ARTICLE |
Correspondence to: Satoshi Waguri, Dept. of Cell Biology and Neuroscience (A1), Osaka U. Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail: waguri@anat1.med.osaka-u.ac.jp
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Summary |
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Two mannose 6-phosphate receptors, cation-dependent and -independent receptors (CDMPR and CIMPR), play an important role in the intracellular transport of lysosomal enzymes. To investigate functional differences between the two in vivo, their distribution was examined in the rat liver using immunohistochemical techniques. Positive signals corresponding to CIMPR were detected intensely in hepatocytes and weakly in sinusoidal Kupffer cells and interstitial cells in Glisson's capsule. In the liver acinus, hepatocytes in the perivenous region showed a more intense immunoreactivity than those in the periportal region. On the other hand, positive staining of CDMPR was detected at a high level in Kupffer cells, epithelial cells of interlobular bile ducts, and fibroblast-like cells, but the corresponding signal was rather weak in hepatocytes. In situ hybridization analysis also revealed a high level of expression of CIMPR mRNAs in hepatocytes and of CDMPR mRNA in Kupffer cells. By double immunostaining, OX6-positive antigen-presenting cells in Glisson's capsule were co-labeled with the CDMPR signal but were only faintly stained with anti-CIMPR. These different distribution patterns of the two MPRs suggest distinct functional properties of each receptor in liver tissue. (J Histochem Cytochem 49:13971405, 2001)
Key Words: mannose 6-phosphate, receptors, immunofluorescence, hepatocytes, Kupffer cells, antigen-presenting cells, rat, liver
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Introduction |
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NEWLY SYNTHESIZED LYSOSOMAL ENZYMES acquire mannose 6-phosphate (M6P) recognition signals that direct them from the trans-Golgi network (TGN) to the prelysosomal compartments. Two receptors, cation dependent- and -independent mannose 6-phosphate receptors (CD- and CIMPR), have been shown to be involved in this sorting function in mammalian cells. To date, differences in the properties of the two MPRs have been extensively studied using biochemical and cell biological approaches. CIMPR, a large transmembrane glycoprotein (Mr z300 kD), is also known as the insulin-like growth factor II (IGFII)/M6P receptor and contains two M6P-binding sites as well as a distinct IGF-II-binding site in the extracytoplasmic domain. On the other hand, CDMPR is a small receptor (Mr z46 kD) and has one M6P-binding site. It is believed that CDMPR exists as a dimer and/or tetramer on the membrane. Both receptors may appear on the cell surface, where they are then internalized. CIMPR, but not CDMPR, binds extracellular ligands (
In contrast to the accumulated biochemical and cell biological data on the MPRs, only a few studies have appeared on the histological distribution of the two receptors (
We have previously studied the distribution of lysosomal cysteine proteinases, cathepsins B, H, and L, in various tissues and found that they show heterogeneous distribution patterns depending on the enzymes, cells, and tissues (
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Materials and Methods |
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Animals and Tissue Preparation
Four adult male Wistar rats (7 weeks of age, 250 g) were deeply anesthetized with pentobarbital (25 mg/kg IP) and perfused via the portal vein first with 5 ml of physiological saline and then with 50 ml of 4% paraformaldehyde buffered with 0.1 M phosphate buffer (PB), pH 7.4, containing 4% sucrose. The liver tissue was excised, cut into small pieces, and immersed in the same fixative for 4 hr. After washing three times with the same buffer containing 7.5% sucrose, cryoprotection was ensured by infusing successively with 15% and 30% sucrose solutions, after which they were embedded with OCT compound. Cryosections were cut in 10-µm sections with a cryostat (Coldtome CM 501; Sakura, Tokyo, Japan) and mounted on gelatin-coated glass slides.
Antibodies
A rabbit polyclonal antibody against rat CIMPR was raised as reported previously (
cDNAs and Probes
The cDNA for rat CDMPR was cloned into the Not I site of Bluescript II SK+ (Stratagene; La Jolla, CA) as previously described (
Immunofluorescence Microscopy
The liver sections were treated with methanol at room temperature (RT) for 15 min and then incubated with 2% normal goat serum at RT for 20 min. They were then incubated with the following primary antibodies at 4C overnight: anti-CIMPR (1.9 µg/ml), anti-CDMPR (1 µg/ml), MSCI-III-7b (1:1000), ED2 (1:500), and OX6 (1:200). Further incubation was performed with FITC-conjugated goat anti-rabbit IgG (1:1000; Seikagaku Kogyo, Tokyo, Japan) or Texas Red-conjugated goat anti-mouse IgG (1:1000; Biomeda; Foster City, CA) at RT for 1 hr. For double immunofluorescence, the sections were immunolabeled first for ED2 or OX6 and then for CIMPR or CDMPR, as described above. After each step, sections were rinsed with 0.01 M phosphate buffered 0.5 M saline (pH 7.2), containing 0.1% Tween-20 (Sigma Chemical; St Louis, MO). For control experiments, sections were incubated either with a non-immunized rabbit serum diluted to 1:1000 or with a dilution buffer, followed by incubation with FITC-conjugated goat anti-rabbit IgG or Texas Red-conjugated goat anti-mouse IgG. For the cultured cells, CDMPR-GFP was transiently expressed in HeLa cells by transfection using the calciumphosphate method as described previously (
In Situ Hybridization
Bluescript II SK+ plasmids encoding each cDNA were linearized by digesting at convenient restriction sites on either side of the inserts. Digoxigenin (DIG)-labeled cRNA probes were then synthesized using an RNA labeling kit (Roche Diagnostics; Mannheim, Germany) according to the manufacturer's recommended protocol.
The liver sections were prehybridized in a hybridization buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 600 mM NaCl, 0.25% SDS, 50% deionized formamide, 10% dextran sulfate, 20 µg/µl tRNA from E. coli, 1 x Denhardt's solution) at 50C for 1 hr, and then hybridized with DIG-labeled probes in the same buffer at 50C for 20 hr. After washing with 50% formamide in 2 x SSC at 50C for 30 min, the sections were treated with 1 µg/ml ribonuclease A at 37C for 30 min. They were then washed once with 2 x SSC at 50C for 20 min and twice with 0.2 x SSC at 50C for 20 min. The signal was detected using a DIG Nucleic Acid Detection Kit (Roche Diagnostics).
Immunoblot Analysis
The rat liver tissue was homogenized with a cell lysis buffer solution (0.05 M Tris-HCl, pH 7.5, 0.15 M NaCl, 1% NP-40, 0.5% deoxycholate, 1 mM EDTA) containing a protease inhibitor cocktail (Roche Diagnostics). After centrifugation at 15,000 x g for 15 min, the supernatant was subjected to 10% SDS-PAGE under non-reduced conditions and the proteins were then transferred onto a PVDF membrane (Immobilon; Millipore, Bedford, MA). The blots were immunolabeled with anti-CIMPR or anti-CDMPR antibody, followed by HRP-conjugated anti-rabbit IgG. The signals were detected using ECL reagents (Amersham Pharmacia Biotech).
Immunoprecipitation
HeLa cells transiently expressing CDMPR-GFP and control cells were incubated with methionine-free DMEM (Life Technologies; Rockville, MD) containing 10% dialyzed FBS for 30 min, followed by an incubation with 10 mBq/ml of Redivue PRO-MIX (Amersham Pharmacia Biotech) in the same medium overnight. After washing three times with PBS, the cells were lysed with cell lysis buffer that contained a protease inhibitor cocktail. The resulting lysates were precleared with protein ASepharose CL4B (Amersham Pharmacia Biotech) for 1 hr at 4C and centrifuged for 15 min. The supernatant was incubated, first with anti-CDMPR or anti-GFP antibody overnight at 4C and then with protein ASepharose CL4B for 1 hr at 4C. After washing the Sepharose, the immune complexes in the SDS gel sampling buffer was boiled for 5 min, and then electrophoresed on a 10% SDS-polyacrylamide gel. The gel was fluorographed and imaged using BAS2000 (Fuji Film; Tokyo, Japan).
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Results |
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Characterization of the Antibodies Used in This Study
The specificity of anti-rat CIMPR antibody has previously been demonstrated by immunoprecipitation and immunofluorescence microscopy (300 kD under non-reduced conditions (Fig 1A). The reduced form of CIMPR was not recognized by this antibody (data not shown). In contrast, anti-CDMPR recognized neither the reduced nor the non-reduced form of CDMPR in our immunoblotting system. To further characterize this antibody, HeLa cells transiently expressing the CDMPR-GFP fusion protein were analyzed by immunoprecipitation. As shown in Fig 1B, the antibody recognized both the endogenous CDMPR (
46 kD) and the transiently expressed CDMPR-GFP (two bands around
70 kD). The lower sharp band corresponding to CDMPR-GFP probably represents the non-palmitoylated form, as has been reported previously (
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Distribution of the Two MPRs in the Rat Liver
As shown in Fig 2A and Fig 2C, a strong granular immunofluorescence signal for CIMPR was detected in hepatocytes, whereas the signal was faint in sinusoidal cells. A weak but distinct signal was also detected on the cell surface of hepatocytes. In the acinus, the intensity was higher in perivenous than in periportal hepatocytes (Fig 2A). Positive staining for CIMPR was also detected in interstitial cells and in the epithelial cells of interlobular bile ducts, which are located in the portal triad, although this intensity was lower than that found in hepatocytes (Fig 2E). Granular staining for CDMPR was detected in both hepatocytes and sinusoidal cells. The latter were much more intensely immunoreactive than the former (Fig 2B and Fig 2D). No clear-cut difference in immunofluorescence intensity was detected in hepatocytes with respect to their location within the acinus (Fig 2B). In the case of Glisson's capsule, a positive signal for CDMPR was detected in the epithelial cells of interlobular bile ducts and the intensity was higher than that in the hepatocytes. Some interstitial cells, including fibroblast-like cells, also showed distinct signals for CDMPR (Fig 2F and Fig 5C). The same staining patterns were obtained when the antibody MSCI-III-7b, which recognizes the entire cytoplasmic domain of CDMPR, was used (data not shown).
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The preferential localization of CIMPR and CDMPR in hepatocytes and sinusoidal cells was also confirmed by ISH. As shown in Fig 3, the majority of the CIMPR transcript was detected in hepatocytes (Fig 3A), whereas the expression level of CDMPR mRNA was higher in sinusoidal cells than in hepatocytes (Fig 3B). The distribution pattern of the CDMPR transcript was similar to that of cathepsin L (Fig 3C), which has been shown to exist at higher levels in Kupffer cells than in hepatocytes (
Identification of CDMPR-positive Cells
We next attempted to identify the CDMPR-positive cells in the sinusoid and Glisson's capsule using the MAbs, ED2 and OX6, which are known to serve as markers for Kupffer cells (
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Discussion |
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The present study demonstrates that CI- and CDMPR are expressed at different levels and locations in the rat liver. The former was distributed mainly in hepatocytes, whereas the latter was localized in non-hepatocytic cells, such as Kupffer cells and antigen-presenting cells (APCs).
In a previous study,
It has been shown that the cathepsin L family of proteinases, such as cathepsins K and S, play important roles in bone absorption and antigen processing in osteoclasts and APCs, respectively (
There have been several studies concerning interactions of both MPRs with M6P. In equilibrium dialysis binding studies,
As stated above, CIMPR is mainly expressed in hepatocytes, suggesting the importance of this receptor in the cells. In addition to its sorting function of M6P-containing lysosomal enzymes in the TGN, CIMPR has also been shown to bind several ligands on the cell surface. It is involved in the downregulation of the extracellular IGFII levels (
The distribution of CIMPR within the liver acinus is also intriguing. Its expression was high in the perivenous region compared to the periportal region. This staining pattern is in agreement with our previous results, in which the same distribution patterns of lysosomal cysteine proteinases, cathepsins B and H, in the rat liver acinus were demonstrated (
Collectively, the present data, which show that the expression of CIMPR was predominant in hepatocytes whereas that of CDMPR was prominent in Kupffer cells and APCs, suggest that the two MPRs possess different functions in the rat liver.
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Acknowledgments |
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Supported by a grant from The Japan Ministry of Education, Science and Culture.
We are grateful to Dr A. HillRehfeld (Georg-August-University, Göttingen), and Dr Ishidoh (Juntendo University, Japan) for providing MSCI-III-7b and cDNA for cathepsin L, respectively. We also thank Dr K. Matsuno (Kumamoto University School of Medicine, Kumamoto) for his valuable suggestions concerning dendritic cells in the rat liver.
Received for publication March 7, 2001; accepted June 13, 2001.
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