靶向肝臟星狀細胞之內質網蛋白TXNDC5抑制非典型乙型轉化生長因子訊號以延緩肝臟纖維化

Targeting Endoplasmic Reticulum Protein TXNDC5 in Hepatic Stellate Cells Mitigates Liver Fibrogenesis by Repressing Non-Canonical Transforming Growth Factor-β Signaling

洪振庭(Chen-Ting Hung)

指導教授 ： 楊鎧鍵

國立臺灣大學/醫學院/藥理學研究所/博士(2022年)

https://doi.org/10.6342/NTU202200366

摘要

背景及目的
肝臟纖維化 (Liver fibrosis, LF) 常發生於肝臟受到長期的損傷像是病毒性肝炎及過度飲酒。然而，至今尚無有效治療肝纖維化的治療方式。 先前，本實驗室找到一個內質網蛋白thioredoxin domain containing 5 (TXNDC5)，亦是蛋白質雙硫鍵異構酶家族的一員，其被證實為造成心臟、肺臟及腎臟纖維化的重要媒介。本實驗中，我們以探討TXNDC5是否也具有潛力作為治療肝纖維化的標的蛋白為目標。
方法
利用組織學及轉錄組分析肝硬化病人肝臟。Col1a1-GFPTg, Alb-Cre;Rosa26-tdTomato 及 Tie2-Cre/ERT2;Rosa26-tdTomato 老鼠被應用於確認肝損傷後TXNDC5於肝臟中各細胞類型中的蛋白表現量。 同時，應用人類肝星狀細胞株中進行體外實驗研究。利用Col1a2-Cre/ERT2;Txndc5fl/fl (Txndc5cKO)及Alb-cre;Txndc5fl/fl (Txndc5Hep-cKO) 老鼠分別在肝星狀細胞(HSC)及肝細胞(Hepatocytes)剔除TXNDC5 基因。於實驗老鼠給予四氯化碳(Carbon tetrachloride, CCl4) 及膽管結紮(Bile duct ligation, BDL) 手術誘導其肝臟纖維化。利用組織染色, second harmonic generation (SHG) 影像以及轉錄/蛋白質分析定量肝臟纖維化程度。
結果
在人類及老鼠纖維化肝臟，TXNDC5顯著地被表現在病灶中，特別是活化態肝星狀細胞。在肝星狀細胞中，TXNDC5媒介TGFβ1刺激造成纖維化反應，包括細胞活化, 增生, 存活及細胞外基質生成。此外，過度表達TXNDC5也足以造成其細胞活化等促纖維化反應。於機制上， TGFβ1 誘導內質網壓力 (ER stress)增加及ATF6媒介的轉錄調節來誘導TXNDC5的表現。 另外，TXNDC5透過自身PDI活性調控下游相關的JNK及STAT3訊號，同時，透過此機制促進肝星狀細胞活化及賦予其抗凋亡能力。專一性地剔除肝纖維化老鼠中肝星狀細胞的TXNDC5可以回復肝臟纖維化。
結語
內質網蛋白TXNDC5透過自身氧化還原相關能力促使肝星狀細胞活化, 增生以及細胞外基質生成，進而導致肝臟纖維化。因此，針對TXNDC5來減緩肝臟纖維化可作為具潛力的治療方針。

關鍵字

TXNDC5 ； 肝纖維化 ； 內質網 ； STAT3 ； JNK

並列摘要

Background Objectives: Liver fibrosis (LF) occurs following chronic liver injuries. Currently, there is no effective therapy for LF. Recently, we identified thioredoxin domain containing 5 (TXNDC5), an ER protein disulfide isomerase (PDI), as a critical mediator of cardiac, lung and kidney fibrosis. Here, we aimed to determine if TXNDC5 also contributes to LF and its potential as a therapeutic target for LF.
Design: Histological and transcriptome analyses on human cirrhotic livers were performed. Col1a1-GFPTg, Alb-Cre;Rosa26-tdTomato and Tie2-Cre/ERT2;Rosa26-tdTomato mice were used to determine the cell type(s) where TXNDC5 was induced following liver injury. In vitro investigations were conducted in human hepatic stellate cells (HSC). Col1a2-Cre/ERT2;Txndc5fl/fl (Txndc5cKO) and Alb-Cre;Txndc5fl/fl (Txndc5Hep-cKO) mice were generated to delete TXNDC5 in HSCs and hepatocytes, respectively. Carbon tetrachloride (CCl4) treatment and bile duct ligation (BDL) surgery were employed to induce liver injury/fibrosis in mice. The extent of liver fibrosis was quantified using histological staining, second harmonic generation (SHG) imaging, and transcript/protein analyses on markers of liver fibrosis.
Results: TXNDC5 was upregulated markedly in human and mouse fibrotic livers, particularly in activated HSC at the fibrotic foci. TXNDC5 was induced by TGF1 in HSCs and it was both required and sufficient for the activation, proliferation, survival and extracellular matrix production of HSC. Mechanistically, TGF1 induces TXNDC5 expression through increased ER stress and ATF6-mediated transcriptional regulation. In addition, TXNDC5 promotes liver fibrosis by redox-dependent JNK and STAT3 activation in HSCs through its PDI activity, activating HSCs and making them resistant to apoptosis. HSC-specific deletion of Txndc5 reverted established LF in mice.
Conclusions: ER protein TXNDC5 promotes LF through redox-dependent HSC activation, proliferation and ECM production. Targeting TXNDC5, therefore, could be a potential novel therapeutic strategy to ameliorate LF.

並列關鍵字

TXNDC5 ； liver fibrosis ； ER ； STAT3 ； JNK

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