Augmenter Of Liver Regeneration Expression Research Articles

Augmenter of liver regeneration promotes the proliferation of HL-7702 cells in carbon tetrachloride-induced acute liver injury via increasing autophagy

Objective: To investigate the protective effect of augmenter of liver regeneration (ALR) against acute liver injury and related mechanisms. Methods: HL-7702 cells were divided into normal control group, carbon tetrachloride (CCl4)-induced acute liver injury group, ALR+CCl4 intervention group, 3-methyladenine (3-MA)+CCl4 intervention group, and ALR+3-MA+CCl4 intervention group. The ALR+CCl4 and ALR+3-MA+CCl4 intervention groups were transfected with ALR plasmids at 8 hours before CCl4 treatment. All groups except the normal control group were treated with CCl4, and 30 minutes later, the 3-MA+CCl4 and ALR+3-MA+CCl4 intervention groups were treated with 3-MA. The cells were collected at 24 hours after CCl4 treatment. The HL-7702 cells and supernatant were collected to measure the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) (IU/L). Western blot was used to measure the levels of ALR, cyclin D, cyclin E, proliferating cell nuclear antigen (PCNA), autophagy-related gene 7 (Atg7), and autophagy genes LC3, p62, and Beclin-1. Quantitative real-time PCR was used to measure the mRNA expression of ALR. A one-way analysis of variance was used for comparison of means between any two groups. Results: The ALR+CCl4 intervention group had significant increases in the protein and mRNA expression of ALR compared with the acute liver injury group (both P < 0.05). The CCl4-induced acute liver injury group had significant increases in the protein and mRNA expression of ALR compared with the normal control group (both P < 0.05). Compared with the CCl4-induced acute liver injury group, the ALR+CCl4 intervention group had significant reductions in ALT (0.73±0.17 IU/L vs 1.43±0.38 IU/L, P < 0.05) and AST (19.85±1.83 IU/L vs 56.73±6.25 IU/L, P < 0.05) in supernatant, significantly increased expression of cyclin D, cyclin E, PCNA, LC3, Atg7, and Beclin-1 in hepatocytes, and significantly reduced expression of p62, which suggested that ALR protected the liver against acute liver injury, promoted the regeneration of hepatocytes, and enhanced the autophagy of hepatocytes. The ALR+3-MA+CCl4 intervention group had a significant reduction in the expression of regeneration-associated proteins compared with the ALR+CCl4 intervention group, while there was no significant difference between the ALR+3-MA+CCl4 intervention group and 3-MA+CCl4 intervention group, which suggested that after the inhibition of autophagy, there were significant reductions in the regeneration of hepatocytes and liver regeneration promoted by ALR. Conclusion: ALR can promote the regeneration of hepatocytes in liver parenchyma, which is achieved by the regulation of autophagy.

Hepatic Deficiency of Augmenter of Liver Regeneration Exacerbates Alcohol-Induced Liver Injury and Promotes Fibrosis in Mice.

Why only a subpopulation (about 15%) of humans develops liver cirrhosis due to alcohol is a critical as yet unanswered question. Liver-specific depletion of augmenter of liver regeneration (ALR) protein in mice causes robust steatosis and hepatocyte apoptosis by 2 weeks; these pathologies regress subsequently with return of ALR expression even at lower than control levels, but the mice develop modest steatohepatitis by 8 weeks. We aimed to investigate whether chronic alcohol ingestion promotes excessive hepatic fibrosis in these ALR-deficient mice. Liver-specific ALR-deficient and wild type (WT) female mice (8–10 weeks old) were placed on 4% alcohol-supplemented or isocaloric diet for 4 weeks. Liver sections were examined for histopathology, and parameters of steatosis and fibrosis were quantified. The mRNA expression of alcohol dehydrogenase-1, acetaldehyde dehydrogenase-1 and cytochrome P450-2E1 increased in WT mice but decreased in ALR-deficient mice upon alcohol ingestion. While alcohol induced steatosis and mild inflammation in WT mice, ALR-deficient mice showed minimal steatosis, strong hepatocellular injury and inflammation, prominent ductular proliferation, and robust fibrosis. Compared to the WT mice, alcohol feeding of ALR-deficient mice resulted in significantly greater increase in hepatic TNFα and TGFβ, and oxidative stress; there was also hepatic iron accumulation, robust lipid peroxidation and mitochondrial DNA damage. Importantly, similar to ALR-deficient mice, lower hepatic ALR levels in human alcoholic liver cirrhosis were associated with increased iron content, reduced expression of alcohol dehydrogenase and acetaldehyde dehydrogenase, and elevated fibrogenic markers. We conclude that ALR deficiency or anomaly can play a critical role in alcohol-induced hepatic fibrosis/cirrhosis, mechanisms of which may involve dysregulation of alcohol metabolism and iron homeostasis, mitochondrial damage and oxidative injury.

Antiapoptotic and antioxidative protein ALR in Cholestatic Liver Diseases – Do bile acids regulate ALR expression via Egr1?

Cholestatic liver diseases result when the excretion of bile acids from the liver is interrupted. It has been shown that inflammation contributes to liver injury during cholestasis and previous studies indicated that bile acids up- regulate early growth response fator-1 (Egr1) through the activation of MAPK signaling pathway. Egr1 is responsible for the development of the inflammation through up-regulation of several pro-inflammatory genes. Augmenter of liver regeneration (ALR) is a hepatotrophic factor with anti- oxidative and anti-apoptotic properties which has been found to be highly expressed in regenerating livers after tissue loss or insults through toxic substances. Nevertheless, little is known about the impact of ALR in cholestasis. Therefore, the aim of our study is to investigate the potential role of bile acids in regulating ALR expression as a protective protein in cholestasis, and to determine which transcription factors might regulate the tissue-specific expression of ALR.

Deceleration of liver regeneration by knockdown of augmenter of liver regeneration gene is associated with impairment of mitochondrial DNA synthesis in mice.

Hepatic stimulator substance, also known as augmenter of liver regeneration (ALR), is a novel hepatic mitogen that stimulates liver regeneration after partial hepatectomy (PH). Recent work has indicated that a lack of ALR expression inhibited liver regeneration in rats, and the mechanism seems to be related to increased cell apoptosis. The mitochondria play an important role during liver regeneration. Adequate ATP supply, which is largely dependent on effective mitochondrial biogenesis, is essential for progress of liver regeneration. However, ALR gene expression during liver regeneration, particularly its function with mitochondrial DNA synthesis, remains poorly understood. In this study, ALR expression in hepatocytes of mice was suppressed with ALR short-hairpin RNA interference or ALR deletion (knockout, KO). The ALR-defective mice underwent PH, and the liver was allowed to regenerate for 1 wk. Analysis of liver growth and its correlation with mitochondrial biogenesis showed that both ALR mRNA and protein levels increased robustly in control mice with a maximum at days 3 and 4 post-PH. However, ALR knockdown inhibited hepatic DNA synthesis and decelerated liver regeneration after PH. Furthermore, both in the ALR-knockdown and ALR-KO mice, expression of mitochondrial transcription factor A and peroxisome proliferator-activated receptor-γ coactivator-1α were reduced, resulting in impaired mitochondrial biogenesis. In conclusion, ALR is apparently required to ensure appropriate liver regeneration following PH in mice, and deletion of the ALR gene may delay liver regeneration in part due to impaired mitochondrial biogenesis.

Knockdown of augmenter of liver regeneration in HK-2 cells inhibits inflammation response via the mitogen-activated protein kinase signaling pathway.

Augmenter of liver regeneration (ALR) is a growth factor that is ubiquitously expressed in multiple forms among eukaryotes. The present study focused on the role of endogenous ALR on the hypoxia/reoxygenation (H/R)-induced inflammatory response in human kidney 2 (HK-2) cells, and the underlying molecular mechanisms. To determine the relationship between exogenous and endogenous ALR, exogenous ALR was administrated to HK-2 cells, and endogenous ALR protein and mRNA expression was examined by Western blotting and quantitative real-time polymerase chain reaction (qPCR), respectively. In order to knockdown endogenous ALR expression, HK-2 cells were infected with lentiviral shRNA/ALR, after which cell viability was determined by the MTS cell viability assay. Cells were subjected to hypoxia for 6 h and reoxygenation for 12 h. Levels of monocyte chemotactic protein (MCP-1), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) were determined by enzyme-linked immunosorbent assay (ELISA) and qPCR. Cells were harvested, and nuclear and phosphorylated protein extracts were prepared from the HK-2 cell lysates. Nuclear factor κB (NF-κB), and phosphorylated extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) were analyzed by Western blotting. The translocation of NF-κB was detected by immunofluorescence. Exogenous ALR inhibited the expression of endogenous ALR. Lentiviral shRNA/ALR markedly downregulated endogenous ALR expression, whereas there were no changes in ALR expression in lentiviral shRNA/control HK-2 cells. The results of the MTS assay showed that silencing ALR expression did not influence cell viability. H/R led to increased production of MCP-1, IL-6, and TNF-α. However, knockdown of ALR attenuated the inflammatory response via inhibition of ERK, p38, and JNK phosphorylation. The translocation of NF-κB into the nucleus was also decreased. These results suggest that there is a negative feedback loop involving ALR in HK-2 cells. Knockdown of ALR exerts anti-inflammatory actions via suppression of the mitogen-activated protein kinase signaling pathway.

Augmenter of liver regeneration plays a protective role against hydrogen peroxide-induced oxidative stress in renal proximal tubule cells.

Oxidative stress plays an important role in cellular destruction. Augmenter of liver regeneration (ALR) is an anti-apoptotic factor that is expressed in all mammalian cells and functions as an anti-oxidant by stimulating the expression of a secretory isoform of clusterin and inhibiting reactive oxygen species (ROS) generation. Previous work from our group showed that ALR expression is upregulated in acute kidney injury (AKI) rats, and recombinant human ALR reduces tubular injury. In the present study, we used small interfering RNA (siRNA) silencing of ALR to examine its role in H2O2 induced mitochondrial injury and apoptosis. Knockdown of ALR increased ROS levels, reduced mitochondrial membrane potential, and increased the release of mitochondrial proteins and the rate of apoptosis in response to H2O2. In addition, the ratio of Bax/Bcl-2 was increased in siRNA/ALR groups treated with H2O2. These data confirm the protective role of ALR against oxidative stress-induced mitochondrial injury and suggest a potential mechanism underlying the protective role of ALR in AKI.

Augmenter of liver regeneration, a protective factor against ROS-induced oxidative damage in muscle tissue of mitochondrial myopathy affected patients

Mitochondria-related myopathies (MM) are a group of different diseases defined by a varying degree of dysfunctions of the mitochondrial respiratory chain which leads to reactive oxygen species (ROS) generation followed by oxidative stress and cellular damage. In mitochondrial myopathy muscle tissue an overexpression of antioxidant enzymes has been documented probably as an attempt to counteract the free radical generation.We previously documented, in human non-pathological muscle fibres, the expression of the augmenter of liver regeneration (ALR), a sulfhydryl oxidase enzyme, whose presence is related to the mitochondria; indeed it has been demonstrated that ALR mainly localizes in the mitochondrial inter-membrane space. Furthermore we reported, in different experimental models, in vivo and in vitro, the anti-apoptotic and anti-oxidative capacities of ALR, achieved by up-regulating Bcl-2 anti-apoptotic family factors and the anti-apoptotic/anti-oxidative secretory isoform of clusterin (sClu).With the present study we aimed to determine ALR, Bcl-2 protein, clusterin and ROS expression in muscle tissue biopsies from MM-affected patients. Non-pathological muscle tissue was used as control. Enzymatic, histochemical, immunohistochemical and immune electron microscopy techniques were performed.The data obtained revealed in MM-derived muscle tissue, compared to non-pathological tissue, the over-expression of ROS, ALR and Bcl-2 and the induction of the nuclear, pro-apoptotic, isoform of clusterin (nCLU).

Nrf2 Activates Augmenter of Liver Regeneration (ALR) via Antioxidant Response Element and Links Oxidative Stress to Liver Regeneration

Liver regeneration can be impaired by permanent oxidative stress and activation of nuclear factor erythroid 2-related factor 2 (Nrf2), known to regulate the cellular antioxidant response, and has been shown to improve the process of liver regeneration. A variety of factors regulate hepatic tissue regeneration, among them augmenter of liver regeneration (ALR), attained great attention as being survival factors for the liver with proproliferative and antiapoptotic properties. Here we determined the Nrf2/antioxidant response element (ARE) regulated expression of ALR and show ALR as a target gene of Nrf2 in vitro and in vivo. The ALR promoter comprises an ARE binding site and, therefore, ALR expression can be induced by ARE-activator tertiary butylhydroquinone (tBHQ) in hepatoma cells and primary human hepatocytes (PHH). Promoter activity and expression of ALR were enhanced after cotransfection of Nrf2 compared with control and dominant negative mutant of Nrf2. Performing partial hepatectomy in livers from Nrf2+/+ mice compared with Nrf2-/- knock-out (KO) mice, we found increased expression of ALR in addition to known antioxidant ARE-regulated genes. Furthermore, we observed increased ALR expression in hepatitis B virus (HBV) compared with hepatitis C virus (HCV) positive hepatoma cells and PHH. Recently, it was demonstrated that HBV infection activates Nrf2 and, now, we add results showing increased ALR expression in liver samples from patients infected with HBV. ALR is regulated by Nrf2, acts as a liver regeneration and antioxidative protein and, therefore, links oxidative stress to hepatic regeneration to ensure survival of damaged cells.

Correlation between augmenter of liver regeneration and IFN-γ expression in graft after rat orthotopic liver transplantation

BackgroundPrevious data suggested that augmenter of liver regeneration (ALR) has immunomodulation function by suppressing liver-resident NK cell activity and reducing IFN-γ expression in human liver diseases. The correlation between ALR and IFN-γ expression in graft after rat orthotopic liver transplantation remains uncertain. MethodsA Lewis-to-BN (allograft group) and BN-to-BN (isograft group) rat liver transplantation model was used to investigate the ALR and IFN-γ expression in liver graft. Graft recipients were sacrificed at days 1, 3, 5, and 7 posttransplantation. The histopathologic changes of grafts were observed under light microscope and the intragraft expression of ALR and IFN-γ mRNA and protein was determined by real-time PCR and immunohistochemistry staining, respectively. Correlation between ALR and IFN-γ expression in graft was evaluated by Spearman rank correlation analysis. ResultsThe light microscope inspection revealed severe acute rejection in the allograft group but not in the isograft group at day 7 after liver transplantation. The intragraft ALR showed slight protein expression at day 1 after liver transplantation in both groups and it was significantly increased at days 3, 5, and 7 (P < 0.05). There was no significant difference in ALR mRNA expression between the allograft group and isograft group at day 1 (1.09 ± 0.12 and 1.13 ± 0.10, respectively; P > 0.05, n = 3). The ALR mRNA level was slightly reduced at day 3 in both groups compared with that at day 1 (0.81 ± 0.11 and 0.59 ± 0.10, respectively, P > 0.05). However, it was markedly increased at day 5 (2.86 ± 0.37) and day 7 (3.19 ± 0.33) in the isograft group and was 1.57 ± 0.27 and 1.98 ± 0.13 in the allograft group at days 5 and 7, respectively. IFN-γ protein and mRNA expression in the allograft group was increased at day 1 posttransplantation and reached a peak at day 3, and then it had a slight tendency of decline at day 5 and day 7. And they in the isograft group were at a low level at all times. The levels of ALR mRNA showed a negative correlation with levels of IFN-γ mRNA in the allograft group (r = −0.86, P < 0.05, y = −0.241x + 0.586), whereas there is no correlation between ALR and IFN-γ mRNA expression in the isograft group. ConclusionThese data revealed an obviously negative correlation between ALR and IFN-γ levels intragraft, which indicated that ALR may participate in immunoregulation of acute rejection.

Augmenter of liver regeneration (ALR) gene therapy attenuates CCl4-induced liver injury and fibrosis in rats

Liver fibrosis represents a process of healing and scarring in response to chronic liver injury. Augmenter of liver regeneration (ALR) has been shown to protect hepatocytes from various toxins. The aim of this study was to investigate the effects of ALR gene therapy on liver injury and fibrosis induced by CCl4 in rats and further explore the underlying mechanisms. Human ALR expression plasmid was delivered via the tail vein. ALR gene therapy might protect the liver from CCl4-induced injury and fibrogenesis by attenuating the mitochondrial dysfunction, suppressing oxidative stress, and inhibiting activation of HSCs. This report demonstrated that ALR gene therapy protected against the ATP loss, increased the activity of ATPase, decreased intrahepatic reactive oxygen species level, and down-regulated transforming growth factor-β1, platelet-derived growth factor-BB, and α-smooth muscle actin expression. Following gene transfer liver function tests were significantly improved. In brief, ALR gene therapy might be an effective therapeutic reagent for liver fibrosis with potential clinical applications.

Nrf2, a transcription factor mainly involved in antioxidant defense, regulates the expression of ALR, Augmenter of Liver Regeneration

The liver is frequently challenged by so called metabolic overload, lipid accumulation, viruses or toxins, which induce the formation of reactive oxygen species (ROS). ROS can damage lipids, protein and DNA by peroxidation leading to complete degradation. A crucial player in the defense against oxidative stress is NF-E2-related factor 2 (Nrf2). The transcription factor Nrf2 is important for protecting cells against oxidative damage. It has been shown that Nrf2 binds to the antioxidant response element (ARE) controlling the expression of antioxidant proteins involved in the detoxification of harmful compounds. Additionally, Augmenter of Liver Regeneration (ALR) was reported to be upregulated in models of liver damage caused by oxidative stress. Recent micro array analysis demonstrated that Nrf2 regulates several cytoprotective proteins including ALR. Furthermore, promoter analysis of ALR predicts the presence of ARE, the potential binding site for Nrf2. Therefore, we aimed to verify whether Nrf2 is involved in the regulation of ALR expression. Treatment of primary human hepatocytes and HepG2 cells with tBHQ, a well known ARE-activator, induced mRNA and protein expression of ALR. Furthermore, the promoter activity of ALR was significantly induced after co-transfection of Nrf2 over control and dominant negative mutant of Nrf2 (dnNrf2). The functional impact of increased promoter activity after Nrf2-transfection was underlined by elevated mRNA and protein levels of ALR. In addition, we could show that ALR expression in livers from Nrf2-/- KO mice compared to Nrf2+/+ mice was attenuated during hepatic regeneration after a two-third hepatectomy. In conclusion, we demonstrated that the anti-oxidant transcription factor Nrf2 regulates the expression of ALR, and this regulation is mediated by activation of ARE. Thus, we speculate that ALR might play an important role as hepatotrophic factor against oxidative stress caused by different insults in the liver.

Foxa2 (HNF-3β) regulates expression of hepatotrophic factor ALR in liver cells

Liver regeneration is a multistep and well-orchestrated process which is initiated by injuries such as tissue loss, infectious or toxic insults. Augmenter of liver regeneration (ALR) is a hepatotrophic growth factor which has been shown to stimulate hepatic regeneration after partial hepatectomy and therefore seems to be regulated during the regenerative process in the liver. Our aim was to analyze how ALR is regulated in hepatic tissues and which transcription factors might regulate its tissue-specific expression. Promoter studies of ALR (−733/+527bp) revealed potential regulatory elements for various transcription factors like Foxa2, IL-6 RE-BP and C/EBPβ. Analysis of the promoter activity by performing luciferase assays revealed that co-transfection with Foxa2 significantly induced the activity of ALR promoter in HepG2 cells. EMSA and Supershift analysis using anti-Foxa2 antibody confirmed the specific binding of Foxa2 to ALR promoter and this binding was inducible when the cells were simultaneously stimulated with IL-6. The increased binding after activation with IL-6 and/or Foxa2 was confirmed by elevated ALR protein levels using Western blot technique. In addition, we could not detect any binding of C/EBPβ and IL-6 RE-BP to the promoter of ALR. In conclusion, these results indicate that ALR is regulated by Foxa2, and this regulation may be amplified by IL-6.

Augmenter of liver regeneration protects kidneys from ischaemia/reperfusion injury in rats

Augmenter of liver regeneration (ALR), which was identified originally for its crucial role in promoting hepatocyte proliferation, is expressed in both the liver and kidney. Protective effects of ALR have been demonstrated in experimental models of acute liver failure. In the present study, we investigated the effect of ALR on renal ischaemia/reperfusion (I/R) injury and the possible mechanisms of its action. Male Sprague-Dawley rats were subjected to renal ischaemia for 60 min and then administered with either saline or recombinant human ALR (rhALR). A sham-operated group served as control. The expression of ALR in the sham-operated and acute kidney injury (AKI) groups was detected by immunohistochemistry and western blotting. Renal dysfunction and injury were assessed by measurement of serum biochemical markers and histological grading. Expression of proliferating cell nuclear antigen (PCNA) was determined by immunohistochemistry. Renal ALR expression increased significantly in rats with ischaemic AKI compared with the sham-operated rats. Serum biochemical parameters showed that renal dysfunction was improved by administration of rhALR. Histological analysis revealed that treatment with rhALR also reduced the extent of kidney injury. Intraperitoneal injection of rhALR enhanced the proliferation of renal tubular cells. Conclusions. Administration of rhALR effectively reduces tubular injury and ameliorates the impairment of renal function. The protective effect of rhALR is associated with enhancement of renal tubular cell regeneration.

Expression of cytosolic ALR in HCC cells and its impact on hepatocellular growth in vitro and in vivo

Progression of HCC depends on different factors like metastasis and invasiveness. Previously, we have shown that the hepatotrophic protein Augmenter of Liver Regeneration (ALR) is highly expressed in cirrhotic livers and HCC. The aim of our study was to gain more insight in to the functional role of differential regulation of ALR in HCC and its impact on cellular growth in vitro and in vivo. Therefore the cytosolic 15 kDa isoform ALR was re-induced in hepatoma cell line HepG2 by stable transfection with a human ALR expression vector containing the 375 bp ALR cDNA under the control of the cytomegalovirus (CMV) promoter (ALR-HepG2). Quantitative RT-PCR revealed a strong induction of ALR mRNA expression in ALR-HepG2 cells, which was confirmed by matched protein levels performing western blot and immunocytochemical analysis. Interestingly, in vitro proliferations studies revealed no difference in cellular growth between wild type and ALR expressing HepG2 cells, whereas previous studies showed an increased proliferation rate after ALR application into culture medium. For analyzing the impact of ALR on tumour growth in vivo wild type and HepG2 cells stably expressing ALR were injected subcutaneously into nude mice. After three weeks body weight and tumour size were determined as well as samples for further analysis were taken. Re-expression of ALR was confirmed by RT-PCR and immunohistochemistry. Tumours originated from stably ALR expressing HCC cells compared to controls resulted in slightly lower average body weight of mice and displayed less necrotic areas, more epithelial-like cell growth as well as less polymorphisms and atypical mitotic figures. On the other hand mock transfected HepG2 derived tumours showed a more cancerous like growth with clear signs of necrosis. Based on these findings and in line with the in vitro results we assume that down regulation of 15kDa isoform of ALR expression in HCC cells supports tumorgenicity and metastatic potential of HCC cells.

ALR expression indicates liver regeneration upon distinct tissue damage

Hepatocellular regeneration requires various factors promoting cellular proliferation. Among them, ALR (augmenter of liver regeneration), was discovered to be highly expressed in vivo under circumstances of liver regeneration and in liver diseases such as cirrhosis and carcinoma. Interestingly, ALR was shown to exhibit its action exclusively on regenerating livers and hepatoma cells. Therefore ALR is an interesting target to analyze liver regeneration. The aim of this study was to investigate the molecular regulation and expression of ALR in murine models of liver damage caused by tissue loss through 70% liver resection, by bile duct ligation, CCl4 intoxication, by fatty liver through a methionine choline deficient diet (MCD) and a high fat diet (lard, cholesterol, Na-cholate) model. ALR mRNA Expression was analyzed by quantitative RT-PCR in liver tissue samples at different time points. ALR mRNA levels were significantly enhanced in liver samples after 2/3 partiel hepatectomy in short (up to 72h) and long term oberservation (12d). Additionally, carbon tetrachloride treatment resulted in a slightly increase of ALR expression within 24 to 72 hours. Bile duct ligation as a model for acute fibrosis displayed intense ALR mRNA levels after 6 weeks. As models for non alcoholic steatohepatits (NASH), MCD and Paigen diet were used representing fatty livers with high and mild fibrotic scores, respectively. In both models liver sample demonstrated high ALR mRNA levels after 3 and 12 weeks with higher expression rates within Paigen mice. In all models a profound inflammation reaction was described and therefore it is likely, that enhanced levels of proflammatory cytokines such as IL-1 are abundant. The latter was shown to activate ALR promoter and ALR expression and might explain our observations. Analysis of ALR expression in different mouse liver disease models might be a useful indicator of an activated process leading to liver regeneration and repair.

Elevated expression of SREBP-2 and ALR in human NASH tissue specimens

Nonalcoholic steatohepatitis (NASH) is a frequently recognized disease which is associated with obesity, insulin resistance and type 2 diabetes mellitus. Despite its frequent contribution, not much is known about the molecular mechanisms involved in NASH genesis. The expression of sterol regulatory element-binding protein 2 (SREBP-2) is an important factor in the pathogenesis of fatty livers. To examine the ability of regeneration of fatty livers, a RNA data base of specimens and corresponding RNA samples derived from patients with clinical and pathological proven NASH was set up and characterized. Aim of this study was to select and characterize tissue specimens of patients undergoing surgical liver resection regarding the correlation of expression patterns of regeneration and lipometabolism. Donors were selected according to body mass index (BMI), alcohol intake and histological features. Patients with excessive alcohol intake, positive serology for hepatitis B, C or a BMI smaller than 30 were excluded from this study. Grading (necroinflammatory activity) and staging (extent of fibrosis) of NASH was done according to Brunt et al. After RNA isolation and cDNA synthesis, samples were analyzed by real-time PCR. The expression patterns of SREBP-2 and augmenter of liver regeneration (ALR), a growth factor that is upregulated in regenerating liver tissue, were detected in NASH specimens and compared to normal controls. The analysis of the clinical data of NASH (n=33) and control samples (n=10) showed an equal distribution in age, medication and previous illnesses. The expression levels of SREBP-2 and ALR were significantly higher in NASH, increasing according to fatty degeneration grade, compared to normal controls. On the basis of the established and characterized RNA data base, a correlation between expression levels of SREBP-2 and ALR was shown in human NASH specimens. Our results indicate that regeneration ability of fatty livers is increased.

Expression and localization of augmenter of liver regeneration in human muscle tissue

Mitochondrial DNA (mt-DNA) disorders and abnormal regulation of nuclear-derived proteins devoted to the cross-talk between the two cellular genomes have recently interested researchers in the field of neuromuscular diseases. We have identified, isolated and sequenced a new gene, augmenter of liver regeneration (ALR) that stimulates in vivo hepatocyte proliferation and up-regulates mt-DNA expression and ATP production. ALR protein (Alrp) is mainly located, in rat, in the mitochondrial inter-membrane space and its mRNA is particularly abundant in brain, muscle, testis and liver, tissues whose activity is mostly dependent on mitochondrial metabolism. Studies on rat Alrp sequence revealed the presence of homologous amino-acid sections into proteins derived from mouse, human, Drosophyla, plants and even DNA viruses. In this article, we evaluated ALR expression in normal human muscular tissues, both as protein and as mRNA. The data, obtained by molecular biology, immunohistochemistry and electron microscopy, demonstrated that: (i) Alrp and ALR mRNA are present in human muscular tissue; (ii) Alrp is particularly expressed in muscular fibres rich in mitochondria; (iii) Alrp is localized in the mitochondrial inter-membrane space or associated to mitochondrial cristae; and (iv) in subjects younger then 35 years of age, ALR mRNA expression is different between male and female subjects. In conclusion, the present data set Alrp, as a factor associated with mitochondria also in human tissue, call for future studies aimed at establishing Alrp as an important factor involved in the molecular events that trigger neuromuscular diseases.

A hepatocyte nuclear factor-3 site in ALR promoter is important for interleukin 6-induced expression

Liver regeneration is an important survival process that occurs after liver injury e.g. viral infection, toxins or hepatectomy. This process involves a variety of specific cytokines, growth factors and transcription factors. In the liver, hepatocyte nuclear factors (HNFs) play a central role during liver regeneration. These include the HNF3 transcription factors, also termed forkhead box (Fox) family. Additionally, it has been shown that Foxa2 (HNF-3β) expression is induced in regenerating mouse livers after partial hepatectomy as well as in livers from patients with hepatocellular carcinoma HCC. Furthermore, we have shown that Augmenter of Liver Regeneration (ALR) is significantly increased in hepatocellular carcinoma. In addition, increased expression levels of ALR were shown in a Foxa2- transgenic mouse model. The aim of our study was to identify transciption factors that regulate the expression of ALR. Promoter studies of ALR revealed potential binding sites for Foxa2, IL-6 RE, CEBP/β, YY1 and HNF-4α. The promoter activity of ALR was significantly induced over the control after co-transfection of Foxa2 in HepG2 cells. To verify whether the putative Foxa2 element is capable of binding Foxa2 protein, Electrophoresis Mobility Shift Assays (EMSA) were performed. Supershift analysis using α-Foxa2 antibody indicates the specific binding of Foxa2 to ALR promoter. This binding was inducible when the cells were simultaneously stimulated with IL-6. This increased binding after Foxa2-transfection was confirmed by elevated ALR protein levels using Western Blot technique. On the other hand, no binding was observed with IL-6 RE, CEBP/β and YY1. In conclusion, we demonstrated that Foxa2 enhances the expression of ALR. We assumed that ALR might be regulated by Foxa2, and this regulation could be amplified by simultaneously activation using IL-6. Taken together, both IL-6 and Foxa2 induce the expression of ALR, and Foxa2 is necessary in the IL-6 response of ALR promoter.

Augmenter of Liver Regeneration inhibits angiogenesis and invasiveness in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant disorders, and in some parts of the world represents the most important neoplastic cause of death. In general, HCC represents a hypervascularized cancer and its progression depends on different factors like angiogenesis and invasiveness. Several studies showed that the transcription factor Hypoxia-Inducible Factor 1-alpha plays a central role in HCC progression, angiogenesis and neovascularization. Hif-1 alpha can directly activate the expression of different pro-angiogenic factors like VEGF, VEGFR and angiopoietins. Furthermore, we have shown that Augmenter of Liver Regeneration (ALR), a hepatotrophic factor with specific liver regeneration enhancing effects, is induced in cirrotic livers and hepatocellular carcinoma. The goal of our study was to find out whether ALR modulate the tumor angiogenesis and invasiveness. Using immunohistochemistry technique, we have shown that the invasiveness of HCC in patients was inversely correlated with ALR expression. Performing Western Blot analysis, using WT and ALR-stable transfected HepG2 cells, we found that Hif1-alpha protein expression was significantly reduced in the ALR-stable transfected cells comparing to the mock control. Migration assays with modified Boyden chambers, we demonstrated that the over-expression of ALR in HepG2 cells inhibits the cancer cell motility (3,3±1,29 cells/hpf) compared to the mock control (15,5±3,85 cells/hpf). Based on these results we hypothesize that ALR might interfere with angiogenesis- and invasiveness-related pathways in hepatocellular carcinomas. Here we show for the first time that increased levels of ALR might have the potential to reduce metastasis diminishing migratory and invasive properties of HCC cells.

IL–6 and IL–1 enhance hepatic expression of ALR (Augmenter of Liver Regeneration)

IL–6 and IL–1 enhance hepatic expression of ALR (Augmenter of Liver Regeneration)