HGF/c-Met: A Key Promoter in Liver Regeneration

1Hepatocyte growth factor (HGF) is secreted by mesenchymal cells and is expressed in different type cells such as hepatic stellate cells (HSC), vascular endothelial cells (ECs), and Kupffer cells (KCs). HGF is a specific ligand for the tyrosine kinase receptor c-Met. The HGF/c-Met pathway plays a crucial role in the protection and regeneration of tissues. In various models built for injury and disease, HGF promotes cell survival and tissue regeneration, and improves chronic inflammation and fibrosis. Simultaneously, HGF is always acted as a potent inducer of lymphangiogenesis, angiogenesis, and tumor growth through promoting tumor cell invasion and metastasis.

2In 1984, Russell et al. identified and purified HGF in rat platelets, and Gohda et al. purified human HGF from the plasma of fulminant hepatic failure patients in 1988. In 1989, Nakamura et al. cloned the cDNA of human HGF and clarified the primary structure of HGF, and identified HGF as a novel growth factor that has a unique structural characteristic. Then, researchers gradually found that the fibroblast-derived factor scatters factor (SF) and HGF have the same structure and function. They finally confirmed that they are identical proteins encoded by a single gene.

3The HGF gene is mapped on the long arm of chromosome 7 of q21.1, and alternative splicing results in multiple transcripts, at least one of which encodes an inactive pre-pro-HGF. Then, the pre-pro-HGF becomes pro-HGF after a cleavage process between Arg494 and Val495. Mesenchymal cells produced HGF, which is a specific paracrine factor. And it is secreted out of the cells in the precursor form (pro-HGF) and also activated by the proteolytic cleavage at the Arg-Val site. The active HGF consists of 697 or 692 amino acids as a heterodimeric composed of α-chain and β-chain. Four kringle domains constitute the α-chain, while serine protease-like structures constitute the β-chain (Figures 1A,B). HGF activators reported previously include serum proteases and cell membrane proteases, such as HGF activator (HGF-A), urokinase-type plasminogen activator (uPA), plasma kallikrein, coagulation factors XII and XI, metalloproteinases and heparin. HGF-A is the central protease responsible for activating pro-HGF in serum. However, the studies in HGF-A knockout mice have shown that HGF-A gene deletion does not affect liver regeneration. In contrast, uPA plays a crucial role in HGF activation during liver regeneration.

4Unlike other growth factors and their receptors, HGF binds to the c-Met as a unique ligand, which is also located on chromosome 7. c-Met is a 190 kDa protein that contains a protein domain that binds to extracellular ligands, a transmembrane domain with tyrosine kinase activity, and a cytoplasmic domain. The c-Met has a 50 kDa β-chain and a 145 kDa α-chain for its mature form. The binding of HGF and c-Met can induce clustering of c-Met and phosphorylation of Y1234 and Y1235, and then phosphorylates Y1349 and Y1356 in the carboxy-terminal region (Figure 1C), which subsequently results in the adaptor molecule binding with it and transmitting downstream signals. HGF has two individual MET-binding interfaces, which include a high affinity NK1 (N-terminal and first kringle domain) binding site and a low affinity β-chain binding site.

5The biological functions of HGF are extensive and diverse. In embryonic development, targeted destruction of HGF or c-Met could lead to mouse death in utero and damage the development of the liver and placenta. Recombinant human HGF (rh-HGF) can significantly inhibit hepatocyte death and stabilize structural and vascular integrity in mice with acute liver failure (ALF). HGF receptor overexpression, mutation, amplification, or changes in its kinase activity is closely related to many different types of tumors.

6HGF/c-Met signaling pathway is indispensable for liver regeneration and regular repair. Studies showed that the lack of c-Met leads to liver necrosis and jaundice, delays regeneration, and high mortality in rodents. In the model of the normal rats and mice, injecting HGF into the portal vein caused hepatocyte proliferation and hepatomegaly. When recombinant human HGF-activator (rh-HGF-activator) was administered via the portal vein, compared with the control group, and the liver regeneration rate in the rh-HGF-activator-treated group was significantly higher. After partial hepatectomy (PHx), the tyrosine phosphorylation of c-Met occurs just in 5 min and increases gradually, then peaks at 60 min. In vitro, the activated HGF binding to the c-Met drives the phosphorylation of c-Met tyrosine residues, then Wnt-independent nuclear translocation of β-catenin is induced by HGF. Subsequently, c-Met is internalized and degraded promptly by the ubiquitin-proteasome degradation pathway. In the initial stage of liver regeneration, HGF activates JAK/STAT3, PI3K/Akt/NF-κB, and Ras/Raf pathways via HGF/c-Met, and initiates cell proliferation.

7In this review, we summarized the physiological and pathological mechanisms of the HGF/c-Met in liver regeneration. Specifically, we highlight the latest research on the HGF/c-Met axis, including the roles of non-coding RNAs in regulating of liver regeneration by HGF.

8PHx in mice or rats is a mature model for the study of the mechanism of liver regeneration. After PHx (>70%) or liver injury, rapid recovery of liver volume and function and strict regulation at the beginning and end stages are unique features of the liver. After PHx on mice or rats, the residual liver tissue recovered to the same level both in mass and volume as before in just 1 week. Both single-chain HGF and two-chain (active form) HGF are present in normal liver, and the single-chain HGF is the dominant form, while the expression of HGF mRNA is almost undetectable. After PHx, the level of HGF in the plasma has increased 10 to 20 times. According to the changes in endogenous HGF expression levels, liver regeneration can be divided into two stages. In phase I, the depletion phase, 0–3 h after PHx, HGF was derived from the transcription of HGF gene in KCs and ECs of normal liver, with a decrease in the level of both single-chain HGF (inactive form) and active two-chain HGF (active form) as characteristic. In phase II, the production phase, from 3 to 48 h or more after PHx, HGF was newly synthesized by ECs and HSCs and characterized by a significant increase in both single-chain and two-chain HGF levels. In addition, only active two-chain HGF was detected in plasma during the first 3 h after PHx. At the end of the liver regeneration process, TGF-β plays a role in the extracellular matrix (ECM) reconstruction. And with the ECM rebuilds, TGF-β binds to HGF, blocking its activation and restoring the hepatocytes to stasis.

9Another earliest biochemical change observed after PHx was the increased activity of uPA. Inactive pro-HGF was activated by uPA in the ECM. How do hemodynamic changes regulate the uPA activation is still unclear. However, uPA activation in such endothelial cells associated with the increased mechanical stress and turbulence have been documented.

10We know that the microenvironment, including cells and ECM plays a crucial role in maintaining liver tissue homeostasis and regeneration. The remodeling of ECM is accompanied by the renewal of many proteins. Urokinase is an activator for matrix remodeling and is detectable in tissues suffering wound healing and liver regeneration. After PHx, the uPA quantity remains relatively constant, but the uPA receptor (uPAR) dramatically increases after PHx, just beginning within 1 min. It enhances the activity of uPA in all remaining liver lobes. Then, the uPAR is induced to activate the fibrinolytic cascade reaction, and the plasminogen is activated to plasmin within 15 min. Plasminase promotes the conversion of matrix metalloproteinases to active metalloproteinases at 30 min after PHx. Both metalloproteinase and plasmin play a role in matrix remodeling and the renewal of ECM. The remodeling of ECM triggers a signal impulse through integrin, which leads to activating the inactive HGF attached to the ECM into its active form. It causes local and systemic HGF receptor c-Met activation within 30 min to 1 h after PHx. So metalloproteinase and metalloproteinase tissue inhibitor levels are essential in regulating the release and activation of HGF during regeneration.

11A great deal of work focusing on the regulation of uPA activity in the initial stage of liver regeneration is underway. Recent reports show that the increased release of preformed HGF in the extracellular matrix during liver regeneration which supports liver regeneration, is due to the up-regulation of uPA by TLR3. And during hepatitis B virus infection, no matter in mouse liver tissues or human liver cell lines, HBx down-regulated expression of uPA by the uPA promoter epigenetic regulation, resulting in damage to liver regeneration. Deletion of nuclear factor ImerC (NFI-C) leads to overexpression of plasminogen activator I (PAI-1) and subsequent inhibition of uPA activity and HGF signal, thus impairing hepatocyte proliferation. The hepatic hemodynamics changed immediately after PHx, and the portal vein pressure increased about three times theoretically. Still, the actual measurement showed that the portal vein pressure after PHx was only twice as much as that before operation. The increase of intravascular mechanical stress and turbulence promotes the activation of uPA in endothelial cells and other cells. This change regulates the production and release of HGF through β-1 integrin and vascular endothelial growth factor receptor 3 (VEGFR3) pathway. In addition, the permeability of the LSEC fenestra is heighten, and the nitric oxide secretion makes hepatocytes sensitive to HGF.

12HGF/c-Met axis has multiple effects in different cell types (Figure 2). The binding of C-Met to HGF initiates the receptor’s innate kinase activity, followed by the formation of a multisubstrate docking site for the intracellular junction protein, which recruits signaling molecules due to homologous dimerization and autophosphorylation at Tyr1234 and Tyr1235 and subsequent phosphorylation at Tyr1349 and Tyr1356 at the c-terminal tail. Gab1 in the Gab family of docking proteins which can directly or indirectly bind to c-Met through Grb2, is the most critical downstream multi-adapter protein of c-Met. The phosphorylated Gab1 creates additional binding sites for most downstream signaling molecules, which activate the different signaling pathways, including JAK/STAT3, phosphoinositide 3-kinase (PI3K)/Akt/NF-κB, Ras/Raf, and mitogen-activated protein kinase (MAPK) cascades.

13Because of its rapid response to PHx, STAT3 is considered to be a trigger for liver regeneration. In addition, HGF promotes the phosphorylation of STAT3 to activate the STAT3 pathway. In mice, the activity of STAT3 DNA binding was observed within 30 min and peaked 3 h after PHx. And this response is IL-6-dependent, and this STAT3 activation is almost inhibited in IL-6 deficient mice. The STAT3 pathway regulates hepatocyte proliferation through holding cyclin D1/p21 and prevents cell death by up-regulating FLIP, Bcl-2, Bcl-xL, Ref1, and MnSOD.

14The PI3K/Akt pathway plays a vital role in regulating cell growth, migration, differentiation, and apoptosis. When c-Met is activated by HGF, PI3K catalyzes the production of phosphatidylinositol-3,4,5-triphosphate after Gab1 coupled to the p85 subunit, which ultimately leads to phosphorylation of c-Met.

15In the Ras pathway, Erk1/2 as two mitogen-activated protein kinases to transmit mitotic signals, and the HGF/c-Met signaling pathway phosphorylates and activates Erk1/2 after PHx. ) found that when the liver regeneration was impaired, the phosphorylation of Erk1/2 was not detected in c-Met mutant mice. The results showed that specific signaling molecules, such as the protein kinase Akt, were fully activated by c-Met and other signaling receptors cooperators. The activation of ERK1/2 kinase was utterly dependent on c-Met during liver regeneration.

16The MAPK signaling is activated in response to growth factors and cytokines to promote liver regeneration effectively and plays a dominant role in cellular responses, including proliferation, migration, and differentiation. HGF in PHx activates MAPK signaling to promote liver regeneration effectively. In addition, studies have shown that during the adult liver progenitor cells (HPC)/oval cells involved in the repair of chronically injured livers, the HGF/c-Met pathway plays a crucial contribution in regulating TGF-β-mediated epithelial-mesenchymal transition (EMT) responses. The coordination and balance of TGF-β and HGF promoted the optimal regenerative potential of HPCs. These data indicate that the HGF/c-Met signaling system is essential for the regeneration of mature organs.

17The HGF/c-Met axis is strictly controlled by various mechanisms in normal tissues, and regulated by the amount of active HGF accurately. Two HGF protease inhibitors have been identified, HGF activator inhibitor (HAI)-1 and/or HAI-2, which control the activation of HGF ligand to limit the production of active HGF. The HGF-specific antagonist NK4, which is similar to HGF, can bind to C-Met and competitively antagonize downstream phosphorylation induced by HGF. In addition, the HGF/c-Met axis is regulated by the phosphorylation of the c-Met receptor. Many studies have shown that multiple pathways can negatively regulate the kinase activity of the c-Met receptor. For example, various protein tyrosine phosphatases negatively regulate the c-Met docking site by dephosphorylating c-Met receptor tyrosine residues. And c-Met monoubiquitination, internalization, and degradation could be triggered by casitas B lineage lymphoma ubiquitin ligase. Other studies have shown that changes in certain factors can regulate the expression of HGF and c-Met to regulate the HGF/c-Met axis. In vitro, the expression of HGF in hepatic stellate cells underwent a dramatic decrease under hypoxia, and the same as c-Met in hepatocytes. Human placental hydrolysate (hPH) treatment increased the secretion of HGF and promoted the proliferation of hepatocytes in rats after PHx. found that cytokine signaling inhibitory factor 1 (SOCS1) regulates hepatocyte proliferation through the HGF/c-Met axis. HGF stimulation-induced c-Met and Gab1 phosphorylation, cell migration, and proliferation in SOCS1 deficient mice hepatocytes. Later studies further showed that SOCS1 not only attenuates HGF/c-Met signaling but also regulates the expression of c-Met. In the model of chronic liver regeneration in rodents, Annalisa Addante et al. found that the bone morphogenetic protein (BMP)9 and HGF/c-Met signaling axes establish a signal crossover through ALK1 by modulating SMAD1 (pro-survival) and p38 mitogen-activated oval in kinase (p38MAPK; pro-apoptotic) determines the fate of oval cells. Another study showed that, the cellular transcription factor late SV40 factor (LSF) which is overexpressed in most of human hepatocellular carcinoma cases, is transcribed up-regulated osteopontin (OPN). Then, c-Met is activated via the potential interaction between OPN and its cell surface receptor CD44.

18Under pathological conditions, many factors regulate cell proliferation by controlling the HGF/c-Met axis. Well known, the liver plays a vital and irreplaceable role in regulating physiological and biochemical balance. It is straightforward to cause pathological liver damage due to various factors such as inflammation and viral infection. found that compared to ALF patients, in patients with acute chronic liver failure (ACLF), liver endothelial cells display a significant loss of CXC chemokine receptor type 7 DNA binding inhibitor 1 (CXCR7-ID1) dependent HGF expression, which correlates with the poor hepatocyte proliferation. Research proves that bacterial hepatocyte growth factor receptor agonist InlB321/15, a virulence factor produced by the pathogenic bacterium Listeria monocytogenes, improved hepatocyte proliferation and stimulated liver regeneration in animals with 70% hepatectomy.

19In addition, some drugs, such as rosiglitazone and GW9662, the peroxisome proliferator-activated receptor gamma (PPARγ) agonist and antagonist, respectively, also regulate cell proliferation through the HGF/c-Met axis. Cheng et al. found that, PPARγ, a nuclear transcription factor with multiple biological functions, plays a important role in regulating cell cycle in liver regeneration. They found that rosiglitazone inhibited liver regeneration while and GW9662 accelerated liver regeneration. Blocking the phosphorylation of c-Met by its inhibitor SGX523 could significantly eliminate the enhanced effect of PPARγ antagonist GW9662 on liver regeneration. Activation of HGF/c-Met pathways by phosphorylation of c-Met and ERK1/2 were inhibited in rosiglitazone-treated mice. It indicated that PPARγ hinders liver growth and hepatocyte proliferation by inhibiting the HGF/c-Met/ERK1/2 pathway. Researchers believe that these pathways may represent potential targets for liver disease in the future. Schisandra Chinensis and Resina Draconis treatment significantly increased the expression levels of HGF, then promoted the recovery of liver function in liver regeneration model mice through the HGF/c-Met signaling pathway, and ameliorated acute liver injury by promoting liver cell proliferation. Another study showed that rapamycin could promote lens epithelial cells apoptosis by inhibiting HGF-induced phosphorylation of AKT/mTOR, ERK, and JAK2/STAT3 signaling molecules. At present, a synthetic small molecule, 1K1 has the effect of anti-fibrotic and promoting liver regeneration in rodents. It is an engineered form of naturally occurring HGF fragment NK1, which has the advantages of better stability and is easier to be produced and might be used as a therapeutic medicine for acute and chronic liver disease. Furthermore, age also plays a role in regulating HGF. It is well known that liver regeneration capacity will decrease with increasing age. assessed 130 patients who underwent hepatectomy and found that HGF and c-Met expression in older patients was significantly lower than in younger patients. The average liver volume increase of young patients after 6 months of PHx is substantially more significant than that of elderly patients.

20In recent years, there have been many reports on the roles of non-coding RNA (ncRNA) in regulating the HGF/c-Met axis, which are mainly focused on tumor-related studies. C-Met is the target gene of multiple miRNAs. These miRNAs mainly include the miR-34 family (including miR-34a, miR-34b, and miR-34c), miR-206, miR-1, and other tumor suppressor miRNAs. They inhibit cell cycle progression, epithelial-mesenchymal transition, metastasis, stem cell transformation, and angiogenesis and promote cell apoptosis by inhibiting the HGF/c-Met axis. On the contrary, most lncRNAs targeting c-Met compete for endogenous RNAs (ceRNAs), decoys, or sponges, and combine with specific miRNAs to prevent the inhibition of c-Met, including ceRNAs: lnc-XIST, lnc-MALAT1, lnc-GAPLINC, lnc-HOTAIR targeting miR-34, LINC00240 targeting miR-4465, LNCRNA ZFAS1 targeting miR-193a-3p, lncRNA UCA1 targeting miR-1271-5p, etc. However, the research on the mechanisms of ncRNAs in regulating the HGF/c-Met axis in the background of liver regeneration is still minimal. Recent studies have shown that HGF can up-regulate the expression of SNHG12 in regular liver cell lines and activate the Wnt/β-catenin signal pathway to promote hepatocyte proliferation and liver regeneration.

21In human liver regeneration, limited by the means and purposes of clinical treatment, the researches on HGF are mainly carried out in the context of living donor liver transplantation rather than liver resection. Studies have shown that HGF levels are significantly increased on days 1, 7, and 14 after living donor liver surgery and are related to the recipient liver volume on day 14. reported that early-phase elevation of serum levels of various growth factors, including HGF, might be associated with the initiation of liver regeneration after hepatectomy in humans. At the same time, Tomiya et al. reported that serum HGF levels were increased after PHx, which was associated with hepatocellular dysfunction, necrosis, and systemic inflammation. On the other hand, proved that the post hepatectomy bile HGF level might be more meaningful for the early assessment of post hepatectomy liver function, bile HGF level rather than serum HGF level is more closely related to the post hepatectomy liver failure (PHLF). also suggested that bile HGF as a potentially functional marker of liver function after hepatectomy can be excreted from the liver at a higher concentration than that in serum. Furthermore, showed that after hepatectomy, effective HSC mobilization and homing depends on HGF, and these findings have important implications for potential treatment strategies.

22The powerful regeneration potential of the liver is legendary, which is attributed to various precise and complicated network regulations. These factors cooperate to restore the metabolic and synthetic functions accurately and timely after liver injury or resection. HGF plays a vital role in the initial stage of liver repair.

23HGF has been confirmed closely relating to liver repair after injury by various causes (chemical poison, infection, ischemia, physical injury or PHx, etc.). In animal experiments, HGF initiates liver regeneration at the beginning of the damage. The plasma HGF concentration changes throughout the repair progress, and HGF is maintained at a high concentration during the continuous regeneration phase until regeneration is complete. In clinical cases, the significant increase in HGF after live donor liver transplantation can be observed and related to the final liver regeneration volume. Without its specific receptor c-Met, HGF cannot play a role in liver regeneration. Furthermore, HGF combines with c-Met to trigger downstream cascade reactions and activate JAK/STAT3, PI3K/Akt/NF-κB, and Ras/Raf pathways, affecting cell proliferation, growth, and survival. The reports on HGF in human liver regeneration are minimal, and the use of HGF for drug development is challenging. Still, the preclinical studies currently have a large amount of data to support its therapeutic effects. Numerous clinical trials using rh-HGF protein or targeting HGF genes for clinical treatment are ongoing, which indicates that HGF may be a promising target for the therapies of human diseases.