Regulation of polyamine synthesis in human hepatocytes by hepatotrophic factor augmenter of liver regeneration

doi:10.1016/j.bbrc.2006.04.040

Biochemical and Biophysical Research Communications

Volume 345, Issue 1, 23 June 2006, Pages 181-187

https://doi.org/10.1016/j.bbrc.2006.04.040 Get rights and content

Abstract

Different stages of liver regeneration are regulated by a variety of factors such as the liver growth associated protein ALR, augmenter of liver regeneration. Furthermore, small molecules like polyamines were proven to be essential for hepatic growth and regeneration. Therefore, using primary human hepatocytes in vitro we investigated the effect of ALR on the biosynthesis of polyamines. We demonstrated by HPLC analysis that recombinant ALR enhanced intracellular hepatic putrescine, spermidine, and spermine levels within 9–12 h. The activation of polyamine biosynthesis was dose dependent with putrescine showing the strongest increase. Additionally, ALR treatment induced mRNA expression of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase, both key enzymes of polyamine biosynthesis. Further, ALR induced c-myc mRNA expression, a regulator of ODC expression, and therefore we assume that ALR exerts its liver regeneration augmenting effects through stimulation of its signalling pathway leading in part to enhanced polyamine synthesis.

Section snippets

Materials and methods

Reagents. Human recombinant HGF was purchased from R&D Systems (Wiesbaden, Germany). Collagenase (type IV), Hepes, and other buffer supplements were purchased from Sigma (Taufkirchen, Germany). Collagen I coated plates (Biocoat) were purchased from BD (Bedford, UK) and fetal calf serum was from Biochrom (Berlin, Germany). Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/l glucose was obtained from Biowhittaker (Verviers, Belgium), and all other media additives were purchased from Serva

ALR enhances [³H]thymidine incorporation in primary human hepatocytes

The potential of ALR to augment liver regeneration was analyzed by determination of DNA synthesis in primary human hepatocytes upon treatment with rhALR in vitro. To perform DNA synthesis induction experiments, recombinant human ALR was expressed and purified protein was verified by SDS gel electrophoresis demonstrating a monomeric and homodimeric structure under reducing and nonreducing conditions, respectively (Fig. 1A). Stimulation of primary hepatocytes with the hepatotrophic factors ALR (50

Discussion

The aim of this study was to elucidate the effects of ALR on the polyamine levels of primary human hepatocytes in vitro and which molecular mechanisms are involved in the regulation of polyamine metabolism upon stimulation with the hepatotrophic factor ALR.

The study is based on the findings that hepatocytes in a normal healthy liver are in a quiescent state, but get easily activated (“primed”) to enter the cell cycle for proliferation after tissue loss e.g., hepatectomy [3]. Further, it was

Acknowledgments

We gratefully acknowledge the technical assistance of S. Laberer, A. Gräbe, and J. Ouart, University of Regensburg, Germany.

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Bile acid synthesis is restricted to hepatocytes and is rate-limited by CYP7A1 (cholesterol 7α hydroxylase). CYP7A1 expression undergoes tight regulation and is repressed after partial hepatectomy to prevent the accumulation of toxic bile acids. Augmenter of Liver Regeneration (ALR) is a hepatotrophic factor shown to support liver regeneration by augmenting cell proliferation and reducing apoptosis. Nevertheless, less is known about ALR's role in protecting hepatocytes from bile acid accumulation and bile acid-induced apoptosis. Therefore, HepG2 and Huh-7 cells were incubated with recombinant human ALR (rALR) and the expression of CYP7A1, bile acid-induced apoptosis as well as potential molecular mechanisms were analyzed. We found that rALR reduces CYP7A1 expression by increasing nuclear NFκB levels. Moreover, rALR reduced glycochenodeoxycholate (GCDC)-induced-apoptosis by decreased expression of pro-apoptotic Bax and enhanced expression of anti-apoptotic Mcl-1, which is regulated by phosphatidylinositol-3-kinase (PI3K)/Akt activation and glycogen synthase kinase-3β (GSK3β) phosphorylation. Inhibitors for PI3K/Akt (GSK690693) and GSK3β (SB415286) confirmed the specificity of rALR treatment for this pathway. In addition, rALR reduces pro-death signaling by decreasing GCDC-induced JNK phosphorylation. Taken all together, rALR might contribute to protecting hepatocytes from toxic concentrations of bile acids by down-regulating their de novo synthesis, attenuating apoptosis by activation of PI3K/Akt - GSK3β pathway and inhibition of JNK signaling. Thereby this suggests a new role of ALR in augmenting the process of liver regeneration.
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Cholestasis is a liver disease associated with retention of bile in the liver, which leads to local hepatic inflammation and severe liver damage. In order to investigate the mode of action of drug-induced cholestasis, in vitro models have shown to be able to recapitulate important elements of this disease. In this study, we applied untargeted metabolomics to investigate the metabolic perturbances in HepaRG® cells exposed for 24 h and 72 h to bosentan, a cholestatic reference toxicant. Intracellular profiles were extracted and analysed with liquid chromatography and accurate-mass spectrometry. Metabolites of interest were selected using partial least-squares discriminant analysis and random forest classifier models. The observed metabolic patterns associated with cholestasis in vitro were complex. Acute (24 h) exposure revealed metabolites related to apoptosis, such as ceramide and triglyceride accumulation, in combination with phosphatidylethanolamine, choline and carnitine depletion. Metabolomic alterations during exposure to lower dosages and a prolonged exposure (72 h) included carnitine upregulation and changes in the polyamine metabolism. These metabolites were linked to changes in phospholipid metabolism, mitochondrial pathways and energy homeostasis. The metabolic changes confirmed the mitotoxic effects of bosentan and revealed the potential involvement of phospholipid metabolism as part of the mode of action of drug-induced cholestasis.
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Citation Excerpt :
This results in the activation of EGF-R pathways via MAPK activation [141] and subsequently AP1 activation [116]. Upon activation of the MAPK pathway, rALR increases polyamines synthesis, a prerequisite for liver regeneration, by increasing the expression of c-myc and ODC (ornithine-decarboxylase), the rate-limiting enzyme of polyamine synthesis [142]. Moreover, after rALR treatment, primary human hepatocytes show increased proliferation, but reduced expression and activity of various cytochrome P450 [143], which might be mediated by activated NFκB [143].
Liver regeneration is a well-orchestrated process that is triggered by tissue loss due to trauma or surgical resection and by hepatocellular death induced by toxins or viral infections. Due to the central role of the liver for body homeostasis, intensive research was conducted to identify factors that might contribute to hepatic growth and regeneration. Using a model of partial hepatectomy several factors including cytokines and growth factors that regulate this process were discovered. Among them, a protein was identified to specifically support liver regeneration and therefore was named ALR (Augmenter of Liver Regeneration). ALR protein is encoded by GFER (growth factor erv1-like) gene and can be regulated by various stimuli. ALR is expressed in different tissues in three isoforms which are associated with multiple functions: The long forms of ALR were found in the inner-mitochondrial space (IMS) and the cytosol. Mitochondrial ALR (23 kDa) was shown to cooperate with Mia40 to insure adequate protein folding during import into IMS. On the other hand short form ALR, located mainly in the cytosol, was attributed with anti-apoptotic and anti-oxidative properties as well as its inflammation and metabolism modulating effects. Although a considerable amount of work has been devoted to summarizing the knowledge on ALR, an investigation of ALR expression in different organs (location, subcellular localization) as well as delineation between the isoforms and function of ALR is still missing. This review provides a comprehensive evaluation of ALR structure and expression of different ALR isoforms. Furthermore, we highlight the functional role of endogenously expressed and exogenously applied ALR, as well as an analysis of the clinical importance of ALR, with emphasis on liver disease and in vivo models, as well as the consequences of mutations in the GFER gene.
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During hepatic regeneration in rodents, ALR was found to be upregulated in hepatocytes and released into serum shortly after partial hepatectomy (Gandhi et al., 1999). Interestingly, we found that ALR plays an important role in the modulation of the hepatic metabolism (Dayoub et al., 2006; Thasler et al., 2006), similar to other well-known growth factors such as EGF (Greuet et al., 1997). Moreover, an interesting study has confirmed that exogenous ALR has a protective function in the kidney by attenuating the inflammatory response in renal cells undergoing hypoxia/deoxygenation injury (Yan et al., 2015a).
The acute-phase response (APR) is an inflammatory process triggered mainly by IL-6 in response to neoplasm, tissue injury, infection or inflammation. Signaling of IL-6 is transduced by activating STAT3 which rapidly results in production of acute-phase proteins (APPs) such as fibrinogen β (FGB) and haptoglobin (HP). Augmenter of liver regeneration (ALR), a hepatotrophic factor supporting liver regeneration, was reported to be upregulated after liver damage. In this study we analyzed the role of ALR for IL-6 signaling and APR. Thus, we investigated the expression and release of APPs in human liver cells under conditions of increased exogenous or endogenous ALR. HepG2 cells and ALR-reexpressing HepG2 cells were treated with IL-6 in the presence or absence of exogenous ALR for different time points. The mRNA expression and release of both FGB and HP were measured by RT-PCR and ELISA. We found that exogenously applied ALR attenuated the IL-6-induced mRNA expression and protein secretion of both FGB and HP. In contrast, IL-6 stimulation in HepG2 cells which re-express ALR, revealed elevated APR shown by increased mRNA expression and secretion of FGB and HP. Furthermore, we found that ALR-mediated regulation of IL-6-induced APP production is accompanied by altered STAT3 activity. While exogenous ALR reduced the IL-6-induced phosphorylation of STAT3, endogenous ALR enhanced STAT3 activity in liver cells. In conclusion, ALR, dependent on its localization, changes APR at least in part, by modifying STAT3 activation. This study shows a dual signaling of ALR and suggests that ALR is pivotal for the regulation of APR, a crucial event in liver injury and regeneration.
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The polyamines (PAs) spermidine, spermine, putrescine and cadaverine are an essential class of metabolites found throughout all kingdoms of life. In this comprehensive review, we discuss their metabolism, their various intracellular functions and their unusual and conserved regulatory features. These include the regulation of translation via upstream open reading frames, the over-reading of stop codons via ribosomal frameshifting, the existence of an antizyme and an antizyme inhibitor, ubiquitin-independent proteasomal degradation, a complex bi-directional membrane transport system and a unique posttranslational modification—hypusination—that is believed to occur on a single protein only (eIF-5A). Many of these features are broadly conserved indicating that PA metabolism is both concentration critical and evolutionary ancient. When PA metabolism is disrupted, a plethora of cellular processes are affected, including transcription, translation, gene expression regulation, autophagy and stress resistance. As a result, the role of PAs has been associated with cell growth, aging, memory performance, neurodegenerative diseases, metabolic disorders and cancer. Despite comprehensive studies addressing PAs, a unifying concept to interpret their molecular role is missing. The precise biochemical function of polyamines is thus one of the remaining mysteries of molecular cell biology.

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¹: These authors contributed equally to this work.

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Regulation of polyamine synthesis in human hepatocytes by hepatotrophic factor augmenter of liver regeneration

Abstract

Section snippets

Materials and methods

ALR enhances [3H]thymidine incorporation in primary human hepatocytes

Discussion

Acknowledgments

Gastroenterology

Hepatology

J. Biol. Chem.

Gastroenterology

J. Biol. Chem.

J. Biol. Chem.

Can. J. Physiol. Pharmacol.

Biochim. Biophys. Acta

J. Hepatol.

Gastroenterology

FEBS Lett.

Exp. Cell Res.

Liver regeneration

Science

Cloning and sequence analysis of the rat augmenter of liver regeneration (ALR) gene: expression of biologically active recombinant ALR and demonstration of tissue distribution

Proc. Natl. Acad. Sci. USA

Augmenter of liver regeneration: its place in the universe of hepatic growth factors

Hepatology

A fresh look at augmenter of liver regeneration in rats

Hepatology

Hepatopoietin acts as an autocrine growth factor in hepatoma cells

DNA Cell Biol.

Augmenter of liver regeneration (ALR) may promote liver regeneration by reducing natural killer (NK) cell activity in human liver diseases

J. Gastroenterol.

Expression of augmenter of liver regeneration (ALR) in human liver cirrhosis and carcinoma

Histopathology

Effect of hepatic stimulator substance administration on tissue regeneration due to thioacetamide-induced liver injury in rats

Scand. J. Gastroenterol.

ALR enhances [³H]thymidine incorporation in primary human hepatocytes