Human Augmenter Of Liver Regeneration Research Articles

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.

Augmenter of liver regeneration causes different kinetics of ERK1/2 and Akt/PKB phosphorylation than EGF and induces hepatocyte proliferation in an EGF receptor independent and liver specific manner

Background/Aim Augmenter of liver regeneration (ALR) is a potent growth factor which supports liver regeneration in experimental animals. The aim of this study was to compare proliferation as well as the kinetics of ERK1/2 and Akt/PKB phosphorylation by recombinant human ALR (rhALR) and EGF in human hepatocytes and extrahepatic cells. Methods Kinetics of ERK1/2 and Akt/PKB phosphorylation were determined in primary human hepatocytes (phh) after stimulation with rhALR and EGF. Induction of proliferation was analyzed in phh and several cell lines of hepatic and extrahepatic origin by the MTT and [ 3H]-thymidine assay. Results The kinetics of ERK phosphorylation showed clear differences, whereby rhALR caused a transient and EGF a permanent increase during the observation period of 60 min. For both, Akt and ERK phosphorylation, EGF caused a faster effect with maximal levels observed already after 2 min, whereas rhALR caused maximal phosphorylation between 10 and 15 min. Using the EGF receptor inhibitor AG1478 we provide evidence of an EGF receptor independent induction of proliferation by rhALR. Furthermore, rhALR induced proliferation only in phh and the human liver derived cell lines HepG2 and Chang. In contrast, EGF enhanced proliferation in all analyzed cell types including cell lines of colon, bronchial, pancreatic and gastric origin (SW480, BC1, L36PL and GC1). Conclusion rhALR and EGF induce different kinetics of ERK and Akt phosphorylation in human hepatocytes. The mitogenic effect of rhALR is liver specific and seems to be at least partially independent from EGF receptor mediated signaling.

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.

OL-059 Effect of blocking expression of human augmenter of liver regeneration (hALR) on the expression of transforming growth factor-α (TGF-α) and epidermal growth factor receptor in HepG2

OL-059 Effect of blocking expression of human augmenter of liver regeneration (hALR) on the expression of transforming growth factor-α (TGF-α) and epidermal growth factor receptor in HepG2

Human augmenter of liver regeneration is important for hepatoma cell viability and resistance to radiation-induced oxidative stress

To gain new insight into the biological function of the human augmenter of liver regeneration (hALR) in HCC, we studied its involvement in radiation-induced damage and recovery of HCC cells. We found that hALR expression was up-regulated in both HCC tissues and multiple hepatoma cell lines and correlated significantly with increased radiation clonogenic survival after radiation treatment. Exogenous expression of hALR increased radiation resistance in SMMC-7721 cells, and the increased survival was accompanied by a decrease in apoptosis and a prolonged G 2–M arrest after irradiation. Overexpression of ALR significantly increased the mitochondrial membrane potential, inhibited cytochrome c release, and opposed the loss of intracellular ATP levels after radiation. Moreover, knockdown of ALR by siRNA resulted in inhibition of viability in the absence of exogenously added oxidative stress and radiation sensitization in HepG2 cells. Importantly, hALR expression was very low in normal hepatocyte L02 cells, and hALR silencing had a minimal effect on L02 viability and radiation sensitivity. These results suggest that human ALR is important for hepatoma cell viability and involved in the protection of hepatoma cells against irradiation-induced damage by its association with mitochondria. Targeting hALR may be a promising novel approach to enhance the radiosensitivity of hepatoma cancers.

Inhibitory effects of recombinant human augmenter of liver regeneration on gene expression of c-fos in hepatic stellate cells of rats

Objective To investigate the effects of recombinant human augmenter of liver regen-eration (hALR) on c-fos gene expression in hepatic stellate cells (HSCs). Methods HSC-T6 cells were cultured in medium containing different concentrations of hALR (200,20,2 μg,/L respectively), and col-lected at different incubation periods (8,24,48,72 h). Total RNA of HSCs was isolated, and the c-fos gene expressions levels were detected by reverse transcription polymerase chain reaction. Results The C-fos gene expression levels of HSCs in high (200 μg/L), middle (20 μg/L) and low (2 μg/L) concentra-tions of hALR groups were significantly lower than those in control group after exposure to hALR for 8,24, 48 and 72 h. The expression of C-fos gene in low,middle,and high hALR groups was 50%-53% ,38%-43% ,and 26%-30% of control group,respectively. Conclusion hALR could inhibit the gene expression of c-fos in HSCs. Key words: Hepatic stellate cell; c-fos; Gene expression

The cloning,expression and pruification of recombinant

Objective To provide a technique of preparation recombinant hALR by gene engineering in larger scale.Methods Construct hALR(human augmenter of liver regeneration) protein expression vector pGEX-3X-hALR,induce the expreesion of hALR,and pruified the protein by affinity chromatography.Results Succeed in constructing the vector pGEX-3X-hALR and obtained the recombinant hALR protein.Conclusion The mothed could be used for preparation recombinant hALR by gene engineering in larger scale. Key words: Human augmenter of Iiver regeneration; Cloning,molecular; Expression; Purification

Genetic Recombinant Expression and Characterization of Human Augmenter of Liver Regeneration

To establish a highly effective prokaryotic recombinant expression system for human augmenter of liver regeneration (hALR) and to characterize the recombinant hALR both in vitro and in vivo. ALR cDNA was synthesized and inserted into expression vector pET28a+, the recombinant plasmid was transformed into BL21, and expression of hALR was induced by IPTG. Recombinant hALR (rhALR) was purified by sequential detergent wash, enterokinase (EK) digestion, gel-filtration, and chelating chromatography. The rhALR was identified by SDS-PAGE, immunoblotting, MALDI-TOF-MS, and N-terminal sequencer. Cell proliferative effect of rhALR on human hepatocytes was analyzed by MTT. The protective effect of rhALR on liver function was observed on CCl(4)-induced intoxicated mice. Recombinant expression plasmid of ALR [pET28(a+)-hALR] was confirmed by restriction enzyme digestion and DNA sequencing. The expressed rhALR constituted 30% of total bacterial protein. Molecular weight was 15,029 for monomer and 30,136 for dimer by mass determination. N-terminal was M-R-T-Q-Q, exactly the same as anticipated for hALR. The purified protein migrating at about 15 KD showed excellent antigenicity in immunoblotting. The rhALR also showed a strong stimulative effect on hepatocyte proliferation. ALT and AST levels, liver histological structure, as well as the survival rate of CCl(4)-intoxicated mice were significantly improved when rALR was administrated at 40 microg/kg or 200 microg/kg. The rhALR is successfully expressed highly effectively with anticipated MW, N-terminal, and antigenicity. It could play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and improving liver function in CCl(4)-intoxicated mice.

304 HUMAN AUGMENTER OF LIVER REGENERATION TRANSGENIC OVINE EMBRYO DERIVED FROM SOMATIC CELL NUCLEAR TRANSFER

The aim of this research was to develop an efficient screening technique to detect transgenic ovine embryos using neomycin resistance (NeoR) and enhanced green fluorescence protein (EGFP) genes as genetic markers. A 0.8-kb fragment of the ovine β-lactoglobulin promoter sequence (BLG) and 1.8 kb of the human augmenter of liver regeneration (ALR) genomic sequence were derived by PCR amplification. These 2 fragments were inserted into the MCS of pGEM-7zf(+) plasmid; this vector was named p7Z-BA. The coding sequence of NeoR was derived by PCR amplification from the plasmid pIRES2-EGFP and was assembled into the MCS of the pIRES2-EGFP plasmid. The resultant vector (pNIE) contained a NeoR gene coding sequence and an EGFP coding sequence linked by an internal ribosome entry site (IRES) sequence downstream of the CMV promoter. The vector pNIE was excised as an NsiI-SspI fragment and inserted into the vector p7Z-BA. In the end, we had a vector named pNEA, which contained the NeoR gene and the EGFP gene regulated by a CMV promoter for expression in a non-tissue specific mode, and the human ALR gene regulated by the BLG promoter for expression specifically in mammary gland. Sheep fetal fibroblast (SFFB) cells were isolated by attachment of tissue pieces from the ear skin of a 1- to 2-month ovine fetus. Karyotypes of the cells at the third passage and after 15 passages were analyzed. The cells proliferated well and more than 72% of the cells maintained a diploid karyotype after 15 passages. Therefore, the SFFB cells are amenable for transgenic cloning manipulations. For transfection, third-passage SFFB cells at 70% confluency were transfected in a 100-mm dish with pNEA (0.5, 1.0, 2.0, 3.0, 5.0, and 7.0 µg) using Lipofectamine 2000 (2, 4, 6, 8, 10, and 12 µL; Invitrogen, Carlsbad, CA). Cells were checked 24 to 48 h after transfection under fluorescence microscopy for GFP expression, and G418 selection (800 µg mL–1) was applied at that time. After 2 weeks, selected colonies were counted and propagated in culture medium containing 300 µg mL–1 G418 for 2 to 3 passages and cryopreserved. A small portion of the cells was analyzed by PCR for gene integration. Bright green fluorescence could be detected 24 to 48 h after transfection. More colonies were selected when transfection parameters were 2 µg of DNA and 10 µL of Lipofectamine. The results of PCR detection showed that the foreign gene was integrated into the genome. A total of 612 oocytes were aspirated from 2- to 5-mm follicles of ovine ovaries collected from an abattoir; 78% of them were matured after 18 h in culture. Four hundred forty-three oocytes were enucleated, and 332 enucleated oocytes were treated for electrofusion with green fluorescence cells. Of these, 180 (54.2%) couplets were fused. A total of 172 reconstructed embryos were stimulated and cultured in vitro, 31 (18%) of which developed to the blastocyst stage, and 19 blastocysts expressed GFP. In conclusion, we established an effective method to select transgenic embryos formed by nuclear transfer using transfected donor cells.

Cloning and expression of the human augmenter of liver regeneration at low temperature in Escherichia coli

Acute and chronic hepatic failure is a devastating illness of varied causes with considerable mortality. Human augmenter of liver regeneration (hALR) is a hepatotrophic protein and the unique cytokine which can specially stimulate hepatic origin cells to grow regardless of genus. It has been proven that ALR can promote regeneration and avoid all kinds of injury in rat and canine models. In this study, the recombinant protein hALR was expressed successfully with recombinant prokaryotic expression vector pET28a(+) in Escherichia coli BL21 (DE3). We constructed the recombinant expression vector pET28a(+)/hALR with a full-length cDNA encoding hALR protein from normal human liver tissue by one-step reverse transcription-polymerase chain reaction and his-tag recognition sequence encoding polyhistidine (6 × His). Under IPTG (isopropyl-β-d-thiogalactopyranoside) induction for 2 h at 37 °C, recombinant protein hALR was expressed. The expression of recombinant polyhistidine-tagged hALR was increased under low temperature and was confirmed that the temperature of 23 °C was the most suitable IPTG induction condition. Under low temperature induction of IPTG, recombinant protein can be expressed as a soluble protein. Recombinant protein hALR was also purified with His Bind Kits and characterized with SDS-PAGE and Western blotting. The results showed that recombinant hALR could be expressed as a soluble protein under low temperature induction of IPTG. The successful expression of ALR in E. coli makes it possible to further study its biological function and purified recombinant hALR could be developed into a new anti-hepatic damage product.

Construction of High Expression Plasmid of Human Augmenter of Liver Regeneration (hALR), Expression and Purification of hALR

Experimental evidence has been presented to suggest that the human augmenter of liver regeneration (hALR) serves as a hepatotrophic growth factor during liver regeneration and as a generalized growth factor during pancreas transplant/regeneration. A prokaryotic expression plasmid, pRSET/6his-c-myc-hALR was constructed, by cloning synthesized hALR cDNA into pRSET/6his-c-myc that was improved on the basis of pRSET B by the group. As a result, the protein was highly expressed in E. coli BL21. The recombinant hALR was over 60% of the total protein in E. coli. Its validity was confirmed by means of Western Blotting. The protein was purified by Ni-NTA affinity chromatography and this FAD-dependent sulfhydryl oxidase activity was measured.

Expression, purification and bioactivity of human augmenter of liver regeneration

To construct the expression vectors for prokaryotic and eukaryotic human augmenter of liver regeneration (hALR) and to study their biological activity. hALRcDNA clone was obtained from plasmid pGEM-T-hALR, and cDNA was subcloned into the prokatyotic expression vector pGEX-4T-2. The recombinant vector and pGEX-4T-2hALR were identified by enzyme digestion and DNA sequencing and transformed into E coli JM109. The positively selected clone was induced by the expression of GST-hALR fusion protein with IPTG, then the fusion protein was purified by glutathine s-transferase (GST) sepharose 4B affinity chromatography, cleaved by thrombin and the hALR monomer was obtained and detected by measuring H thymidine incorporation. The product of PCR from plasmid pGEM-T-hALR was examined by 1.5% sepharose electrophoresis. The specific strap was coincident with the theoretical one. The sequence was accurate and pGEX-4T-hALP digested by enzymes was coincident with the theoretical one. The sequence was accurate and the fragment was inserted in the positive direction. The recombinant vector was transformed into E coli JM109. SDS-PAGE proved that the induced expressive fusion protein showed a single band with a molecular weight of 41 kDa. The product was purified and cleaved. The molecular weights of GST and hALR were 26 kDa, 15 kDa respectively. The recombinant fusion protein accounted for 31% of the total soluble protein of bacterial lysate. HALR added to the culture medium of adult rat hepatocytes in primary culture and HepG2 cell line could significantly enhance the rate of DNA synthesis compared to the relevant control groups (P < 0.01). Purified hALR has the ability to stimulate DNA synthesis of adult rat hepatocytes in primary culture and HepG2 cells in vitro, and can provide evidence for its clinical application.

Determination of the Distance between the Two Neutral Flavin Radicals in Augmenter of Liver Regeneration by Pulsed ELDOR

Pulsed electron-electron double resonance (ELDOR) has been used to obtain structural information from a FAD-dependent sulfhydryl oxidase, Augmenter of Liver Regeneration (ALR). ALR is a homodimer with each subunit containing a noncovalently bound FAD cofactor. Both FADs may be converted into the blue neutral radical form by aerobic treatment with DTT. From three-pulse and four-pulse ELDOR experiments, a distance of 26.1 +/- 0.8 A could be determined between the FAD cofactors in human ALR. Taking into account the electron spin density distribution in a neutral flavin radical obtained from density functional theory calculations, a distance of 26.9 A could be estimated for the separation of the spin centers in the X-ray structure of rat ALR. The good agreement confirms that rat ALR may be used as a model for mechanistic discussions of human ALR. The experiments also demonstrate that neutral flavin radicals have the appropriate properties to be used as intrinsic spin labels for distance determinations in proteins.

365 Human augmenter of liver regeneration is highly expressed in regenerative nodules of liver cirrhosis

365 Human augmenter of liver regeneration is highly expressed in regenerative nodules of liver cirrhosis

Expression of human augmenter of liver regeneration in pichia pastoris yeast and its bioactivity in vitro.

To construct a yeast expression system of human augmenter of liver regeneration (hALR) and to examine its bioactivity in vitro. With PCR and gene recombination techniques, cDNA of open reading frame of hALR was obtained from recombinant plasmid pcDNA3.1-hALR and inserted into plasmid pPIC9. The cDNA of hALR from recombinant plasmid pPIC9-hALR demonstrated by sequencing was subcloned into plasmid pPIC9K. The recombinant plasmid pPIC9K-hALR was transformed into GS115 with electroporation. hALR was expressed by GS115 under the induction of 5 mL/L methanol and purified with ultrafiltration after it was analyzed by 15% SDS-PAGE and Western blot. The effects of hALR on in vitro proliferation of QGY and HepG(2) cells were evaluated by (3)H-TdR methods. The correctness and integrity of recombinant plasmids pPIC9-hALR and pPIC9K-hALR were identified by restriction digestion, PCR and sequencing methods, respectively. hALR as a secretive protein was successfully expressed by GS115. Its molecular weight was about 15 ku and the target protein was about 60% of the total protein in the supernatant from GS115 with plasmid pPIC9K-hALR. The results of Western blot of hALR showed the specific band. The high qualitative hALR was obtained through ultrafiltration. hALR could stimulate in vitro proliferation of QGY and HepG(2) cells in a dose-dependent manner, but there was a difference in reactivity to hALR between QGY and HepG(2). The hALR as a secretive protein can be successfully expressed by GS115. It may stimulate in vitro proliferation of QGY and HepG(2) cells at a dose-dependent manner. But QGY and HepG(2) cells have different reactivities to hALR.

Purification, crystallization and preliminary X-ray studies of human augmenter of liver regeneration.

Human augmenter of liver regeneration has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group C222, with unit-cell parameters a=51.7 A, b=78.8 A, c=63.7 A. Diffraction data were collected to 2.80 A with a completeness of 99.9% (99.9% for the last shell), a R(sym) value of 0.092(0.236) and an I/sigma(I) value of 6.2(2.7).

Cloning and sequence analysis of human genomic DNA of augmenter of liver regeneration.

The liver is one of the organs, which have potential regenerative capability in mammalian animal[1]. The study of the canine model indicated that the liver could regenerate to original size after 70% hepatectomy in only two weeks[2]. So it is a hot research topic for the cellular and molecular mechanism of liver regeneration. Accumulated results demonstrated that the hepatocyte growth factor (HGF)[3], insulin-like growth factor I and II (IGF-I, II)[4], epidermal growth factor (EGF), transforming growth factor alpha (TG F alpha)[5] and insulin[6] are among the most important growth factors for liver regenerative regulation. In recent years, a heat-stable protein in the serum of the patients with various liver diseases has been noted for its potential stimulation effects on the liver regeneration, and this growth factor is called hepatocyte-stimulatory substance (HSS). Gradient purification and sequence analysis of HSS protein indicated that the HSS protein itself is the augmenter of liver regeneration (ALR)[7], or called hepatopoietin (HPO)[8]. The immunohistochemical staining indicated that the expression of the ALR mainly existed in platelets and the sperm cells in testes, and ALR also could be found in the liver and the spleen which contain many platelets[9]. The analysis of the protein structure of the human and mouse ALR indicated that the primary protein structure of ALR does not contain a typical signal peptide sequence, and it is unknown if a specific receptor is necessary for the effect of the ALR. Therefore, it is important to clone the genomic DNA sequence of the ALR and it is also very helpful for the analysis of the structure of the ALR genomic DNA and regulation at the transcriptional and post-transcriptional levels.

Highly divergent amino termini of the homologous human ALR and yeast scERV1 gene products define species specific differences in cellular localization

The yeast scERV1 gene product is involved in the biogenesis of mitochondria and is indispensable for viability and regulation of the cell cycle. Recently the general importance of this gene for the eukaryotic cell was shown by the identification of a structural and functional human homologue. The homologous mammalian ALR (Augmenter of Liver Regeneration) genes from man, mouse and rat are involved in the phenomenon of liver regeneration. A low expression rate of the genes is found in all investigated cells and mammalian tissues but it is specifically induced after damage of liver organs and is especially high during spermatogenesis. The alignment of the different proteins identifies a highly conserved carboxy terminus with more than 40% identical amino acids between yeast and mammals. The conserved carboxy terminus is functionally interchangeable between distantly related species like yeast and man. In contrast, the amino terminal parts of the proteins display a high degree of variability and significant differences even among closely related species. This finding leads to the problem whether the amino termini have comparable or divergent functions in different species. In this study we demonstrate by heterologous complementation experiments in yeast that the complete human ALR protein with its own amino terminus is not able to substitute for the yeast scERV1 protein. Fusion proteins of Alrp and scErv1p with the green fluorescence protein were created to investigate the respective subcellular localizations of these homologous proteins in yeast and human cells. In yeast cells human Alrp accumulates in the cytoplasm in contrast to yeast scErv1p that is preferentially associated with yeast mitochondria. Comparable studies with human cells clearly show that the homologous human Alrp is located in the cytosol of these cells. Fractionation experiments and antibody tests with yeast and human mitochondria and cellular extracts verify these findings.

High expression of human augmenter of liver regeneration inE. coli

Heat-stable hepatocyte stimulatory activity has been described in the liver of weanling rats and pigs. This growth factor is called hepatocyte stimulator substance (HSS)[1]. Hagiya et al[2] reported the complete amino acid sequence of a 30KDa band from their purified product of rat liver and the cloning and sequence analysis of its cDNA. They called it augmenter of liver regeneration (ALR). Our previous study[3] demonstrated that human ALR had been cloned and sequenced. To further study the bioactivity of human ALR (hALR), we constructed a highly expressed vector pBV-hALR in E. coli and about 20% of somatic protein of rhALR was expressed.

Human augmenter of liver regeneration: Molecular cloning, biological activity and roles in liver regeneration

The complete amino acid sequence of human augmenter of liver regeneration (hALR) was reported by deduction from nucleotide sequence of its complementary DNA. The cDNA for hALR was isolated by screening a human fetal liver cDNA library and the sequencing of this insert revealed an open reading frame encoding a protein with 125aa and highly homologous (87%) with rat ALR encoding sequence. The recombinant hALR expressed from its cDNA in transient expression experiments in cos-7 cells could stimulate DNA synthesis of HTC hepatoma cell in the dose-dependent and heat-resistant way. Northern blot analysis with rat ALR cDNA as probe confirmed that ALR mRNA was expressed in the normal rat liver at low level and that dramatically increased in the regenerating liver after partial hepatectomied rat. This size of hALR mRNA is 1.4 kb long and expressed in human fetal liver, kidney and testis. These findings indicated that liver itself may be the resource of ALR and suggested that ALR seems to be an important paracrined regulator of liver regeneration.