Hepatocyte Growth Factor Activator Research Articles

Deregulated hepsin protease activity confers oncogenicity by concomitantly augmenting HGF/MET signalling and disrupting epithelial cohesion.

Hepsin belongs to a family of cell-surface serine proteases, which have sparked interest as therapeutic targets because of the accessibility of extracellular protease domain for inhibitors. Hepsin is frequently amplified and/or overexpressed in epithelial cancers, but it is not clear how enhanced hepsin expression confers a potential for oncogenicity. We show that hepsin is consistently overexpressed in more than 40% of examined breast cancers, including all major biological subtypes. The effects of doxycycline-induced hepsin overexpression were examined in mammary epithelial organoids, and we found that induced hepsin acutely downmodulates its cognate inhibitor, hepatocyte growth factor (HGF) activator inhibitor type 1 (HAI-1). Hepsin-induced depletion of cellular HAI-1 led to a sharp increase in pericellular serine protease activity. The derepressed hepsin proteolytically activated downstream serine proteases, augmented HGF/MET signalling and caused deterioration of desmosomes and hemidesmosomes; structures important for cell cohesion and cell-basement membrane interaction. Moreover, chronic induction of hepsin considerably shortened the latency of Myc-dependent tumourigenesis in the mouse mammary gland. The serine protease and uPA system inhibitor WX-UK1, identified as a micromolar range hepsin inhibitor, prevented hepsin from augmenting HGF/MET signalling and disrupting desmosomes and hemidesmosomes. The findings suggest that the oncogenic activity of hepsin arises not only from elevated expression level but also from depletion of HAI-1, events which together trigger gain-of-function activity impacting HGF/MET signalling and epithelial cohesion. Thus, hepsin overexpression is a major oncogenic conferrer to a serine protease activity involved in breast cancer dissemination.

Matrix metalloproteinase 9 modulates collagen matrices and wound repair

Acute and chronic injuries are characterized by leukocyte infiltration into tissues. Although matrix metalloproteinase 9 (Mmp9) has been implicated in both conditions, its role in wound repair remains unclear. We previously reported a zebrafish chronic inflammation mutant caused by an insertion in the hepatocyte growth factor activator inhibitor gene 1 (hai1; also known as spint1) that is characterized by epithelial extrusions and neutrophil infiltration into the fin. Here, we performed a microarray analysis and found increased inflammatory gene expression in the mutant larvae, including a marked increase in mmp9 expression. Depletion of mmp9 partially rescued the chronic inflammation and epithelial phenotypes, in addition to restoring collagen fiber organization, as detected by second-harmonic generation imaging. Additionally, we found that acute wounding induces epithelial cell mmp9 expression and is associated with a thickening of collagen fibers. Interestingly, depletion of mmp9 impaired this collagen fiber reorganization. Moreover, mmp9 depletion impaired tissue regeneration after tail transection, implicating Mmp9 in acute wound repair. Thus, Mmp9 regulates both acute and chronic tissue damage and plays an essential role in collagen reorganization during wound repair.

Novel Role for γ-Catenin in the Regulation of Cancer Cell Migration via the Induction of Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1)

γ-Catenin (Plakoglobin), a well-described structural protein functioning at the adherens junctions and desmosomes, was shown to be either lost or weakly expressed in non-small cell lung cancer (NSCLC) cells and tumor tissues. However, the tumor suppressive affects of γ-catenin were not fully understood. In this study, we have identified a novel role for the affects of γ-catenin on non-small cell lung cancer (NSCLC) cell migration. Expression of γ-catenin in NSCLC cells resulted in reduced cell migration as determined by both scratch assays and trans-well cell migration assays. Moreover, the affects of γ-catenin on cell migration were observed to be p53-dependent. Mechanistically, the anti-migratory effects seen via γ-catenin were driven by the expression of hepatocyte growth factor activator inhibitor Type I (HAI-1 or SPINT-1), an upstream inhibitor of the c-MET signaling pathway. Furthermore, the re-expression of γ-catenin sensitized NSCLC cells to c-MET inhibitor-mediated growth inhibition. Taken together, we identify γ-catenin as a novel regulator of HAI-1, which is a critical regulator of HGF/c-MET signaling. Therefore, targeting γ-catenin-mediated HAI-1 expression might be a useful strategy to sensitize NSCLC to c-MET inhibitors.

Matrix metalloproteinase 9 modulates collagen matrices and wound repair.

Acute and chronic injuries are characterized by leukocyte infiltration into tissues. Although matrix metalloproteinase 9 (Mmp9) has been implicated in both conditions, its role in wound repair remains unclear. We previously reported a zebrafish chronic inflammation mutant caused by an insertion in the hepatocyte growth factor activator inhibitor gene 1 (hai1; also known as spint1) that is characterized by epithelial extrusions and neutrophil infiltration into the fin. Here, we performed a microarray analysis and found increased inflammatory gene expression in the mutant larvae, including a marked increase in mmp9 expression. Depletion of mmp9 partially rescued the chronic inflammation and epithelial phenotypes, in addition to restoring collagen fiber organization, as detected by second-harmonic generation imaging. Additionally, we found that acute wounding induces epithelial cell mmp9 expression and is associated with a thickening of collagen fibers. Interestingly, depletion of mmp9 impaired this collagen fiber reorganization. Moreover, mmp9 depletion impaired tissue regeneration after tail transection, implicating Mmp9 in acute wound repair. Thus, Mmp9 regulates both acute and chronic tissue damage and plays an essential role in collagen reorganization during wound repair.

Regulation of hepatocyte growth factor in mice with pneumonia by peptidases and trans-alveolar flux.

Hepatocyte growth factor (HGF) promotes lung epithelial repair after injury. Because prior studies established that human neutrophil proteases inactivate HGF in vitro, we predicted that HGF levels decrease in lungs infiltrated with neutrophils and that injury is less severe in lungs lacking HGF-inactivating proteases. After establishing that mouse neutrophil elastase cleaves mouse HGF in vitro, we tested our predictions in vivo by examining lung pathology and HGF in mice infected with Mycoplasma pulmonis, which causes neutrophilic tracheobronchitis and pneumonia. Unexpectedly, pneumonia severity was similar in wild type and dipeptidylpeptidase I-deficient (Dppi-/-) mice lacking neutrophil serine protease activity. To assess how this finding related to our prediction that Dppi-activated proteases regulate HGF levels, we measured HGF in serum, bronchoalveolar lavage fluid, and lung tissue from Dppi+/+ and Dppi-/- mice. Contrary to prediction, HGF levels were higher in lavage fluid from infected mice. However, serum and tissue concentrations were not different in infected and uninfected mice, and HGF lung transcript levels did not change. Increased HGF correlated with increased albumin in lavage fluid from infected mice, and immunostaining failed to detect increased lung tissue expression of HGF in infected mice. These findings are consistent with trans-alveolar flux rather than local production as the source of increased HGF in lavage fluid. However, levels of intact HGF from infected mice, normalized for albumin concentration, were two-fold higher in Dppi-/- versus Dppi+/+ lavage fluid, suggesting regulation by Dppi-activated proteases. Consistent with the presence of active HGF, increased expression of activated receptor c-Met was observed in infected tissues. These data suggest that HGF entering alveoli from the bloodstream during pneumonia compensates for destruction by Dppi-activated inflammatory proteases to allow HGF to contribute to epithelial repair.

Expression of human kallikrein 1-related peptidase 4 (KLK4) and MET phosphorylation in prostate cancer tissue: immunohistochemical analysis

Resistance to or relapse after androgen deprivation therapy (ADT) remains a significant problem in patients with prostate cancer. Several studies have hypothesized that the overexpression of MET and the activating signaling axis in prostate cancer cells are associated with castration-resistant prostate cancer (CRPC). On the other hand, the expression of human kallikrein 1-related peptidase (KLK) 4, which activates plasma HGF activator zymogen, in prostate cancer patients with bone metastasis or advanced stage has also been reported. In this study, we analyzed the expression and phosphorylation of MET along with KLK4 by immunohistochemistry in 62 formalin-fixed paraffin-embedded sections of prostate cancer collected by needle biopsy at our hospital between 2006 and 2011. As a result, the phosphorylation of MET was observed in 56% (35 of 62 cases) and positively correlated with worsening PSA relapse rate in a group of ADT-treated patients (P = 0.0445), suggesting significant correlation with CRPC. Overexpression of KLK4 was observed in patients with high T stage (P = 0.0001) and high Gleason score (P = 0.0146), and the expression was correlated with the phosphorylation of MET (P = 0.0002). Pathologically, strong MET phosphorylation observed in specific architectures in prostate cancer, such as cribriform structures, ill-defined glands or solid patterns, suggested the significance of MET activation in promoting the architectural formation of prostate cancer. In addition, high positivity rate (80%) of phospho-MET staining at high-grade prostatic intraepithelial neoplasia (HGPIN) may indicate the importance of the activating signaling axis in the carcinogenesis of prostate cancer.

Structure-based discovery of small molecule hepsin and HGFA protease inhibitors: Evaluation of potency and selectivity derived from distinct binding pockets

Hepatocyte growth factor activator (HGFA), matriptase and hepsin are all S1 trypsin-like serine endopeptidases. HGFA is a plasma protease while hepsin and matriptase are type II transmembrane proteases (TTSPs). Upregulated expression and activity of all three proteases is associated with aberrant cancer cell signaling through c-MET and RON tyrosine kinase cell-signaling pathways in cancer. We modeled known benzamidine protease inhibitor scaffolds into the active sites of matriptase, hepsin and HGFA to design new non-peptide inhibitors of hepsin and HGFA. First, we used a docking model of the irreversible inhibitor, Nafamostat, bound to the active site of HGFA in order to explore structure activity relationships (SAR). Compounds were screened for inhibition of HGFA activity in a kinetic enzyme assay using a chromogenic substrate. Next, we designed matched pair compound libraries of 3-amidino and 4-amidino phenylalanine (benzamidine) arginine peptidomimetics based on the structure of matriptase inhibitor, CJ-672. Compounds were screened for inhibition of HGFA, matriptase, and hepsin enzyme activity using fluorogenic substrates. Using this strategy we have discovered the first reported non-peptide small molecule inhibitors of both HGFA and hepsin. These inhibitors have differential potency and selectivity towards all three proteases. A subset of piperazinyl ureas highlighted by 25a, have excellent potency and selectivity for hepsin over matriptase and HGFA.

Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma

A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases. It is of interest if the single-domain Kazal-type inhibitor controls the activity of turkey seminal plasma proteinases. Pure preparations of the Kazal-type inhibitor and anti–Kazal-type inhibitor monospecific immunoglobulin Gs were used as ligands in affinity chromatography for proteinase isolation from turkey seminal plasma. Gene expression and the immunohistochemical detection of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. The hepatocyte growth factor activator (HGFA) was identified in the binding fraction in affinity chromatography. Hepatocyte growth factor activator activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner. This protease was a primary physiological target for the single-domain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma, and phosphorylation was the primary posttranslational modification of HGFA. In addition to HGFA, acrosin was a target proteinase for the single-domain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and was not present as a free enzyme. The single-domain Kazal-type inhibitor was specific for the reproductive tract. The germ cell–specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis; secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in the changes in sperm membranes during maturation and in the maintenance of the microenvironment in which sperm maturation occurred and sperm was stored. The role of HGFA in these processes remains to be established.

Foretinib inhibits angiogenesis, lymphangiogenesis and tumor growth of pancreatic cancer in vivo by decreasing VEGFR-2/3 and TIE-2 signaling.

Foretinib, a multiple kinase inhibitor undergoing clinical trials, could suppress the activity of hepatocyte growth factor (HGF) receptor c-MET and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, Foretinib may inhibit two critical lymphangiogenic signaling receptors VEGFR-3 and TIE-2. However, the effect of Foretinib on lymphatic endothelial cells (LECs) in vitro and lymphangiogenesis in vivo is still unknown. We found Foretinib decreased basal- and HGF-induced c-MET activity at low concentrations. However, Foretinib only reduced the proliferation of pancreatic cancer cells at high concentration reflecting the intrinsic chemoresistance of pancreatic cancer cells. Foretinib inhibited VEGF-A, VEGF-C and Angiopoetin-2 (ANG-2)-stimulated tube formation and sprouting of LECs by reducing VEGFR-2, VEGFR-3 and TIE-2 activation and increased apoptosis of LECs. In xenograft animal study, Foretinib suppressed tumor growth by inhibiting proliferation, angiogenesis and lymphangiogenesis. Additionally, Foretinib inhibited angiogenesis and lymphangiogenesis more significantly and exhibited low detrimental effect in orthotopic animal study. Collectively, we suggested that Foretinib simultaneously inhibits cancer cells and LECs to reduce pancreatic tumor growth in vivo and demonstrated for the first time that Foretinib suppresses angiogenesis and lymphangiogenesis by blocking VEGFR-2/3 and TIE-2 signaling.

Hepatocyte Growth Factor Activator Inhibitor Type 1 Maintains the Assembly of Keratin into Desmosomes in Keratinocytes by Regulating Protease-Activated Receptor 2–Dependent p38 Signaling

Hepatocyte growth factor activator inhibitor type 1 (HAI-1; official symbol SPINT1) is a membrane-associated serine proteinase inhibitor abundantly expressed in epithelial tissues. Genetically engineered mouse models demonstrated that HAI-1 is critical for epidermal function, possibly through direct and indirect regulation of cell surface proteases, such as matriptase and prostasin. To obtain a better understanding of the role of HAI-1 in maintaining epidermal integrity, we performed ultrastructural analysis of Spint1-deleted mouse epidermis and organotypic culture of an HAI-1 knockdown (KD) human keratinocyte cell line, HaCaT. We found that the aggregation of tonofilaments to desmosomes was significantly reduced in HAI-1-deficient mouse epidermis with decreased desmosome number. Similar findings were observed in HAI-1 KD HaCaT organotypic cultures. Immunoblot and immunohistochemical analyses revealed that p38 mitogen-activated protein kinase was activated in response to HAI-1 insufficiency. Treatment of HAI-1 KD HaCaT cells with a p38 inhibitor abrogated the above-observed ultrastructural abnormalities. The activation of p38 induced by the loss of HAI-1 likely resulted from enhanced signaling of protease-activated receptor-2 (PAR-2), because its silencing abrogated the enhanced activation of p38. Consequently, treatment of HAI-1 KD HaCaT cells with a serine protease inhibitor, aprotinin, or PAR-2 antagonist alleviated the abnormal ultrastructural phenotype in organotypic culture. These results suggest that HAI-1 may have a critical role in maintaining normal keratinocyte morphology through regulation of PAR-2-dependent p38 mitogen-activated protein kinase signaling.

Induction of tumor initiation is dependent on CD44s in c-Met⁺ hepatocellular carcinoma.

BackgroundHepatocellular carcinoma (HCC) patients with active hepatocyte growth factor (HGF)/c-Met signaling have a significantly worse prognosis. c-Met, a high affinity receptor for HGF, plays a critical role in cancer growth, invasion and metastasis. c-Met and CD44 have been utilized as cell surface markers to identify mesenchymal tumor-initiating stem-like cells (TISC) in several cancers including HCC. In this work, we examine the complex relationship between c-Met and CD44s (standard form), and investigate the specific role of CD44s as a tumor initiator and stemness marker in HCC.MethodsGene and protein expression assays were utilized to investigate the relationship between CD44s and c-Met in HCC cell lines. Tumor-sphere assays and in vivo tumor assays were performed to investigate the role of CD44+ cells as TISCs. Student’s t-test or one-way ANOVA with Tukeys post-hoc test was performed to test for differences amongst groups with a p < .05 as significant.ResultsIn an immunohistochemical and immunoblot analysis of human HCC samples, we observed that more than 39% of human HCC samples express c-Met and CD44. To study the relationship between c-Met and CD44, we used MHCC97-H cells, which are CD44+/c-Met+. The knockdown of c-Met in MHCC97-H cells decreased CD44s, reduced TISC characteristics and decreased tumorsphere formation. Furthermore, we demonstrate that the inhibition of PI3K/AKT signaling decreased CD44s expression and subsequently decreased tumorsphere formation. The down-regulation of CD44s leads to a significant loss of a TISC and mesenchymal phenotype. Finally, the down-regulation of CD44s in MHCC97-H cells decreased tumor initiation in vivo compared with the scrambled control.ConclusionsIn summary, our data suggest that CD44s is modulated by the c-Met-PI3K-AKT signaling cascade to support a mesenchymal and TISC phenotype in HCC cells. Moreover, c-Met could be a potential therapeutic drug for targeting HCC cells with TISC and mesenchymal phenotypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1166-4) contains supplementary material, which is available to authorized users.

Hepatocyte growth factor and Met in drug discovery.

Activation of the hepatocyte growth factor (HGF)-Met pathway evokes dynamic biological responses that support the morphogenesis, regeneration and survival of cells and tissues. A characterization of conditional Met knockout mice indicates that the HGF-Met pathway plays important roles in the regeneration, protection and homeostasis of cells such as hepatocytes, renal tubular cells and neurons. Preclinical studies in disease models have indicated that recombinant HGF protein and expression plasmid for HGF are biological drug candidates for the treatment of patients with diseases or injuries that involve impaired tissue function. The phase-I and phase-I/II clinical trials of the intrathecal administration of HGF protein for the treatment of patients with amyotrophic lateral sclerosis and spinal cord injury, respectively, are ongoing. Biological actions of HGF that promote the dynamic movement, morphogenesis and survival of cells also closely participate in invasion-metastasis and resistance to the molecular-targeted drugs in tumour cells. Different types of HGF-Met pathway inhibitors are now in clinical trials for treatment of malignant tumours. Basic research on HGF and Met has lead to drug discoveries in regenerative medicine and tumour biology.

The solution structure of the MANEC-type domain from hepatocyte growth factor activator inhibitor-1 reveals an unexpected PAN/apple domain-type fold.

A decade ago, motif at N-terminus with eight-cysteines (MANEC) was defined as a new protein domain family. This domain is found exclusively at the N-terminus of >400 multi-domain type-1 transmembrane proteins from animals. Despite the large number of MANEC-containing proteins, only one has been characterized at the protein level: hepatocyte growth factor activator inhibitor-1 (HAI-1). HAI-1 is an essential protein, as knockout mice die in utero due to placental defects. HAI-1 is an inhibitor of matriptase, hepsin and hepatocyte growth factor (HGF) activator, all serine proteases with important roles in epithelial development, cell growth and homoeostasis. Dysregulation of these proteases has been causatively implicated in pathological conditions such as skin diseases and cancer. Detailed functional understanding of HAI-1 and other MANEC-containing proteins is hampered by the lack of structural information on MANEC. Although many MANEC sequences exist, sequence-based database searches fail to predict structural homology. In the present paper, we present the NMR solution structure of the MANEC domain from HAI-1, the first three-dimensional (3D) structure from the MANEC domain family. Unexpectedly, MANEC is a new subclass of the PAN/apple domain family, with its own unifying features, such as two additional disulfide bonds, two extended loop regions and additional α-helical elements. As shown for other PAN/apple domain-containing proteins, we propose a similar active role of the MANEC domain in intramolecular and intermolecular interactions. The structure provides a tool for the further elucidation of HAI-1 function as well as a reference for the study of other MANEC-containing proteins.

Studies of hepatocyte growth factor in bronchoalveolar lavage fluid in chronic interstitial lung diseases.

Previous studies have suggested that hepatocyte growth factor (HGF) inhibits lung fibrosis as an antagonist of transforming growth factor β (TGF‑β). We assessed HGF expression levels in the lower airways of patients with selected interstitial lung diseases. HGF levels were examined by an enzyme‑linked immunosorbent assay in bronchoalveolar lavage (BAL) fluid supernatants from patients with pulmonary sarcoidosis (PS, n = 52), idiopathic pulmonary fibrosis (IPF, n = 23), nonspecific interstitial pneumonia (NSIP, n = 14), extrinsic allergic alveolitis (EAA, n = 6), bronchiolitis obliterans organizing pneumonia (BOOP, n = 8), chronic eosinophilic pneumonia (EP, n = 6), and in control subjects (n = 13). Intracellular HGF expression in BAL cells was evaluated by flow cytometry. HGF concentrations were elevated in BAL fluid from nonsmokers with IPF (261 ±204 pg/ml, P <0.02), smokers with IPF (220 ±13 pg/ml, P <0.001), and smokers with PS (172 ±33 pg/ml, P <0.02), as compared with controls (148 ±17 pg/ml for nonsmokers; 137 ±9 pg/ml for smokers). HGF levels were positively correlated with TGF‑β concentrations in BAL fluid (r = 0.3; P = 0.02) and negatively-with vital capacity (r = -0.2; P = 0.02). BAL neutrophils, and, for the first time, BAL lymphocytes, were identified as intracellular HGF‑positive cells. Our results do not support evidence for strong antifibrotic HGF activity. The highest HGF concentrations were observed in BAL fluid from patients with IPF, and they were also positively correlated with TGF‑β levels. Thus, although the local protective mechanisms such as the HGF expression are upregulated in chronic interstitial lung diseases, they are not enough to prevent lung fibrosis.

Hepatocyte growth factor mimetic protects lateral line hair cells from aminoglycoside exposure.

Loss of sensory hair cells from exposure to certain licit drugs (e.g., aminoglycoside antibiotics, platinum-based chemotherapy agents) can result in permanent hearing loss. Here we ask if allosteric activation of the hepatocyte growth factor (HGF) cascade via Dihexa, a small molecule drug candidate, can protect hair cells from aminoglycoside toxicity. Unlike native HGF, Dihexa is chemically stable and blood-brain barrier permeable. As a synthetic HGF mimetic, it forms a functional ligand by dimerizing with endogenous HGF to activate the HGF receptor and downstream signaling cascades. To evaluate Dihexa as a potential hair cell protectant, we used the larval zebrafish lateral line, which possesses hair cells that are homologous to mammalian inner ear hair cells and show similar responses to toxins. A dose-response relationship for Dihexa protection was established using two ototoxins, neomycin and gentamicin. We found that a Dihexa concentration of 1 μM confers optimal protection from acute treatment with either ototoxin. Pretreatment with Dihexa does not affect the amount of fluorescently tagged gentamicin that enters hair cells, indicating that Dihexa’s protection is likely mediated by intracellular events and not by inhibiting aminoglycoside entry. Dihexa-mediated protection is attenuated by co-treatment with the HGF antagonist 6-AH, further evidence that HGF activation is a component of the observed protection. Additionally, Dihexa’s robust protection is partially attenuated by co-treatment with inhibitors of the downstream HGF targets Akt, TOR and MEK. Addition of an amino group to the N-terminal of Dihexa also attenuates the protective response, suggesting that even small substitutions greatly alter the specificity of Dihexa for its target. Our data suggest that Dihexa confers protection of hair cells through an HGF-mediated mechanism and that Dihexa holds clinical potential for mitigating chemical ototoxicity.

Proinflammatory Cytokines (IL-1α, IL-6) and Hepatocyte Growth Factor in Patients with Alcoholic Liver Cirrhosis.

Background. The aim of the study was to assess the activity of interleukin-1α, interleukin-6, and hepatocyte growth factor protein (HGF) in serum of patients with alcoholic liver cirrhosis. Materials and Methods. Sixty patients with alcoholic liver cirrhosis treated in various hospitals were randomly enrolled. The stage of cirrhosis was assessed according to the Child-Turcotte-Pugh scoring system. The control group consisted of ten healthy persons without liver disease, who did not drink alcohol. Additionally, the group of alcoholics without liver cirrhosis was included in the study. The activity of interleukin-1α, interleukin-6, and HGF in blood plasma of patients and controls was measured using the sandwich enzyme immunoassay technique with commercially available quantitative ELISA test kits. Results. Higher concentrations of HGF protein were demonstrated in patients with Child class B and Child class C liver cirrhosis, compared to controls and alcoholics without liver cirrhosis. Moreover, significantly higher concentrations of HGF protein were found in patients with Child class C liver cirrhosis compared to patients with Child class A liver cirrhosis (p < 0.05). The concentrations of interleukin-1α in patients with Child class B and Child class C liver cirrhosis were significantly higher in comparison with controls. Significantly higher concentrations of interleukin-6 were demonstrated in Child class C, compared to Child class A.

Alteration of angiogenesis in Helicobacter heilmannii‐induced mucosa‐associated lymphoid tissue lymphoma: Interaction with c‐Met and hepatocyte growth factor

The hepatocyte growth factor (HGF)/c-Met pathway has attracted attention in the formation of malignant tumors, as HGF secreted from the microcirculatory components as well as residing macrophages has been suggested to act on the c-Met receptors of cancer cells to decrease apoptosis and increase proliferation, invasion, and metastasis. The present study was undertaken to elucidate the interaction of the gastric, hepatic, and pulmonary mucosa-associated lymphoid tissue (MALT) lymphoma induced by Helicobacter heilmannii infection with c-Met and HGF. C57BL/6 female mice, infected with H. heilmannii for 3 months were used. The localization of the HGF, c-Met, and HGF activator immunoreactivities was observed by the indirect immunohistochemical methods. In addition, the effect of c-Met antibody and c-Met inhibitor, PHA-665752, was also investigated. c-Met immunoreactivity was found in the lymphocytes composing the MALT lymphoma, and HGF immunoreactivity was recognized mostly in the endothelial cells and macrophages in the MALT lymphoma. HGFA was localized on mesenchymal cells other than the lymphocytes. The administration of the antibody against c-Met or the c-Met inhibitor to the infected mice induced the significant suppression of hepatic and pulmonary MALT lymphoma, while the gastric MALT lymphoma showed only a tendency to decrease in size, while the active caspase 3 positive cells markedly decreased in the gastric, hepatic, and pulmonary MALT lymphoma after the treatment with the c-Met antibody or the c-Met antagonist. HGF and c-Met pathway were suggested to contribute to the lymphomagenesis in the MALT lymphoma after H. heilmannii infection.

Sequence and Conformational Specificity in Substrate Recognition: SEVERAL HUMAN KUNITZ PROTEASE INHIBITOR DOMAINS ARE SPECIFIC SUBSTRATES OF MESOTRYPSIN

Mesotrypsin is an isoform of trypsin that is uniquely resistant to polypeptide trypsin inhibitors and can cleave some inhibitors rapidly. Previous studies have shown that the amyloid precursor protein Kunitz protease inhibitor domain (APPI) is a specific substrate of mesotrypsin and that stabilization of the APPI cleavage site in a canonical conformation contributes to recognition by mesotrypsin. We hypothesized that other proteins possessing potential cleavage sites stabilized in a similar conformation might also be mesotrypsin substrates. Here we evaluated a series of candidate substrates, including human Kunitz protease inhibitor domains from amyloid precursor-like protein 2 (APLP2), bikunin, hepatocyte growth factor activator inhibitor type 2 (HAI2), tissue factor pathway inhibitor-1 (TFPI1), and tissue factor pathway inhibitor-2 (TFPI2), as well as E-selectin, an unrelated protein possessing a potential cleavage site displaying canonical conformation. We find that Kunitz domains within APLP2, bikunin, and HAI2 are cleaved by mesotrypsin with kinetic profiles of specific substrates. TFPI1 and TFPI2 Kunitz domains are cleaved less efficiently by mesotrypsin, and E-selectin is not cleaved at the anticipated site. Cocrystal structures of mesotrypsin with HAI2 and bikunin Kunitz domains reveal the mode of mesotrypsin interaction with its canonical substrates. Our data suggest that major determinants of mesotrypsin substrate specificity include sequence preferences at the P1 and P'2 positions along with conformational stabilization of the cleavage site in the canonical conformation. Mesotrypsin up-regulation has been implicated previously in cancer progression, and proteolytic clearance of Kunitz protease inhibitors offers potential mechanisms by which mesotrypsin may mediate pathological effects in cancer.

HGF-Met Pathway in Regeneration and Drug Discovery.

Hepatocyte growth factor (HGF) is composed of an α-chain and a β-chain, and these chains contain four kringle domains and a serine protease-like structure, respectively. Activation of the HGF–Met pathway evokes dynamic biological responses that support morphogenesis (e.g., epithelial tubulogenesis), regeneration, and the survival of cells and tissues. Characterizations of conditional Met knockout mice have indicated that the HGF–Met pathway plays important roles in regeneration, protection, and homeostasis in various cells and tissues, which includes hepatocytes, renal tubular cells, and neurons. Preclinical studies designed to address the therapeutic significance of HGF have been performed on injury/disease models, including acute tissue injury, chronic fibrosis, and cardiovascular and neurodegenerative diseases. The promotion of cell growth, survival, migration, and morphogenesis that is associated with extracellular matrix proteolysis are the biological activities that underlie the therapeutic actions of HGF. Recombinant HGF protein and the expression vectors for HGF are biological drug candidates for the treatment of patients with diseases and injuries that are associated with impaired tissue function. The intravenous/systemic administration of recombinant HGF protein has been well tolerated in phase I/II clinical trials. The phase-I and phase-I/II clinical trials of the intrathecal administration of HGF protein for the treatment of patients with amyotrophic lateral sclerosis and spinal cord injury, respectively, are ongoing.

Hepatocyte growth factor activator inhibitor type-1 in cancer: Advances and perspectives (Review)

Cancer is one of the most common diseases, with high morbidity and mortality rates. Large‑scale efforts have been made to understand the pathogenesis of the disease, particularly in the advanced stages, in order to develop effective therapeutic approaches. Hepatocyte growth factor activator inhibitor type-1 (HAI-1), also known as serine protease inhibitor Kunitz type 1, inhibits the activity of several trypsin-like serine proteases. In particular, HAI-1 suppresses hepatocyte growth factor (HGF) activator and matriptase, resulting in subsequent inhibition of HGF/scatter factor and macrophage‑stimulating protein (MSP). HGF and MSP are involved in cancer development and progression, via the receptors Met receptor tyrosine kinase (RTK) and Ron RTK, respectively. Therefore, HAI-1-mediated downregulation of HGF and MSP signaling may suppress tumorigenesis and progression in certain types of cancers. Abnormal HAI-1 expression levels have been observed in various types of human cancer. The exact function of HAI-1 in cancer pathogenesis, however, has not been fully elucidated. In this review, the focus is on the potential impact of aberrant HAI-1 expression levels on tumorigenesis and progression, the underlying mechanisms, and areas that require further investigation to clarify the precise role of HAI-1 in cancer.