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CD73 regulates hepatic stellate cells activation and proliferation through Wnt/β-catenin signaling pathway

https://doi.org/10.1016/j.ejphar.2020.173667Get rights and content

Highlights

  • High expression of CD73 in ethanol plus CCl4-induced alcoholic liver tissues, primary HSCs and activated HSC-T6 cells.

  • Inhibition of CD73 attenuates ethanol plus CCl4-induced alcoholic liver fibrosis in vivo.

  • CD73 promotes the activation and proliferation of acetaldehyde-stimulated HSC-T6 cells.

  • CD73 silencing facilitates the apoptosis of acetaldehyde-stimulated HSC-T6 cells.

  • CD73 regulates the activation and proliferation of HSCs through Wnt/β-catenin signaling pathway.

Abstract

Alcoholic liver fibrosis (ALF) is commonly associated with long-term alcohol consumption and the activation of hepatic stellate cells (HSCs). Inhibiting the activation and proliferation of HSCs is a critical step to alleviate liver fibrosis. Increasing evidence indicates that ecto-5′-nucleotidase (CD73) plays a vital role in liver disease as a critical component of extracellular adenosine pathway. However, the regulatory role of CD73 in ALF has not been elucidated. In this study, both ethanol plus CCl4-induced liver fibrosis mice model and acetaldehyde-activated HSC-T6 cell model were employed and the expression of CD73 was consistently elevated in vivo and in vitro. C57BL/6 J mice were intraperitoneally injected with CD73 inhibitor Adenosine 5′-(α, β-methylene) diphosphate sodium salt (APCP) from 5th week to the 8th week in the development of ALF. The results showed APCP could inhibit the activation of HSCs, reduce fibrogenesis marker expression and thus alleviate ALF. Silencing of CD73 inhibited the activation of HSC-T6 cells and promoted apoptosis of activated HSC-T6 cells. What's more, the proliferation of HSC-T6 cells was inhibited, which was characterized by decreased cell viability and cycle arrest. Mechanistically, Wnt/β-catenin pathway was activated in acetaldehyde-activated HSC-T6 cells and CD73 silencing or overexpression could regulate Wnt/β-catenin signaling pathway. Collectively, our study unveils the role of CD73 in HSCs activation, and Wnt/β-catenin signaling pathway might be involved in this progression.

Introduction

Alcoholic liver fibrosis (ALF), a sustained scarring response, caused by long-term alcohol consumption, is characterized by the accumulation of excessive amounts of extracellular matrix (ECM). Persisting liver fibrogenesis may progress to liver cirrhosis or hepatocellular carcinoma (Liu et al., 2019; Xu et al., 2019). Hepatic stellate cells (HSCs) activation, the critical event of hepatic fibrogenesis, leads to the abundant synthesis and secretion of ECM and exerts an important role in the up-regulation of α-smooth muscle actin (α-SMA) (Zhang et al., 2018). Given the important role of HSCs in liver fibrogenesis, promoting the apoptosis of activated HSCs is a critical step in fibrosis reversion (Ding et al., 2019; Peverill et al., 2014). Accumulating evidence illustrates that the development of ALF in alcoholics is mainly due to the metabolism of ethanol to the highly reactive compound acetaldehyde (Mello et al., 2008; Wang et al., 2015). Acetaldehyde, acting on HSCs in a paracrine manner, can directly increase the expression of Col1α1 and deposition of ECM by activating multiple signaling pathways and transcription factors (Gao and Bataller, 2011; Setshedi et al., 2010). Recent years have yielded a wealth of data implicating that the activation of HSCs stimulated with acetaldehyde in vitro closely mimics the activation of HSCs in vivo and ultimately results in ALF (Wang et al., 2014; Yang et al., 2016).

Ecto-5′-nucleotidase (CD73), a membrane-bound nucleotide that breakdowns extracellular ATP into adenosine, has emerged as an important regulator of pathophysiologic responses related to inflammation, tissue fibrosis, and cancer (Beavis et al., 2012; Buchheiser et al., 2011). Previous studies have identified high expression of CD73 in a multitude of cancer cell types including hepatocellular carcinoma and the favorable role of tumor growth and metastasis (Ma et al., 2019; Zhang, 2010, 2014). Furthermore, blocking CD73 can effectively alleviate suppression of the adaptive immune system and inhibit the growth and metastasis of tumors (Koszałka et al., 2015). In prior work, we found that blockade of adenosine A1 and A2A receptors prevented the development of ALF, indicating that extracellular adenosine and its receptors play a vital role in the pathophysiology of ALF with complex mechanisms (Wang et al., 2020; Yang et al., 2015). Moreover, CD73 catalyzes the terminal step of ATP formation to extracellular adenosine and functions in modulating adenosine signaling (Zimmermann et al., 2012). Therefore, it is meaningful for the study to explore the role of CD73 in the progression of ALF.

Numerous reports have confirmed that Wnt/β-catenin pathway is a pivotal regulator of cell growth and proliferation and is activated in many human organ fibrosis (Guo et al., 2012; Nishikawa et al., 2018). Other studies have demonstrated that Wnt/β-catenin pathway is closely related to the proliferation of HSCs and liver fibrosis (Yu et al., 2019). Mechanically, previous studies identified that CD73 could be involved in adenosine metabolism via interacting with Wnt/β-catenin pathway (Spychala, 2000; Spychala and Kitajewski, 2004). Therefore, whether CD73 regulates the proliferation and activation of HSCs through Wnt/β-catenin pathway has captured the attention of our team.

Section snippets

Mouse model of ALF

C57BL/6 J mice (male, 8–10 weeks of age, 21–24 g) were purchased from the Animal Experiment Center of Anhui Medical University. All experiments were conducted as required of the Ethics Committee and Animal Experimental Committee at Anhui Medical University. We replicated the mouse model of ALF based on the study by B. Gao et al. (Kwon et al., 2014) and used APCP, an inhibitor of CD73, to knockdown CD73 expression in vivo. The mice were randomly divided into six groups of 12 animals: vehicle

Upregulation of CD73 expression in HSCs may be associated with ethanol plus CCl4-induced liver fibrosis

To define whether CD73 expression is associated with ALF, ethanol plus CCl4-induced mouse liver fibrosis model was developed for histopathological examination. First, Liver injury in liver fibrosis mice was evaluated by H&E staining. Masson and Sirius red staining were used to indicate the collagen area, and the activated HSCs were highlighted by α-SMA positivity (Fig. 1A). Moreover, Serum levels of AST and ALT were higher (P < 0.01) in ethanol plus CCl4-induced liver fibrosis mice compared

Discussion

With the striking increase in alcohol consumption, ALF has been widely recognized as a vital global health issue and the research on its pathogenesis and therapeutic targets has become the hotspot of global focus (Bala and Szabo, 2012; Huang et al., 2018). Fortunately, ALF is a reversible process that strikes a balance between the synthesis and degradation of ECM that leads to abnormal deposition of fibrous connective tissue in response to various liver injuries (Huang et al., 2020). Activated

Data availability

All data included in this study are available upon request by contact with the corresponding author.

CRediT authorship contribution statement

Wen-qian Jia: designed the research and wrote the main manuscript text. Tao-cheng Zhou: directed . Jing-wen Dai: directed . Zhen-ni Liu: directed . Ya-fei Zhang: directed the data analysis, All authors read and approved the manuscript. Dan-dan Zang: designed the research and wrote the main manuscript text, directed the data analysis, All authors read and approved the manuscript.

Declaration of competing interest

The authors declare no conflicts of interest.

Acknowledgments

This study was supported by the National Natural Science Foundation of China (Grant No. 81270498 and Grant No. 81970518). The authors thank the Center for Scientific Research of Anhui Medical University for valuable help in our experiment.

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