第二型轉麸胺酶透過β型轉化性生長因子抑制單鈉尿酸鹽結晶引起之發炎反應

研究目的：痛風的成因是由於血液裡尿酸濃度過高造成單鈉尿酸鹽(monosodium urate；MSU）沉積於體內關節及關節周邊所引起的發炎性疾病。在先前的文獻指出，在由MSU crystal所引起的小鼠腹腔發炎模式中，第二型轉麩胺酶 (transglutaminase 2；TG2) 會調節凋亡細胞的清除而抑制發炎。本實驗主要探討TG2於MSU crystal誘發之發炎反應中所扮演之角色。研究方法及資料：蒐集痛風病人關節之滑膜組織以及關節液以分離單核球。分別利用組織免疫染色(Immunohistochemistry)與RT-PCR (Reverse Transcriptase-PCR) 檢測TG2的表現量。接著利用MSU crystal引發小鼠巨噬細胞株(RAW 264.7)產生發炎反應，使用RT-PCR 與ELISA (enzyme-linked immunosorbent assay) 檢測IL-1 (Interleukin- 1)、TNF-α (Tumor necrosis factor-alpha) 與TGF-β1 (Transforming growth factor β1)。為了進一步探討TG2參與MSU crystal誘發發炎之相關性，利用TG2過度表現之RAW264.7細胞株(TG2 o/e)以及抑制TG2之小干擾RNA (TG2 siRNA) 來研究TG2在MSU crystal引起發炎的角色。另外，使用西方墨點法探討MSU crystal在TG2基因踢除之小鼠胚胎纖維母細胞(TG2 KO MEF cells)中對JAK2之磷酸化影響。轉移相關基因1 (Metastasis associated gene 1, MTA1) 也使用小干擾RNA來研究其在MSU crystal誘發發炎之相關性。最後，利用TG2缺失小鼠檢測TG2於活體內參與MSU crystal 誘發之發炎效應。研究結果：在痛風病人的關節液及滑膜當中發現TG2表現量增加。MSU crystal刺激RAW 264.7細胞株之後IL-1β、TNF-α、TGF-β1和TG2之mRNA及蛋白產生皆增加。在TG2遭抑制之TG2-siRNA RAW 264.7細胞株及TG2 KO MEF細胞株中，IL-1β的mRNA和細胞激素表現量提高且TGF-β1的mRNA和細胞激素表現量降低。說明TG2參與TGF-β1之合成分泌。相反地，在TG2 o/e RAW 264.7細胞株中，MSU crystal所誘發之IL-1β及TNF-α之mRNA表現量會較野生型RAW 264.7細胞株減少；而TGF-β1的mRNA表現會提高。而TG2在MSU crystal誘發發炎之訊號路徑上，結果顯示TG2過度表達降低JAK2之磷酸化，而透過小干擾RNA抑制TG2結果剛好相反。最後，在TG2之轉錄因子MTA1以小干擾RNA抑制時，細胞激素IL-1β與TNF-α表現量提高且TGF-β1的表現量降低。以外加合成之TG2蛋白則可扭轉小干擾RNA抑制MTA1此一結果。小鼠活體內，實驗結果發現TG2缺乏將導致小鼠腹腔內MSU crystal誘發之發炎效應加劇。結論：實驗發現MTA1可調控TG2之生成最後導致TGF-β1產生而抑制且自我緩解MSU crystal誘發之發炎。同時，在訊號路徑上，TG2扮演一個限制JAK2 磷酸化之角色，從而抑制JAK-STAT訊號傳遞而調控發炎現象。

ABSTRACTObjective: Transglutaminase 2 (TG2), a protein crosslinking enzyme with multiple biochemical functions, has been involved in bacterial lipopolysaccharide (LPS) induced inflammation through nuclear factor-kappa B (NF-kappaB). In this study, the specific role of TG2 in monosodium urate (MSU) crystal–induced inflammation was studied.Method: Immunohistochemistry, reverse transcription (RT)-PCR were performed to detect TG2 expression in synovial fluid mononuclear cells (SFMCs) and synovial tissue from patients with GA. MSU crystals stimulated RAW264.7 mouse macrophages were analyzed for IL-1β, tumor necrosis factor (TNF-α), transforming growth factor β1 (TGF-β1) and TG2 expression by RT-PCR and ELISA. TG2 small interfering (si) RNA-mediated knockdown and overexpression in RAW264.7 cells were used to evaluate the role of TG2 in resolving MSU crystal-induced inflammation. MSU crystal–induced JAK2 phosphorylation was detected in TG2 null mouse embryonic fibroblasts (MEFs) by immunoblotting. In addition, the role of metastatic tumor antigen 1 (MTA1), a master chromatin modifier, was investigated by MTA1small interfering (si) RNA-mediated knockdown. In addition, the inflammatory responses were followed in wild type and TG2 null mice after being challenged with MSU crystals in an in vivo peritonitis model.Results: TG2 expression was up-regulated in the synovium tissue and SFMCs from patient with GA. The level of IL-1β, TNF-α, TGF-β1 and TG2 were consistently increased in MSU crystals-stimulated RAW 264.7 cells. The absence of TG2 resulted in increased IL-1β and TNF-α but reduced TGF-β1 production in MSU crystal-stimulated RAW264.7 and TG2 null MEF cells. TG2 is needed for the production of TGF-β1. On the contrary, TG2 overexpression dramatically suppressed IL-1β and TNF-α but significantly enhanced TGF-β1. Meanwhile, MSU crystals induced phosphorylation of JAK2 was decreased in TG2 overexpression cells and TG2 knockout caused overactivation of JAK2. While studying the impact of MTA1 status on MSU crystal–induced inflammation, we found that si-MTA1 impairs the basal as well as the MSU crystal-induced expression of TG2 and TGF-β1 but increased IL-1β and TNF-α. In contrast, recombinant TG2 reversed the effect of MTA1 knockdown on MSU crystal-induced inflammation. Finally, TG2-deficient mice exhibited hyper inflammatory responses after being challenged with MSU crystals in an in vivo peritonitis model.Conclusions: These findings reveal an inherent regulatory role of TG2 upon theexpression of MTA1 and suggest that TG2 regulation of TGF-β1 may contribute to self-limitation and resolution of MSU crystal–induced inflammation. TG2 is also required for restriction against JAK2 phosphorylation and thus regulates MSU crystal–induced inflammation.