Endothelial nitric oxide synthase is a key mediator of hepatocyte proliferation in response to partial hepatectomy in mice

Endothelial nitric oxide synthase (eNOS) is a critical modulator of vascular tone and blood flow and plays major roles in liver physiology and pathophysiology. Nitric oxide (NO) is widely recognized as one of the key humoral factors important for the initiation of liver regeneration in response to partial hepatectomy. Liver regeneration in response to partial hepatectomy is dependent on the efficiency of growth factor-mediated cell-cycle progression. Epidermal growth factor receptor (EGFR) is a critical mediator of multiple hepatic mitogens, such as epidermal growth factor (EGF), transforming growth factor alpha, amphiregulin, and heparin-binding EGF in regenerating livers. However, the functional significance of endothelial nitric oxide synthase (eNOS) expressed in hepatocytes, and its potential role in EGFR-mediated hepatocyte proliferation, remains unexplored. We sought to determine whether eNOS is essential for hepatocyte proliferation in response to partial hepatectomy (PH). Our studies with eNOS knockout (eNOS(-/-) ) mice suggest that eNOS activation is essential for the efficient induction of early events and elicitation of a robust hepatocyte proliferative response to PH. Moreover, eNOS expression is essential for the efficient early induction of matrix metalloprotease-9, a known mediator of extracellular matrix remodeling and growth factor activation in regenerating livers. Our in vitro studies suggest that eNOS is a critical mediator of EGF-induced hepatocyte proliferation, potentially via its influence on the induction of early growth response-1 (Egr-1) and phosphorylation of c-Jun--known mediators of cell-cycle progression. EGF-induced eNOS phosphorylation at Ser 1177 is dependent on the phosphorylation and activation of EGFR/PI3 kinase/AKT signaling in hepatocytes. CONCLUSION: Collectively, these results highlight a hitherto unrecognized role for eNOS activation in hepatocyte proliferation with implications for targeted therapies to enhance liver regenerative response in chronic disorders.     

Intact signaling by transforming growth factor beta is not required for termination of liver regeneration in mice

Transforming growth factor beta (TGF-beta) is a potent inhibitor of hepatocyte proliferation in vitro and is suggested to be a key negative regulator of liver growth. To directly address the role of TGF-beta signaling in liver regeneration in vivo, the TGF-beta type II receptor gene (Tgfbr2) was selectively deleted in hepatocytes by crossing "floxed" Tgfbr2 conditional knockout mice with transgenic mice expressing Cre under control of the albumin promoter. Hepatocytes isolated from liver-specific Tgfbr2 knockout (R2LivKO) mice were refractory to the growth inhibitory effects of TGF-beta1. The peak of DNA synthesis after 70% partial hepatectomy occurred earlier (36 vs. 48 hours) and was 1.7-fold higher in R2LivKO mice compared with controls. Accelerated S-phase entry by proliferating R2LivKO hepatocytes coincided with the hyperphosphorylation of Rb protein and the early upregulation of cyclin D1 and cyclin E. However, by 120 hours after partial hepatectomy, hepatocyte proliferation was back to baseline in both control and R2LivKO liver. Regenerating R2LivKO liver showed evidence of increased signaling by activin A and persistent activity of the Smad pathway. Blockage of activin A signaling by the specific inhibitor follistatin resulted in increased hepatocyte proliferation at 120 hours, particularly in R2LivKO livers. In conclusion, TGF-beta regulates G(1) to S phase transition of hepatocytes, but intact signaling by TGF-beta is not required for termination of liver regeneration. Increased signaling by activin A may compensate to regulate liver regeneration when signaling through the TGF-beta pathway is abolished, and may be a principal factor in the termination of liver regeneration.     

Expression of Notch-1 and its ligand Jagged-1 in rat liver during liver regeneration

The Notch/Jagged signaling pathway is important for cellular differentiation and proliferation. Its dysfunction is associated with human pathologies in several tissues including liver. Point mutations in Jagged-1 gene are the cause for Alagille syndrome, associated with paucity of intrahepatic bile ducts. To determine the putative role of the trans-membrane receptor Notch and its ligand Jagged-1 in liver regeneration, we investigated the expression of Notch and Jagged-1 in rat liver following 2/3 partial hepatectomy. Immunohistochemical staining of normal rat liver showed that Notch was expressed in hepatocytes, bile duct cells and endothelial cells, whereas Jagged-1 was expressed in bile duct cells and hepatocytes. Both Notch-1 and Jagged-1 proteins were upregulated in hepatocytes after partial hepatectomy up to day 4. After partial hepatectomy, nuclear translocation of the intracellular cytoplasmic domain of Notch (NICD) increased and peaked within 15 minutes, indicating the activation of Notch. Expression of the Notch-dependent target gene (HES-1) expression increased within 30-60 minutes. Addition of recombinant Jagged-1 protein to primary cultures of hepatocytes stimulated hepatocyte DNA synthesis. Furthermore, injection of silencing RNA for Notch and Jagged-1 to livers 2 days before partial hepatectomy significantly suppressed proliferation of hepatocytes at days 2 to 4 of the regenerative response. In conclusion, Notch/Jagged signaling pathway is activated during liver regeneration and is potentially contributing to signals affecting cell growth and differentiation.     

Review: Regulation of liver regeneration by pro-inflammatory cytokines

The liver has tremendous regenerative capacity. This distinguishes it from other vital organs (e.g. the brain, heart and lungs) that cannot replace functional tissue once it has been destroyed. Although hepatocytes rarely proliferate in the healthy adult liver, virtually all surviving hepatocytes replicate at least once after 70% partial hepatectomy. Therefore, partial liver resection has been used to characterize mechanisms that regulate liver regeneration. Residual hepatocytes up-regulate both proliferative and liver-specific gene expression in order to preserve tissue specific function. In addition, hepatocyte proliferation is tightly co-ordinated to complement regenerative responses in hepatic nonparenchymal cells (e.g. endothelia, biliary epithelia, stellate and Kupffer cells), so that the entire organ can be reconstituted within days. Studies with neutralizing antibodies to tumour necrosis factor-α (TNF) clearly demonstrate that, after partial hepatectomy, TNF promotes liver cell proliferation. The present review focuses on the regulation of the hepatocyte proliferative response by pro-inflammatory cytokines.     

S-adenosylmethionine regulates cytoplasmic HuR via AMP-activated kinase

BACKGROUND & AIMS: After liver injury, hepatic S-adenosylmethionine (SAM) content decreases, and the blockage this molecule imposes on hepatocyte proliferation is released, facilitating liver regeneration. This activity of SAM is important for normal liver function because mice deficient in hepatic SAM display abnormal liver regeneration and develop hepatocellular carcinoma. How SAM regulates hepatocyte growth is unclear, but because SAM blocks hepatocyte growth factor (HGF)-induced cyclin D1 expression and DNA synthesis without affecting HGF-induced extracellular signal-regulated kinase phosphorylation, the mitogen-activated protein kinase (MAPK) pathway is probably not the target. METHODS: The effects of SAM on AMPK, HuR localization were assessed in rat hepatocytes after HGF, AICAR, and SAM treatment. RESULTS: We show here that HGF and 5-aminoimidazole-4-carboxamide-riboside (AICAR), an activator of AMP-activated protein kinase (AMPK), induce the phosphorylation of AMPK in hepatocytes and that SAM blocks this process. We also show that HGF- and AICAR-induced AMPK activation stimulate the transport from nucleus to cytoplasm of HuR, an RNA-binding protein that increases the half-life of target mRNA such as cyclin A2, and that SAM blocks this process. We found that, in hepatocytes, AICAR increases HuR binding to cyclin A2 messenger RNA (mRNA) as well as the expression and stability of this mRNA and that SAM blocks these events. Consistently, we found that AICAR induces hepatocyte proliferation and that SAM blocks this effect. Finally, we found that liver AMPK phosphorylation, cytoplasmic HuR, and binding of HuR to HuR-target mRNA and the steady-state levels of these mRNA are increased in knockout mice deficient in hepatic SAM. CONCLUSIONS: Our results yield novel insights about the mechanism by which SAM inhibits cell-cycle progression in the liver.     

Hallmarks of postoperative liver regeneration: An updated insight on the regulatory mechanisms

Performance and advances in liver surgery makes remarkable progress of the understanding of liver regeneration. Liver regeneration after liver resection has been widely researched, and the underlying mechanism mostly concerns proliferation of hepatocytes and the influence by inflammation through activation of Kupffer cells and the other parenchymal cells, the second regenerative pathway by hepatic progenitor cells (HPCs), inducing angiogenesis, remodeling of a extracellular matrix (ECM), and termination mechanisms. New clinical surgeries and the updated multiomics analysis are exploiting the remarkable progress, especially in immune regulation and metabolic process of two emerging hallmarks. This review briefly represents a systemic outline of eight hallmarks, including hepatocyte proliferation, contribution of hepatic progenitor cells, inducing angiogenesis, reprogramming of the extracellular matrix, apoptosis and termination of proliferation, inflammation, immune and metabolic regulation, which are set as organizing characteristics of postoperative liver regeneration and future directions of refining treatment targets.     

Purinergic P2Y2 receptors promote hepatocyte resistance to hypoxia

BACKGROUND/AIMS: ATP stimulation of purinergic P2 receptors (P2YR and P2XR) regulates several hepatic functions. Here we report the involvement of ATP-mediated signals in enhancing hepatocyte tolerance to lethal stress. METHODS: The protection given by purinergic agonists was investigated in rat hepatocytes exposed to hypoxia. RESULTS: ATP released after hypotonic stress (200 mOsm/L) as well as P2YR agonists prevented hepatocyte killing by hypoxia with efficiency ranking UTP > ATPgammaS > ADPbetaS, whereas the P2XR agonist, methylene-adenosine-5'-triphosphate, was ineffective. Adenosine-5'-O-3-thiotriphosphate (ATPgammaS; 100 micromol/L) also prevented Na+ -overload in hypoxic cells by inhibiting the Na+/H+ exchanger, without interfering with hypoxic acidosis. ATPgammaS activated Src and promoted a Src-dependent stimulation of both ERK1/2 and p38MAPK. Blocking p38MAPK with SB203580 reverted the protection given by ATPgammaS on both cell viability and Na+ accumulation, whereas ERK1/2 inhibition with PD98058 was ineffective. An increased phosphorylation of ERK1/2 was also evident in untreated hypoxic hepatocytes. PD98058 ameliorated Na+ accumulation and cell death caused by hypoxia. Hepatocyte pre-treatment with ATPgammaS reverted ERK1/2 activation in hypoxic cells. SB203580 blocked the effects of ATPgammaS on both ERK1/2 and Na+/H+ exchanger. CONCLUSIONS: The activation of p38MAPK by P2Y2R increases hepatocyte resistance to hypoxia by down-modulating ERK1/2-mediated signals that promote Na+ influx through the Na+/H+ exchanger.     

Hepatocyte growth factor as well as vascular endothelial growth factor gene induction effectively promotes liver regeneration after hepatectomy in Solt-Farber rats

BACKGROUND/AIMS: Hepatic oval cells play an important role in liver regeneration when proliferation of mature hepatocytes is inhibited. The aim of this study was to examine the effect of hepatocyte growth factor (HGF), or vascular endothelial growth factor (VEGF) on proliferation of oval cells in the Solt-Farber rat model. METHODOLOGY: One hour after 70% partial hepatectomy, 2-acetyl-aminofluorene-induced damaged rats were infected intravenously with recombinant adenoviral vectors, encoding rat HGF or human VEGF, or Escherichia coli beta-galactosidase as a control. RESULTS: The plasma HGF concentrations in the HGF-transferred rats were elevated compared with the other groups at 4 and 7 days after hepatectomy. Oval cells were confirmed by positive staining of both cytokeratin-19 and alpha-fetoprotein. Oval cells around the portal tracts in the HGF or VEGF-transferred rats increased in number compared with the control rats at 7 and 9 days after hepatectomy. The proliferating cell nuclear antigen labeling indices of oval cells and the hepatic regeneration rate after hepatectomy were significantly augmented by the HGF or VEGF treatment. Moreover, cyclin E expression was elevated in the HGF-treated rats. CONCLUSIONS: In the Solt-Farber rat model, HGF or VEGF gene injection effectively promoted liver regeneration after hepatectomy mainly with increased proliferation of hepatic oval cells.     

Adenosine triphosphate release and purinergic regulation of cholangiocyte transport

The discovery of purinergic receptors on almost every cell type studied to date suggests that purinergic signaling is a fundamental process regulating cell and organ level functions. Purinergic receptors have been found on all principal liver cell types, including liver parenchymal cells, or hepatocytes, and biliary epithelial cells, or cholangiocytes. Both hepatocytes and cholangiocytes are capable of the regulated release of adenosine triphosphate (ATP), and both cell types express a range of purinergic receptors to mediate cellular processes. The role of extracellular nucleotides in liver function is presently being elucidated. Extracellular ATP, in addition to autocrine regulation of liver cell volume, has recently been shown to play an important role in paracrine signaling to coordinate specific hepatocyte and cholangiocyte cellular responses. The findings that (1) cholangiocytes are capable of the regulated release of ATP into bile, (2) ATP is present in bile in concentrations capable of stimulating purinergic receptors, and (3) P2 receptor stimulation results in brisk Cl(-) channel activation and fluid secretion suggest an important role of extracellular ATP in the regulation of bile formation. This article highlights important developments in our understanding of the role of purinergic signaling in cholangiocyte transport and bile formation.     

Eosinophils secrete IL-4 to facilitate liver regeneration

The liver is a central organ for the synthesis and storage of nutrients, production of serum proteins and hormones, and breakdown of toxins and metabolites. Because the liver is susceptible to toxin- or pathogen-mediated injury, it maintains a remarkable capacity to regenerate by compensatory growth. Specifically, in response to injury, quiescent hepatocytes enter the cell cycle and undergo DNA replication to promote liver regrowth. Despite the elucidation of a number of regenerative factors, the mechanisms by which liver injury triggers hepatocyte proliferation are incompletely understood. We demonstrate here that eosinophils stimulate liver regeneration after partial hepatectomy and toxin-mediated injury. Liver injury results in rapid recruitment of eosinophils, which secrete IL-4 to promote the proliferation of quiescent hepatocytes. Surprisingly, signaling via the IL-4Rα in macrophages, which have been implicated in tissue repair, is dispensable for hepatocyte proliferation and liver regrowth after injury. Instead, IL-4 exerts its proliferative actions via IL-4Rα in hepatocytes. Our findings thus provide a unique mechanism by which eosinophil-derived IL-4 stimulates hepatocyte proliferation in regenerating liver.     

Thrombospondin-1 is a novel negative regulator of liver regeneration after partial hepatectomy through transforming growth factor-beta1 activation in mice

The matricellular protein, thrombospondin-1 (TSP-1), is prominently expressed during tissue repair. TSP-1 binds to matrix components, proteases, cytokines, and growth factors and activates intracellular signals through its multiple domains. TSP-1 converts latent transforming growth factor-beta1 (TGF-β1) complexes into their biologically active form. TGF-β plays significant roles in cell-cycle regulation, modulation of differentiation, and induction of apoptosis. Although TGF-β1 is a major inhibitor of proliferation in cultured hepatocytes, the functional requirement of TGF-β1 during liver regeneration remains to be defined in vivo. We generated a TSP-1-deficient mouse model of a partial hepatectomy (PH) and explored TSP-1 induction, progression of liver regeneration, and TGF-β-mediated signaling during the repair process after hepatectomy. We show here that TSP-1-mediated TGF-β1 activation plays an important role in suppressing hepatocyte proliferation. TSP-1 expression was induced in endothelial cells (ECs) as an immediate early gene in response to PH. TSP-1 deficiency resulted in significantly reduced TGF-β/Smad signaling and accelerated hepatocyte proliferation through down-regulation of p21 protein expression. TSP-1 induced in ECs by reactive oxygen species (ROS) modulated TGF-β/Smad signaling and proliferation in hepatocytes in vitro, suggesting that the immediately and transiently produced ROS in the regenerating liver were the responsible factor for TSP-1 induction. CONCLUSIONS: We have identified TSP-1 as an inhibitory element in regulating liver regeneration by TGF-β1 activation. Our work defines TSP-1 as a novel immediate early gene that could be a potential therapeutic target to accelerate liver regeneration.     

Pathological roles of purinergic signaling in the liver

Purinergic signaling has been postulated as a mechanism of cellular signaling since the early 1970s. Cellular responses triggered by extracellular nucleotides and nucleosides occur by defined adenosine (P1) and ATP (P2) receptors, respectively, and play a prominent role in many aspects of health and disease, including those involving the liver. In normal physiology, extracellular nucleotides modulate many of the normal biologic and hepatic metabolic processes such as gluconeogenesis and insulin responsiveness. Further, in multiple disease states, ATP and certain nucleotides serve as danger signals and are involved in heightened purinergic receptor activation in a myriad of pathologic processes. Recently, others and we have shown the regulation of purinergic signaling by ectonucleotidases to play an important role in the acute vascular pathobiology of liver inflammation, regeneration, and immunity, as in ischemia reperfusion and transplantation. Increased understanding into mechanisms of extracellular ATP metabolism by such ecto enzymes has also led to novel insights into the exquisite balance of nucleotide P2-receptor and adenosinergic P1-receptor signaling in those chronic hepatic diseases characterized by steatosis, fibrosis, and malignancy. This review will explore the developing role of purinergic signaling in the pathophysiology of liver disease and comment on potential future clinical applications.