Dev Som, Dong Yixuan, Hamilton James P
Department of Biochemistry, All India Institute of Medical Sciences, Kalyani, West Bengal, 741245, India.
Department of Physiology, Johns Hopkins University, School of Medicine, Baltimore, MD, USA.
J Mol Med (Berl). 2025 May;103(5):531-545. doi: 10.1007/s00109-025-02535-y. Epub 2025 Mar 26.
Wilson disease (WD) is a potentially fatal metabolic disorder caused by the inactivation of the copper (Cu) transporter ATP7B, resulting in systemic Cu overload and fibroinflammatory liver disease. The molecular mechanism and effects of elevated Cu on cytoskeletal dynamics in liver fibrogenesis are not clear. Here, we tested the regulation of hepatic cytoskeleton and fibrogenesis with respect to Cu overload in WD. Atp7b (knockout) mice with established liver disease, hepatocyte-specific Atp7b△ knockout mice without fibroinflammatory disease, and the age-and sex-matched controls were compared using Western blotting, real-time quantitative reverse transcription PCR (qRT-PCR), immunohistochemical (IHC) staining and transcriptomics (RNA-sequencing) analysis. In Atp7b mice with developed liver disease, there is a significant increase in cytoskeletal protein expression with a reduction in α-tubulin acetylation. In these mice before the onset of liver pathology, no significant changes in cytoskeletal nor hepatic stellate cell activation are observed. As hepatic copper levels rise, an increase in cytoskeletal proteins with a decrease in acetylated-α-tubulin/α-tubulin ratio occurs. RNA-sequencing, qRT-PCR, and immunostaining confirm that the tubulin is upregulated at the transcriptional level and hepatocytes are the primary source of early tubulin increases before fibrosis. An increase in α-tubulin with a decrease in α-tubulin acetylation via Hdac6 and Sirt2 induction facilitates fibrosis as reflected by concomitant increases in desmin and α-SMA immunostaining in Atp7b mice at 20 weeks. Moreover, strongly positive correlations between α-tubulin and α-tubulin deacetylase with the expression of liver fibrosis markers are observed in animal and human WD. Hepatocyte-specific Atp7b△ mice lack significant changes in tubulin as well as fibrosis despite hepatic steatosis. This study provides evidence that microtubule destabilization causes cytoskeletal rearrangement and facilitates hepatic stellate cell (HSC) activation and fibrosis in the murine model of WD. KEY MESSAGES: Hepatic cytoskeleton system is induced in Wilson disease. Hepatic microtubules acetylation is dysregulated in murine Wilson disease. Microtubules destabilization is positively associated with liver fibrosis in Wilson disease. Microtubules destabilization concomitant with fibrogenesis exacerbates WD progression.
威尔逊病（WD）是一种潜在致命的代谢紊乱疾病，由铜（Cu）转运蛋白ATP7B失活引起，导致全身铜过载和纤维炎性肝病。铜升高对肝纤维化过程中细胞骨架动力学的分子机制和影响尚不清楚。在此，我们研究了WD中铜过载对肝细胞骨架和纤维化的调控作用。使用蛋白质免疫印迹法、实时定量逆转录聚合酶链反应（qRT-PCR）、免疫组织化学（IHC）染色和转录组学（RNA测序）分析，对已患肝病的Atp7b（基因敲除）小鼠、无纤维炎性疾病的肝细胞特异性Atp7b△基因敲除小鼠以及年龄和性别匹配的对照小鼠进行了比较。在已患肝病的Atp7b小鼠中，细胞骨架蛋白表达显著增加，α-微管蛋白乙酰化水平降低。在这些小鼠肝脏病变发作前，未观察到细胞骨架及肝星状细胞激活有显著变化。随着肝脏铜水平升高，细胞骨架蛋白增加，乙酰化-α-微管蛋白/α-微管蛋白比值降低。RNA测序、qRT-PCR和免疫染色证实，微管蛋白在转录水平上调，肝细胞是纤维化前早期微管蛋白增加的主要来源。通过Hdac6和Sirt2诱导，α-微管蛋白增加而α-微管蛋白乙酰化减少，促进了纤维化，这在20周龄的Atp7b小鼠中表现为结蛋白和α-SMA免疫染色同时增加。此外，在动物和人类WD中，观察到α-微管蛋白和α-微管蛋白去乙酰化酶与肝纤维化标志物表达之间存在强正相关。肝细胞特异性Atp7b△小鼠尽管有肝脂肪变性，但微管蛋白及纤维化无显著变化。本研究提供了证据表明，在WD小鼠模型中，微管不稳定导致细胞骨架重排，促进肝星状细胞（HSC）激活和纤维化。关键信息：威尔逊病中肝细胞骨架系统被诱导。小鼠威尔逊病中肝微管乙酰化失调。微管不稳定与威尔逊病中的肝纤维化呈正相关。微管不稳定与纤维化同时发生会加剧WD进展。
