ScRNA-seq can more accurately analyze gene expression variability in different cell types. We utilized this technology to detect gene expression differences in non-parenchymal liver cells and found that USP18 expression was significantly reduced in activated HSCs (myofibroblasts) when compared to quiescent HSCs. Our previous research focused on the role of deubiquitination in liver disease, hence we were interested in alterations to USP18 during HSC activation, and speculated that USP18 may play an important role in maintaining HSC quiescence. Many studies have demonstrated that USP18 contributes to cell signaling, response to viral and bacterial infections, the development of various malignant tumors, and the development of autoimmune diseases16. Studies have also shown that USP18 inhibits cardiac remodeling and prevents the development of heart failure. It has also been suggested to serve as a biomarker of cardiac remodeling. The expression of USP18 was found to be significantly higher in damaged cardiomyocytes compared to healthy cells. It was postulated that the cardioprotective effect of USP18 was the result of inhibition of the TAK1-p38-/JNK1/2 signaling pathway, an example of a MAPK dependent pathway. In addition, in mouse models, it inhibits cell hypertrophy and fibrosis and delays the onset of heart failure17. One study also showed that USP18 protects against hepatic steatosis and insulin resistance10. As predicted, in primary HSCs, we confirmed that USP18 expression is significantly reduced during HSC activation, and restoration of USP18 expression inhibits HSCs activation. Therefore, USP18 plays different roles in different tissues, and even in different cell types of the same tissue.
As an important gene transcription regulator involved in immune and inflammatory responses, NF-κB is widely present in the cytoplasm of many cell types and can be activated by a variety of cytokines, lipopolysaccharides, oxygen radicals and ultraviolet rays. Previous studies on chronic viral hepatitis B have found that the expression of NF-κB p65 in liver tissue of such patients is positively correlated with the expression of type I and III collagen and the staging of liver fibrosis. This further indicates that NF-κB p65 is closely related to the occurrence and development of liver fibrosis18. It has also been reported that USP18 interacts with transforming growth factor β (TGF-β) activated kinase 1 (TAK1)/TAK-binding protein 1 (TAB1) complexes and deubiquitinates TAK1/TAB1 complexes, resulting in negative regulation of the nuclear factor κB (NF-κB) signaling pathway in T cells19. TAK1, a mitogen-activated protein kinase activated by transforming growth factor-β, phosphorylates IKKβ to activate NF-kB signaling. Relevant studies have also found that TAK1 activated by upstream regulatory factors not only aggravates the steatosis and ischemia/reperfusion injury of hepatocytes, but also mediates myocardial hypertrophy of cardiomyocytes20–22. In a follow-up mechanism study, we found that USP18 inhibits HSC activation by blocking the NF-κB signaling pathway. Based on previous research10, we showed in primary HSCs that USP18 directly binds to TAK1 and this binding gradually weakens with HSC activation. Subsequently, USP18 overexpression led to decreased phosphorylation of TAK1. In previous research, no evidence was provided to elucidate the deubiquitination site of TAK1 by USP18. We transfected primary HSCs with plasmids bearing different lysine site mutations of ubiquitin, and demonstrated that USP18 overexpression removed the k63-linked polyubiquitin chain. Thus, USP18 inhibits TAK1 activity by interfering with the k63 ubiquitination modification of TAK1.
It is known that inflammation, one of the initiating factors of liver fibrosis, affects the occurrence of fibrosis through the interactions between inflammatory cells, cytokines and related signaling pathways23. Hepatic fibrosis is a complex disease involving both repair and damage, and coregulated by a variety of cell signaling transduction pathways. For example, studies have confirmed that Nestin can affect the degradation rate of TβRI by regulating Caveolin1 and plays an important role in the activation of the TGFβ-Smad2/3 signaling pathway, a main pro-fibrotic pathway. Downregulation of Nestin can significantly alleviate tissue fibrosis, providing a potential target for reversing liver fibrosis24. Some studies have successfully transfected β-catenin siRNA molecules into HSCs, leading to silencing of β-catenin expression, inhibition of HSC proliferation and reduced collagen fiber synthesis; indicating that β-catenin inhibits HSCs activation and alleviates liver fibrosis by down-regulating the Wnt/β-catenin signaling pathway25. Our study has confirmed that stable expression of USP18 can inhibit HSC activation. Moreover, in the liver fibrosis model, USP18 overexpression can significantly alleviate liver fibrosis. The onset and development of liver fibrosis is a dynamic process. At present, it has been confirmed that early liver fibrosis is reversible, but without timely and effective intervention, it can evolve into cirrhosis and possibly, hepatocellular carcinoma. Therefore, delaying or reversing the occurrence and development of liver fibrosis has very important clinical significance. Despite more research on the etiology and mechanisms underlying liver fibrosis in recent years, there is currently no effective clinical treatment available. Only by clarifying the cell signal transduction pathway related to liver fibrosis and its mechanism, can we discover new therapeutic targets that can effectively block and/or reverse liver fibrosis in the clinical setting. Our research has given new insights into the pathogenesis of liver fibrosis, and may provide a new strategy for disease prevention and treatment.