Regulation of pericellular proteolysis by hepatocyte growth factor activator inhibitor type 1 (HAI-1) in trophoblast cells

Hepatocyte growth factor activator inhibitor type 1/serine protease inhibitor Kunitz type 1 (HAI-1/SPINT1) is a membrane-bound Kunitz-type serine protease inhibitor that is abundantly expressed on the surface of cytotrophoblasts, and is critically required for the formation of the placenta labyrinth in mice. HAI-1/SPINT1 regulates several membrane-associated cell surface serine proteases, with matriptase being the most cognate target. Matriptase degrades extracellular matrix protein such as laminin and activates other cell surface proteases including prostasin. This study aimed to analyze the role of HAI-1/SPINT1 in pericellular proteolysis of trophoblasts. In HAI-1/SPINT1-deficient mouse placenta, laminin immunoreactivity around trophoblasts was irregular and occasionally showed an intense punctate pattern, which differed significantly from the linear distribution along the basement membrane observed in wild-type placenta. To explore the molecular mechanism underlying this observation, we analyzed the effect of HAI-1/SPINT1 knock down (KD) on pericellular proteolysis in the human trophoblast cell line, BeWo. HAI-1/SPINT1-KD BeWo cells had increased amounts of cellular laminin protein and decreased laminin degradation activity in the culture supernatant. Subsequent analysis indicated that cell-associated matriptase was significantly decreased in KD cells whereas its mRNA level was not altered, suggesting an enhanced release and/or dislocation of matriptase in the absence of HAI-1/SPINT1. Moreover, prostasin activation and pericellular total serine protease activities were significantly suppressed by HAI-1/SPINT1 KD. These observations suggest that HAI-1/SPINT1 is critically required for the cell surface localization of matriptase in trophoblasts, and, in the absence of HAI-1/SPINT1, physiological activation of prostasin and other protease(s) initiated by cell surface matriptase may be impaired.     

Tissue injury alters the site of expression of hepatocyte growth factor activator inhibitor type 1 in bronchial epithelial cells

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a Kunitz-type transmembrane serine proteinase inhibitor that inhibits trypsin-like serine proteinases, such as hepatocyte growth factor activator, matriptase, hepsin and prostasin. HAI-1 is expressed in polarized epithelial cells, in which HAI-1 is mainly located on the basolateral membrane. In the present study, we analyzed the expression and distribution of HAI-1 in respiratory epithelium. We found that HAI-1 is expressed by the bronchial respiratory epithelium with basal or basolateral localization and also by the alveolar epithelium. Bronchial expression of HAI-1 was also confirmed using cultured human bronchial epithelial cells. The epithelial expression of HAI-1 was augmented in response to tissue injury such as cancer invasion and inflammation. Surprisingly, in the injured pulmonary tissue, HAI-1 showed distinct apical translocation in ciliated epithelial cells of the bronchiole. We suggest that, in addition to its basolateral surface localization, HAI-1 can transiently localize to the apical surface of respiratory ciliated epithelial cells under conditions of severe inflammation, possibly interacting with a specific cellular proteinase on the apical surface.     

Immunohistochemical localization of histamine N-methyltransferase in guinea pig tissues.

Histamine plays important roles in gastric acid secretion, inflammation, and allergic response. Histamine N-methyltransferase (HMT; EC 2.1.1.8) is crucial to the inactivation of histamine in tissues. In this study we investigated the immunohistochemical localization of this enzyme in guinea pig tissues using a rabbit polyclonal antibody against bovine HMT. The specificity of the antibody for guinea pig HMT was confirmed by Western blotting and the lack of any staining using antiserum preabsorbed with purified HMT. There was strong HMT-like immunoreactivity (HMT-LI) in the epithelial cells in the gastrointestinal tract, especially in the gastric body, duodenum, and jejunum. The columnar epithelium in the gallbladder was also strongly positive. Almost all the myenteric plexus from the stomach to the colon was stained whereas the submucous plexus was not. Other strongly immunoreactive cells included the ciliated cells in the trachea and the transitional epithelium of the bladder. Intermediately immunoreactive cells included islets of Langerhans, epidermal cells of the skin, alveolar cells in the lung, urinary tubules in the kidney, and epithelium of semiferous tubules. HMT-LI was present in specific structures in the guinea pig tissues. The widespread distribution of HMT-LI suggests that histamine has several roles in different tissues.     

Localization of hepatocyte growth factor activator inhibitor type 1 in Langhans' cells of human placenta

Activation of hepatocyte growth factor (HGF) is a crucial limiting step in HGF-induced signaling pathway. The HGF activator inhibitor type 1 (HAI-1) was identified as a potent inhibitor of HGF activator (HGFA), a serine proteinase that is responsible for the activation of HGF in vivo. HAI-1 is an integral membrane Kunitz-type serine proteinase inhibitor, and its mRNA has been reported to be most abundant in the placenta. In this report, specific antibody to HAI-1 was used in an immunohistochemical procedure to determine the localization of HAI-I in human placenta. HAI-1 was expressed in cytotrophoblasts (Langhans' cells) of the double-layered trophoblastic epithelium of chorionic villi tissue, and syncytiotrophoblasts were almost negative. On the other hand, extravillous trophoblasts of cytotrophoblastic columns showed markedly decreased immunoreactivity, and those infiltrating into the superficial decidua membrane of early placenta were hardly stainable. The amnionic epithelial cells were also immunostained intensely. The presence of HAI-1 mRNA was also confirmed in a cultured human cytotrophoblastic cell line. In addition to HAI-1, low but distinct expression of HGFA mRNA was observed in the placenta tissue and cultured cytotrophoblasts by using a sensitive RT-PCR method. Since HGF plays an essential role in the placenta development, expression of HAI-1 and HGFA may have an important regulatory role in the placenta. The localization of HAI-I in the proliferating trophoblastic stem cells (Langhans' cells), but not in syncytiotrophoblasts and extravillous trophoblasts, suggest a possible role of HAI-1 in the proliferation of trophoblasts.     

Characterization of matriptase expression in normal human tissues.

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.     

HAI-1 regulates activation and expression of matriptase, a membrane-bound serine protease

Hepatocyte growth factor activator inhibitor-1 (HAI-1) was initially identified as cognate inhibitor of matriptase, a membrane-bound serine protease. Paradoxically, HAI-1 is also required for matriptase activation, a process that requires sphingosine 1-phosphate (S1P)-mediated translocation of the protease to cell-cell junctions in human mammary epithelial cells. In the present study, we further explored how HAI-1 regulates this protease. First, we observed that after S1P treatment HAI-1 was cotranslocated with matriptase to cell-cell junctions and that the cellular ratio of HAI-1 to matriptase was maintained during this process. However, when this ratio was changed by cell treatment with HAI-1 small interfering RNA or anti-HAI-1 MAb M19, spontaneous activation of matriptase occurred in the absence of S1P-induced translocation; S1P-induced matriptase activation was also enhanced. These results support a role for HAI-1 in protection of cell from uncontrolled matriptase activation. We next expressed matriptase, either alone or with HAI-1 in breast cancer cells that do not endogenously express either protein. A defect in matriptase trafficking to the cell surface occurred if wild-type matriptase was expressed in the absence of HAI-1; this defect appeared to result from matriptase toxicity to cells. Coexpression with matriptase of wild-type HAI-1, but not HAI-1 mutants altered in its Kunitz domain 1, corrected the trafficking defect. In contrast, catalytically defective matriptase mutants were normal in their trafficking in the absence of HAI-1. These results are also consistent with a role for HAI-1 to prevent inappropriate matriptase proteolytic activity during its protein synthesis and trafficking. Taken together, these results support multiple roles for HAI-1 to regulate matriptase, including its proper expression, intracellular trafficking, activation, and inhibition. protease-activated receptor-2; hepatocyte growth factor; urokinase; sphingosine 1-phosphate; Kunitz domain     

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is required for branching morphogenesis in the chorioallantoic placenta

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-associated Kunitz-type serine proteinase inhibitor that was initially identified as a potent inhibitor of hepatocyte growth factor activator. HAI-1 is also a cognate inhibitor of matriptase, a membrane-associated serine proteinase. HAI-1 is expressed predominantly in epithelial cells in the human body. Its mRNA is also abundant in human placenta, with HAI-1 specifically expressed by villous cytotrophoblasts. In order to address the precise roles of HAI-1 in vivo, we generated HAI-1 mutant mice by homozygous recombination. Heterozygous HAI-1+/- mice underwent normal organ development. However, homozygous HAI-1-/- mice experienced embryonic lethality which became evident at embryonic day 10.5 postcoitum (E10.5). As early as E9.5, HAI-1-/- embryos showed growth retardation that did not reflect impaired cell proliferation but resulted instead from failed placental development and function. Histological analysis revealed severely impaired formation of the labyrinth layer, in contrast all other placental layers, such as the spongiotrophoblast layer and giant cell layer, which were formed. Our results indicate that mouse HAI-1 is essential for branching morphogenesis in the chorioallantoic placenta and lack of HAI-1 function may result in placental failure.     

Immunohistochemical analysis of ZnT1, 4, 5, 6, and 7 in the mouse gastrointestinal tract.

Expression of five zinc transporters (ZnT1, 4, 5, 6, and 7) of the Slc30 family in the mouse gastrointestinal tract was studied by immunohistochemical analysis. Results demonstrated unique expression patterns, levels, and cellular localization among ZnT proteins in the mouse gastrointestinal tract with some overlapping. ZnT1 was abundantly expressed in the epithelium of the esophagus, duodenum of the small intestine, and cecum of the large intestine. ZnT4 was predominantly detected in the large intestine. ZnT5 was mainly expressed in the parietal cell of the stomach and in the absorptive epithelium of the duodenum and jejunum. ZnT6 was predominantly detected in the chief cell of the stomach, columnar epithelial cells of the jejunum, cecum, colon, and rectum. Lastly, ZnT7 was observed in all epithelia of the mouse gastrointestinal tract with the highest expression in the small intestine. Expression of ZnT proteins in the absorptive epithelial cell of the gastrointestinal tract suggests that ZnT proteins may play important roles in zinc absorption and endogenous zinc secretion.     

Down-regulation of HAI-1 is associated with poor-differentiation status of colorectal cancer

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type serine protease inhibitor which is widely expressed in epithelial cells. The purpose of this study is to investigate the relationship between HAI-1 expression and differentiation status of colorectal epithelia. The expression of HAI-1 in clinical samples of both cancerous and normal colorectal tissues was evaluated by immunohistochemical staining. An in vitro epithelial differentiation model of Caco-2 cell was established, and the characteristics of differentiation of Caco-2 cells were observed by transmission electron microscopy. The expression of HAI-1 in Caco-2 cells at different differentiation stages was examined by western blot. Immunohistochemical staining of 52 human colorectal cancer tissues showed a definite correlation between HAI-1 expression and differentiation status: IHC score (mean ± SE) of HAI-1 was higher for well- or moderately-differentiated colorectal cancer tissues than for poorly-differentiated colorectal cancer tissues, with significant differences in HAI-1 positive rate (P < 0.01 and P < 0.05 for well-differentiated vs. poorly-differentiated and moderately-differentiated vs. poorly-differentiated, respectively). Immunohistochemical staining of normal colorectal tissues showed positive HAI-1 expression in well-differentiated epithelial cells whereas the under-differentiated crypt cells showed very weak HAI-1 staining signals. The result of western blot also showed the gradual increasing of HAI-1 expression during the process of Caco-2 differentiation in vitro. HAI-1 expression correlates with the differentiation status of colorectal epithelia and could serve as a differentiation marker.     

The Protease Inhibitor HAI-2, but Not HAI-1, Regulates Matriptase Activation and Shedding through Prostasin

The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating the intracellular trafficking and activation of matriptase. In this study, we, therefore, used genetically engineered mice to determine the effect of ablation of endogenous HAI-1 and endogenous HAI-2 on endogenous matriptase expression, subcellular localization, and activation in polarized intestinal epithelial cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase was mechanistically linked to accelerated activation and shedding of the protease caused by loss of prostasin regulation by HAI-2. Taken together, these data indicate that HAI-1 regulates the activity of activated matriptase, whereas HAI-2 has an essential role in regulating prostasin-dependent matriptase zymogen activation.     

Localization of type 7 17beta-hydroxysteroid dehydrogenase in mouse tissues. In situ hybridization studies

The enzyme type 7 17beta-hydroxysteroid dehydrogenase (17beta-HSD) selectively catalyzes the conversion of estrone (E1) into estradiol (E2). In order to obtain detailed information about the exact sites of action of type 7 17beta-HSD, we have studied the cellular localization of type 7 17beta-HSD mRNA in mouse tissues using in situ hybridization (ISH). In parallel studies, we also measured the enzyme mRNA levels by quantitative real time (RT)-PCR. In the ovary, strong hybridization signal was restricted to corpus luteum cells. In the female mammary gland, type 7 17beta-HSD mRNA was found to be expressed in stromal cells surrounding the ducts. In the clitoral and preputial glands, specific labeling was observed in the epithelial cells of both acini and small ducts. In the adrenal gland, hybridization signal was observed in the zona fasciculata and reticularis in the cortex. In the liver, hybridization signal was found in all the hepatocytes. In the colon, type 7 17beta-HSD mRNA expression was restricted to epithelial cells of the mucosa. From the results obtained with quantitative real time RT-PCR, it appears, with a very few exceptions, that in tissues exhibiting low mRNA expression no ISH signal could be detected. The present data suggest that E2 can be formed through the action of type 7 17beta-HSD in specific cell types in the ovary and peripheral tissues, in addition to type 1 17beta-HSD, thus providing tissues with an alternative route of formation of E2.     

Human kallikrein 10 expression in normal tissues by immunohistochemistry.

The normal epithelial cell-specific 1 (NES1) gene (official name kallikrein gene 10, KLK10) was recently cloned and encodes for a putative secreted serine protease (human kallikrein 10, hK10). Several studies have confirmed that hK10 shares many similarities with the other kallikrein members at the DNA, mRNA, and protein levels. The enzyme was found in biological fluids, tissue extracts, and serum. Here we report the first detailed immunohistochemical (IHC) localization of hK10 in normal human tissues. We used the streptavidin-biotin method with two hK10-specific antibodies, a polyclonal rabbit and a monoclonal mouse antibody, developed in house. We analyzed 184 paraffin blocks from archival, current, and autopsy material, prepared from almost every normal human tissue. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. Previously, we reported the expression of another novel human kallikrein, hK6, by using similar techniques. The IHC expression of hK10 was generally cytoplasmic and not organ-specific. A variety of normal human tissues expressed the protein. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, epididymis, endometrium, fallopian tubes, gastrointestinal tract, bronchus, salivary glands, bile ducts, and gallbladder. The choroid plexus epithelium, the peripheral nerves, and some neuroendocrine organs (including the islets of Langerhans, cells of the adenohypophysis, the adrenal medulla, and Leydig cells) expressed the protein strongly and diffusely. The spermatic epithelium of the testis expressed the protein moderately. A characteristic immunostaining was observed in Hassall's corpuscles of the thymus, oxyphilic cells of the thyroid and parathyroid glands, and chondrocytes. Comparing these results with those of hK6, we observed that both kallikreins had a similar IHC expression pattern.     

Immunolocalization of a novel collectin CL-K1 in murine tissues.

We have recently identified a novel collectin, CL-K1, that may play a role in innate immunity as a member of the collectin family. In this study using mice, we investigated the tissue distribution of CL-K1 for better understanding of its pathophysiological relevance. Real-time PCR analyses demonstrated that CL-K1 mRNA was expressed in all tissues tested. Immunohistochemical analyses demonstrated that CL-K1 was expressed in proximal tubules of kidney, in mucosa of the gastrointestinal tract, and in bronchial glands of bronchioles similar to the localization of SP-A and SP-D in these pulmonary structures. Immunohistochemistry also showed that CL-K1 was highly expressed in hepatocytes around the central veins in liver, which suggests that murine CL-K1 may be mainly produced in the liver and secreted into the blood stream as is human CL-K1. CL-K1 was especially detected in vascular smooth muscle in several types of tissues. In addition, it was also expressed in intestinal Paneth cells, in mesangial cells of kidney, in pancreatic islet D cells, and in neurons of the brain. It is of interest that this profile of CL-K1 expression is unique among the collectins. Together these histological findings may be useful for understanding the biological function of this novel collectin.     

Androgen receptor expression in human tissues: an immunohistochemical study.

The cellular localization of the human androgen receptor was visualized immunohistochemically using a mouse monoclonal antibody (MAb) F39.4, directed against a fragment of the N-terminal domain of the androgen receptor. The nuclear immunoreactivity of various human tissues with F39.4 was generally consistent with earlier biochemical and autoradiographic data. However, previously suggested androgen receptor expression in thyroid, pancreatic, gastrointestinal, and bladder tissues was not confirmed immunohistochemically. Stratified squamous epithelia of vagina and cervix showed selective immunostaining of the basal cell layer, whereas in the preputial epithelium the intensity of immunoreactivity decreased gradually with maturation. In contrast, glandular epithelia of the sweat glands, male accessory sex organs, and female breast showed nearly exclusive F39.4 staining of the inner cylindric layer. In the testis, Sertoli cells, peritubular myoid cells, and interstitial cells were immunoreactive with MAb F39.4. Expression of the androgen receptor by smooth muscle tissue was largely confined to the male reproductive organs. The specificity and sensitivity of this simple and rapidly performed immunohistochemical technique in the detection of the human androgen receptor at the cellular and subcellular level makes it worthwhile to study tissue androgen receptor expression by immunohistochemistry in physiological and pathological states.     

In situ hybridization and immunohistochemical analysis of cytochrome P450 1B1 expression in human normal tissues.

Cytochrome P450 1B1 (CYP1B1) is a recently cloned dioxin-inducible form of the cytochrome P450 supergene family of xenobiotic-metabolizing enzymes. CYP1B1 is constitutively expressed mainly in extrahepatic tissues and is inducible by aryl hydrocarbon receptor ligands. Human CYP1B1 is involved in activation of chemically diverse human procarcinogens, including polycyclic aromatic hydrocarbons and some aromatic amines, as well as the endogenous hormone 17 beta-estradiol. The metabolism of 17 beta-estradiol by CYP1B1 forms 4-hydroxyestradiol, a product believed to be important in estrogen-induced carcinogenesis. Although the distribution of CYP1B1 mRNA and protein in a number of human normal tissues has been well documented, neither the cells expressing CYP1B1 in individual tissue nor the intracellular localization of the enzyme has been thoroughly characterized. In this study, using nonradioactive in situ hybridization and immunohistochemistry, we examined the cellular localization of CYP1B1 mRNA and protein in a range of human normal tissues. CYP1B1 mRNA and protein were expressed in most samples of parenchymal and stromal tissue from brain, kidney, prostate, breast, cervix, uterus, ovary, and lymph nodes. In most tissues, CYP1B1 immunostaining was nuclear. However, in tubule cells of kidney and secretory cells of mammary gland, immunoreactivity for CYP1B1 protein was found in both nucleus and cytoplasm. This study demonstrates for the first time the nuclear localization of CYP1B1 protein. Moreover, the constitutive expression and wide distribution of CYP1B1 mRNA and protein in many human normal tissues suggest functional roles for CYP1B1 in the bioactivation of xenobiotic procarcinogens and endogenous substrates such as estrogens. (J Histochem Cytochem 49:229-236, 2001)     

Cytochrome P-450 localization in normal human adult and foetal liver by immunocytochemistry using a monoclonal antibody against human cytochrome P-450

Immunocytochemical studies with a monoclonal antibody (MAb-HL3), which recognises a major isozyme of human hepatic cytochrome P-450, have demonstrated this cytochrome in both cryostat and formalin-fixed paraffin-embedded sections of normal human adult liver. Prior trypsin digestion of the formalin-fixed sections prevented staining. There was a zonal distribution of immunoreactive cytochrome P-450, with localization predominantly in the hepatocytes of zone 3 of the hepatic acinus (the centrilobular region). Cytochrome P-450 was also demonstrated in foetal liver, but all foetal hepatocytes contained immunoreactive cytochrome P-450 and there was no zonal distribution of the protein. The biliary epithelium of adult liver contained a small amount of immunoreactive cytochrome P-450 whereas there was no immunoreactivity in the epithelium of foetal bile ducts.