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J. Biol. Chem., Vol. 276, Issue 48, 44512-44520, November 30, 2001

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Apolipoprotein A-V

A NOVEL APOLIPOPROTEIN ASSOCIATED WITH AN EARLY PHASE OF LIVER REGENERATION^*

Hendrik N. van der Vliet, Martijn Groenink Sammels, Aad C. J. Leegwater^§, Johannes H. M. Levels^§, Pieter H. Reitsma^§, Willem Boers, and Robert A. F. M. Chamuleau^¶

From the Departments of  Experimental Hepatology and ^§ Experimental Internal Medicine, Academic Medical Center, University of Amsterdam, Meibergdreef 9, Amsterdam 1105 AZ, The Netherlands

Received for publication, July 20, 2001, and in revised form, September 12, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Liver regeneration in response to various forms of liver injury is a complex process, which ultimately results in restoration of the original liver mass and function. Because the underlying mechanisms that initiate this response are still incompletely defined, this study was aimed to identify novel factors. Liver genes that were up-regulated 6 h after 70% hepatectomy (PHx) in the rat were selected by cDNA subtractive hybridization. Besides known genes associated with cell proliferation, several novel genes were isolated. The novel gene that was most up-regulated was further studied. Its mRNA showed a liver-specific expression and encoded a protein comprising 367 amino acids. The mouse and human cDNA analogues were also isolated and appeared to be highly homologous. The human gene analogue was located at an apolipoprotein gene cluster on chromosome 11q23. The protein encoded by this gene had appreciable homology with apolipoproteins A-I and A-IV. Maximal expression of the gene in the rat liver and its gene product in rat plasma was observed 6 h after PHx. The protein was present in plasma fractions containing high density lipoprotein particles. Therefore, we have identified a novel apolipoprotein, designated apolipoprotein A-V, that is associated with an early phase of liver regeneration.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The liver possesses the unique capacity to restore damaged or lost liver cell mass by organized cell proliferation (1, 2). This process of liver regeneration has been studied extensively in a rat model of 70% partial hepatectomy (PHx),¹ a model first described by Higgins et al. (3). After PHx, the remaining differentiated liver cells re-enter the cell cycle by undergoing a transition from the quiescent G₀ phase to the G₁ phase at 1-4 h after PHx (priming phase). DNA synthesis in rat hepatocytes starts at 12-16 h after PHx and increases to a peak around 22-24 h. Within 7 days of PHx, the rat liver has regained its original mass.

To elucidate the mechanisms responsible for the regenerative response, research has focused on the molecular changes during the G₀/G₁ transition. Over 70 immediate-early genes have been identified, the expression of which is up-regulated during the priming phase (4, 5). These genes include for example the proto-oncogenes c-fos, c-jun, and c-myc. In addition, studies in knockout mice have revealed an essential role for the cytokines TNF and IL-6 in the initiation of liver cell proliferation. In both TNF receptor type-1 and IL-6 knockout mice, liver regeneration was impaired after PHx, whereas IL-6 injected at the time of the operation restored proliferative capacity (6, 7). Also a rapid induction of DNA binding by the transcription factors NFB and STAT3 was demonstrated during the immediate-early response to PHx (8, 9).

However, the factors mentioned alone are not sufficient to induce liver regeneration. In inducible nitric-oxide synthase knockout mice after PHx, liver regeneration was impaired, but IL-6 and TNF levels and STAT3 binding were not altered (10). Moreover, c-fos, c-jun, c-myc, NFB, STAT3, and IL-6 were induced to a similar extent in the ligated and nonligated liver lobes after portal branch ligation whereas only the nonligated lobes underwent compensatory regeneration (11). Besides, based on the application of a technique of temporary partial hepatectomy, the proliferative response did not appear to be determined during the priming phase, but later during mid to late G₁ (12).

These findings suggest that, although the immediate-early response after PHx is necessary for liver regeneration, a further later response is also required. Factors in the delayed response during G₁ might be triggers that are essential for the regeneration process. Some genes that contribute to the delayed response have already been identified, such as p53, c-ras, and various cyclins (13-15).

To identify additional liver-specific factors present during the delayed response, we characterized gene expression profiles in rat liver at 6 h after PHx. To identify and isolate up-regulated genes, a cDNA subtraction technique was applied to mRNA from rats after PHx and sham surgery. This paper describes the isolation and characterization of a novel gene that undergoes appreciable up-regulation after PHx on both mRNA and protein level, and encodes for a novel apolipoprotein, designated apolipoprotein A-V.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Rat Tissue Preparation-- Rats were subjected to 70% PHx as previously described (3). Briefly, male Wistar rats (200-225 g) were anesthetized and subjected to midventral laparotomy. Subsequently, the left lateral and the median liver lobes were removed. In sham-operated animals, the liver was exposed and manipulated before closing the abdomen. Animals were sacrificed at various times after PHx or sham surgery. At sacrifice heparin plasma was collected, and the remaining regenerating liver was harvested. For determination of the tissue specificity of gene expression, various tissues were isolated from a female Wistar rat (175 g). Tissues and plasma were stored at 80 °C. Tissue specimens of the regenerating liver were also fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) and imbedded in paraffin as previously described (16). Experiments were carried out in accordance with the guidelines for animal care and experimentation of the University of Amsterdam.

RNA Isolation-- Total RNA was isolated from tissue using the TRIzol reagent kit (Life Technologies). Liver poly(A)⁺ RNA was isolated from total liver RNA using oligo(dT)-cellulose (Roche Molecular Biochemicals) affinity chromatography as previously described (17). To obtain highly purified poly(A)⁺ RNA populations, the oligo(dT)-cellulose step was performed twice.

cDNA Subtractive Hybridization-- The PCR-select cDNA subtraction kit (CLONTECH) was used to selectively amplify genes that were differentially expressed during liver regeneration. The liver mRNA population 6 h after PHx was compared with that 6 h after sham operation. The cDNA subtractive hybridization was performed according to the manufacturer's protocol. Resulting cDNA fragments were amplified and ligated into the pCR II vector (Invitrogen, Groningen, The Netherlands). The fragments were characterized by nucleotide sequencing. For gene identification, the sequences were compared with those reported in the data bases maintained at the National Center for Biotechnology Information (Bethesda, MD) using the BLAST search program.

Northern Analysis-- For the Northern analysis of mRNA expression in rat tissues, total RNA (20 µg) or poly(A)⁺ RNA (0.8 µg) was electrophoresed in a 0.22 M formaldehyde-1% agarose gel and transferred and fixed to Hybond-N nylon membrane (Amersham Pharmacia Biotech). Northern analysis of RAP3 mRNA expression in human tissues was performed using the human 12-lane Multi Tissue Northern blot (CLONTECH, Heidelberg, Germany). 25 ng of cDNA was labeled with ³²P according to the random primed labeling method (18) and used as a hybridization probe. Hybridization was carried out according to standard techniques (17). The amount of hybridization was analyzed and quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA). Constancy of mRNA loading was monitored by methylene blue staining of the blots (19) or by hybridization with a rat heavy chain ferritin cDNA. The transcription of the heavy chain of ferritin is not affected by partial hepatectomy or laparotomy (20).

In Situ Hybridization-- In situ hybridization was performed on serial paraffin sections of regenerating rat liver after PHx using the method described by Moorman et al. (16). Single-stranded antisense RNA probes, labeled with [³⁵S]CTP, were made by in vitro RNA transcription, as described by Van Kempen et al. (21), using the pBluescript SK(±) vector containing the inserts of interest as template. The RAP3 insert of the pET-15b expression construct (see "Protein Expression and Raising of Antibodies") was cloned into the pBluescript vector using the XbaI and XhoI sites.

Isolation of Full-length cDNAs-- Full-length cDNA of rat and human RAP3 were obtained by library screening according to standard hybridization techniques (17). A rat liver cDNA library was constructed from poly(A)⁺ RNA isolated from rat liver 6 h after 70% hepatectomy using the Great Lengths cDNA synthesis kit (CLONTECH) according to the manufacturer's protocol. The adaptor-ligated full-length cDNA inserts were cloned into the pCI vector at the EcoRI restriction site and transformed into DH10B cells (Life Technologies, Inc., Breda, The Netherlands). A human liver cDNA library, transformed into DH12S cells, was purchased from Life Technologies. To detect rat and human RAP3 cDNAs, probes of the rat RAP3 cDNA fragment and the full-length rat RAP3 cDNA were used, respectively. Full-length cDNA of mouse RAP3 was purchased as an IMAGE Consortium cDNA clone (22), known in the GenBank^TM data base of expressed sequence tags (ESTs) under accession number AA987093. All the obtained full-length RAP3 cDNAs were sequenced bi-directionally.

Rapid Amplification of cDNA Ends-- Both 5'- and 3'-RACE reactions were carried out for rat RAP3 using the Marathon cDNA amplification kit (CLONTECH). The starting material was poly(A)⁺ RNA isolated from rat liver 6 h after PHx. Adaptor-ligated cDNA was created according to the manufacturer's protocol. The 5'-RACE PCR was carried out with the adaptor-ligated cDNA and a combination of the adaptor primer and the internal reverse primer 5'-CAGGCTCTCTCAAGGGTCCC-3' or 5'-CTGTGGCTAGGCGGGGGTGG-3'. The 3'-RACE PCR reaction was carried out using a combination of the adaptor primer and the internal forward primer 5'-GTGGTCCTGCTGGGGGATCA-3' or 5'-AGTACCTTCATCCGTGTCAG-3'. The resulting 5'- and 3'-RACE fragments were ligated into the pCR II vector, and the sequences of the 5'- and 3'-cDNA ends of RAP3 were determined by nucleotide sequencing.

Software Analysis-- The nucleotide and amino acid residue sequences of RAP3 were analyzed using various software applications. Homology data base searches were carried out using the BLAST program (23). Using GCG DNA software (Genetics Computer Group (GCG), Madison, WI), the nucleotide sequences were translated into the corresponding amino acid sequences. The helical wheel function of the GCG DNA software was applied to examine the amphipathic nature of -helical peptide segments (Wisconsin Package Version 10.0, GCG). To predict protein secondary structure, the Predator software tool at the server of the European Molecular Biology Laboratory (Heidelberg, Germany) was used. The ExPASy molecular biology server from the Swiss Institute of Bioinformatics was used to calculate the theoretical molecular mass of the RAP3 protein (24). Predictions of signal sequences and transmembrane segments were generated using PSORT II software (25). The PROSITE data base was searched for known motifs in the RAP3 amino acid sequence (26). Multiple alignments were assessed using ClustalW software (27).

Protein Expression and Raising of Antibodies-- The insert encoding the rat RAP3 protein was amplified from the adaptor-ligated cDNA used in the RACE experiment. PCR reactions were carried out with the forward primer 5'-CGGAATTCATATGAGGAAGAGCTTCTGGGAGT-3' and the reverse primer 5'-CGGAATTCATATGTTAACCTGAGTGACCCTCA-3'. The resulting insert fragment was cloned into the pET-15b expression vector (Novagen) in the sense orientation at the NdeI site, thereby introducing an additional His tag sequence at the N-terminal part of the protein. Recombinant RAP3 protein was expressed in BL21(DE3) cells (Novagen) and purified under denaturing conditions using HisBind affinity chromatography. Expression and purification were carried out according to the manufacturer's protocol (Novagen). The buffer of the purified protein was replaced by PBS/6 M urea using a PD-10 column (Amersham Pharmacia Biotech), and the solution was diluted to a protein concentration of about 200 µg/ml. Polyclonal antibodies against the purified recombinant RAP3 protein were raised in New Zealand White rabbits following subcutaneous injection of 200 µg of antigen combined with Freund's complete adjuvant and subsequent booster injections of antigen. For protein identification, the His tag was removed using thrombin (Novagen). Subsequently, the N-terminal part of the protein was sequenced by the Protein Research Facility of the E.C. Slater Institute in Amsterdam under the supervision of Dr. T. Muijsers.

Western Blotting-- Western blotting was carried out for the immunodetection of RAP3 protein. Samples, containing 0.8 µl of rat plasma or 0.2 ng of the purified recombinant RAP3 protein, were electrophoresed in a 10% gel for sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were electrophoretically transferred to a polyvinylidene fluoride membrane using Tris-glycine SDS-polyacrylamide gel electrophoresis buffer containing 20% methanol. The blots were incubated overnight in PBS containing 0.1% Tween-20 and 3% Protifar (Nutricia, Zoetermeer, The Netherlands). The blots were washed with PBS containing 0.1% Tween 20 and 0.2% Protifar. Subsequently, the membranes were incubated for 1 h with rabbit serum containing polyclonal antibodies against RAP3 diluted in wash buffer. The blots were washed and incubated for 1 h with a second antibody (goat anti-rabbit immunoglobulin labeled with horseradish peroxidase (DAKO, Glostrup, Denmark) diluted in wash buffer). The blots were washed and incubated for 5 min with Lumi-Light Western blotting substrate (Roche Molecular Biochemicals). RAP3 was visualized and quantified by chemiluminescence measurement using a Lumi Imager and LumiAnalyst software (Roche Molecular Biochemicals).

Analysis of Plasma by Gel-filtration Chromatography-- Mouse plasma fractions isolated by standard fast-protein liquid chromatography techniques and analyzed for their cholesterol contents were generously provided by Prof. Dr. F. Kuijpers (Department of Pediatrics, Academic Hospital Groningen, The Netherlands). Their preparation and analysis have been described elsewhere (28). The plasma had been obtained from five wild type mice with FVB background (29). The protein and cholesterol profiles of rat plasma were determined as described previously (30). 60 µl of plasma, diluted 1:1 with Tris-buffered saline (TBS)/Tween elution buffer, was analyzed using a Superose 6 HR 10/30 column (Amersham Pharmacia Biotech) and TBS containing 0.005% (v/v) Tween-20, pH 7.4, as eluent.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Identification of Up-regulated Genes after PHx-- To identify genes that are up-regulated in the rat liver 6 h after PHx, a cDNA subtractive hybridization was performed. The liver mRNA population 6 h after PHx was compared with that at 6 h after sham operation. By using sham liver mRNA rather than normal liver mRNA, the two isolated mRNA populations were comparable with respect to acute phase mRNAs induced by surgery. 12 genes, isolated by the subtraction, were at least 1.5-fold up-regulated in rat liver 6 h after PHx relative to corresponding rat liver from sham-operated controls (Table I). Most of these genes have been reported to be associated with liver regeneration or with cell proliferation in general (Table I). Among the up-regulated genes there were three unknown genes, which were designated regeneration-associated proteins (RAP) 1, 2, and 3. The identity of the most up-regulated novel gene RAP3 and its relation to liver regeneration were studied in more detail.

Expression of RAP3 after PHx and Its Tissue Specificity-- To examine the relation of RAP3 to liver regeneration, its gene expression in rat liver was studied after PHx. The mRNA expression pattern of RAP3 at 3, 6, 12, 18, 24, and 30 h after laparotomy and PHx is shown in Fig. 1a. Two RAP3 mRNA bands appeared on Northern blot. RAP3 expression was not altered by sham operation. However, its expression was increased from 3 to 12 h after PHx with a peak at 6 h. In situ hybridization showed the same pattern of gene expression and showed that RAP3 was expressed in hepatocytes. 3 h after PHx, expression of RAP3 mRNA was increased in the periportal but not in the pericentral areas of the hepatic acinus. This zonation of expression was less apparent at the peak of gene expression 6 h after PHx, when the expression was also increased pericentrally (data not shown). RAP3 mRNA levels were also determined in normal rat tissues. RAP3 mRNA was detected in the liver but not in any of the other tissues examined (Fig. 1b).

View larger version (80K): [in this window] [in a new window]   Fig. 1.   Expression of RAP3 mRNA (a) in the rat liver up to 30 h after PHx, (b) in various rat tissues, and (c) in various human tissues. Northern blots (a) of total RNA isolated from rat liver removed 3, 6, 12, 18, 24, and 30 h after PHx and sham operation, (b) of total RNA isolated from various rat tissues, and (c) of poly(A)+ RNA isolated from various human tissues. Rat RAP3 cDNA (a and b) and human RAP3 cDNA (c) were used as probes. Equality of mRNA loading was monitored by methylene blue staining of the blot.

RAP3 Gene-- The identity of RAP3 was studied further using its full-length cDNA, which was obtained by screening a rat liver cDNA library. The complete nucleotide sequence was obtained by bi-directional sequencing (Fig. 2). The start and end of the full-length cDNA were confirmed by both 5'- and 3'-RACE reactions. Two cDNAs of RAP3 were detected: one having 1282 bp and the other 1834 bp. They were identical, except the latter contained 552 additional base pairs at the 3'-end. Data base searching revealed that the RAP3 sequence was about 90% homologous with murine EST clones of the mouse liver and fetus (Washington University-Howard Hughes Medical Institute Mouse EST project). The larger mouse cDNA of RAP3 was isolated by sequencing the EST clone with GenBank^TM accession number AA987093. Its cDNA of 1814 bp had 90% homology with its rat analogue. No appreciable similarity of murine RAP3 to known genes was found, although it was about 80% homologous with parts of the human chromosome 11q23 (GenBank^TM accession number AC007707). To find the cDNA of this human analogue of RAP3, a human liver cDNA library was screened. The human RAP3 cDNA had characteristics similar to those of the murine cDNAs. Its two transcripts were somewhat larger, specifically 1324 and 1896 bp. They were 77 and 76% homologous with their rat analogues. Like in the rat, the expression of RAP3 mRNA was restricted to the liver as shown on a more extensive human tissue blot (Fig. 1c).

View larger version (51K): [in this window] [in a new window]   Fig. 2.   Nucleotide and deduced amino acid sequences of rat RAP3 cDNA and protein. The letters and numbers for cDNA are in normal font and for protein are in italics. RAP3 has two cDNAs, which differ in the beginning of the poly(A) tail. The larger cDNA comprising 1832 bp is depicted here. The smaller cDNA ends at base pair 1282; its last three bases (ggc) are shown in boldface and underlined. The 20 amino acids forming the signal peptide of the protein are also underlined.

The human cDNA was identical to parts of the human chromosome 11q23. This locus is also the site of an apolipoprotein gene cluster, which contains the genes for apolipoproteins A-I, A-IV, and C-III (Fig. 3). Fig. 3 also depicts the exon/intron organization of the human RAP3 gene. This gene consists of three exons having 58, 111, and 1679 bp, respectively. The third exon of the smaller construct consists of 1137 bp. The two introns were 112 and 517 bp in length.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Genomic organization of the human RAP3 locus on chromosome 11q23. a, schematic representation of the apolipoprotein cluster at locus 11q23 in relation to the RAP3 locus. The arrows denote the transcription directions and the distances between the genes are indicated. b, schematic representation of the genomic structure of the human RAP3 gene. Translated regions are given in black. The two sizes of the 3'-untranslated region (3'-UTR) depict the different sizes of the two RAP3 transcripts. c, exon/intron boundaries of the human RAP3 gene. Intron sequences are shown in lowercase, and exon sequences are in uppercase. Both exon and intron sizes are indicated. The ag/gt consensus splice sequences are in boldface.

All of the mentioned RAP3 nucleotide sequences have been submitted to the DJB/EMBL/GenBank^TM data bases under GenBank^TM accession numbers AF202887 (larger rat cDNA), AF202888 (smaller rat cDNA), AF327059 (mouse cDNA), AF202889 (larger human cDNA), and AF202890 (smaller human cDNA).

RAP3 Protein-- The characteristics of the proteins encoded by the murine and human RAP3 genes were studied to elucidate the identity of RAP3. The nucleotide sequences were translated into the corresponding amino acid sequences, and, by analyzing the six reading frames, the largest and most likely proteins were chosen as RAP3 proteins. The amino acid sequence of rat RAP3 is shown in Fig. 2. The two cDNAs of the rat RAP3 gene both encoded the same protein. The same holds true for the human RAP3 cDNAs. The predicted rat and mouse RAP3 proteins, having 367 and 368 amino acids, respectively, were somewhat larger than their human analogue, which comprised 363 amino acids. The RAP3 protein analogues were 73% homologous (Fig. 4a). Comparison with other amino acid sequences showed that RAP3 was a hitherto unknown protein. RAP3 protein had an appreciable homology (20-28%) with apoA-IV and apoA-I of various species. The comparison of human RAP3 protein and human apoA-IV is illustrated in Fig. 4b. The apoA-IV protein is characterized by 13 tandem repetitions of 22-amino acid segments that have the propensity to form amphipathic -helices. Most of these repeats are punctuated by proline residues (31). The secondary structure of the RAP3 protein was predicted to have an overall -helical content of about 60%. When analyzing the sequence segments of human RAP3 that correlate with the tandem repeats of human apoA-IV, a few segments can be recognized that tend to form amphipathic alpha helices (data not shown).

View larger version (44K): [in this window] [in a new window]   Fig. 4.   Multiple alignments (a) of the RAP3 amino acid sequences from different species and (b) of the human amino acid sequences of RAP3 and apoA-IV. The sequences were compared using ClustalW software. Asterisks, fully conserved residues; colons, strongly conserved residues; periods, weakly conserved residues.

RAP3 protein was predicted to have a cleavable signal peptide, comprising the first 20 amino acid residues of the protein sequence (Fig. 2). No other transmembrane segments were identified, because no other hydrophobic segments were present in the protein. If RAP3 protein has a cleavable signal peptide, the calculated molecular masses of the precursor and mature RAP3 proteins would be 41 and 39 kDa, respectively. No characteristic functional patterns were found, apart from some common phosphorylation sites. Furthermore, no glycosylation or lipid modification sites were present in the RAP3 proteins.

Because of the presence of a cleavable signal peptide and, thus, a possible secretion of RAP3 protein into blood, the presence of the protein in rat plasma was investigated using polyclonal antibodies that were raised against recombinant RAP3. The RAP3 antiserum recognized this antigen on Western blot, whereas pre-immune serum gave no RAP3 signal (data not shown). The RAP3 antiserum was now used to identify RAP3 in plasma of normal rats and rats at 3, 6, 12, 18, 24, 36, and 48 after PHx or sham operation (Fig. 5). RAP3 was present in normal rat plasma, and its concentration was increased 3 and 6 h after PHx. At 12 h after PHx, the concentration was still elevated in one rat but almost normal in another rat. At 18 h after PHx the plasma RAP3 level had decreased to a subnormal level. The plasma RAP3 concentration in sham-operated rats remained constant. When related to the amount of recombinant protein on blot, the concentration of RAP3 in normal rat plasma was estimated to be about 1 µg/ml. This concentration was increased five times at 3 and 6 h after PHx.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   RAP3 protein levels in rat plasma after PHx. Western blots of 0.8-µl plasma samples from rats 0, 3, 6, 12, 18, 24, 36, and 48 h after PHx or sham operation. The blots were incubated with RAP3 antiserum at a 1:6000 dilution. For each time point after PHx and sham surgery, blots for two different rats are shown. The positive control is the recombinant RAP3 protein that contains 20 additional amino acids.

Because the RAP3 amino acid sequence has homology with apolipoproteins, the presence of RAP3 protein in plasma apolipoprotein particles was sought. Using the rat RAP3 antiserum, the distribution of RAP3 in mouse plasma fractions, which had been isolated by gel-filtration chromatography, was determined. The cholesterol profile of the fractions and the Western blot results of the fractions positive for RAP3 are shown in Fig. 6. RAP3 was found in plasma fractions containing the larger HDL particles but not in the fractions containing the LDL or VLDL particles. To investigate whether the enhanced levels of RAP3 protein after PHx merely reflected an up-regulation of HDL, protein and cholesterol profiles of normal rat plasma and rat plasma 6 h after PHx were determined (Fig. 7). The level of HDL-protein was decreased 6 h after PHx, whereas the level of HDL-cholesterol remained constant.

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Presence of RAP3 protein in mouse plasma fractions. A pool of plasma from five mice was fractionated by gel-filtration chromatography. The cholesterol profile of the fractions is shown in the upper panel (28). The three peaks represent three subclasses of the various lipoprotein particles: VLDL, LDL, and HDL. In the lower panel, the RAP3 protein bands on Western blot are given from fractions 24-30. The positive control (pos. contr.) is RAP3 in normal mouse plasma.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Protein (a) and cholesterol (b) profiles of normal rat plasma and rat plasma 6 h after PHx. Plasma was fractionated by gel-filtration chromatography. a, protein profiles. The albumin and HDL-protein peaks are indicated by arrows. b, cholesterol profiles. The HDL-cholesterol peaks are indicated.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

After PHx, DNA synthesis and cell proliferation are stimulated and coordinated until the original liver mass has been restored. This process involves the remodeling of complete liver architecture, including extracellular matrix, vasculature, and the biliary tree. The mechanisms regulating this regenerative process have been incompletely elucidated. However, several important factors controlling the process, including growth factors, cytokines, and transcription factors, have been identified (32). Four different phases of gene activation have been identified in the regenerating rat liver after PHx: The first is the immediate-early phase (0-4 h) during which cells are primed to G₁; the second or delayed phase (4-8 h) precedes activation of cell cycle genes in the third phase (8-20 h); and, finally, in the fourth phase (20-48 h) DNA replication and mitosis occur (32).

The immediate-early phase is associated with up-regulation of more than 70 genes (4, 5). However, after priming of the cell, other factors are needed for regeneration to proceed. Our hypothesis is that in the second phase a decisive event occurs, which is necessary to trigger continuation of regeneration. Accordingly, we looked for genes that are up-regulated 6 h after PHx in the rat.

Twelve genes (nine known and three unknown) were found to be up-regulated in rat liver 6 h after PHx relative to control livers (Table I). Although the most up-regulated gene (amyloid A) might reflect an acute phase reaction (33), all the other eight known genes have been associated with liver regeneration or with cell proliferation in general: fibronectin (34), an intracisternal-A particle element (35), gamma-actin (36), and ribophorin I (37) with liver regeneration; 2-macroglobulin with hepatocyte proliferation in vitro (38); ribosomal protein S5 with protein synthesis (39); ribosomal protein L13 (which is similar to the human breast basic conserved 1 gene) with breast cancer (40); and the chaperonin-containing TCP-1 gamma subunit with protein folding (41). The associations of these genes with cell proliferation indicate that the applied subtraction technique was a valid method for identifying regeneration-related genes.

We were interested in identifying novel factors essential for liver regeneration. Because RAP3 showed the greatest degree of up-regulation among the three isolated novel genes, this gene and its relationship to liver regeneration were studied in more detail.

Full-length cDNAs of rat, mouse, and human RAP3 were isolated and sequenced. Rat and mouse cDNA sequences showed 90% homology, and the murine sequences had 75% homology with human RAP3. Thus, the RAP3 gene is moderately conserved in different species. Furthermore, by using 3'-RACE reaction and Northern blotting it was found that RAP3 is transcribed as two mRNAs. Because all three exons of RAP3 in the human genome are represented in the transcripts, the presence of two transcripts cannot be explained by alternative splicing but must be due to two alternative polyadenylation sites.

However, the two mRNAs encode the same protein. By software analysis of the RAP3 amino acid sequence, only one characteristic was found: a cleavable signal peptide that directs the protein into the secretory pathway of the cell (42). No other transmembrane segments were identified. These findings are consistent with the detection of RAP3 protein in plasma. 6 h after PHx in the rat both the liver RAP3 mRNA level and the plasma concentration of RAP3 protein appeared to be maximally increased. This coincidence indicates either rapid synthesis and secretion of the protein or increased RAP3 mRNA synthesis in response to release of RAP3 protein from a pre-existing intracellular pool. The first possibility seems to be the more likely. In addition, because the increased plasma concentration of RAP3 protein normalized rapidly, the half-life of circulating RAP3 protein would appear to be short.

Because both the peaks of liver RAP3 mRNA expression and of levels of RAP3 protein in plasma occurred 6 h after PHx, we postulate that RAP3 plays a role in the delayed-early phase of liver regeneration. Both RAP3 mRNA and RAP3 protein levels remained constant after sham operation, indicating that RAP3 is not an acute phase protein. The rapid decreases of both hepatic RAP3 mRNA expression and RAP3 protein levels in plasma, shortly after their peaks 6 h after PHx, suggest a role for RAP3 in the process of transition of the primed hepatocyte into a proliferative hepatocyte. The results of in situ hybridization also favor a role of RAP3 in the regenerative process, because they are in accordance with previous observations that indicate that the process of liver regeneration starts periportally and subsequently spreads to involve the pericentral region of the acinus (43). In addition, a transient increase of RAP3 protein concentration was detected in the plasma of a patient 1 day after a 60% hepatectomy.²

Data base searching revealed an appreciable homology (20-28%) between RAP3 and apolipoproteins A-I and A-IV. Like these apolipoproteins, RAP3 protein is predicted to have mainly an -helical secondary structure. The -helical secondary structures of apoA-I and apoA-IV are characterized by their organization in 22-amino acid repeats that have an amphipathic nature (31). Although this organization in RAP3 protein is much less pronounced, a few comparable repeats can be distinguished. Furthermore, the human RAP3 gene is located on chromosome 11q23, where the genes for apoA-I, apoC-III, and apoA-IV are also located (44). A regulatory relationship between the genes for apolipoprotein subtypes in this cluster has been suggested (44). In addition, under fasting conditions mRNA levels of apoA-I, apoC-III, and apoA-IV in the intestine were shown to be co-regulated (45). The RAP3 gene may belong to the same apolipoprotein gene cluster. Consistent with this hypothesis is the observation that the genomic organization of the RAP3 gene (Fig. 3) resembles that of apoA-IV, which also contains only two small and one long exons (46). Like apoA-IV, it lacks an intron, which is found in most other human apolipoprotein genes, in the area encoding the 5'-nontranslated region of its mRNA.

The murine cDNA sequences of RAP3 were about 75% identical to their human analogue. The same moderate conservation is also observed for the corresponding sequences of apoA-I and apoA-IV. Like most apolipoproteins (47), RAP3 is expressed in the liver. In contrast to apoA-I and apoA-IV, RAP3 mRNA is not expressed in the intestine. The association of RAP3 with apolipoproteins is further supported by our finding that RAP3 is present in mouse plasma in protein fractions that contain HDL particles. Therefore, we postulate that RAP3 protein is a novel apolipoprotein constituent of HDL. Accordingly, we propose that RAP3 be classified as apolipoprotein A-V (apoA-V).

The concentration of apoA-V in normal rat plasma was estimated to be about 1 µg/ml. The rat plasma concentrations of apoA-I and apoA-IV are about 400 and 160 µg/ml, respectively (48), which makes apoA-V a minor constituent of HDL.

The issue arises whether apoA-V plays an essential role in the process of liver regeneration or whether its up-regulation on mRNA and protein levels is only an epiphenomenon of liver regeneration that is associated with lipoprotein metabolism. The latter possibility seems to be unlikely, because 6 h after PHx rat plasma did not show a significant increase in HDL-cholesterol concentration and the HDL-protein level was even subnormal (Fig. 7). These findings are in agreement with data presented by Kurumiya et al. (49), which showed that, 6 h after 70% PHx in the rat, the mRNA expression of apoA-I (the major constituent of HDL) and the blood concentration of HDL-cholesterol were unchanged. Because the elevated plasma concentration of apoA-V is not accompanied by a concomitant increase in the concentration of HDL-cholesterol and HDL-protein, it is concluded that apoA-V may have a specific function during the early phase of liver regeneration.

Such a function would most likely involve lipid transport. During regeneration, lipids are essential constituents of newly formed membranes. One possible function could be that apoA-V stimulates lipid uptake by the liver, to support the de novo synthesis of membranes. However, the concentration of apoA-V in plasma peaks already at 6 h after PHx, whereas mitosis starts only 20 h later. At the time of mitosis, the apoA-V plasma concentration has even a subnormal level (Fig. 5). Therefore, we speculate that apoA-V might act as an antagonist of lipid uptake by the liver to protect the reduced-sized liver against a possible lipid overload in the early phase of liver regeneration. However, the exact function of apoA-V remains to be definitively elucidated.

	ACKNOWLEDGEMENTS

We thank Adrie Maas and Joost Daalhuysen for performing the rat surgery. We also thank the Department of Anatomy & Embryology, Academic Medical Center, and especially Theo Hakvoort and Jacqueline Vermeulen for their help in carrying out the in situ hybridization experiments. We are grateful to Dr. E. A. Jones for careful reading of the manuscript.

	FOOTNOTES

^* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF202887, AF202888, AF327059, AF202889, and AF202890.

^¶ To whom correspondence should be addressed: Dept. of Gastroenterology and Hepatology, Room C2-331, Academic Medical Center, University of Amsterdam, P.O. Box 22660, Amsterdam 1100 DD, The Netherlands. Tel.: 31-20-566-2422; Fax: 31-20-691-7033; E-mail: r.a. chamuleau@amc.uva.nl.

Published, JBC Papers in Press, September 27, 2001, DOI 10.1074/jbc.M106888200

² H. N. van der Vliet, M. Groenink Sammels, A. C. J. Leegwater, J. H. M. Levels, P. H. Reitsma, W. Boers, and R. A. F. M. Chamuleau, unpublished observation.

	ABBREVIATIONS

The abbreviations used are: PHx, 70% partial hepatectomy; apo, apolipoprotein; EST, expressed sequence tag; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends; RAP3, regeneration associated protein 3; TNF, tumor necrosis factor; IL-6, interleukin-6; STAT, signal transducers and activators of transcription; TBS, Tris-buffered saline; bp, base pair(s).

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