Underlying Roles Of Wnt2b In The Progression Of Liver Fibrosis And Related Mechanisms

BackgroundLiver fibrosis is clinically associated with the development of cirrhosis and hepatocellular carcinoma (HCC), and has become one of the most significant public health concerns worldwide. Liver fibrosis is a wound-healing response to repeated liver injury and chronic liver inflammation of different etiologies. Fibrogenesis is usually considered as a dynamic process, with the potential to regress by cessation of injury, or to progress into cirrhosis if the key pathways involved in this process were not interrupted successfully at the right time. The activation of hepatic stellate cells(HSCs) is the pivotal event in liver fibrosis. Following the onset of liver injury, the quiescent vitamin A-rich HSCs are activated and differentiate to pro-fibrogenic myofibroblasts (MFBs), which are considered to be the main source of extracellular matrix (ECM) and the major effectors during fibrogenesis. Although innovations have been made recent years, the intricate mechanisms underlying HSCs activation in hepatic fibrogenesis are not fully clarified and no anti-fibrotic therapy has yet been approved by FDA.Wnt2b, also referred to as Wnt13, is a highly conserved secretary glycoprotein of the Wingless-type MMTV integration site (Wnt) family. The functions of Wnt2b in liver physiology have been well demonstrated. Impaired function of Wnt2b in embryos results in a complete absence or severe decrease in hepatic tissue.Additionally,genetic evidence supported a central role of Wnt2b in coordinating early liver development from the multipotent foregut endoderm. The conversion of cholangiocytes to hepatocytes in zebrafish with Wnt2b mutants was impaired following substantial hepatocyte depletion. Shackel et al. observed that Wnt2b mRNA increased in human primary biliary cirrhosis (PBC) by using cDNA array analysis.However, the significance of Wnt2b in hepatic inflammation and fibrosis-related liver diseases is still unclear. Therefore,the aim of the current work was to determine the role of Wnt2b in the pathogenesis of liver fibrosis.We firstly confirmed that Wnt2b expression was elevated in the fibrotic liver tissues and then identifed the main cellular source of Wnt2b during fibrogenesis. Next, by employing the hydrodynamic injection (HD) of shRNA targeting mouse Wnt2b (sh-Wnt2b) andWnt2b-overexpression plasmid (pRK5-mWnt2b), we evaluated whether the level of hepatic Wnt2b expression influenced the degree of liver fibrosis. An immortalized human HSC line LX2 was used to confirm the influence of Wnt2b on HSCs in vitro.Lastly, molecular mechanistic studies were carried out both in vitro and in vivo.Aims1. To determine the hepatic expressions of Wnt2b in clinical fibrotic subjects and in liver fibrosis mouse models, and the major celluar source of Wnt2b during fibrogenesis;2. To investigate whether the level of hepatic Wnt2b expression influenced the degree of liver fibrosis, and analyze the influence of Wnt2b on HSCs activation;3. To explore the molecular mechanisms underlying the regulatory effect of Wnt2b on fibrosis pregression and HSCs activation.Methods1. The expressions of Wnt2b in fibrotic subjects and normal controls were evaluated by immunohistochemistry analysis;2. Two liver fibrosis mouse models were established by repeated administration ofthioacetamide (TAA) or carbon tetrachloride (CCI4);3. The response of primary hepatocytes under hepatic stress was mimicked by challenging with hepatotoxic chemicals in vitro;4. The expressions of Wnt2b in rodent fibrotic livers were detected by immunohistochemistry, RT-PCR, Western blotting;5. The amount of Wnt2b in the liver homogenate was evaluated by ELISA assays;6. The cellular localization of Wnt2b in both normal and fibrotic liver tissues was assayed by immunofluorescence staining;7. Mouse livers were perfused in situ with collagenase type ?, and then the expression levels of Wnt2b in hepatocytes and non-parenchymal cells (NPCs)were compared by RT-PCR, Western blotting assays;8. The levels of Wnt2b in HSCs isolated from fibrotic mice and controls were assayed by immunofluorescence;9. The changes of Wnt2b during the process of HSCs activation were detected by using RT-PCR and comparing the level of Wnt2b in quiescent HSCs with HSCs cultured for 14 days in vitro;10. The level of Wnt2b in livers from TAA-challenged mice was knockdown or upregulated by hydrodynamic injection (HD) of shRNA targeting mouse Wnt2b(sh-Wnt2b) and Wnt2b-overexpression plasmid (pRK5-mWnt2b), respectively;11. Hepatic collagen deposition, HSCs activation and CD45 positive cell infiltration in mice challenged with TAA combined with HD injection of pRK5-mWnt2b/sh-Wnt2b construct or control vector were measured by H&E, Sirus Red Staining,Western blotting, FACS and ALT assays;12. The receptors for Wnt signaling were confirmed to be presented in HSCs by using RT-PCR; the changes of receptors during the process of HSCs activation were detected by comparing the level of Wnt receptors in quiescent HSCs with HSCs cultured for 14 days in vitro;13. The influence of Wnt2b on HSCs in vitro was confirmed by assayed the expressions of a-SMA and Collagen-I in LX2 cells cultured with the conditioned medium (CM) from Wnt2b-overexpressing HEK293 cells, and in LX2 cells transfected with Wnt2b-overexpressing vector;14. The expressions of ?-catenin in mice fibrotic livers were detected by immunohistochemistry, RT-PCR, Western blotting;15. The levels of ?-catenin in HSCs isolated from fibrotic mice and controls were assayed by RT-PCR and immunofluorescence staining;16. The changes of ?-catenin during the process of HSCs activation were detected by using RT-PCR and immunofluorescence staining, and comparing the level of?-catenin in quiescent HSCs with HSCs cultured for 14 days in vitro;17. The influence of Wnt2b on ?-catenin expression was confirmed by assayed the expressions of ?-catenin in LX2 cells cultured with the conditioned medium (CM)from Wnt2b-overexpressing HEK293 cells, and in LX2 cells transfected with Wnt2b-overexpressing vector;18. Effects of ?-catenin shRNA on a-SMA and Collagen-I protein levels in LX2 cells cultured with Wnt2b-conditioned medium and Wnt2b-overexpressing LX2 cells were determined by western blotting;19. The expressions of TLR4 in mice fibrotic livers were detected by immunohistochemistry, RT-PCR, Western blotting;20. The levels of TLR4 in HSCs isolated from fibrotic mice and controls were assayed by RT-PCR and immunofluorescence staining;21. Influence of TAK242,a TLR4 inhibitor,on the exacerbated fibrosis effect mediated by silencing Wnt2b was determined by Western blotting and Sirius Red staining;22. Influence of Wnt2b on the pro-fibrogenic effects of LPS, as well as on the sensitized effect to TGF-? mediated by TLR4 activation, were assayed by Western blotting for a-SMA and Collagen-I;23. The expression of TLR4 and TLR4-related signaling intermediates in LX2 cells cultured in Wnt2b-CM/transfected with Wnt2b-overexpressing vector, as well as in liver tissues from mice challenged with TAA combined with HD injection of pRK5-mWnt2b/sh-Wnt2b construct, were detected by Western blotting;24. The expression and nuclear translocation of NF-?B in HSCs isolated from mice challenged with TAA combined with HD injection of pRK5-mWnt2b / sh-Wnt2b construct were assayed by immunofluorescence staining.Results1. Wnt2b expression is elevated in fibrotic liversCompared to normal controls, the fibrotic liver tissues exhibited higher levels of Wnt2b as demonstrated by immunohistochemistry analysis. Consistently, in two liver fibrosis mouse models established by repeated administration of thioacetamide (TAA)or CCl4, hepatic Wnt2b expression was also markedly increased compared to healthy controls. These findings suggested that Wnt2b might be involved in the pathogenesis of liver fibrosis.2. Wnt2b is mainly produced by hepatocytes during liver fibrogenesisThe expression of Wnt2b was markedly up-regulated in liver parenchyma obtained from fibrotic mice compared to controls, while Wnt2b was slight elevated in NPCs(non-parenchymal cells). Moreover, when primary hepatocytes isolated from healthy control mice were challenged with hepatotoxic chemicals in vitro, both the mRNA and protein levels of Wnt2b were increased in a dose-dependent manner, suggesting hepatocytes were the primary cellular source of Wnt2b within fibrotic livers. The level of Wnt2b in HSCs obtained from fibrotic mice was higher than controls. As quiescent HSCs isolated from healthy control mice were cultured for 14 days in vitro,these self-activated HSCs exhibited higher expression of Wnt2b than the quiescent phenotype,indicating the levels of Wnt2b in activated HSCs were also increased during liver fibrogenesis.3. Wnt2b attenuates the progression of liver fibrosisHydrodynamic injection with sh-Wnt2b and Wnt2b-overexpression plasmid could efficiently decrease or increase the expression of Wnt2b in livers from TAA-challenged mice, respectively. We found that sh-Wnt2b markedly promoted total hepatic collagen deposition, HSCs activation, and CD45 positive cell infiltration,indicating that the decrease in Wnt2b expression accelerated fibrosis progression. In contrast to Wnt2b silencing, Wnt2b overexpression resulted in decreased hepatic collagen deposition, HSCs activation, and CD45 positive cell infiltration. These results collectively suggested that Wnt2b attenuates the progression of liver fibrosis.4. Wnt2b exerts a direct inhibitory effect on HSCs activationMost of the receptors for Wnt signaling were confirmed to be presented in HSCs.Fzd4 and Fzd7 receptors were increased in activated HSCs compared to the quiescent phenotype, indicating a direct regulation of Wnt2b signaling on HSCs. The expressions of a-SMA and Collagen-I were markedly inhibited in LX2 cells cultured with the conditioned medium (CM) from Wnt2b-overexpressing HEK293 cells.Additionally, as LX2 cells transfected with Wnt2b-overexpression vector, both?-SMA and Collagen-I were down-regulated. These findings indicated a direct inhibitory effect of Wnt2b on HSCs activation.5. ?-catenin expression is elevated in fibrotic liversCompared to normal controls, the fibrotic liver tissues exhibited higher levels of?-catenin as demonstrated by immunohistochemistry analysis, RT-PCR and Western blotting. The expression and nuclear distribution of ?-catenin were enhanced in HSCs obtained from fibrotic mice than in control mice. Consistently, as quiescent HSCs isolated from healthy control mice were cultured for 14 days in vitro, these self-activated HSCs exhibited higher expression of ?-catenin than the quiescent phenotype.6. Wnt2b is able to promote the expression of ?-cateninCompared to controls, hydrodynamic injection with Wnt2b-overexpression plasmid markedly promotes the hepatic expression of ?-catenin. Moreover, the expression of?-catenin was up-regulated in LX2 cells cultured with Wnt2b-conditioned medium and in LX2 cells transfected with Wnt2b-overexpression vector, suggesting that Wnt2b positively regulate the expression of ?-catenin7. The inhibitory effects of Wnt2b on HSCs activation is ?-catenin-independentAlthough Wnt2b was shown to induce the expression of ?-catenin, silencing P-catenin did not abrogate Wnt2b-mediated inhibitory effects on HSCs,suggesting Wnt2b exerted the inhibitory effects on the activation of HSCs was not dependent on the canonical Wnt/?-catenin pathway, alternative mechanism might be involved.8. TLR4 expression is elevated in fibrotic liversCompared to normal controls, hepatic TLR4 expression was markedly higher in TAA-treated mice as demonstrated by immunohistochemistry analysis, RT-PCR and Western blotting. The expressions of TLR4 were enhanced in HSCs obtained from fibrotic mice than in control mice. Additionally, the small intestinal bacterialovergrowth (SIBO) and bacterial translocation were observed in mice fibrotic models.These results collectively implied an involvement of TLR4 signaling in liver fibrosis.9. Wnt2b suppresses TLR4 activation-mediated pro-fibrogenic effectsLiver fibrosis was more difficult to be induced in TLR4-/- mice than in WT mice,characterized by lower deposition of collagen. Notably, the inhibition of TLR4 pathway with TAK242 abrogated the exacerbated fibrosis effect mediated by silencing Wnt2b. In vitro studies, LPS exposure increased a-SMA and Collagen-I expressions in LX2 cells in a dose-dependent manner, and such pro-fibrogenic effects were markedly attenuated by the presence of Wnt2b. In addition,TLR4 signaling was shown to sensitize HSCs to TGF-? (transforming growth factor beta) stimulation. At the suboptimal dose, TGF-? alone could not induce marked activation of HSCs, while the pre-treatment of LPS enhanced the response of LX2 cells to TGF-?,resulting in strong induction of a-SMA and Collagen-I. Importantly, Wnt2b afforded the resistance of LX2 cells to LPS-mediated TGF-? sensitization,demonstrating a negative regulation of Wnt2b on TLR4-mediated pro-fibrogenic effects.10. Wnt2b disturbs TLR4 signaling transductionThe expressions of TLR4 and NF-?B signal transduction were reduced in LX2 cells treated with Wnt2b-CM, as well as in LX2 cells transfected with Wnt2b-overexpression vector. Furthermore, the phosphorylation of TLR4 signaling transduction-associated molecules mitogen-activated protein (MAP) kinases was also suppressed in the presence of Wnt2b. In the parallel in vivo experiments, the expressions of TLR4 and TLR4-related signaling intermediates in tissues from fibrotic Wnt2b-overexpressing livers were down-regulated compared to WT controls,whereas TLR4 signaling transduction was augmented in Wnt2b-silencing livers.Meanwhile, Wnt2b overexpression inhibited the expression and nuclear translocation of NF-?B in HSCs compared to controls, whereas Wnt2b silencing promoted the activation of NF-?B in HSCs, suggesting that Wnt2b disturbed TLR4 signaling transduction though modulating the expression of TLR4 as well as the activation of TLR4-related signaling intermediates.Conclusions1. Wnt2b was up-regulated in fibrotic liver tissues. Hepatocytes were the main celluar source of Wnt2b during liver fibrogenesis,and activated HSCs were also able to increase the expression of Wnt2b to some extent within fibrotic livers.Wnt2b exhibited protective effects against fibrosis progression and HSCs activation.2. Despite the expression of ?-catenin in the fibrotic liver tissues was higher than in normal control mice, and Wnt2b was shown to induce the expression of ?-catenin,silencing ?-catenin did not abrogate Wnt2b-mediated inhibitory effects on HSCs ,suggesting Wnt2b exerted the inhibitory effects against fibrosis progression was not dependent on the canonical Wnt/?-catenin pathway.3. A negative regulation of Wnt2b was indentified on the toll-like receptor 4 (TLR4)signaling, characterized by inhibiting the expression of TLR4 as well as the activation of NF-?B and MAPKs. Wnt2b was shown not only to directly suppress LPS-induced HSCs activation, but also to inhibit TLR4-enhanced the sensitivity o HSCs to TGF-?.Taken together, the current study provided new insights into the pathophysiology of liver fibrosis by characterizing Wnt2b as a potential endogenous suppressor of TLR4 signaling, exerting inhibitory effects on HSCs activation and fibrosis progression.These findings suggested a novel mechanism for maintaining tissue homeostasis during the early stage of liver disease, and indicated a possible role for Wnt2b in sterile inflammation and inflammation-related oncogenesis.