ARTICLE |
Correspondence to: Masashi Koono, Second Dept. Pathology, Miyazaki Medical College, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan.
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Summary |
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We used a specific monoclonal antibody to human hepatocyte growth factor activator inhibitor type 1 (HAI-1) in immunohistochemical procedures to determine the distribution and localization of HAI-1 in human tissues. In normal adult tissues, HAI-1 was predominantly expressed in the simple columnar epithelium of the ducts, tubules, and mucosal surface of various organs. In all cases, HAI-1 was localized predominantly on the cellular lateral (or basolateral) surface. By contrast, hepatocytes, acinar cells, endocrine cells, stromal mesenchymal cells, and inflammatory cells were hardly stainable with the antibody, and stratified squamous epithelium showed only faint immunoreactivity on the surface of cells of the basal layer. In the gastrointestinal tract, the surface epithelium was strongly stained. RNA blot analysis confirmed the presence of specific mRNA transcript in the gastrointestinal mucosa, and in situ hybridization revealed that HAI-1 mRNA showed a similar cellular distribution pattern. Although HAI-1 was not expressed in normal hepatocytes, strong immunoreactivity was observed on the epithelium of pseudo-bile ducts and on the surface of scattered hepatocytes in fulminant hepatitis. The enhanced expression was also noted in regenerating tubule epithelial cells of the kidney after infarction. We conclude that HAI-1 is preferentially expressed in the simple columnar epithelium of the mucosal surface and duct, that the predominant localization of HAI-1 is the cell surface, and that the expression of HAI-1 can be modulated by tissue injury and regeneration. (J Histochem Cytochem 47:673682, 1999)
Key Words: hepatocyte growth factor (HGF), HGF activator, HGF activator inhibitor, immunohistochemistry
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Introduction |
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Hepatocyte growth factor (HGF) is a pleiotropic factor initially identified as a growth factor for hepatocytes (
HGFA is a blood coagulation factor XIIa-like serine proteinase and is secreted by the liver as an inactive zymogen, circulating in the blood in this form (
Mature HAI-1 has two well-defined Kunitz domains. The first domain appears to be responsible for the inhibition of HGFA (
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Materials and Methods |
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Monoclonal Antibody
A Balb/c mouse was immunized with purified HAI-1 protein prepared from conditioned medium of MKN-45 cells as described () was suitable to stain formalin-fixed, paraffin-embedded tissue specimen and was used for this study. The epitope of this antibody was around the second Kunitz domain of HAI-1 protein (
Immunohistochemistry
All human tissues were obtained from surgical specimens or at autopsy. Formalin-fixed, paraffin-embedded tissue specimens were prepared according to the routine procedure. Sections were dewaxed in xylene and rehydrated in decreasing ethanol solutions and water. After antigen retrieval (5 min of autoclaving in 10 mM citrate buffer, pH 6.0), the sections were treated with 3% H2O2 in PBS for 10 min and washed in PBS twice, followed by blocking in 3% bovine serum albumin (BSA) in PBS for 1 hr at room temperature (RT). Then the sections were incubated with the primary antibody (1N7; 10 µg/ml of PBS containing 1% BSA) for 16 hr at 4C. Negative controls consisted of omission of the primary antibody, and as positive control a colon mucosal tissue in which HAI-1 mRNA expression was confirmed previously was used (
Immunoelectron Microscopy
Human stomach surgical specimens fixed with 4% paraformaldehyde in 0.1 M phosphate buffer were carried through 5%, 10%, 15%, and 20% sucrose in PBS for 2 hr each and finally immersed in 20% sucrose containing 50% OCT compound (Lab Tek; Naperville, IL) in PBS overnight, and then rapidly frozen in liquid nitrogen. The frozen tissues were sectioned at 6 µm with a cryostat and dried on glass slides. Before the immunostaining, we treated sections with 50 mM Tris-HCl buffer, pH 9.0 or pH 10.0, for 48 hr at RT. We confirmed that pretreatment with a higher pH solution influenced the degree of unmasking of the epitope (
Immunoblot Analysis
Subconfluent MKN-28 gastric adenocarcinoma and CCK-81 colon adenocarcinoma cells on 100-mm dishes were washed three times with PBS and immediately scraped into 2 ml of 10% trichroloacetic acid on ice. MKN-28 and CCK-81 were obtained from IBL (Fujioka, Japan) and Japanese Cancer Research Resources Bank, respectively. The precipitated proteins were harvested by centrifugation (14,000 rpm, 3 min) and the pellet was extracted with 200 µl of 7 M urea/2% Triton X-100 /5% 2-mercaptoethanol, followed by centrifugation (14,000 rpm, 3 min). To prepare a tissue extract specimen, fresh human non-neoplastic gastric mucosa (0.16 g), which was obtained from the surgically resected stomach of a gastric cancer patient, was immediately frozen in liquid nitrogen, crushed, and homogenized in an extraction buffer containing 50 mM Tris-HCl (pH 7.5)/150 mM NaCl/5 mM EDTA/2 mM phenylmethyl sulfonyl fluoride/5 µg/ml leupeptin/2% Triton X-100/2% Nonidet P-40, followed by centrifugation (14,000 rpm, 3 min). The resultant supernatants were mixed with SDS sample buffer and boiled for 3 min. SDS-PAGE was performed under reducing conditions using a 412% gradient gel. After electrophoresis, the proteins were transferred electrophoretically onto Immobilon membrane (Millipore; Bedford, MA). After blocking the nonspecific binding sites with 5% nonfat dry milk in 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.05% Tween 20 (TBS-T), the membrane was incubated with MAb 1N7 (1 µg/ml in TBS-T containing 1% BSA) at 4C overnight, followed by washing in TBS-T four times and incubation with peroxidase-conjugated swine anti-rabbit immunoglobulin IgG (Bio-Rad; Hercules, CA), diluted 1:5000 in TBS-T with 1% BSA for 1 hr at RT. The labeled proteins were visualized with a chemiluminescence reagent (NEN Life Science; Boston, MA).
In Situ Hybridization Analysis of HAI-1 mRNA
For the probe, a 622-BP cDNA fragment of HAI-1 corresponding to bases 144765 of HAI-1 cDNA sequence was generated by the polymerase chain reaction (PCR), using a plasmid harboring full-length HAI-1 cDNA as a template (
RNA Blot Analysis
Total RNA was extracted using Trisol reagent (Gibco BRL) from gastrointestinal mucosa and from cultured cells. Twenty µg of total RNA was electrophoresed on a 1% formaldehyde agarose gel and transblotted onto Hybond-N+ nylon membrane (Amersham; Poole, UK) and RNA was UV-crosslinked onto the membrane. Hybridization was performed in a mixed solution of 50% formamide, 5 x Denhardt's solution, 25 mM PB (pH 6.5), 0.1% SDS, 100 µg/ml of sonicated and heat-denatured salmon sperm DNA, and 5 x SSC at 42C for 16 hr. The blots were washed as follows: three times in 0.1% SDS in 1 x SSC for 15 min at RT and twice in the same solution for 20 min at 65C. The membranes were autoradiographed with Kodak XR-5 film at -80C for 6 hr or 24 hr. Human HAI-1 cDNA (-32P]-CTP.
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Results |
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Specificity of MAb 1N7
Before we started the immunohistochemical study, the specificity of MAb 1N7 was assessed by immunoblot analysis using whole extracts of cultured MKN-28 and CCK-81 cells and normal human gastric mucosa. As shown in Figure 1A, a single major band of MW around 66 kD and 64 kD was detected in the cultured cells and gastric mucosa, respectively, indicating the specificity of MAb 1N7. The tumor cell-derived HAI-1 showed a slightly higher MW than that derived from normal mucosa, and this difference may reflect an altered glycosylation of HAI-1 in tumor cells. RNA blot analysis revealed that HAI-1 mRNA was in fact present in MKN-28 and CCK-81 cells and in normal gastrointestinal mucosa (Figure 1B and Figure 1C). The levels of HAI-1 mRNA were correlated with the amounts of immunoreactive cellular HAI-1 protein (Figure 1A and Figure 1B). Furthermore, the immunohistochemical reaction was adsorbed by an excess amount of recombinant HAI-1. This was performed by using a normal colon mucosal specimen (Figure 2A and Figure 2B) in which the presence of HAI-1-specific mRNA and of proteins had been previously confirmed (
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Tissue and Cellular Distribution of HAI-1
Subsequent immunohistochemical studies showed that HAI-1 immunoreactivity was evident in the surface epithelium of the gastrointestinal tracts. In the stomach, the immunoreactivity was largely restricted to the surface epithelial and pit-lining cells, and both fundic and pyloric glandular epithelia were hardly stained (Figure 2C). In the positive cells, the cell surface was predominantly stained (Figure 2D). The stromal components, such as fibroblasts, smooth muscle cells, and inflammatory cells, were negative. In situ hybridization revealed a similar distribution of HAI-1 mRNA in the gastric mucosa (Figure 2F), supporting the specificity of the immunostaining. In contrast, the esophageal stratified squamous epithelium was poorly stained, and only very faint immunoreactivity was detectable, preferentially in the cells of the basal layer (Figure 2G and Figure 2H).
In the liver, the bile duct epithelium was stained, whereas the hepatocytes and hepatic sinusoidal cells were negative (Figure 2I and Figure 2J). The epithelia of the extrahepatic bile ducts and the gallbladder were also stained. In the pancreas, the epithelium of the duct system was stained. In particular, the cells of centroacini, intercalated ducts, and intralobular ducts were strongly stained. By contrast, the acinar cells and cells of the islets were negative (Figure 2K and Figure 2L).
In the kidney, HAI-1 immunoreactivity was observed in the tubule epithelium. The immunoreactivity was strong in the distal tubules and collecting ducts. On the other hand, the proximal tubules were only faintly stained. The glomerular cells were largely negative, and weak signals were occasionally detectable in the glomerular epithelial cells. The transitional epithelial cells of the renal pelvis, ureter, and urinary bladder were hardly stainable. The prostate glandular epithelium was also positively stained, and the immunoreactivity was predominantly observed in the secretory compartment of the epithelium compared with the basal compartment (Figure 2M). The endocervical epithelium of the uterus was strongly stained and the endometrial epithelium was also positive, whereas myometrial tissues were completely negative. In the endocervix, the surface epithelium was stained more strongly than the glandular epithelium (Figure 2N and Figure 2O). The cervical stratified squamous epithelium was only faintly focally stained.
The results of immunostaining are summarized in Table 1. In general, HAI-1 was detectable in the simple columnar epithelium covering the mucosal surface or ducts. Predominant cellular surface localization was suggested, and the lateral surfaces of the cells were preferentially stained (Figure 2D and Figure 2O). It should be noted that the organ distribution shown in Table 1 was in accordance with the previous RNA blot studies (
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Immunoelectron Microscopy
We confirmed that pretreatment of cryostat sections with Tris buffer, pH 9.0 or pH 10.0, remarkably enhanced HAI-1 immunostaining by MAb 1N7, although the ultrastructures were damaged to a lesser extent by the alkaline treatment. In the body portions of human stomach, the immunostaining for HAI-1 was observed on the surface of the basolateral plasma membrane of the surface epithelial cells. The lateral plasma membrane generally showed intenser immunoreaction than that of the basal plasma membrane (Figure 3). These results were quite compatible with the light microscopic findings above. The control experiments did not exhibit any immunoreactions (not shown).
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Enhanced Expression of HAI-1 Protein in Injured Tissues
Because the generation of active HGFA is believed to occur exclusively in injured tissue through proteolytic activation of pro-HGFA by thrombin (
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Discussion |
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This work presents the first systematic immunohistochemical study using an MAb directed against HAI-1 protein in human tissues. Specificity of the immunohistochemical reaction was verified in the following manner. (a) Staining of positive and negative control tissue sections in each assay was prepared. (b) The immunoreactivity was adsorbed by a recombinant HAI-1 protein. (c) In situ hybridization using specific antisense riboprobe for HAI-1 mRNA was followed by a stringent ribonuclease A treatment, resulting in a distribution pattern of the specific signal similar to the immunohistochemistry results. (d) The antibody recognized specifically HAI-1 protein in the immunoblot analysis. Finally (e), the immunostaining pattern of HAI-1 in paraffin sections using antigen retrieval is comparable with that obtained in frozen section (
HAI-1 protein was predominantly expressed on the surface of the simple columnar epithelium covering the mucosal surface and duct lumen. In contrast, stratified squamous epithelia of various organs, transitional epithelia of the urinary tract, ciliated bronchial epithelium, and hepatocytes were largely negative or were stained only very weakly. In addition to the columnar epithelium, Schwann cells were also stained for HAI-1. The predominant cellular surface localization of HAI-1 confirmed in this report is consistent with the molecular structure of HAI-1 protein deduced by the mRNA sequence, i.e., the presence of a presumed transmembrane domain in the HAI-1 molecule (
At present, the exact biological role of HAI-1 in vivo is uncertain. Because HAI-1 is an efficient endogenous inhibitor of HGFA, the expression of HAI-1 may have an important role in regulation of the activation of HGF/SF in local tissues. This hypothesis may agree with the fact that the cells which expressed HAI-1 protein in this study are susceptible to HGF/SF. For example, HGF/SF is an important mitogen for the gastric, bile duct, and renal tubule epithelium (
Of particular interest was the observation that immunoreactivity to HAI-1 protein was upregulated in injured and regenerating tissues. In the injured tissues, proteolytic activation of zymogen of HGFA would occur through thrombin generation, generating a considerable amount of active HGFA (
In summary, the distribution and cellular localization of HAI-1 protein were examined in normal adult tissues. Although the exact function and role of HAI-1 in vivo remain to be clarified, its distribution suggests that this protein may have an important function in the surface columnar epithelium of various organs. We also hypothesize that HAI-1 may be somehow involved in tissue responses to injury and in regenerative processes, both of which require further investigation.
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Acknowledgments |
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We are grateful to Dr K. Denda (Department of Life Science, Tokyo Institute of Technology) for recombinant HAI-1, to Dr H. Tsubouchi (Second Department of Internal Medicine, Miyazaki Medical College) for helpful suggestions, and to Mr S. Ide and Ms N. Iwakiri for excellent technical assistance.
Received for publication October 2, 1998; accepted December 9, 1998.
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