Tan et al. Cell Death and Disease (2021) 12:474 https://doi.org/10.1038/s41419-021-03749-x Cell Death & Disease ARTICLE Open Access Fas/FasL mediates NF-κBp65/PUMA-modulated hepatocytes apoptosis via autophagy to drive liver fibrosis Siwei Tan 1,2,XianzhiLiu1, Lingjun Chen1, Xiaoqin Wu1,LiTao1,XuemeiPan1,ShuyanTan1, Huiling Liu1,JieJiang1 and Bin Wu 1,2 Abstract Fas/Fas ligand (FasL)-mediated cell apoptosis involves a variety of physiological and pathological processes including chronic hepatic diseases, and hepatocytes apoptosis contributes to the development of liver fibrosis following various causes. However, the mechanism of the Fas/FasL signaling and hepatocytes apoptosis in liver fibrogenesis remains unclear. The Fas/FasL signaling and hepatocytes apoptosis in liver samples from both human sections and mouse models were investigated. NF-κBp65 wild-type mice (p65f/f), hepatocytes specific NF-κBp65 deletion mice (p65Δhepa), p53-upregulated modulator of apoptosis (PUMA) wild-type (PUMA-WT) and PUMA knockout (PUMA-KO) littermate models, and primary hepatic stellate cells (HSCs) were also used. The mechanism underlying Fas/FasL-regulated hepatocytes apoptosis to drive HSCs activation in fibrosis was further analyzed. We found Fas/FasL promoted PUMA- mediated hepatocytes apoptosis via regulating autophagy signaling and NF-κBp65 phosphorylation, while inhibition of autophagy or PUMA deficiency attenuated Fas/FasL-modulated hepatocytes apoptosis and liver fibrosis. Furthermore, NF-κBp65 in hepatocytes repressed PUMA-mediated hepatocytes apoptosis via regulating the Bcl-2 κ fi 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; family, while NF- Bp65 de ciency in hepatocytes promoted PUMA-mediated hepatocytes apoptosis and enhanced apoptosis-linked inflammatory response, which contributed to the activation of HSCs and liver fibrogenesis. These results suggest that Fas/FasL contributes to NF-κBp65/PUMA-modulated hepatocytes apoptosis via autophagy to enhance liver fibrogenesis, and this network could be a potential therapeutic target for liver fibrosis. Introduction organization of the liver6.It’s widely accepted that the Liver fibrosis represents one of the major consequences progression of hepatic fibrosis is associated with con- of morbidity and mortality worldwide1,2, and the activa- siderable injury and loss of hepatocytes, which may pre- tion of hepatic stellate cells (HSCs), which is regulated by sent as a main inflammatory stimulus for HSCs multiple cell populations or soluble mediators, is the activation7,8. The apoptotic cells could release the – major source of extracellular matrix substances (ECM)3 5. nucleotides ATP and UTP, which bind to purinergic Hepatocytes are the major parenchymal cells of the liver receptors (especially the P2Y2 receptor) on macrophages and essential for maintaining the function and and HSCs, leading to their activation9,10. As a classic apoptosis modulator, Fas, following Fas ligand (FasL) engagement, leads to the recruitment of Fas-associated Correspondence: Siwei Tan ([email protected]) or Bin Wu (wubin6@mail. proteins having death domains and the initiators into a sysu.edu.cn) 1Department of Gastroenterology, the Third Affiliated Hospital of Sun Yat-Sen death-inducing signaling complex (DISC) to cause apop- – University, Guangzhou, Guangdong Province 510630, China tosis11 13, and the Fas/FasL has been demonstrated to 2 Guangdong Provincial Key Laboratory of Liver Disease Research, Guangzhou, participate in the pathological processes of hepatic Guangdong Province 510630, China These authors contributed equally: Siwei Tan, Xianzhi Liu Edited by B. Zhivotovsky © The Author(s) 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Official journal of the Cell Death Differentiation Association Tan et al. Cell Death and Disease (2021) 12:474 Page 2 of 18 fibrosis/cirrhosis, acute/chronic hepatitis and hepato- hepatocytes apoptosis and liver fibrosis. Furthermore, – carcinoma14 16. hepatocytes deletion of NF-κBp65 promoted PUMA- Autophagy is an evolutionarily conserved and catabolic mediated hepatocytes apoptosis via regulating the Bcl-2 process that targets cytosolic material, organelles and family, and the enhanced hepatocytes apoptosis linked long-lived proteins to lysosomes to be degraded for sur- inflammatory action to drive HSCs activation and liver – vival, development, differentiation, and homeostasis17 19. fibrogenesis. Thus, we suggest that Fas/FasL contributes Hepatocytes have been revealed to own a high level of to NF-κBp65/PUMA-regulated hepatocytes apoptosis via autophagic flux because of their increased abundance of autophagy to enhance liver fibrogenesis. lysosomes and lysosomal enzymes, and the enhanced autophagy could modulate the progression to hepatocytes Results death20, and our previous study has suggested autophagy Fas/FasL-mediated apoptosis involved in liver fibrogenesis was required for liver fibrogenesis21. Nuclear factor-κB To evaluate the role of Fas/FasL in liver fibrosis, liver (NF-κB), as a ubiquitous and inducible transcription fac- specimens from healthy volunteers and liver fibrosis tor responsible for mediating the expression of a large patients were analyzed. Histological staining presented a number of genes involved in differentiation, apoptosis, loss of preserved architecture and excess deposition of and proliferation22, and NF-κBp65, as the main functional ECM, associated with obvious hepatic apoptosis and element, involves in various physiological and pathologi- upregulated Fas/FasL expression, were observed in the cal events and influences the survival of hepatocytes and liver fibrotic tissues compared with the normal samples activation of HSCs23. Activation of NF-κB in non- (Fig. 1a, b). Western blotting also confirmed a similar parenchymal cells promotes inflammation, fibrosis, and condition (Fig. 1c, d). By using mouse model, we found hepatocarcinogenesis in the liver, whereas suppression of that CCl4 injection-induced prominent liver fibrosis in NF-κB in parenchymal cells enhances hepatocarcinogen- mice as shown by α-SMA and COL-I staining, which was esis in some cases and retards hepatocarcinogenesis in accompanied with obvious apoptosis and Fas/FasL upre- others10,24. The activation of NF-κB in HSCs appears to gulation (Fig. 1a, b), and western blotting further repre- promote hepatic fibrosis via modulating fibrogenic effects sented that the levels of Fas, FasL, α-SMA, COL-I, COL- and anti-apoptotic effects9. While decreased or absent IV, and cleaved caspase-3 were enhanced in the fibrotic NF-κB activity in hepatocytes might lead to subsequent sections (Fig. 1c, d). These results indicated that Fas/FasL fibrosis by regulating hepatocytes injury and the primary involved in hepatic apoptosis in liver fibrogenesis. – trigger of fibrogenic responses in liver23 26. Considerable death and loss of hepatocytes induced by Autophagy participated in Fas/FasL-mediated hepatic different etiologies are always associated with the initia- apoptosis in liver fibrosis tion and progression of hepatic fibrosis1,27. p53- Our preceding data has testified that autophagy was upregulated modulator of apoptosis (PUMA), is one of required for liver fibrosis21. In the current study, we the most potent mediators of apoptosis induced by var- found that the autophagic element BECN1 was enhanced ious stimuli28. PUMA deficiency could block cell apop- in liver fibrosis, which was associated with the upregu- totic responses to p53 activation, DNA-damaging agents, lated α-SMA and COL-I expressions and apoptotic sig- and hypoxia in hepatocytes29. In response to multiple naling (Fig. 2a), and ultrastructural analysis showed the insults, PUMA functions through other Bcl-2 family typical autophagosomes in the hepatocytes of mouse and members, including Bcl-2, Mcl-1, and Bcl-xL, to induce human liver fibrotic sections but scarce in the normal mitochondrial dysfunction and caspases activation30,31. tissues (Fig. 2b). We pretreated mice with autophagic PUMA-mediated apoptotic response in hepatocytes is a inhibitor 3-methyladenine (3-MA) and found 3-MA direct cause of compensatory proliferation and carcino- treatment alleviated the degree of apoptosis and liver genesis in the liver32. Mice deficient in NF-κB signaling or fibrosis in mice, and did not affect the levels of Fas and the anti-apoptotic Bcl-2 family members developed FasL (Fig. 2a, c and Supplementary Fig. 1a). To sum up, spontaneous hepatocellular carcinoma secondary to these data suggested that autophagy participated in Fas/ hepatocytes death33, and PUMA has been demonstrated FasL-mediated hepatic apoptosis in liver fibrosis. to be a target of NF-κBp65 and a critical mediator of TNF-α-induced hepatocytes apoptosis34. However, the PUMA responded to Fas/FasL/autophagy-regulated