomarkers in chondrocytes. Moreover, NIBR189, an EBI2 antagonist, ameliorated the 7α,25-DHC-induced proteoglycan depletion in the ex vivo organ cultured explants of articular cartilage. These data suggest that antagonism of EBI2 might have an anti-catabolic effect to prevent the progressive degeneration of articular cartilage through the counteraction against to 25-HC-7α,25-DHC-induced chondrocyte oxiapoptophagy in the synovial joint under inflammatory conditions. Hence, it was hypothesized that the inhibition of EBI2 might counteract the IL-1β-induced arthritis through the suppression of chondrocyte oxiapoptophagy. NIBR189 counteracted significantly against to IL-1β-induced severe catabolic effects such as the proteoglycan depletion in ex vivo organ cultured explants of articular cartilage, the suppression of ECM components, the expression and activation of cartilage degrading enzymes, and the upregulation of inflammatory mediators. Furthermore, NIBR189 did not only ameliorate the IL-1β-induced cytotoxicity and caspase dependent apoptosis in chondrocytes, but also counteract against to IL-1β-induced autophagy. Taken together, these results suggest that antagonism of the 7α,25-DHC receptor EBI2 can inhibit 7α,25-DHC-induced chondrocyte oxiapoptophagy. Finally, as a result of this study, it is thought that the application of EBI2 inhibition can provide directions for arthritis prevention and arthritis treatment strategies.