ARTICLE |
Correspondence to: Ichiei Iida, Inst. of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Shinshu University Graduate School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Japan. E-mail: i2davh@coral.ocn.ne.jp
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Summary |
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Hepatocyte growth factor activator (HGFA) can activate the single-chain hepatocyte growth factor (HGF) required for embryonic development. We studied the immunohistochemical (IHC) localization of HGFA in adult mouse liver and its developmental changes from embryonic day 12 to postnatal day 30. A heterogeneous distribution of HGFA was observed in adult liver tissues. The hepatocytes around the hepatic veins were preferentially positive for HGFA, whereas those in other areas were negative. Depending on the vascular diameter, the hepatic veins were bordered by a one- to three-cell-thick layer of hepatocytes positive for HGFA, which showed evidence of cellcell heterogeneity in staining intensity. Immunoelectron microscopy detected ubiquitous distribution of the gold particle reaction product for HGFA in the cytoplasm of these hepatocytes, especially in the rough endoplasmic reticulum. Developmental analysis indicated that there was hardly any staining of HGFA until postnatal day 0 and that noticeable staining was initially detected in the pericentral hepatocytes on postnatal day 3. Subsequently, immunoreactivity increased and the distinct staining pattern had been established by postnatal day 30. These results suggest that HGFA proteins are produced in the hepatocytes surrounding the efferent hepatic veins in the mouse and that development of the unique distributing pattern takes place postnatally. (J Histochem Cytochem 51:11391149, 2003)
Key Words: hepatocyte growth factor, activator, liver, development, immunohistochemistry, mouse
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Introduction |
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HGFA, an activator of HGF, has been purified from fetal bovine (
HGF has been identified a tissue organizer with multiple biological activities and has been isolated as the growth factor of hepatocytes (
HGFA mRNA is mainly expressed in parenchymal cells of adult liver and its protein is secreted into the blood. A recent study reported that the expression of the HGFA gene was detected not only in the liver but also in the gastrointestinal tract of normal adult mice. In these mice, HGFA mRNA was strongly expressed in the liver as well as colon, moderately in the stomach, duodenum, jejunum, and ileum, weakly in the kidney, and almost imperceptibly in the brain, heart, testis, and ovary (
To gain a better understanding of the role of HGFA in liver function and organogenesis, we employed monospecific antibodies to investigate the IHC distribution of HGFA in adult and developing mouse liver tissues.
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Materials and Methods |
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Animals and Tissues
Liver tissues of ICR mice of different ages were examined: embryonic day 12, postnatal days 0, 3, 14, and 30, and postnatal week 8. Five embryos irrespective of gender and five male mice from each postnatal group were used. Adult male C57BL/6J mice were also used for verifying strain differences. All animals were purchased from SLC (Hamamatsu, Japan). They were sacrificed under deep anesthesia with pentobarbital sodium solution (0.04 mg/g body weight) in accordance with the Shinshu University School of Medicine Animal Care Committee guidelines.
Antibodies for HGFA
Three antibodies against HGFA were used for our study. N-19 goat polyclonal antibody for human HGFA was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A-1 and P1-4 mouse monoclonal antibodies (MAbs) raised against human HGFA (
Reverse Transcription-polymerase Chain Reaction (RT-PCR)
Total RNA from mouse liver obtained on postnatal day 8 was extracted with the RNeasy mini kit (Qiagen; Valencia, CA) according to the manufacturer's instructions. cDNA was synthesized from 1 µg of total RNA by means of SuperScript II reverse transcriptase (Invitrogen; Carlsbad, CA). cDNA samples were subjected to RT-PCR amplification using the GeneAmp PCR System 9700 (Applied Biosystems; Foster City, CA) with a primer set selective for mouse HGFA: the forward primer was 5'-CGGAGGCACCTGCCACC-3', and the reverse primer was 5'-GCAGCGGTGCTGGCCAC-3' (150 bp). Amplification consisted of a hot start at 94C for 3 min followed by 50 cycles of denaturation at 94C for 30 sec, annealing at 60C for 30 sec, and extension at 72C for 30 sec with a final 7-min extension at 72C. The PCR products were size-fractionated by 3% agarose gel electrophoresis and visualized with ethidium bromide staining. Sequencing used the ABI PRISM 310 Genetic Analyzer (Applied Biosystems) to confirm that the PCR product corresponded to the mouse HGFA cDNA sequence (
Immunoblotting Assay
ICR mouse plasma (Cosmo Bio; Tokyo, Japan) was heated at 100C for 4 min in a sample buffer solution containing 1% SDS under reducing conditions. It was then subjected to SDS-polyacrylamide slab gel electrophoresis with a small slab gel (6 x 7 cm) followed by immunoblotting analysis. After transfer to nitrocellulose membranes, blots were blocked with 5% bovine serum albumin for 1 hr and then incubated overnight at 4C with goat polyclonal antibody against HGFA diluted to 1:100 with 3% bovine serum albumin/150 mM sodium phosphate, pH 7.4. Blots were washed three times in PBSTween and incubated with appropriate peroxidase-labeled secondary antibodies diluted 1:200 for 30 min. Visualization was attained with the reaction using 4-chloro-1-naphthol.
Light Microscopic Immunohistochemistry
Small pieces of liver tissue were fixed for 24 hr in a Methacarn solution (methanolchloroformacetic acid 6:3:1) at room temperature or in 4% paraformaldehyde (PFA)/100 mM sodium phosphate, pH 7.4, at 4C, and embedded in paraffin. After deparaffinization and rehydration, 5-µm-thick serial sections were immunostained with anti-HGFA antibodies by employing the ABC method followed by DAB reaction. Before immunostaining on PFA-fixed specimens with the N-19 antibody, they were pretreated for antigen retrieval in 10 mM Na citrate, pH 6.0, in a microwave at 100C for 5 min (
Confocal Laser Scanning Microscopy (CLSM)
The sections of adult mouse liver tissues treated as described above were doubly stained with a mixture of antibodies for HGFA (N-19) and GS. The former antibody was detected by means of donkey anti-goat IgG conjugated with Alexa Fluor 488 and, after blocking treatment with normal goat serum, the latter was detected with the aid of goat anti-rabbit IgG conjugated with Alexa Fluor 568. Fluorescent labeled antibodies were purchased from Molecular Probes (Eugene, OR). Specimens were observed with an Olympus Fluo View CLSM (Olympus; Tokyo, Japan) equipped with Ar and He/Ne lasers.
Electron Microscopic Immunohistochemistry
Small pieces of liver tissue (approximately 1 mm x 0.5 mm x 0.5 mm) were fixed in 4% paraformaldehyde, 0.1% glutaraldehyde, and 100 mM sodium phosphate, pH 7.4, at 4C. After rinsing in a cold solution consisting of 100 mM lysine, 100 mM sodium phosphate, pH 7.4, and 150 mM NaCl and dehydration in a graded series of cold ethanol, they were embedded in Lowicryl K4M at -20C (
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Results |
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Immunoblotting Assay
Immunoblot analysis demonstrated that the N-19 and A-1 antibodies were reactive with mouse HGFA (Fig 1A). These antibodies produced a single signal of 96 kD in response to mouse plasma, which suggests that the unactivated HGFA precursor in the circulation was detected. No discernible signal was obtained with the P1-4 antibody (data not shown).
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HGFA in Adult Liver
RT-PCR analysis demonstrated expression of HGFA mRNA in adult mouse livers. The predicted 150-bp fragment was consistently amplified from liver cDNA (Fig 1B). The PCR product was ascertained to correspond to the mouse HGFA cDNA sequence (data not shown). Light microscopic IHC using the N-19 antibody showed heterogeneous distribution of HGFA in the adult liver tissues at postnatal week 8 (Fig 2). Positive staining was restricted to the cytoplasm of hepatocytes adjacent to the efferent hepatic veins, including the central veins in the hepatic lobules, sublobular veins, and collecting veins (Fig 2A), but hepatocytes were mostly negative for HGFA in the other regions of liver tissue, including the periportal area. The largest hepatic veins were bordered by a one-cell-thick layer of HGFA-positive hepatocytes interspersed with HGFA-negative hepatocytes (Fig 2A and Fig 2B). Around smaller efferent hepatic veins, only a one- or two-cell-thick layer of hepatocytes stained for HGFA, thus forming a lining of positive cells (Fig 2A and Fig 2C). In the hepatic lobules, on the other hand, HGFA-positive hepatocytes were observed in a one- to three-cell-thick layer around the central veins (Fig 2A and Fig 2D). The HGFA-positive hepatocytes showed cellcell heterogeneity in staining intensity, ranging from faint to strong, with the strongly positive hepatocytes preferentially distributed near the hepatic veins. The control experiment using the absorbed antibody exhibited a marked reduction in staining intensity, thus confirming the specificity of the N-19 antibody (Fig 2E and Fig 2F). All these findings were the same for both the ICR and C57BL/6J strains. Use of two other MAbs, A-1 and P1-4, resulted in the same staining pattern as that of N-19 antibody in PFA-fixed liver specimens (Fig 3). Electron microscopic IHC showed that the gold particles were ubiquitously distributed in the cytoplasm of the HGFA-positive hepatocytes, principally in the rough endoplasmic reticulum, whereas the nucleoplasm and cell organelles such as mitochondria and lysosomes of these cells were mostly negative (Fig 4).
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HGFA in Liver Development
Immunoreactivity to HGFA and its localization were observed to change during liver development, while positive staining was not detected in the developing livers until postnatal day 0 (Fig 5A and Fig 5B). On postnatal day 3, positive staining became somewhat detectable around the central veins in the hepatic lobules (Fig 5C and Fig 5D). After this the immunoreactivity increased, and weak but detectable staining was observed on postnatal day 14 (Fig 5E and Fig 5F). On postnatal day 30, positive staining was clearly observed in the hepatocytes surrounding the efferent hepatic veins (Fig 5G and Fig 5H), similar to that seen in the adult liver. Throughout development of the liver, however, the periportal hepatocytes remained negative for HGFA.
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During liver development, HGFA exhibited a similar distribution pattern to that of GS, a representative enzyme synthesized by the pericentral hepatocytes (Fig 6A6C). However, CLSM observation of adult liver tissues showed that the two antigens did not display a completely identical distribution, i.e., some pericentral hepatocytes were positive for GS but not for HGFA (Fig 6D6F), indicating that HGFA has an intrinsic distribution pattern in the liver tissue different from that of GS.
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Discussion |
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The study presented here showed that the localization of HGFA in the adult mouse liver was confined to the hepatocytes surrounding the efferent hepatic veins. The thickness of the HGFA-positive cell layer varied depending on the diameter of the hepatic vein branches. HGFA immunoreactivity was mostly negative in the fetal and neonatal livers, became detectable in the hepatocytes adjacent to the pericentral veins by postnatal day 3, and was clearly observable by postnatal day 30. Use of the N-19 antibody cannot help determine whether the HGFA detected here is active or inactive, because both isoforms contain a long-chain subunit. Moreover, N-19 is an affinity-purified goat polyclonal antibody raised against an epitope mapping at the amino terminus of the long-chain subunit of human HGFA, while the peptide used to raise this antibody is identical in mouse and human HGFA (
A similar localization pattern for HGFA was detected in GS of adult mouse livers, consistent with previously reported results for mouse and rat livers (-1-antitrypsin are synthesized by all hepatocytes. The cytochrome P450, NADPH reductase, and glutathione-S-transferases, all enzymes involved in detoxification, are present either exclusively or predominantly in the 610-cell-deep layers of hepatocytes around the central vein (
A viable reason for the absence of detectable staining for HGFA in fetal and neonatal mouse livers is that HGFA is synthesized at too low a level for detection by the IHC technique employed. Another study found that the HGF mRNA level in the liver was very low during late gestation, increased remarkably in neonates, and reached a maximum 2 weeks after birth (
The liver regenerates in a variety of proliferative patterns. In acute liver injuries of mice after CCl4 administration, the appearance of necrotic hepatocytes around the central veins was found to result in the random presence of proliferative hepatocytes in the lobulus. When mice are subjected to hepatectomy, the manner of hepatocyte proliferation varies depending on the amount of liver tissue removed. After 70% partial hepatectomy, a marked, random DNA synthesis by hepatocytes was observed in the lobulus, while after 30% partial hepatectomy, the cells undergoing DNA synthesis were located at the periportal zone of the lobulus (
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Acknowledgments |
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Supported in part by the Kazato Research Foundation.
Mouse monoclonal antibodies for HGFA, P1-4, and A-1 were a generous gift from Dr Keiji Miyazawa (Department of Molecular Pathology, Graduate School of Medicine, University of Tokyo).
Received for publication July 2, 2002; accepted April 10, 2003.
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