Statin可經由調控成骨細胞Sirtuin/FoxO3a/Cyr61反應途徑來減緩膠原蛋白所誘導之關節炎的發展

Abstract

類風濕性關節炎(Rheumatoid arthritis,RA)是由於人體產生自體免疫導致在關節處產生慢性發炎所造成的現象。關節處不斷的發炎反應會導致滑膜組織(synovial tissue)不斷的增生，逐漸增大向其他位置侵襲，造成骨頭的損失。另外慢性發炎反應的持續進行，也會吸引巨噬細胞等免疫細胞浸潤達發炎處，促進蝕骨細胞(osteoclasts)的形成，進行骨吸收的作用。目前對於其詳細的研究機制則仍待更進一步的探討。 Cyr61 ( Cysteine- rich angiogenic inducer 61)，CCN家族成員之一，目前已知在細胞生長、血管新生、腫瘤生長、發炎反應等等有關，相關文獻發現在關節炎患者中的B細胞中，Cyr61會不正常的大量表現，且Cyr61也會促進血管新生以及巨噬細胞的吸引，因此Cyr61在關節炎中可能扮演一個重要的角色。有鑒於關節炎的致病機制還不清楚，我們希望能夠透過了解Cyr61在關節炎中扮演的地位，在治療此疾病上能夠提供更有力的方法。 我們初步的研究顯示，在Collagen-Induced Arthritis(CIA)的大鼠模式中，發現受侵蝕的骨頭周圍有大量Cyr61的表現。此大量表現Cyr61蛋白質的位置恰好符合巨噬細胞的所在，初步推論Cyr61蛋白質可以吸引巨噬細胞到發炎處並進一步的破壞骨質。為了更進一步了解其中的調控機制，我們嘗試一種在治療高膽固醇疾病方面的藥物: statin。近年來，statin在治療關節炎方面的確有其療效，但對其治療效果卻有很大的差異。我們利用simvastain藥物以matrigel包裹注射至老鼠膝關節，使之穩定緩慢的釋放出來，發現經由simvastatin治療後，對關節炎的進展有舒緩的效果，證明statin在治療關節炎上極具潛力。另外在in vitro方面，發現simvastatin會經由促進FoxO3a的核轉移(nuclear translocation)，使FoxO3a活化後，對下游的Cyr61啟動子進行轉錄抑制，造成Cyr61蛋白表現量的減少。為了更進一步的了解simvastatin是透過何種機制來促進FoxO3a的活性，參考文獻中發現，FoxO3a主要是透過乙醯化(acetylation)及磷酸化(phosphorylation)的方式影響其核轉移，其中乙醯化可能是扮演FoxO3a的重要調控機制之一。另外，我們的研究也指出SIRT在冷光酶分析法(luciferase assay)中可以看到對Cyr61啟動子的抑制，而且SIRT1與FoxO3a蛋白質的交互作用，在共同免疫沉澱法(co-immunoprecipitation)實驗中，的確有結合的現象。顯示出SIRT1在FoxO3a活性的調控中，可能扮演另一個重要的角色。 未來，我們除了在simvastatin所引起下游SIRT1、FoxO3a、Cyr61的分子機制需要更清楚的釐清外。這些蛋白質在膠原蛋白所誘導的關節炎大鼠模式中，也待更進一步的了解其所扮演的地位。希望能夠建立一個完整的研究機制，試圖解釋Simvastatin是透過SIRT1來促進FoxO3a的活性，進而抑制下游基因Cyr61蛋白的表現，使得關節炎中骨頭的骨吸收程度有明顯的緩和作用。利用此完整的機制，對於未來在治療類風濕性關節炎中具有良好的效果。

Rheumatoid arthritis is chronic, inflammatory, systemic, autoimmune diseases characterized by chronic arthritis leading to progressive joint erosions. Inflammation recruits immune cells such as macrophage to the site leading to bone erosion. The recruitment of inflammatory cell is the major risk factor in the arthritis and the mechanism is needed more research. Cyr61, the CCN family, named CCN1 has been reported plays an important role in RA patients. Cyr61 mRNA has a significant increase in RA patients and promotes macrophage migration. In our collagen-induced arthritis(CIA) model, Cyr61 expressed and seems to recruit macrophage to bone resorption area means that it may promote macrophage infiltration and resulted in CIA progression. In vitro, treatment of Cyr61 recombinant protein direct promoted macrophage migration and enhanced CCL2 chemokine expression in U2OS. Thus, Cyr61 has the ability to deteriorate RA progression. Statin, a cholesterol synthesis inhibitor, has been reported to have a potential in therapy of arthritis. To investigate the effect of arthritis, we administrated simvastatin to joint space and led to alleviate CIA progression. By local injection of simvastain, this way of administration can avoid the liver-specific characteristic. In U2OS cell, treatment of simvastatin decreased Cyr61 expression via activation FoxO3a. Simvastatin activated FoxO3a through promoting its nuclear translocation. FoxO3a activity is mediated by post-transcriptional modification that includes phosphorylation and acetylation. Phosphorylation at Thr32, Ser253 and Ser315 leads to nucleus export and acetylation attenuates FoxO3a DNA binding affinity. Simvastain activated FoxO3a through decreasing its phosphorylation and acetylation. Thus, nuclear FoxO3a bound to Cyr61 promoter and repressed transcription. Deacetylation of FoxO3a seems to SIRT1-dependent manner. Treatment of Simvastatin enhanced SIRT1 activity and promoted FoxO3a interaction. The references related to statins on FoxO3a phosphorylation are much clear than acetylation. We found a way that statins mediate FoxO3a acetylation In summary, simvastatin represses Cyr61 expression through promoting FoxO3a deacetylation and nucleus translocation in vitro. In animal model, simvstatin alleviates CIA progression, decreases Cyr61 expression and promotes FoxO3a nuclear translocation that correlated with in vitro data. By confirming the SIRT1/FoxO3a/Cyr61 pathway, it will be a potential target in therapy of RA.