GSK-3|[beta]| suppresses the proliferation of rat hepatic oval cells through modulating Wnt/|[beta]|-catenin signaling pathway

The liver has a remarkable capacity to regenerate after toxic injury or surgical resection. Oval cells are facultative stem cells in the adult liver but are activated only if hepatocyte proliferation is inhibited1,2,3. These cells can differentiate into hepatocytes and bile duct cells and can thus restore the architecture and function of the damaged liver tissue4,5,6,7. Oval cells are usually present in normal fetal livers but have been implicated in hepatic carcinogenesis in a variety of hepatic pathologies8,9. Glycogen synthase kinase-3β (GSK-3β) is a constitutively active serine-threonine kinase that was originally identified as a key regulatory enzyme in glucose metabolism10. It was subsequently found to be involved in a variety of cellular pathways, depending on its substrates10,11,12. Regulation of the Wnt/β-catenin signaling is only one of its diverse functions10,11. GSK-3β activity is increased by the site-specific phosphorylation of Tyr216, whereas the phosphorylation of Ser9 inhibits GSK-3β activity12. A growing body of research has identified a critical role of GSK-3β signaling in various aspects of hepatic biology, including liver development, regeneration, proliferation, and hepatocellular carcinoma (HCC) pathogenesis13,14,15,16,17,18. However, there have been relatively few reports that connect GSK-3β signaling with liver oval cells. WB-F344 cells are hepatic stem-like epithelial cells of rat origin and were first isolated from the liver of an adult male Fischer-344 rat. After transplantation into the liver of adult syngeneic German strain Fischer-344 rats deficient in the bile canalicular enzyme dipeptidyl peptidase IV (DPP-IV), WB-F344 cells can be integrated into hepatic plates and differentiate into mature hepatocytes19. When treated with sodium butyrate and cultured on Matrigel, WB-F344 cells differentiate into the biliary phenotype20. For these reasons, WB-F344 cells have been used as an in vitro representative of oval cells21,22. In the present study, we examined the effects of GSK-3β on the growth of cultured WB-F344 cells and investigated changes in the downstream targets of GSK-3β. The effects of GSK-3β manipulation in liver regeneration were also examined in rats using 2-acetylaminofluorine and a partial hepatectomy (2-AAF/PH)23. For the construction and production of a lentivirus overexpressing GSK-3β, cDNA for GSK-3β was amplified using primers containing the restriction site for Age I (sense: 5′-GAGGATCCCCGGGTACCGGTCGCCACCATGTCGGGGCGACCGAGAACC-3′, antisense: 5′-TCACCATGGTGGCGACCGGGGTAGAGTTGGAGGCTGATG-3′). After digestion with Age I, the cDNA was inserted into the pGC-FU vector. The recombinant vector, and vectors pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells to produce GC-GSK-3βLV. GC-FU-GFP was used as a negative control. For 2-AAF/PH, rats received a daily dose of 2-AAF (Sigma; 20 mg/kg, by gavage) suspended in corn oil (1%) for 4 consecutive days. PH (70%) was carried out on the next day. The day after PH, treatment with 2-AAF was resumed and lasted for 7 additional days. Treatment with SB216763 (1 or 2 mg/kg, intraperitoneally) or the vehicle (200 μL 75% DMSO) started the day before PH and then on d 1, 3, and 5 after PH (n=3 per condition). A group of three rats receiving 200 μL of normal saline (NS) was included as an additional control. Animals were euthanized on d 7 after PH24 and liver weight and femur length were measured. Immunoblotting assays showed that GSK-3βRNAiLV markedly suppressed the expression of GSK-3β in WB-F344 cells (Figure 2A–2C). GC-GSK-3βLV increased the expression of GSK-3β (Figure 2D and 2E). CCK-8 assays showed that the downregulation of GSK-3β with GSK-3βRNAiLV caused a significant increase in the proliferation of WB-F344 cells (Figure 2F). In contrast, GSK-3β overexpression markedly suppressed the proliferation of WB-F344 cells (Figure 2F). The adult liver harbors stem cells that can be activated upon severe liver injury to give rise to both hepatocytic and biliary epithelial cell lineages25. The present study demonstrated that GSK-3β signaling plays an important role in the proliferation of oval cells. Previous studies in mice have shown that disruption of the GSK-3β gene results in embryonic lethality because of severe mid-gestation liver degeneration13. The inhibition of GSK-3β also impairs the regeneration of adult rat liver after PH15,17. These findings suggest that GSK-3β is essential for liver development and regeneration in vivo. However, Ito et al reported that the concurrent inhibition of GSK-3β and TGF-β can induce the proliferation of mature hepatocytes in vitro18. The present study showed a negative regulatory role for GSK-3β in liver oval cell proliferation. We use the pharmacological GSK-3β-specific inhibitor SB216763 to inhibit the activity of GSK-3β in WB-F344 cells. SB216763 inhibits the activity of GSK-3β by phosphorylating Ser9 at the N-terminus26 but does not affect the total level of GSK-3β. GSK-3β inhibition significantly increased the proliferation of WB-F344 cells. The downregulation of GSK-3β using a lentivirus also promoted WB-F344 cell proliferation. Consistently, overexpressing GSK-3β with a lentivirus inhibited cell proliferation. Finally, we assessed the effect of GSK-3β inhibition on oval cell proliferation in vivo. The results in rats with PH were consistent with the in vitro findings: treatment with the GSK-3β inhibitor dramatically increased the expression of phospho-Ser9-GSK-3β, and promoted the proliferation of oval cells and liver regeneration. The different regulatory roles for GSK-3β between hepatocytes and oval cells suggest that GSK-3β modulates the proliferation of these cells through different signaling pathways. GSK-3β is known to modulate cell survival and apoptosis through multiple intracellular signaling pathways27. GSK-3β also plays an important role in canonical Wnt signalling pathway, which is essential for self-renewal of many stem cells, including hepatic progenitors28. GSK-3β exists in a multimeric complex with APC, axin and β-catenin, where GSK-3β phosphorylates the N terminal Ser/Thr of β-catenin leading to its degradation mediated by ubiquitin/proteasomes29,30. Activation of Wnt signalling leads to GSK-3β inactivation resulting in dephosphorylation and stabilization of β-catenin protein in the cytosol. The increased levels of β-catenin lead to its nuclear translocation, and its interaction with transcription factors LEF/TCF (lymphoid enhancer factor/T cell factor) activate the expression of target genes like c-myc and cyclin D1, leading to an increase in cell proliferation27,31. Several recent studies in rodents have elucidated the role of Wnt/β-catenin signaling in oval cell activation and proliferation. Wnt/β-catenin signaling is induced in the livers of mice exposed to DDC, and oval cells isolated from DDC-exposed livers display nuclear localization of β-catenin in response to Wnt3a stimulation32. In rats, Wnt/β-catenin signaling is also activated by 2AAF/PH33. Moreover, the activation of β-catenin signaling in response to Wnt ligands was recently observed in WB-F344 cells, resulting in their proliferation34. In another study, the significant upregulation of several Wnt genes was accompanied by increased levels of active β-catenin in mice exposed to DDC. The activation of the Wnt/β-catenin signaling pathway in vitro is sufficient to induce proliferation of cultured hepatic stem/progenitor cells35. GSK-3β has been implicated in the regulation of the Wnt/β-catenin signaling pathway36. In canonical Wnt signaling, the extracellular ligand Wnt causes the inactivation of GSK-3β, resulting in β-catenin activation37,38. Wnt signaling can also be activated by direct intracellular inhibition of GSK-3 function using specific inhibitors39,40. Our results demonstrate that inhibiting the activity of GSK-3β with a pharmacological inhibitor or downregulating GSK-3β expression with a lentivirus can increase the level of β-catenin and cyclin D1. In contrast, overexpressing GSK-3β with a lentivirus in cultured WB-F344 cells downregulates β-catenin and cyclin D1. It was reported that the proliferation of WB-F344 cells is severely impaired in the absence of β-catenin and that they show a decreased expression of downstream targets such as cyclin D141. Together, these findings lead us to hypothesize that GSK-3β modulates rat hepatic oval cell proliferation through Wnt/β-catenin signaling. In conclusion, the current study shows that GSK-3β plays an important role in the proliferation of rat hepatic oval cells, which is complementary to Wnt/β-catenin signaling in modulating oval cell proliferation. These findings indicate the potential for developing GSK-3β inhibitors to promote liver regeneration. Qi-yu ZHANG and Yun-feng SHAN designed the research; Xiao-ke JI, Yuan-kang XIE, and Jun-qiao ZHONG performed the research; Qi-gang XU and Qi-qiang ZENG analyzed the data; and Xiao-ke JI and Yang WANG wrote the paper. This study was supported by the National Natural Science Foundation of China (No 30700800) and Zhejiang Extremely Key Subject of Surgery. We thank Ka-te HUANG and Li WAN for their technical assistance with the immunohistochemistry.