ARTICLE |
Correspondence to: Chen-Yong Lin, Lombardi Cancer Center, New Research Building W412, Georgetown U. Medical Center, 3970 Reservoir Rd. NW, Box 571469, Washington, DC 20057-1421. E-mail: lincy@georgetown.edu
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Summary |
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Matriptase is a type II transmembrane serine protease that has been implicated in the progression of epithelium-derived tumors. The role of this protease in the biology of normal epithelial cells remains to be elucidated. Matriptase mRNA has been detected by Northern analysis in tissues rich in epithelial cells, and the protein is expressed in vivo in normal and cancerous breast, ovarian, and colon tissues. However, a systematic analysis of the distribution of matriptase protein and mRNA in normal human tissues rich in epithelium has not been reported. In this study we characterized the expression of the protease in a wide variety of normal human tissues using a tissue microarray and whole tissue specimens. Significant immunoreactivity and mRNA expression were detected in the epithelial components of most epithelium-containing tissues. Matriptase expression was found in all types of epithelium, including columnar, pseudostratified columnar, cuboidal, and squamous. Distinct spatial distributions of reactivity were observed in the microanatomy of certain tissues, however. This suggests that although matriptase is broadly expressed among many types of epithelial cells, its activity within a tissue may be regulated in part at the protein and mRNA levels during the differentiation of selected epithelia.
(J Histochem Cytochem 51:10171025, 2003)
Key Words: matriptase, transmembrane serine, protease, hepatocyte growth factor, activator inhibitor-1 (HAI-1), immunohistochemistry
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Introduction |
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MATRIPTASE, also known as membrane-type serine protease-1 (MT-SP1) and tumor-associated differentially expressed gene-15 (TADG-15), is a multi domain serine protease of the S1 chymotrypsin-like family (
Matriptase has been shown in vitro to activate the latent forms of HGF, uPA, and PAR-2, and to cleave extracellular matrix (ECM) components such as laminin and fibronectin (
The relationship between matriptase expression in cancer cells vs their normal counterparts has not been extensively studied. However, the expression of matriptase has been preliminarily characterized in several cancer sites. In ovarian cancer,
In normal human tissues, the expression of matriptase has been detected by Northern blotting in tissues rich in epithelium, including the prostate, stomach, small intestine, colon, lung, and kidney, as well as in placenta and peripheral blood leukocytes (
The function of matriptase in vivo is currently unknown. The mouse orthologue of matriptase, termed epithin, was cloned from mouse thymic epithelial cells, and may play a role in epithelial cell-directed thymic T-cell maturation (
In this study we have examined the expression of matriptase protein and mRNA in a broad range of normal human tissues using a tissue microarray and selected additional whole tissue sections. We wished to determine whether matriptase is expressed broadly among epithelial tissues or is more limited to one histological type of epithelium vs another (e.g., columnar vs stratified squamous). We also wanted to determine whether the expression level was spatially graded in the microanatomy of epithelium-containing tissues, as has been described for matriptase in the human breast and for rat matriptase along the cryptvillous axis in the small intestine. Finally, we sought to determine whether matriptase expression in normal human tissues correlates with the expression pattern of HAI-1, the matriptase inhibitor, which would further support a functional link between matriptase and HAI-1 in vivo. A description of the spatial distribution of matriptase expression in normal tissues could provide a framework for understanding the function of matriptase in normal cell biology and tissue homeostasis. Such information could furthermore be useful in understanding how matriptase expression may be deregulated in human pathology, such as carcinogenesis.
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Materials and Methods |
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Tissue Specimens
Normal human tissues were obtained as a normal human tissue microarray from Imgenex (San Diego, CA) and as whole tissue sections from the Histopathology and Tissue Shared Resource at the Lombardi Cancer Center, Georgetown University. The Imgenex tissue microarray contains 59 cores of normal human tissues representing all of the major human organ systems. Each section is an approximately 1-mm-diameter disk of paraffin-embedded, formalin-fixed tissue. All specimens on the Imgenex slide and the slides obtained from the Histopathology and Tissue Shared Resource were processed within 4 hr of removal, fixed with 10% neutral formalin, paraffin-embedded according to standard protocols, cut to 45-µm thickness, and placed on glass slides for immunohistochemistry (IHC) or in situ hybridization (ISH).
Monoclonal Antibody
The specificity of the anti-matriptase monoclonal antibody (MAb) S5 used for IHC has been described elsewhere (
Immunohistochemistry
IHC was performed using the Vectastain ABC kit (Vector Laboratories; Burlingame, CA) with minor modifications to the manufacturer's protocol. Sections were stained using the matriptase-specific MAb clone S5 (IgG1) (
In Situ Hybridization
Probes for ISH were prepared by generating matriptase-specific digoxigenin-labeled sense and antisense RNA riboprobes using the Dig-RNA labeling kit (BoehringerMannheim; Mannheim, Germany) according to a modified manufacturer's protocol as previously described (
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Results |
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Tissue and Cell Distribution of Matriptase
In all of the organs examined, matriptase was predominately expressed in epithelial cells of the surface-lining epithelium. Immunoreactivity was absent from most non-epithelial cell types, including stromal fibroblasts, although expression is observed in endothelial cells and isolated tissue leukocytes (data not shown). The pattern of matriptase expression is discussed below in the context of organ systems. Representative immunohistochemical and in situ staining is shown in Fig 1 Fig 2 Fig 3 Fig 4 Fig 5 Fig 6. Results of immunohistochemical staining are summarized in Table 1.
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Respiratory System. Strong matriptase expression was found in the respiratory mucosa in the nasal and bronchial lining (Fig 1A and Fig 1B). The glandular epithelium of serous and mucous glands in the nasal mucosa also stained positively (Fig 1A). In the lung, negative staining was observed in the alveolar epithelium (Fig 1C). Staining was also seen in bronchial seromucous glandular epithelium, but not in chondrocytes within cartilage (not shown) nor in stromal fibroblasts.
Digestive and Hepatobilliary System.
The digestive tract epithelium showed strong matriptase immunoreactivity. In the salivary gland, strong staining was observed in excretory duct epithelium, but staining was negative in serous and mucous secretory cells (Fig 1D). Both squamous epithelium and underlying submucous glands were positive in the esophagus (Fig 2A). The basal and intermediate layers of the squamous esophageal strata showed stronger reactivity than luminal layers. ISH confirmed the production of matriptase mRNA primarily in the intermediate esophageal stratum (Fig 2B and Fig 2C). In the stomach, the mucous neck glands and deeper gastric glands stained strongly, and moderate staining was observed in mucinous glandular epithelia (Fig 2D); ISH revealed strong staining in the deeper gastric glands (Fig 2E and Fig 2F). In the duodenum and small intestine, a gradient of matriptase immunoreactivity was observed along the cryptvillous axis (Fig 1E1G). Relatively moderate reactivity was seen in crypt cells, and progressively stronger reactivity appeared towards the villous tip. This observation is similar to that reported for the rat homologue of matriptase in the small intestine (
Genitourinary and Reproductive System.
Kidney tubule epithelial cells showed strong reactivity, whereas cells of the glomerular apparatus were negative (Fig 3A and Fig 3B). Transitional epithelium of the urinary bladder was positive (Fig 3C), as was the epithelium of the ureter (not shown). Strong reactivity was observed in seminal vesicle epithelium (Fig 4A) and was confirmed by ISH (Fig 4B and Fig 4C). All cell types in the testis were negative, including Sertoli and Leydig cells, fibromyocytes, and spermatogonia (Fig 3D). The epithelial lining of the epididymis, however, was positive (Fig 4D), and was confirmed as such by ISH (Fig 4E and Fig 4F). In the endometrium, a difference was observed in matriptase immunoreactivity in epithelium during the proliferative and secretory stages of the menstrual cycle (Fig 3E and Fig 4F). The endometrial lining showed moderate positivity during the proliferative phase, but weak staining during the secretory phase. The stromal elements showed no reactivity. Moderate reactivity was seen in the mucous-secreting glandular epithelium of the cervix (Fig 3G). As previously reported (
Endocrine System. The adrenal gland displayed a distinct pattern of matriptase staining (Fig 5A5C). All secretory epithelial cells of the zona fasciculata and zona reticularis of the adrenal cortex stained strongly. However, cells of the zona granulosa showed mostly weak or negatively stained cells, with moderate staining appearing in some cells. The adrenal medulla, a neural crest-derived tissue, lacked reactivity (Fig 5C). As previously described, cells of the islets of Langerhans of the endocrine pancreas were negative. Thyroid follicular epithelium, however, showed matriptase staining (not shown).
Skin and Subcutaneous Tissues.
Matriptase expression was detected in squamous epithelial cells in the epidermal layer of the skin (Fig 6A). No staining was seen in dermal fibroblasts. Matriptase immunostaining in the skin was confirmed by ISH (Fig 6B and Fig 6C). As previously reported, breast duct epithelia demonstrated matriptase immunostaining, in contrast to underlying tissue stromal fibroblasts and adipocytes (
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Discussion |
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Using a normal human tissue microarray and selected normal human tissues, we examined the expression of matriptase in a broad range of epithelium-containing tissues. IHC and ISH analysis indicated that matriptase is broadly expressed in the surface epithelial lining of almost all tissues examined and was absent from most mesenchymal components. Immunoreactivity was detected in epithelia of many types: simple columnar, pseudo-stratified columnar, cuboidal, and stratified squamous. Therefore, matriptase expression is not limited to epithelia of any particular histological type, indicating that matriptase plays a role common to the function of all epithelia. However, the intensity of matriptase immunoreactivity did show distinct spatial distributions in the epithelial components of several tissues. For example, the intensity in the esophagus was strongest in the intermediate layer and less intense in the surface layer. In the intestine, a gradient of intensity was observed in the cryptvillous axis, where matriptase immunoreactivity increased towards the villous tip, consistent with the pattern previously seen for rat matriptase in the rat small intestine (
The expression pattern of matriptase closely correlated with that previously described for the matriptase inhibitor HAI-1, which is broadly localized to surface lining epithelia (
To date, several in vitro substrates for matriptase have been identified and suggest a role for the protease in matrix remodeling and the regulation of cell growth and survival, cell motility, and cell morphogenesis. However, without proven knowledge of the true in vivo substrates of the enzyme, it is difficult to predict the physiological role of matriptase in normal human tissues. Therefore, an important future goal must be the demonstration of the in vivo cleavage and activation of putative matriptase substrates. In addition, it will be essential to characterize how matriptase protease activity is regulated physiologically. This information, together with the above description of the relevant cell types that express matriptase in vivo, will help pave the way for understanding the role of matriptase in the biology of normal epithelia, and how deregulation of its activity may promote pathological states in multiple organ systems.
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Acknowledgments |
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Supported by the Susan G. Komen Breast Cancer Foundation DRA99-003037 and BCTR0100345 and DOD grants DAMD 17-01-1-0252, DAMD 17-02-1-0391, and DAMD 17-00-1-0264.
We acknowledge the Histopathology and Tissue Shared Resource of the Lombardi Cancer Center at Georgetown University Medical Center for help in selecting tissue specimens. We would also like to acknowledge the assistance of the Tissue Culture Shared Resource and the Microscopy and Imaging Resource. These resources are supported by NIH grants P50-CA58185 (TCSR) and P30-CA51008 (TCSR and MIR).
Received for publication October 31, 2002; accepted February 19, 2003.
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