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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

S-adenosylmethionine (SAMe) is involved in numerous complex hepatic processes such as hepatocyte proliferation, death, inflammatory responses, and antioxidant defense. One of the most relevant actions of SAMe is the inhibition of hepatocyte proliferation during liver regeneration. In hepatocytes, SAMe regulates the levels of cytoplasmic HuR, an RNA-binding protein that increases the half-life of target messenger RNAs such as cyclin D1 and A2 via inhibition of hepatocyte growth factor (HGF)-mediated adenosine monophosphate–activated protein kinase (AMPK) phosphorylation. Because AMPK is activated by the tumor suppressor kinase LKB1, and AMPK activates endothelial nitric oxide (NO) synthase (eNOS), and NO synthesis is of great importance for hepatocyte proliferation, we hypothesized that in hepatocytes HGF may induce the phosphorylation of LKB1, AMPK, and eNOS through a process regulated by SAMe, and that this cascade might be crucial for hepatocyte growth. We demonstrate that the proliferative response of hepatocytes involves eNOS phosphorylation via HGF-mediated LKB1 and AMPK phosphorylation, and that this process is regulated by SAMe and NO. We also show that knockdown of LKB1, AMPK, or eNOS with specific interference RNA (iRNA) inhibits HGF-mediated hepatocyte proliferation. Finally, we found that the LKB1/AMPK/eNOS cascade is activated during liver regeneration after partial hepatectomy and that this process is impaired in mice treated with SAMe before hepatectomy, in knockout mice deficient in hepatic SAMe, and in eNOS knockout mice. Conclusion: We have identified an LKB1/AMPK/eNOS cascade regulated by HGF, SAMe, and NO that functions as a critical determinant of hepatocyte proliferation during liver regeneration after partial hepatectomy. (HEPATOLOGY 2009;49:608–617.)

Hepatocyte growth factor (HGF) induces hepatocyte proliferation and plays an essential role in liver regeneration.1, 2 HGF activates the MET receptor signaling cascade, which includes the activation of the Ras/extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK), PI3K/Akt, Rac/Pak, and Crk/Rap1 pathways.3, 4 Activation of the Ras/ERK/MAPK response is of great importance for HGF-induced hepatocyte proliferation.5, 6 We have uncovered an alternative noncanonical pathway for HGF signaling associated with adenosine monophosphate–activated protein kinase (AMPK), which is also of great importance for HGF-induced hepatocyte proliferation.7 AMPK is a serine/threonine kinase that was initially characterized as a sensor of cellular energy status that is activated in response to cellular stresses that deplete energy stores (such as hypoxia) switching on metabolic pathways that generate adenosine triphosphate while switching off metabolic pathways that consume adenosine triphosphate.8 More recent studies have implied AMPK in a wider role of cellular processes including cell proliferation, angiogenesis, and inflammation.8 AMPK is activated by phosphorylation by at least two upstream kinases, the tumor suppressor kinase LKB1 and Ca2+/calmodulin-dependent protein kinase kinase.9–11 We have found that activation of AMPK by HGF in hepatocytes stimulates the transport from nucleus to cytoplasm of HuR,7 an RNA-binding protein that increases the half-life of target messenger RNAs (mRNAs) such as cyclin A2 and D1.12, 13 We found also that S-adenosylmethionine (SAMe), the main biological methyl donor and a precursor of glutathione synthesis,14, 15 inhibits these effects of HGF on AMPK activation and HuR translocation, as well as HGF-induced cyclin D1 and D2 expression, DNA synthesis and hepatocyte proliferation, through a process that involves the association of PP2A to AMPK.7

SAMe biosynthesis is catalyzed by methionine adenosyltransferase (MAT), the first step in methionine metabolism.14, 15 Of the two genes (MAT1A, MAT2A) that encode MAT, MAT1A is expressed in the adult liver.14, 15 MAT1A knockout (MAT1A-KO) mice have chronic hepatic SAMe deficiency, display increased proliferation, and spontaneously develop hepatocellular carcinoma.16, 17 We found that in mice lacking MAT1A, liver AMPK is hyperphosphorylated.7 We also observed that HuR's cytoplasmic content, the binding of HuR to cyclin A2 and D1, and the steady-state levels of these two mRNA were all increased in MAT1A-KO animals compared with livers of wild-type (WT) mice.7

Cellular SAMe is related to the growth status of the hepatocytes. Thus, quiescent and proliferating hepatocytes display different SAMe contents, being lower in the growing cells.18 This has been observed in rat liver following partial hepatectomy (PH), a commonly used experimental model for the analysis of liver regeneration,2, 5, 6 after which the content of SAMe is reduced subsequent to the intervention, coinciding with the induction of early-response genes and the onset of DNA synthesis.19–21 When the decrease in liver SAMe after PH is prevented by the administration of exogenous SAMe, the regenerative process is also impaired as a result of an insufficient proliferative response.21 The decrease in hepatic SAMe during liver regeneration is mediated by the peak in nitric oxide (NO) production that occurs in the liver after PH.22–24 NO regulates hepatocyte SAMe content through specific inactivation of hepatic MAT via S-nitrosylation of a cysteine residue (C121) of the enzyme.25, 26

In endothelial cells, AMPK has been shown to phosphorylate and activate endothelial NO synthase (eNOS)27–30 and, also in vascular endothelial cells, HGF stimulates NO production through eNOS phosphorylation.31, 32 NO is important for the proliferative response of the liver, because inhibition of NOS activity with Nω-nitro-L-arginine methyl ester (L-NAME) results in decreased hepatocyte proliferation after PH33 and prevents hepatocyte proliferation in response to HGF.34 Moreover, liver regeneration is impaired in inducible NOS (iNOS) knockout mice.24 eNOS has been identified at the plasma membrane, at the rough endoplasmic reticulum, and in the nuclei of rat hepatocytes, and lysophosphatidic acid (LPA) has been shown to stimulate the phosphorylation of eNOS and NO production as well as the induction of iNOS gene expression.35 This increase in iNOS expression was abrogated in eNOS knockout (eNOS-KO) mice, indicating that endogenous NO synthesized by eNOS mediates iNOS induction upon stimulation with LPA.35

We demonstrate that in mice the proliferative response of hepatocytes involves eNOS phosphorylation and NO production via HGF-mediated LKB1 and AMPK phosphorylation, and that this process is regulated by SAMe. Thus, PH may provoke liver injury, rather than liver regeneration, when eNOS activation is prevented.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Isolation and Culture of Hepatocytes.

Hepatocytes were isolated from male C57BL6 mice via collagenase perfusion as described.36 Two hours after cells were plated, the medium was replaced by modified Eagle's medium supplemented with 5% fetal bovine serum (FBS), and treatments were performed after 2 hours. (Additional details are available in the Supplemental Materials and Methods online.)

Immunoblot Analysis.

Samples were separated via sodium dodecyl sulfate–polyacrylamide gel electrophoresis and analyzed with immunoblotting using commercial antibodies.

Confocal Microscopy.

Hepatocytes were plated onto round cover slips at 1 × 106 cells/60-mm dish. After 2 hours, hepatocytes were washed twice with phosphate-buffered saline (PBS) and HGF (25 ng/mL) and/or SAMe (4 mM) was added. One hour later, cells were fixed in ethanol 100% for 10 minutes at room temperature, rinsed twice with PBS, and kept in PBS-sodium azide. (Additional details are available in the Supplemental Materials and Methods online.)

Gene Silencing.

MLP29 cells, a hepatocyte cell line,37 were transfected with iRNA (Qiagen) for gene silencing using Lipofectamine 2000 (Invitrogen). After 16 hours, the medium was replaced with fresh Dulbecco's modified Eagle's medium (DMEM), 5% FBS for another 8 hours and left overnight in DMEM, 0.1% FBS. Cells were then treated with HGF (25 ng/mL) for 4 hours for western blot assay. (Additional details are available in the Supplemental Materials and Methods online.)

DNA Synthesis.

MLP29 cells were transfected with iRNA as described above. For each transfection, 5,000 cells were placed in a 96-well plate. Medium was replaced 24 hours after transfection with fresh DMEM and 5% FBS. After 4 hours, medium was replaced with DMEM and 0.1% FBS and left overnight at 37°C. Cells were treated with HGF (25 ng/mL), and for the last 10 hours (3H)-thymidine (0.5 μCi/well) was added. Cells were harvested and thymidine incorporation was determined in a scintillation counter. For nontransfected cell experiments, 1,000 cells were plated, and the protocol used was as described above.

Partial Hepatectomy Experiments.

Two-thirds PH was performed using the method of Higgins and Andersen,39 in 2- to 3-month-old male WT mice, WT animals pretreated with SAMe (50 mg/kg intraperitoneal SAMe, three times at 12-hour intervals, last dose 30 minutes before two-thirds PH), MAT1A-KO mice,16 and eNOS-KO38 mice as described.45 Two hours before sacrifice, mice were injected intraperitoneally with bromodeoxyuridine (BrdU) (100 mg/kg body weight) to assess hepatocyte DNA synthesis as described.45 (Additional details are available in the Supplemental Materials and Methods online.)

BrdU Immunohistochemistry.

Frozen liver tissue sections were fixed with acetone for 1 minute at room temperature followed by treatment with 2 M HCl at 37°C for 20 minutes. The sections were then neutralized with 0.1 M sodium borate for 10 minutes, and mouse monoclonal anti-BrdU antibody (Roche Dianostics, UK) was applied overnight at 4°C followed by goat anti-mouse Rhodamine antibody (Cappel, PA) and Hoescht nuclear dye. The number of BrdU-positive cells was counted and expressed as a percentage of the total number of cells, as visualizedw with Hoescht labeling.

NO and SAMe Measurement.

NO was determined in the culture medium of hepatocytes or MLP29 cells using a Nitric Oxide Assay Kit (Assay Designs). Hepatocytes and MLP29 cells were plated for 12 hours in modified Eagle's medium containing 0.1% FBS. At the end of this period, cells were incubated for 4 hours in the presence or absence of HGF (25 ng/mL) and NO was determined in the culture medium. Hepatic SAMe was determined as described.40

Statistical Analysis.

The Student t test was used to evaluate statistical significance. Values of P < 0.05 were considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

HGF Induces LKB1/AMPK/eNOS Phosphorylation Through a Process Inhibited by SAMe.

We have previously demonstrated that, in hepatocytes, HGF induces the phosphorylation and activation of AMPK and that SAMe blocks this process probably via the activation of phosphoprotein phosphatases (Ppases).7 Because AMPK is activated by the tumor suppressor kinase LKB1,8 we hypothesized that in hepatocytes HGF may also induce the phosphorylation of LKB1. We found that 1 hour after the addition of HGF to hepatocytes, the phosphorylation of LKB1 and AMPK was induced without affecting the protein content of both kinases and that this effect remained for at least 4 hours (Fig. 1A). Because AMPK has been found to mediate the phosphorylation of eNOS27–30 and this enzyme is expressed in rat hepatocytes,35 we also examined the phosphorylation of eNOS in response to HGF in hepatocytes. We observed that 1 hour after the addition of HGF, the phosphorylation of eNOS was increased without affecting the protein content of the enzyme and that this effect remained for at least 4 hours (Fig. 1A). Because ERK can phosphorylate LKB1 via p90RSK,41 we used U0126, an ERK1/2 inhibitor,42 to determine whether LKB1, AMPK, and/or eNOS are targets of MAPK or another MET-associated kinase. U0126 addition to hepatocytes inhibited both basal and HGF-induced ERK1/2 phosphorylation (Fig. 1B). On the contrary, U0126 did not inhibit but increased basal LKB1/AMPK/eNOS phosphorylation (Fig. 1B). HGF-mediated phosphorylation of the LKB1/AMPK/eNOS pathway was less pronounced in U0126-treated cells than in control cells (Fig. 1B); this is as expected, because this pathway was already activated in U0126-treated cells before the addition of HGF. We also found that treatment with SAMe blocked the effect of HGF on LKB1/AMPK/eNOS phosphorylation (Fig. 1C). Furthermore, we confirmed by confocal microscopy that within 1 hour, HGF induced eNOS phosphorylation in hepatocytes, and SAMe treatment blocked this effect (Fig. 2). These results support the hypothesis that the LKB1 pathway is not downstream of ERK but is an alternative pathway, downstream of MET, that is distinct from the Ras/ERK/MAPK pathway and is regulated by SAMe.

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Figure 1. HGF induces the phosphorylation of LKB1, AMPK, and eNOS through a process inhibited by SAMe. (A) Mouse hepatocytes were incubated for 1 or 4 hours in the presence or absence of HGF (25 ng/mL). At the end of this period, cell extracts (30 μg per lane) were analyzed via western blotting with the indicated antibodies. (B) Mouse hepatocytes were preincubated in the presence or absence of the ERK1/2 inhibitor U0126 (50 μmol/L) for 2 hours. At the end of this period, cells were treated with buffer only or HGF (25 ng/mL). Cell extracts were collected 1 hour later and analyzed via western blotting with the indicated antibodies. (C) Mouse hepatocytes were incubated for 4 hours with HGF (25 ng/mL), HGF + SAMe (4 mmol/L), SAMe alone, or HGF + L-NAME (2 mmol/L). Cell extracts were collected and analyzed via western blotting with the indicated antibodies. Incubation of mouse hepatocytes in (B) was performed as described in (C). Calyculin A (5 nmol/L), an inhibitor of PP2A and PP1, was added 30 minutes before other additives, and the cell extract was analyzed via western blotting with the indicated antibodies. Data are representative of an experiment performed four times.

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Figure 2. Identification of P-eNOS in primary mouse hepatocytes using confocal microscopy. Hepatocytes were incubated with HGF (25 ng/mL) and/or SAMe (4 mM) for 1 hour. Cells were fixed with ethanol and incubated overnight with a P-eNOS Ser 1177 antibody (red). Nuclei were stained with Dapi (blue).

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We have previously shown that the inhibitory effect of SAMe on HGF-induced AMPK phosphorylation in mouse hepatocytes is prevented by calyculin A, an inhibitor of PP2A and PP1.7 Here we found that the inhibitory effect of SAMe on HGF-induced LKB1 and eNOS phosphorylation is also blocked by calyculin A (Fig. 1D). We also found that 2.5 nM okadaic acid, another inhibitor of PP2A and PP1 activity,7 also blocked the inhibitory effect of SAMe on HGF-induced LKB1 and eNOS phosphorylation (data not shown). Taken together, these results support the concept that SAMe prevents HGF-mediated LKB1/AMPK/eNOS phosphorylation through a process that involves the activation of PPases.

HGF-Induced LKB1/AMPK/eNOS Phosphorylation and Proliferation Is Regulated by NO.

NO is important for the proliferative response of the liver, because inhibition of NOS activity with L-NAME results in decreased hepatocyte proliferation after PH33 and prevents hepatocyte proliferation in response to HGF.34 Moreover, liver regeneration after PH is impaired in iNOS knockout mice,24 and SAMe, which blocks HGF-dependent hepatocyte proliferation,34 inhibits iNOS expression.43 eNOS has been identified at the plasma membrane, rough endoplasmic reticulum, and in the nuclei of rat hepatocytes, and the activation of liver eNOS by LPA has been found to induce iNOS expression and NO production.35 Because NO reduces SAMe content in hepatocytes through inhibition of its synthesis,25, 26 and treatment with L-NAME results in increased SAMe synthesis and content in these cells,34 we hypothesized that NOS inhibition prevents both HGF-mediated LKB1/AMPK/eNOS phosphorylation and proliferation. Accordingly, we observed that in mouse hepatocytes, L-NAME and N-iminoethyl-L-ornithine (NIO), another inhibitor of NOS activity,44 both inhibited HGF-mediated phosphorylation of LKB1/AMPK/eNOS and proliferation (Figs. 1C and 3A,B). These results indicate that, in hepatocytes, NO regulates HGF-mediated LKB1/AMPK/eNOS phosphorylation and that eNOS activity is important for hepatocyte proliferation.

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Figure 3. HGF-mediated LKB1/AMPK/eNOS phosphorylation and proliferation in hepatocytes is inhibited by NIO. (A) Mouse hepatocytes were incubated for 4 hours with HGF (25 ng/mL), HGF + NIO (1 mmol/L), or NIO alone. The cell extract (30 μg per lane) was collected and analyzed via western blotting with the indicated antibodies. Data are representative of an experiment performed four times. (B) Mouse hepatocytes were treated with buffer only (control), HGF (25 ng/mL), HGF + L-NAME (2 mmol/L), or HGF + NIO (1 mmol/L) for another 24 hours, the last 10 hours also with (3H)-thymidine, and the synthesis of DNA was determined. *P < 0.05 versus (A) control or (B) HGF-treated hepatocytes. Data are representative of four experiments performed in triplicate.

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Inhibition of HGF-Mediated Hepatocyte Proliferation via Knockdown of LKBI, AMPK, and eNOS.

To examine if the proliferative response of hepatocytes involves eNOS phosphorylation via HGF-mediated LKB1 and AMPK phosphorylation, we used iRNA to reduce the expression of LKB1, the AMPKα1 catalytic subunit, and eNOS. For these experiments, we used MLP29 cells, which are easier to transfect with iRNA than primary cultures of hepatocytes.7 First we observed that transfection with LKB1-, AMPKα1-, and eNOS-specific iRNA led to a marked reduction in LKB1, AMPKα1, and eNOS protein levels as compared with cells transfected with control iRNA (Fig. 4A). We then observed that whereas in MLP29 cells transfected with control iRNA the addition of HGF induced AMPK and eNOS phosphorylation, in cells transfected with LKB-specific iRNA the effect of HGF on AMPK and eNOS phosphorylation was prevented (Fig. 4B). Similarly, transfection with AMPKα1-specific iRNA prevented the phosphorylation of eNOS induced by HGF but had no effect on LKB1 phosphorylation, supporting the hypothesis that LKB1 is upstream of AMPK (Fig. 4B). Finally, we observed that whereas HGF induced the proliferation of MLP29 cells transfected with control iRNA, in cells transfected with LKB1-, AMPKα1-, or eNOS-specific iRNA, the proliferative response induced by HGF was prevented (Fig. 5). In endothelial cells, HGF stimulates NO production through phosphorylation of eNOS.32, 33 Accordingly, we found that in MLP29 cells transfected with control iRNA, HGF (25 ng/mL) induced the production of NO to the culture medium (from 6.4 ± 0.3 μmol/L in control cells to 9.2 ± 0.1 μmol/L in cells treated with HGF; P < 0.05 [n = 4]) and that this effect was blocked in MLP29 cells transfected with eNOS-specific iRNA (from 6.4 + 0.2 μmol/L in control cells to 6.5 ± 0.1 μmol/L in cells treated with HGF [n = 4]). In hepatocytes, the production of NO to the culture medium was also induced by HGF (from 48.9 ± 0.3 μmol/L in control cells to 73.7 ± 0.7 μmol/L in cells treated with HGF; P < 0.05 [n = 4]). Taken together, these results indicate that HGF induces the activation of an LKB1/AMPK/eNOS cascade that may be of great importance for HGF-induced hepatocyte proliferation.

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Figure 4. HGF-mediated eNOS phosphorylation is downstream of LKB1 and AMPK phosphorylation. MLP29 cells were transfected with 100 nmol/L control iRNA or with LKB1-, AMPKα1-, or eNOS-specific iRNA using oligofectamine reagent for 24 hours. (A) Immunoblots of LKB1, AMPKα1, and eNOS protein expression. (B) Forty-eight hours after transfection, cells were treated with buffer only (control) or with HGF (25 ng/mL) for 4 hours. The cell extract (30 μg per lane) was collected and analyzed via western blotting with the indicated antibodies. Data are representative of an experiment performed four times.

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Figure 5. Knockdown of LKBI, AMPK, or eNOS with iRNA inhibits HGF-mediated proliferation. MLP29 cells were transfected with 100 nmol/L control iRNA or with LKB1-, AMPKα1-, or eNOS-specific iRNA using oligofectamine reagent for 24 hours as in Fig. 4. Twenty-four hours after transfection, cells were treated with buffer only (control) or with HGF (25 ng/mL) for another 24 hours, the last 14 hours also with (3H)-thymidine, and the synthesis of DNA was determined. Figure shows the average of four experiments in triplicate. *P < 0.05 versus MLP29 cells transfected with control iRNA and stimulated with HGF.

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Phosphorylation of LKB1, AMPK, and eNOS During Liver Regeneration After PH: Inhibition by SAMe.

To strengthen the concept that HGF-mediated phosphorylation of the LKB1/AMPK/eNOS cascade is of great importance for hepatocyte proliferation and plays an essential role in liver regeneration, we have examined liver LKB1/AMPK/eNOS phosphorylation following PH in control mice, in mice treated with SAMe, to prevent the reduction in hepatic SAMe after PH,20 and in MAT1A-KO animals deficient in hepatic SAMe.16 It has been reported that HGF levels rise significantly in the blood within 5 minutes after PH and that MET receptor phosphorylation occurs within 30 minutes and is high 30 minutes to 1 hour after PH.5, 6 Consistent with this finding, we found that the LKB1/AMPK/eNOS cascade is phosphorylated within 30 minutes after PH and that this effect was maintained during the next 24 hours (Fig. 6A), suggesting that this pathway is part of the early response during liver regeneration.

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Figure 6. Phosphorylation of LKB1, AMPK, and eNOS during liver regeneration after PH. (A) PH was performed in WT mice. Liver samples were obtained before, 30 minutes, 1 hour, and 24 hours after PH and analyzed (30 μg/lane) via western blotting with the indicated antibodies. Data are representative of an experiment performed four times. (B) PH was performed in WT mice, MAT1A-KO mice deficient in hepatic SAMe, WT animals pretreated with SAMe (WT + SAMe; 50 mg/kg intraperitoneal SAMe, 3 times at 12-hour intervals, last dose 30 minutes before hepatectomy), and in eNOS-KO mice. Liver samples were obtained before and 24 hours after PH and analyzed (30 μg per lane) via western blotting with the indicated antibodies. Data are representative of an experiment performed four times. (C) Hepatocyte proliferation after PH as assessed by BrdU incorporation. The number of BrdU-positive cells at 0 hours and 48 hours after PH were counted and expressed as a percentage of the total number of cells present per section. The ratio between percentage at 48 hours and 0 hours was calculated for WT ± SAMe, eNOS-KO, and WT mice. Data represent the average of four experiments performed in triplicate. *P < 0.05 versus WT liver samples 48 hours after PH.

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Previous studies have shown that liver regeneration after PH is impaired both in SAMe-treated animals21 and in SAMe-deficient MAT1A-KO mice.45 We found that phosphorylation of the LKB1/AMPK/eNOS cascade was blocked in SAMe-treated animals (Fig. 6B). SAMe treatment, however, had no effect on the expression levels of liver MET (data not shown), which is consistent with previous results showing that, in hepatocytes, SAMe has no effect on HGF-dependent MET or ERK1/2 phosphorylation.34, 46 In MAT1A-KO mice deficient in hepatic SAMe, basal liver phosphorylation of LKB1 and AMPK was augmented but failed to increase further following PH (Fig. 6B). We also observed that although basal eNOS phosphorylation was normal in the livers of MAT1A-KO mice, it failed to increase after PH (Fig. 6B). Finally, we observed that in eNOS-KO mice, LKB1 and AMPK phosphorylation following PH was prevented (Fig. 6B). We also observed that, after PH, hepatocyte growth as measured via BrdU incorporation was markedly reduced in WT mice treated with SAMe and in eNOS-KO mice (Fig. 6C).

We have demonstrated previously that liver SAMe decreases after PH in WT mice but not in MAT1A-KO animals, whereas before PH, SAMe content is already about half that measured in control animals.45 Before PH, the hepatic SAMe content was similar in WT and eNOS-KO mice (0.36 ± 0.04 and 0.35 + 0.04 nmol/mg protein, respectively [n = 4]) but was about 2-fold higher in SAMe-treated animals (0.68 ± 0.09 nmol/mg protein [n = 4]). Twenty-four hours after PH, the hepatic SAMe content decreased in WT and eNOS-KO mice (0.23 ± 0.03 and 0.19 ± 0.02 nmol/mg protein, respectively [n = 4]), and although it decreased in SAMe-treated animals as well (0.34 ± 0.03 nmol/mg protein [n = 4]), the levels in this model essentially remained the same as those in WT mice before PH, indicating that there is a correlation between SAMe levels and the inhibitory effects observed on the LKB1/AMPK/eNOS pathway.

These results suggest that activation of the LKB1/AMPK/eNOS cascade is of great importance for hepatocyte proliferation during liver regeneration and stresses the crucial role of SAMe in this process.

Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Previous studies have emphasized the role of activation of the Ras/ERK/MAPK pathway in HGF-induced hepatocyte proliferation and liver regeneration after PH.1–6 The present study has uncovered a noncanonical pathway for HGF-induced hepatocyte proliferation. We have identified an LKB1/AMPK/eNOS cascade regulated by HGF, SAMe, and NO that functions as a critical determinant of hepatocyte proliferation during liver regeneration after PH. This conclusion is based primarily on the finding that HGF induces the phosphorylation of LKB1, AMPK, and eNOS and on the observation that knockdown of LKB1, AMPK, or eNOS prevents HGF-induced hepatocyte proliferation. The finding that the inhibition of the Ras/ERK/MAPK pathway with U0126 did not inhibit but rather increased basal LKB1/AMPK/eNOS phosphorylation suggests that this cascade is not downstream of ERK, but is an alternative pathway downstream of MET that is distinct from the Ras/ERK/MAPK pathway. Our results strengthen our hypothesis that SAMe is a central regulator of hepatocyte proliferation14, 15 and point to the LKB1/AMPK/eNOS cascade as its main site of action. Our results also demonstrate that, in hepatocytes, SAMe prevents HGF-mediated phosphorylation of the LKB1/AMPK/eNOS cascade through a mechanism that probably involves PPase activation. Accordingly, we observed that phosphorylation of the LKB1/AMPK/eNOS cascade in the mouse liver increases during liver regeneration after PH, but that this increase is prevented by the administration of SAMe to mice prior to hepatectomy, which agrees with previous findings showing that SAMe treatment impairs the liver regenerative process.21 We found also that LKB1 and AMPK are hyperphosphorylated in MAT1A-KO mouse livers, which agrees with our observation that the content of cytoplasmic HuR, a target of AMPK in the liver, the binding of HuR to cyclin A2 and cyclin D1, the steady-state levels of these two mRNA, and DNA synthesis are all increased in the livers of MAT1A-KO animals compared with those of WT mice.7 In MAT1A-KO mice, the phosphorylation of LKB1, AMPK, and eNOS failed to increase after PH, which is in agreement with the finding that hepatic SAMe content did not fall further in MAT1A-KO mice compared with WT mice.45 This finding is also consistent with our previous observation showing that liver regeneration after PH is impaired in MAT1A-KO mice, as well as the finding that hepatocytes isolated from these knockout animals do not respond to HGF.45

NO production plays an important role in liver regeneration. After PH, liver regeneration is impaired in mice lacking iNOS.24 Additionally, the inhibition of NOS activity with L-NAME reduces hepatocyte proliferation after PH33 and prevents hepatocyte proliferation in response to HGF.34 It has been shown recently that the hepatic increase in iNOS expression elicited by LPA is abrogated in eNOS-KO mice, indicating that endogenous NO synthesized by eNOS mediates iNOS induction.35 Additionally, it has been found that HuR binds and stabilizes iNOS in hepatocytes47, 48 and that SAMe prevents iNOS induction in the liver and in hepatocytes in response to bacterial lipopolysaccharide and proinflammatory cytokines.43 We found that HGF induces eNOS phosphorylation in hepatocytes and that after eNOS knockdown with specific iRNA, hepatocyte proliferation in response to HGF was markedly reduced. These findings support a model (Fig. 7) in which NO, generated by the combined action of eNOS and iNOS, inhibits hepatic SAMe synthesis25, 26 and releases the blocking effect this molecule exerts on the LKB1/AMPK/eNOS pathway. Consequently, it stimulates the translocation of HuR from the nucleus to the cytoplasm leading to cell cycle progression and hepatocyte proliferation, as well as to further stimulation of NO production. The observation that inhibition of NO synthesis with L-NAME or NIO prevents HGF-induced LKB1/AMPK/eNOS phosphorylation and proliferation agrees with this model because, in the absence of an increased production of NO synthesis in response to HGF, SAMe synthesis will not be inhibited and the blockade this molecule exerts on LKB1/AMPK/eNOS phosphorylation will be maintained. Supporting this view, and the importance of eNOS-generated NO in the regulation of the LKB1/AMPK/eNOS cascade by HGF, in eNOS-KO mice after PH the phosphorylation of LKB1 and AMPK failed to increase and liver regeneration was markedly impaired. The mechanism by which HGF/MET signaling increases LKB1 phosphorylation is not understood. MET might associate with and phosphorylate LKB1, or do so through another intermediate kinase (other than ERK). Interestingly, protein kinase C activates LKB1 in endothelial cells,49 and protein kinase C inhibition impairs HGF-induced MAPK activation in hepatocytes50. Alternatively, it is also possible that MET inactivates a phosphatase allowing for increased LKB1 phosphorylation. Interestingly, PP2A associates with MET in A549 cells.51

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Figure 7. Model of an LKB1/AMPK/eNOS cascade regulated by HGF, SAMe, and NO implicated in hepatocyte proliferation. HGF/MET induces the phosphorylation and activation of LKB1, AMPK, and eNOS. AMPK phosphorylation induces the translocation to cytoplasm of HuR (HuRc), an RNA-binding protein that induces cell cycle progression and hepatocyte proliferation by increasing the half-life of target mRNAs such as cyclin A2 and cyclin D1. HuR also binds and stabilizes iNOS. eNOS-dependent NO production activates iNOS induction, which further contributes to NO synthesis, and MAT I/III inactivation, the enzyme primarily responsible for hepatic SAMe synthesis. This reduction in hepatic SAMe synthesis prevents the activation of PPase, which further increases the phosphorylation and activation of the LKB1/AMPK/eNOS cascade and hepatocyte proliferation. NIO and L-NAME inhibit HGF-mediated hepatocyte proliferation via inhibition of NO synthesis and of the blocking effect this molecule exerts on SAMe production, PPase activation, and the inactivation of LKB1 and AMPK. SAMe inhibits HGF-mediated hepatocyte proliferation via activation of PPase and inactivation of LKB1, AMPK, and eNOS.

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In conclusion, we demonstrate that the proliferative response of hepatocytes involves eNOS phosphorylation and NO production via HGF-mediated LKB1 and AMPK phosphorylation, and that this process is regulated by SAMe. In addition to its mitogenic effect, HGF regulates many other signaling pathways including motility, survival, and morphogenesis.6 Whether the LKB1/AMPK/eNOS cascade plays a role in these other pathways needs to be investigated.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

We thank Begoña Rodríguez and Noemi Magan-Marchal for technical assistance.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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hep_22660_sm_SupMethods.rtf19KSupplementary Methods

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