FAK deletion accelerates liver regeneration after two-thirds partial hepatectomy

www.nature.com/scientificreports OPEN received: 10 May 2016 accepted: 12 September 2016 Published: 28 September 2016 FAK deletion accelerates liver regeneration after two-thirds partial hepatectomy Na Shang1, Maribel Arteaga1, Lennox Chitsike1, Fang Wang1, Navin Viswakarma2, Peter Breslin3,4 & Wei Qiu1 Understanding the molecular mechanisms of liver regeneration is essential to improve the survival rate of patients after surgical resection of large amounts of liver tissue. Focal adhesion kinase (FAK) regulates different cellular functions, including cell survival, proliferation and cell migration. The role of FAK in liver regeneration remains unknown. In this study, we found that Fak is activated and induced during liver regeneration after two-thirds partial hepatectomy (PHx). We used mice with liver-specific deletion of Fak and investigated the role of Fak in liver regeneration in 2/3 PHx model (removal of 2/3 of the liver). We found that specific deletion of Fak accelerates liver regeneration. Fak deletion enhances hepatocyte proliferation prior to day 3 post-PHx but attenuates hepatocyte proliferation 3 days after PHx. Moreover, we demonstrated that the deletion of Fak in liver transiently increases EGFR activation by regulating the TNFα/HB-EGF axis during liver regeneration. Furthermore, we found more apoptosis in Fak-deficient mouse livers compared to WT mouse livers after PHx. Conclusion: Our data suggest that Fak is involved in the process of liver regeneration, and inhibition of FAK may be a promising strategy to accelerate liver regeneration in recipients after liver transplantation. Liver regeneration is a well-orchestrated and tightly regulated biological response to hepatocellular injury or loss involving a complex network of inflammatory, proliferative, and metabolic signals1. Liver regeneration is essential to improve the survival rate of patients after surgical resection of large amounts of liver tissue. In addition, the improvement of the liver regenerative process would reduce the amount of liver tissue required for liver transplantation. This will reduce the risk for the donor and would also enhance the growth of the transplant within the recipient. Two-thirds partial hepatectomy (PHx) in rodents has become a useful paradigm for studying liver regeneration2. With this model, molecular mechanisms of liver regeneration have been emerging. Hepatocyte growth factor (HGF), a major hepatocyte mitogen, is important for liver regeneration through its activation of c-MET3,4. Activation of epidermal growth factor receptor (EGFR) signaling is required for efficient liver regeneration. Mice lacking the EGFR in the liver after PHx showed reduced hepatocyte proliferation and delayed liver regeneration, resulting from a defective entry into the G1–S phase of the cell cycle5. Consistently, a number of kinases that are targets of HGF/c-MET and EGFR signaling, such as protein kinase B (PKB or AKT)6, extracellular receptor kinase (ERK)7 or signal transducer and activator of transcription 3 (Stat3)8, also play important roles in liver regeneration. Integrin pathways have been shown to function in the liver regenerative process. Knockdown and knockout of β1-integrin in hepatocytes impairs liver regeneration through inhibition of EGFR and c-MET activation9, suggesting that integrin signaling is required for liver regeneration. However, deletion of integrin-linked kinase (ILK), an important downstream target of integrin, enhances liver regeneration and enlarges liver mass after PHx10. Focal adhesion kinase (FAK) is another important protein involved in the transmission of integrin signals11,12. Activation of FAK can target multiple downstream signaling pathways (e.g., AKT, ERK and Ras-related C3 botulinum toxin substrate (Rac)), thereby regulating different cellular functions, including cell survival, proliferation and migration13. We have recently shown that FAK is required for c-MET/β-catenin-induced 1 Departments of Surgery and Oncology Institute and Stritch School of Medicine, 2160 South 1st Avenue, Maywood, IL 60153, USA. 2Departments of Molecular/Cellular Physiology, Stritch School of Medicine, 2160 South 1st Avenue, Maywood, IL 60153, USA. 3Department of Biology, Loyola University Chicago, 1032 W. Sheridan Rd., Chicago, IL 60660, USA. 4Department of Surgery, University of Illinois at Chicago 840 South Wood Street Chicago, IL 60612, USA. Correspondence and requests for materials should be addressed to W.Q. (email: ) Scientific Reports | 6:34316 | DOI: 10.1038/srep34316 1 www.nature.com/scientificreports/ Figure 1. Deletion of Fak accelerates liver regeneration after PHx. (A) Left, expression of Fak protein, p-FAK (Y397), p-FAK (Y 576/577) and β-actin in whole livers of WT C57BL/6 mice 0, 1, 2, 3 and 5 days after PHx. Right, quantification of Western blotting by Image J software. (B) Liver weight/body weight ratio was analyzed in Alb-Cre (HepWT) and Alb-Cre; Fakflox/flox (Hep∆Fak) mice after PHx (5 mice per strain at each time point). hepatocarcinogenesis by activation of AKT and ERK14. Since FAK is important in cell proliferation, it is reasonable to suspect that a role exists for FAK in liver regeneration. However, such a role has as yet to be determined. In this study, we investigated the role of FAK in liver regeneration in 2/3 PHx model (resection of two-thirds of the mass of liver tissue). Interestingly, we found that specific deletion of Fak in mouse liver accelerates liver regeneration after 2/3 PHx. Consistently, Fak deletion enhances hepatocyte proliferation prior to day 3 after PHx but attenuates hepatocyte proliferation 3 days after PHx. Intriguingly, we found that the deletion of Fak in mouse liver significantly increases EGFR activation but decreases c-MET activation during liver regeneration. Furthermore, we found that Fak deficiency increases HB-EGF (a ligand of EGFR) during liver regeneration and a specific HB-EGF inhibitor abrogates accelerated liver regeneration and enhanced EGFR activation after PHx. Moreover, we discovered that Fak deficiency increases TNFαexpression after PHx and a neutralizing TNFαantibody suppresses accelerated liver regeneration, enhanced HB-EGF expression and EGFR activation in Fak-deficient mice. In addition, more apoptosis was found in Fak-deficient mouse livers compared to WT mouse livers after PHx. In general, our data suggest that Fak deletion accelerates the liver regenerative process by regulating the TNFα/ HB-EGF/EGFR axis. Inhibition of FAK may be a promising strategy to accelerate liver regeneration in recipients following liver transplantation. Results FAK is activated and induced during liver regeneration after 2/3 PHx. The kinase activity of FAK plays a critical role in its functions15,16. Phosphorylation of FAK on Tyr397 is required for its activation17,18. To study the role of FAK in liver regeneration, we first examined whether FAK is activated during liver regeneration after 2/3 PHx. We found that (...truncated)