Targeted deletion of HAI-1 increases prostasin proteolysis but decreases matriptase proteolysis in human keratinocytes.

Epidermal differentiation and barrier function require well-controlled matriptase and prostasin proteolysis, in which the Kunitz-type serine protease inhibitor HAI-1 represents the primary enzymatic inhibitor for both proteases. HAI-1, however, also functions as a chaperone-like protein necessary for normal matriptase synthesis and intracellular trafficking. Furthermore, other protease inhibitors, such as antithrombin and HAI-2, can also inhibit matriptase and prostasin in solution or in keratinocytes. It remains unclear, therefore, whether aberrant increases in matriptase and prostasin enzymatic activity would be the consequence of targeted deletion of HAI-1 and so subsequently contribute to the epidermal defects observed in HAI-1 knockout mice. The impact of HAI-1 deficiency on matriptase and prostasin proteolysis was, here, investigated in HaCaT human keratinocytes. Our results show that HAI-1 deficiency causes an increase in prostasin proteolysis via increased protein expression and zymogen activation. It remains unclear, however, whether HAI-1 deficiency increases "net" prostasin enzymatic activity because all of the activated prostasin was detected in complexes with HAI-2, suggesting that prostasin enzymatic activity is still under tight control in HAI-1-deficient keratinocytes. Matriptase proteolysis is, however, unexpectedly suppressed by HAI-1 deficiency, as manifested by decreases in zymogen activation, shedding of active matriptase, and matriptase-dependent prostasin zymogen activation. This suppressed proteolysis results mainly from the reduced ability of HAI-1-deficient HaCaT cells to activate matriptase and the rapid inhibition of nascent active matriptase by HAI-2 and other yet-to-be-identified protease inhibitors. Our study provides novel insights with opposite impacts by HAI-1 deficiency on matriptase versus prostasin proteolysis in keratinocytes.

HAI-1 regulates placental folds development by influencing trophoblast cell proliferation and invasion in pigs.

Fetal development is critically dependent on the efficiency of the placenta. Porcine trophoblast cell proliferation and invasion have crucial roles in placental fold development, which is one of the essential events determining placental efficiency. The membrane serine proteinase inhibitor hepatocyte growth factor activator inhibitor-1 (HAI-1) can regulate cellular invasion and motility in different types of epithelial cells, including trophoblast cells in mice. This work used quantitative polymerase chain reaction (qPCR) and immunohistochemistry to compare the expression level and location of HAI-1 in the placenta on gestational days 26, 50, and 95 in Yorkshire and Meishan pigs. The role of HAI-1 in porcine trophoblast cell (PTr2) proliferation, invasion, and migration in vitro was investigated by analyzing the effects of HAI-1 gene silencing or overexpression. Polymorphism in the HAI-1gene was detected to determine associations between the genotype and piglet birth weight in 400 healthy pure-bred Yorkshire piglets. qPCR results showed that HAI-1 mRNA levels significantly increased (P < 0.01) between gestational days 26 and 50 and then decreased (P < 0.01) between days 50 and 95 in both Meishan and Yorkshire pigs. Immunohistochemical analysis showed that HAI-1 protein was strongly expressed by the high columnar trophoblast cells located at the top of the placental folds with low proliferative and invasion capacities. However, it was expressed at very low levels in cuboidal trophoblast cells located at the side and base of the placental folds with high proliferative and invasion capacities. In vitro experiments indicated that HAI-1 had the ability to reduce the proliferation, invasion and migration of trophoblast cells. In addition, one single-nucleotide polymorphism (SNP) of HAI-1 showed a significant association (P < 0.05) with piglet birth weight. These results revealed that HAI-1 could be a vital molecule in placental folds development by regulating trophoblast proliferation and invasion in pigs.

Epididymal protease inhibitor (EPPIN) is a protein hub for seminal vesicle-secreted protein SVS2 binding in mouse spermatozoa.

EPPIN is a sperm-surface drug target for male contraception. Here we investigated EPPIN-interacting proteins in mouse spermatozoa. We showed that EPPIN is an androgen-dependent gene, expressed in the testis and epididymis, but also present in the vas deferens, seminal vesicle and adrenal gland. Mature spermatozoa presented EPPIN staining on the head and flagellum. Immunoprecipitation of EPPIN from spermatozoa pre-incubated with seminal vesicle fluid (SVF) followed by LC-MS/MS or Western blot revealed the co-immunoprecipitation of SVS2, SVS3A, SVS5 and SVS6. In silico and Far-Western blot approaches demonstrated that EPPIN binds SVS2 in a protein network with other SVS proteins. Immunofluorescence using spermatozoa pre-incubated with SVF or recombinant SVS2 demonstrated the co-localization of EPPIN and SVS2 both on sperm head and flagellum. Our data show that EPPIN's roles in sperm function are conserved between mouse and human, demonstrating that the mouse is a suitable experimental model for translational studies on EPPIN.

ITIH3 and ITIH4 polymorphisms and depressive symptoms during pregnancy in Japan: the Kyushu Okinawa Maternal and Child Health Study.

ITIH3 and ITIH4 are involved in the stabilization of the extracellular matrix. Several genome-wide association studies and case-control studies regarding psychiatric disorders have identified ITIH3 and ITIH4 single nucleotide polymorphisms (SNPs). The present case-control study examined the relationship between ITIH3 SNPs rs2535629 and rs736408 and ITIH4 SNPs rs3821831 and rs2239547 and depressive symptoms during pregnancy in Japan. Cases comprised 273 women with depressive symptoms during pregnancy defined as a Center for Epidemiological Studies Depression Scale (CES-D) score ≥ 16. Control subjects comprised 1176 women without depressive symptoms during pregnancy, according to the CES-D criteria, who had not been diagnosed with depression by a doctor. Adjustment was made for age, gestation at baseline, region of residence, the presence of children, family structure, smoking, employment, and education. Compared with the TT genotype of ITIH4 SNP rs2239547, the CC genotype was significantly related to a reduced risk of depressive symptoms during pregnancy: the adjusted odds ratio (95% CI) was 0.84 (0.63-1.11) for the TC genotype and 0.57 (0.36-0.91) for the CC genotype. ITIH3 SNPs rs2535629 and rs736408 and ITIH4 SNP rs3821831 were not related to depressive symptoms during pregnancy. The GCCT haplotype of rs2535629, rs736408, rs3821831, and rs2239547 was significantly positively associated with depressive symptoms during pregnancy. A significant interaction was found between rs2239547 and the presence of children. This is the first study to show significant associations of ITIH4 SNP rs2239547 and the GCCT haplotype with depressive symptoms during pregnancy. The effect of the presence of children might depend on rs2239547.

The Role of Neutrophil Elastase Inhibitors in Lung Diseases.

In many respiratory diseases characterized by an intense inflammatory response, the balance between proteolytic enzymes (proteases, including elastases) and their inhibitors (proteinases inhibitors) is not neutral. Excess activity of neutrophil elastase (NE) and similar proteases has been reported to cause tissue damage and to alter the remodeling process in many clinical conditions such as pneumonia, respiratory distress, and acute lung injury (ALI). Several experimental NE inhibitors have been tested in preclinical and clinical studies of different conditions of inflammatory lung injury such as ALI and pneumonia, with contrasting results. This study reviews the literature regarding NE inhibitors in the field of respiratory diseases and reflects on possible future developments. In particular, we highlight potential gaps in the scientific evidence and discuss potential strategies for focusing investigation on antielastases in clinical practice through the selection of targeted populations and proper outcomes.

Incomplete KLK7 Secretion and Upregulated LEKTI Expression Underlie Hyperkeratotic Stratum Corneum in Atopic Dermatitis.

Atopic dermatitis (AD) is a common inflammatory skin disorder. Chronic AD lesions present hyperkeratosis, indicating a disturbed desquamation process. KLK7 is a serine protease involved in the proteolysis of extracellular corneodesmosome components, including desmocollin 1 and corneodesmosin, which leads to desquamation. KLK7 is secreted by lamellar granules and upregulated in AD lesional skin. However, despite increased KLK7 protein levels, immunostaining and electron microscopy indicated numerous corneodesmosomes remaining in the uppermost layer of the stratum corneum from AD lesions. We aimed to clarify the discrepancy between KLK7 overexpression and retention of corneodesmosomes on AD corneocytes. Western blot analysis indicated abnormal corneodesmosin degradation patterns in stratum corneum from AD lesions. The KLK activity of tape-stripped corneocytes from AD lesions was not significantly elevated in in situ zymography, which was our new attempt to detect the protease activity more precisely than conventional assays. This ineffective KLK activation was associated with impaired KLK7 secretion from lamellar granules and increased expression of LEKTI in AD. Such imbalances in protease-protease inhibitor interactions could lead to abnormal proteolysis of corneodesmosomes and compact hyperkeratosis. Upregulated expression of LEKTI might be a compensatory mechanism to prevent further barrier dysfunction in AD.

Tissue factor pathway inhibitor-2 is critical in zebrafish cardiogenesis.

Human tissue factor pathway inhibitor-2 (Tfpi-2) is an extracellular matrix-associated Kunitz-type serine proteinase inhibitor and plays an important role in various cellular processes. We have previously shown that zebrafish Tfpi-2 (zTfpi-2) mainly expressed in the brain and heart of zebrafish, and it is involved in the development of central nervous system. Here, we identified zTfpi-2 as an evolutionarily conserved protein essential for zebrafish heart development, as embryos depleted of zTfpi-2 failed to undergo cardiogenesis. Changes of cardiogenic markers, vmhc, amhc and bmp4, confirmed zTfpi-2 knockdown caused cardiac defects, including retrenched ventricle, enlarged atrium and malformation of atrioventricular boundary. The sarcomeric organization was also disrupted by embryonic depletion of zTfpi-2, thus establishing the functional role of zTfpi-2 in cardiac contractility. In addition, hematopoietic defects were detected in the zTfpi-2-deficiency embryos. Importantly, injection of ztfpi-2 mRNA attenuated those cardiac and hematopoietic defects. Taken together, this study demonstrated a critical role of zTfpi-2 during embryonic cardiac development, as well as an important regulator of hematopoiesis.

Tissue factor pathway inhibitor-2: a novel gene involved in zebrafish central nervous system development.

Tissue factor pathway inhibitor-2 (Tfpi-2) is an important serine protease inhibitor in the extracellular matrix (ECM), but its precise physiological significance remains unknown. This work is part of a series of studies intended to investigate functional roles of Tfpi-2 and explore the underlying molecular mechanisms. First, we cloned and identified zebrafish Tfpi-2 (zTfpi-2) as an evolutionarily conserved protein essential for zebrafish development. We also demonstrated that ztfpi-2 is mainly expressed in the central nervous system (CNS) of zebrafish, and embryonic depletion of ztfpi-2 caused severe CNS defects. In addition, changes of neural markers, including pax2a, egr2b, huC, ngn1, gfap and olig2, confirmed the presence of developmental abnormalities in the relevant regions of ztfpi-2 morphants. Using microarray analysis, we found that members of the Notch pathway, especially her4 and mib, which mediate lateral inhibition in CNS development, were also downregulated. Intriguingly, both her4 and mib were able to partially rescue the ztfpi-2 morphant phenotype. Furthermore, Morpholino knockdown of ztfpi-2 resulted in upregulation of neuronal markers while downregulation of glial markers, providing evidence that the Notch pathway is probably involved in ztfpi-2-mediated CNS development.

Spink13, an epididymis-specific gene of the Kazal-type serine protease inhibitor (SPINK) family, is essential for the acrosomal integrity and male fertility.

Sperm maturation involves numerous surface modifications by a variety of secreted proteins from epididymal epithelia. The sperm surface architecture depends on correct localization of its components and highlights the importance of the sequence of the proteolytic processing of the sperm surface in the epididymal duct. The presence of several protease inhibitors from different families is consistent with the hypothesis that correctly timed epididymal protein processing is essential for proper sperm maturation. Here we show that the rat (Rattus norvegicus) epididymis-specific gene Spink13, an androgen-responsive serine protease inhibitor, could bind to the sperm acrosome region. Furthermore, knockdown of Spink13 in vivo dramatically enhanced the acrosomal exocytosis during the process of capacitation and thus led to a significant reduction in male fertility, indicating that Spink13 was essential for sperm maturation. We conclude that blockade of SPINK13 may provide a new putative target for post-testicular male contraceptives.

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.

The identification of a new role for LEKTI in the skin: The use of protein 'bait' arrays to detect defective trafficking of dermcidin in the skin of patients with Netherton syndrome.

Lympho-Epithelial Kazal-Type-related Inhibitor (LEKTI) has been demonstrated to be an inhibitor of various kallikreins and is thought to play a role in the regulation of skin desquamation. In order to identify and investigate the potential of LEKTI to interact with other proteins, a method was developed using immobilised proteins onto arrays and nanoUPLC/MALDI-TOF MS. Using various domains of LEKTI, we demonstrated that these domains bound a number of kallikreins (5, 13 and 14) to varied extents on the array surface. Inhibitory assays confirmed that binding on the protein array surface corresponded directly to levels of inhibition. The method was then tested using skin epidermal extracts. All forms of rLEKTI with the exception of rLEKTI 12-15, demonstrated the binding of several potential candidate proteins. Surprisingly, the major binding partners of LEKTI were found to be the antimicrobial peptide dermcidin and the serine protease cathepsin G and no kallikreins. Using confocal microscopy and Netherton syndrome skin sections, we confirmed the co-localisation of LEKTI with dermcidin and demonstrated altered trafficking of dermcidin in these patients. This potential new role for LEKTI as a multifunctional protein in the protection and transport of proteins in the epidermis and its role in disease are discussed.

The role of SPINK5 in asthma related physiological events in the airway epithelium.

BACKGROUND: Genetic studies have shown that variants in SPINK5 may be associated with atopic diseases and asthma. However, the functional role of SPINK5 protein in asthma has not been elucidated. OBJECTIVES: To determine the effects of SPINK5 on asthma related physiological events such as apoptosis, mucus and cytokine production by epithelial cells. METHODS: A549 cells were transfected with SPINK5 expression vector and stimulated with increasing doses of hydrogen peroxide and neutrophil elastase (NE) for measurement of cell viability or apoptosis and analysis of mucus production. Cell viability was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5- diphenyl-tetrazolium bromide) assay and apoptosis by Annexin V/PI staining. Levels of IL-4, IL-6, IL-8, IL-12, IL-13, IFNγ, IL-1ß and RANTES were determined by ELISA in cell culture supernatants. Mucus production was determined by RT-PCR of the MUC5AC gene and PAS staining in NE treated cells. RESULTS: Epithelial cells transfected with SPINK5 expression vector produced more IL-6, IL-8 and RANTES compared to non-transfected cells (p < 0.001, p = 0.003, p < 0.001, respectively). Even though cells transfected with SPINK5 vector displayed significantly higher cell death, we have not observed any clear effect of SPINK5 on apoptosis. PAS staining showed that SPINK5 slightly decreased the mucin production induced by neutrophil elastase in A549 cells. However, SPINK5 had no effect on MUC5AC transcription. CONCLUSION: SPINK5 is an important molecule in asthma. Its role extends beyond its well known protease inhibitor properties.

Antimicrobial peptides in periodontal innate defense.

The development of oral biofilms and the host response to biofilm bacteria and their toxins are important factors in the development of periodontal disease. An early component of the host response is the secretion of antimicrobial proteins and peptides (AMPs) by salivary glands, oral epithelial cells and neutrophils. Over 45 AMPs have been identified in the oral cavity. All are found in saliva and several are also present in gingival crevicular fluid. Of these, 13 are up regulated in periodontal disease while 11 are downregulated. However, the concentrations of most AMPs found in oral fluids are below the effective in vitro concentrations, suggesting that local concentrations must be higher for effect or that additional biological functions are important in the oral cavity. Thus, in addition to direct antibacterial activity (e.g. bactericidal activity, bacterial agglutination), AMPs may affect the course of periodontal disease by inactivating bacterial or host proteases (e.g. secretory leukoprotease inhibitor) or bind bacterial toxins, including lipopolysaccharides (e.g. LL-37). Several AMPs (e.g. defensins) also act as immune system alarmins, i.e. endogenous mediators that recruit and activate antigen-presenting cells to enhance innate and adaptive immune responses. The differential regulation of AMP expression in periodontal disease suggests that AMP panels, including up- and downregulated proteins, can be used in oral fluid diagnosis of periodontal disease and to monitor treatment outcome.

The role of hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2 in endometrial cancer.

Hepatocyte growth factor activator inhibitors (HAI-1 and HAI-2) are Kunitz-type serine protease inhibitors that have a broad inhibitory spectrum against serine proteases. This is the first study to investigate the role of HAI-1 and HAI-2 in endometrial cancer. We investigated the biological functions of HAI-1 and HAI-2 using KLE and HEC-251 endometrial cancer cell lines, thus HAI-1 and HAI-2 were examined in uterine normal endometrium, endometrial hyperplasia and cancer specimens by immunohistochemistry. HAI-1 and HAI-2 showed potential inhibitory effects on cell proliferation, migration and cellular invasion by reduction of matriptase and hepsin expression. This in turn led to an increase in the levels of E-cadherin and Slug, and a reduction in the levels of Vimentin, SIP1, Snail and Twist, and hence ER and PR signal transduction in endometrial cancer cells. The levels of HAI-1 and HAI-2 expression were significantly decreased in endometrial cancer specimens relative to the corresponding normal endometrium specimens. Low HAI-1 and HAI-2 expression was a significant predictor for a poor prognosis compared with high HAI-1 and HAI-2 expression. These findings indicate that HAI-1 and HAI-2 could be considered as therapeutic targets and used as favorable prognosis markers for endometrial cancer.

Increased matriptase zymogen activation in inflammatory skin disorders.

Matriptase, a type 2 transmembrane serine protease, and its inhibitor hepatocyte growth factor activator inhibitor (HAI)-1 are required for normal epidermal barrier function, and matriptase activity is tightly regulated during this process. We therefore hypothesized that this protease system might be deregulated in skin disease. To test this, we examined the level and activation state of matriptase in examples of 23 human skin disorders. We first examined matriptase and HAI-1 protein distribution in normal epidermis. Matriptase was detected at high levels at cell-cell junctions in the basal layer and spinous layers but was present at minimal levels in the granular layer. HAI-1 was distributed in a similar pattern, except that high-level expression was retained in the granular layer. This pattern of expression was retained in most skin disorders. We next examined the distribution of activated matriptase. Although activated matriptase is not detected in normal epidermis, a dramatic increase is seen in keratinocytes at the site of inflammation in 16 different skin diseases. To gain further evidence that activation is associated with inflammatory stimuli, we challenged HaCaT cells with acidic pH or H(2)O(2) and observed matriptase activation. These findings suggest that inflammation-associated reactive oxygen species and tissue acidity may enhance matriptase activation in some skin diseases.

The effect of anti-eppin antibodies on ionophore A23187-induced calcium influx and acrosome reaction of human spermatozoa.

BACKGROUND: Before a spermatozoon can fertilize an oocyte it must undergo a cascade of biochemical and physiological changes that facilitate its binding and penetration into the oocyte. Epididymal protease inhibitor (eppin) has been found to play a critical role in male fertility through an immunological approach. METHODS: In this study, we used an anti-eppin antibody to clarify the effect of eppin on human sperm functions during fertilization. Immunofluorescence studies were performed on ejaculated human spermatozoa in uncapacitated, capacitated and ionophore-treated states. Human spermatozoa were incubated in the presence or absence of anti-eppin antibody under capacitating conditions and with A23187. The effects of the antibody were evaluated on sperm motility, protein phosphotyrosine content and free intracellular calcium. RESULTS: Immunofluorescence results demonstrated that eppin is located on the acrosome and tail. After the acrosome reaction eppin is found on the equatorial segment and tail. We found that blocking eppin with antibodies significantly inhibited the human sperm acrosome reaction induced by A23187 in a dose-dependent manner. Finally, fluo-3 analysis demonstrated that the A23187-induced elevation of sperm intracellular calcium concentration was markedly reduced after incubation with anti-eppin antibody. However, the tyrosine phosphorylation of sperm proteins did not change. CONCLUSION: These results demonstrate that eppin can modulate intracellular calcium concentrations and subsequently affect the calcium ionophore A23187-induced acrosome reaction.

The role of hepatocyte growth factor activator inhibitor-1 (HAI-1) as a prognostic indicator in cervical cancer.

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is a Kunitz-type serine protease inhibitor that has a broad inhibitory spectrum against serine proteases. This is the first study to investigate the role of HAI-1 and its clinical importance in cervical cancer. We attempted to investigate the inhibitory effects of HAI-1 using cervical carcinoma cell lines SiHa with integrated human papillomavirus (HPV) 16 and HeLa with integrated HPV 18. HAI-1 expression in cervical cancer (n=91) were assessed by immunohistochemistry. HAI-1 was found to be a potential inhibitory effects mediated by reduction of hepsin, matriptase and prostasin expression. This led to apoptosis through a reduction in the levels of Bcl-2, Bcl-xL, MUPP-1 and MAGI-3 in cervical cancer cell lines. There were significant correlations between HAI-1 expression and stage (p=0.013), tumor size (p=0.002), stromal invasion (p<0.001), vaginal invasion (p=0.031), parametrial invasion (p=0.012), lymph-node metastasis (p=0.019), and LVS involvement (p=0.002). The disease-free and overall survival rates of patients exhibiting high HAI-1 expression were significantly higher than those of patients exhibiting low HAI-1 expression (p=0.022 and p=0.011, respectively). The present study proposes that these findings may be considered HAI-1 as a therapeutic target for treatment and identify as a favorable prognostic marker for cancer patients of cervical cancer.

LEKTI-1 in sickness and in health.

The stratum corneum (SC) is a biosensor that mediates responses to a variety of exogenous insults through various signalling mechanisms, including the activation of SC serine proteases (SP) kallikrein cascade. The SPINK5 gene encodes an SP inhibitor, the lympho-epithelial-Kazal-type-1 inhibitor (LEKTI-1), which in turn will buffer the excess of SP cascade initiation, key in the maintenance of permeability barrier homeostasis. We demonstrate that LEKTI processing can occur within the SC after secretion from stratum granulosum keratinocytes at least partially by klk7, an SC-specific chymotryptic SP. Unlike the recently described LEKTI-2, neither recombinant full-length LEKTI-1 nor recombinant LEKTI-1 fragments exhibit antimicrobial activity. Finally, we discuss the pathophysiological implications of LEKTI-1 in skin biology as well as its contribution to the pathogenesis of Netherton Syndrome and its potential involvement in atopic dermatitis.

Hepatocyte growth factor activator inhibitor type 1 regulates epithelial to mesenchymal transition through membrane-bound serine proteinases.

Hepatocyte growth factor activator inhibitor-1 (HAI-1), encoded by the serine protease inhibitor Kunitz type 1 (SPINT1) gene, is a membrane-associated proteinase inhibitor that potently inhibits a variety of serine proteinases, including those that are membrane bound. Although HAI-1/SPINT1 is widely expressed by epithelial cells and cancer cells, its functional role is still unclear, particularly in cancer. Here, we show that stable knockdown of HAI-1/SPINT1 in the human pancreatic cancer cell line SUIT-2 induces an elongated spindle-like morphology associated with accelerated invasion, thereby mimicking an epithelial to mesenchymal transition (EMT). We found that HAI-1/SPINT1 knockdown significantly reduced the expression of E-cadherin and was accompanied by up-regulation of Smad-interacting protein 1 (SIP1), an E-cadherin transcriptional repressor. In addition, matrix metalloproteinase-9 (MMP-9) was up-regulated. Similar results were obtained in the HLC-1 lung carcinoma cell line. Moreover, a metastatic variant of SUIT-2 (S2-CP8) that showed loss of E-cadherin expression also showed a significantly reduced level of HAI-1/SPINT1. Engineered overexpression of HAI-1/SPINT1 in S2-CP8 resulted in reversion of E-cadherin expression and SIP1 down-regulation, which accompanied reestablishment of epithelial morphology in culture. The EMT caused by HAI-1/SPINT1 knockdown seemed to be mediated, at least partly, by membrane-bound serine proteinases, matriptase/ST14 and TMPRSS4, as knockdown of matriptase/ST14 or TMPRSS4 in HAI-1/SPINT1 knockdown SUIT-2 cells and HLC-1 cells resulted in reversion of SIP1 and/or MMP-9 expression levels. We suggest that interactions between HAI-1/SPINT1 and membrane-bound serine proteinases contribute to transcriptional and functional changes involved in EMT in certain carcinoma cells.

Hepatocyte growth factor regulation: an integral part of why wounds become chronic.

Hepatocyte growth factor (HGF) is a cytokine known to play multiple roles during the various stages of wound healing. This study addresses the ongoing key questions regarding the role of HGF in wound healing, namely: are HGF and its regulators expressed differently in chronic and acute wounds? Biopsies from normal skin (n=10), acute (n=10), and chronic wounds (n=17) were analyzed by immunohistochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), and quantitative real-time RT-PCR for the presence of HGF, its receptor cMet, its activators and its inhibitors. Immunohistochemical staining for HGF, HGF activators, and HGF inhibitors was similar, with expression being greater in chronic wound dermis compared with acute wound dermis. While expression of cMet in chronic wound dermis was less than in the acute wound dermis. PCR quantification of these proteins showed similar trends although the differences did not reach statistical significance. The results of this study provide important data confirming the role of HGF in wound healing. In addition, we have demonstrated the aberrant expression of the HGF receptor cMet and activation inhibitors (HAI-1 and HAI-2) in chronic wounds. This aberrant expression may have a value in predicting the process of healing and nonhealing wounds and constitute important targets of future therapies.