Tsuyoshi Fukushima

Silencing of Insulin-like Growth Factor-binding Protein-2 in Human Glioblastoma Cells Reduces Both Invasiveness and Expression of Progression-associated Gene CD24

Glioblastoma multiforme (GBM) is a malignant brain tumor characterized by rapid growth and extensive invasiveness. Overexpression of insulin-like growth factor-binding protein-2 (IGFBP-2) has been reported in GBM. However, it remains to be determined how IGFBP-2 is involved in the progression of GBM. We utilized short hairpin-RNA (shRNA) expression retroviral vectors to inactivate the IGFBP-2 gene permanently in two human GBM cell lines, U251 and YKG-1. The stable knockdown of IGFBP-2 resulted in decreased invasiveness, decreased saturation density of the cells in vitro, and decreased tumorigenicity in nude mice. Transcriptional profiling of both lines revealed several genes that were significantly down-regulated by inactivation of IGFBP-2. One such gene was CD24, which has been implicated in progression of various cancers. Indeed, CD24 was expressed in most GBM cases and the inactivation of CD24 in GBM cells suppressed cellular invasiveness, as was the case for IGFBP-2. Forced overexpression of CD24 led to increased invasiveness of both IGFBP-2-inactivated GBM cell lines and also A172, a human GBM cell line with low endogenous CD24. Further supporting the inter-relationship between IGFBP-2 and CD24, knockdown of IGFBP-2 suppressed the CD24 promoter activity. Moreover, both CD24 promoter activity and in vitro invasiveness were restored in knockdown cells by transfection with an IGFBP-2 expression plasmid. These results indicate that CD24 is modulated by IGFBP-2 and contributes to IGFBP-2-enhanced invasiveness of GBM cells.

Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1) Is Required for Branching Morphogenesis in the Chorioallantoic Placenta

ABSTRACT 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.

Clinical relevance of hepsin and hepatocyte growth factor activator inhibitor type 2 expression in renal cell carcinoma

Cell surface proteolysis is important for the generation of bioactive proteins mediating tumor progression. Recent studies suggest that the membrane‐anchored cell surface proteinases matriptase and hepsin have significant roles in tumors. We analyzed the expression and clinical relevance of matriptase and hepsin, and their inhibitors hepatocyte growth factor activator inhibitor type 1 (HAI‐1) and type 2 (HAI‐2) in 66 cases of conventional renal cell carcinomas (RCC). The mRNA level was evaluated in paired samples from tumor and non‐tumorous renal tissues by real‐time reverse transcription–polymerase chain reaction. As matriptase and hepsin potently activate the proform of hepatocyte growth factor (HGF), the expression of HGF and its receptor, c‐Met, was also analyzed. Although upregulation of matriptase was observed occasionally in RCC, the expression level was not associated with prognostic parameters. Hepsin was downregulated in RCC, particularly in early stage disease, but upregulated in advanced stages. There was a trend of higher hepsin expression in RCC with distant metastasis, and Kaplan–Meier survival curves showed that high hepsin expression was associated with reduced overall survival (P < 0.01, log‐rank test). Moreover, multivariate analysis indicated that hepsin was an independent prognostic factor. Overexpression of HGF or c‐Met also showed reduced overall survival. We also observed a tendency of low HAI‐2 expression with reduced overall survival and a statistical association between high hepsin and low HAI‐2 level. No associations were observed between matriptase and HAI‐1 and HAI‐2. Our findings suggest that the balance between hepsin and its inhibitor, HAI‐2, may have prognostic value in RCC. (Cancer Sci 2007; 98: 491–498)

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.

Defect of Hepatocyte Growth Factor Activator Inhibitor Type 1/Serine Protease Inhibitor, Kunitz Type 1 (Hai-1/Spint1) Leads to Ichthyosis-Like Condition and Abnormal Hair Development in Mice

Hepatocyte growth factor activator inhibitor type 1 (HAI-1)/serine protease inhibitor, Kunitz type 1 (SPINT1) is a membrane-bound, serine proteinase inhibitor initially identified as an inhibitor of hepatocyte growth factor activator. It also inhibits matriptase and prostasin, both of which are membrane-bound serine proteinases that have critical roles in epidermal differentiation and function. In this study, skin and hair phenotypes of mice lacking the Hai-1/Spint1 gene were characterized. Previously, we reported that the homozygous deletion of Hai-1/Spint1 in mice resulted in embryonic lethality attributable to impaired placental development. To test the role of Hai-1/Spint1 in mice, the placental function of Hai-1/Spint1-mutant mice was rescued. Injection of Hai-1/Spint1(+/+) blastocysts with Hai-1/Spint1(-/-) embryonic stem cells successfully generated high-chimeric Hai-1/Spint1(-/-) embryos (B6Hai-1(-/-High)) with normal placentas. These embryos were delivered without apparent developmental abnormalities, confirming that embryonic lethality of Hai-1/Spint1(-/-) mice was caused by placental dysfunction. However, newborn B6Hai-1(-/-High) mice showed growth retardation and died by 16 days. These mice developed scaly skin because of hyperkeratinization, reminiscent of ichthyosis, and abnormal hair shafts that showed loss of regular cuticular septation. The interfollicular epidermis showed acanthosis with enhanced Akt phosphorylation. Immunoblot analysis revealed altered proteolytic processing of profilaggrin in Hai-1/Spint1-deleted skin with impaired generation of filaggrin monomers. These findings indicate that Hai-1/Spint1 has critical roles in the regulated keratinization of the epidermis and hair development.

Activation of hepatocyte growth factor activator zymogen (pro-HGFA) by human kallikrein 1-related peptidases

Hepatocyte growth factor activator (HGFA) is a serine protease and a potent activator of prohepatocyte growth factor/scatter factor (pro‐HGF/SF), a multifunctional growth factor that is critically involved in tissue morphogenesis, regeneration, and tumor progression. HGFA circulates as a zymogen (pro‐HGFA) and is activated in response to tissue injury. Although thrombin is considered to be an activator of pro‐HGFA, alternative pro‐HGFA activation pathways in tumor microenvironments remain to be identified. In this study, we examined the effects of kallikrein 1‐related peptidases (KLKs), a family of extracellular serine proteases, on the activation of pro‐HGFA. Among the KLKs examined (KLK2, KLK3, KLK4 and KLK5), we identified KLK4 and KLK5 as novel activators of pro‐HGFA. Using N‐terminal sequencing, the cleavage site was identified as the normal processing site, Arg407–Ile408. The activation of pro‐HGFA by KLK5 required a negatively charged substance such as dextran sulfate, whereas KLK4 could process pro‐HGFA without dextran sulfate. KLK5 showed more efficient pro‐HGFA processing than KLK4, and was expressed in 50% (13/25) of the tumor cell lines examined. HGFA processed by these KLKs efficiently activated pro‐HGF/SF, and led to cellular scattering and invasion in vitro. The activities of both KLK4 and KLK5 were strongly inhibited by HGFA inhibitor type 1, an integral membrane Kunitz‐type serine protease inhibitor that inhibits HGFA and other pro‐HGF/SF‐activating proteases. These data suggest that KLK4 and KLK5 mediate HGFA‐induced activation of pro‐HGF/SF within tumor tissue, which may thereafter trigger a series of events leading to tumor progression via the MET receptor.

Hepatocyte growth factor activator is a serum activator of single-chain precursor macrophage-stimulating protein

Macrophage‐stimulating protein (MSP) is a plasma protein that circulates as a single‐chain proform. It acquires biological activity after proteolytic cleavage at the Arg483–Val484 bond, a process in which serum and cell surface serine proteinases have been implicated. In this article, we report that hepatocyte growth factor activator (HGFA), a serum proteinase which activates hepatocyte growth factor in response to tissue injury, may have a critical role in the activation of pro‐MSP. In vitro analysis has revealed that human HGFA efficiently cleaves human pro‐MSP at the physiological activation site without further degradation, resulting in biologically active MSP, as measured by the chemotactic response and MSP‐induced morphological change of peritoneal macrophages. The processing of pro‐MSP by HGFA is 10‐fold more efficient than processing by factor XIa. To search for a role of HGFA in pro‐MSP activation, we analyzed the processing of mouse pro‐MSP in sera from HGFA‐knockout (HGFA−/−) mice. The proform of MSP was the predominant molecular form in the plasma of both wild‐type and HGFA−/− mice. In wild‐type sera, endogenous pro‐MSP was progressively converted to the mature two‐chain form during incubation at 37 °C. However, this conversion was significantly impaired in sera from HGFA−/− mice. The addition of recombinant HGFA to HGFA‐deficient serum restored pro‐MSP convertase activity in a dose‐dependent manner, and a neutralizing antibody to HGFA significantly reduced the conversion of pro‐MSP in wild‐type serum. Moreover, initial infiltration of macrophages into the site of mechanical skin injury was delayed in HGFA−/− mice. We suggest that HGFA is a major serum activator of pro‐MSP.

Dickkopf-1 is overexpressed in human pancreatic ductal adenocarcinoma cells and is involved in invasive growth

The protein products of the Dickkopf (DKK) genes are antagonists of Wnt glycoproteins, which participate in tumor development and progression by binding to frizzled receptors. In this study, the expression of DKK‐1 was analyzed in a panel of 43 human cultured carcinoma cell lines. DKK‐1 expression was consistently and significantly upregulated in pancreatic carcinoma cell lines. Low level of DKK‐3 expression was also seen. In contrast, the expression of DKK‐2 and ‐4 was not detectable in most pancreatic carcinoma cell lines. The overexpression of DKK‐1 was confirmed in surgically resected human pancreatic cancer tissues, in which the mRNA level was evaluated in paired samples from cancerous and noncancerous pancreatic tissues. In ductal adenocarcinomas (23 cases), DKK‐1 mRNA levels were significantly upregulated compared to corresponding noncancerous tissues in a statistically significant level. To test the biological role of DKK‐1 in pancreatic carcinoma cells, we performed a knockdown of DKK‐1 in SUIT‐2 human pancreatic adenocarcinoma cell line and S2‐CP8, its metastatic subline, using a retroviral short hairpin RNA expression vector. DKK‐1 knockdown resulted in reduced migratory activity of SUIT‐2 in vitro. The in vitro growth rate and Matrigel invasion were also suppressed by DKK‐1 knockdown in S2‐CP8 cells. Collectively, the evidence suggests that, despite of its presumed antagonistic role in Wnt signaling, DKK‐1 may have a role in the aggressiveness of pancreatic carcinoma cells and could, therefore, serve as a novel biomarker of pancreatic cancer.

Role of hepatocyte growth factor activator (HGF activator) in invasive growth of human glioblastoma cells in vivo

Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional growth factor that is involved in invasive growth of tumor cells via its receptor MET, a protein product of c‐met proto‐oncogene. HGF activator (HGFA) is a serine proteinase responsible for the activation of proform of HGF/SF (proHGF/SF). In our study, we examined the effects of engineered expression of HGFA on 2 human glioblastoma cell lines (YKG‐1 and U251). Both cells expressed MET, while only YKG‐1 expressed endogenous proHGF/SF. Enhanced MET phosphorylation and increased migratory activity were induced by the expression of HGFA in YKG‐1 cells in vitro in the presence of thrombin, which is a known activator of proHGFA. In contrast, MET phosphorylation was consistently observed in U251 that lacked endogenous HGF/SF, suggesting ligand‐independent activation of MET in this cell line. Consequently, the expression of HGFA in U251 did not enhance the MET phosphorylation and following cellular response even with the thrombin treatment. However, addition of exogenous proHGF/SF resulted in enhanced migratory activity of HGFA‐expressing U251 cells in the presence of thrombin in vitro. The engineered HGFA expression resulted in significantly enhanced tumor growth with increased vascular density in vivo when YKG‐1 cells were implanted in nude mouse brain. This effect was not observed in U251 lacking endogenous proHGF/SF. These results indicate the possible existence of multiple mechanisms of MET activation in glioblastomas and that the activation system of proHGF/SF is important in progression of glioblastomas that express endogenous proHGF/SF and require ligand‐dependent MET activation.

Membrane-Bound Serine Protease Inhibitor HAI-1 Is Required for Maintenance of Intestinal Epithelial Integrity

Hepatocyte growth factor activator inhibitor type 1 (HAI-1), encoded by the serine protease inhibitor Kunitz type 1 (SPINT1) gene, is a membrane-bound serine protease inhibitor expressed in epithelial tissues. Mutant mouse models revealed that HAI-1/SPINT1 is essential for placental labyrinth formation and is critically involved in regulating epidermal keratinization through interaction with its cognate cell surface protease, matriptase. HAI-1/SPINT1 is abundantly expressed in both human and mouse intestinal epithelium; therefore, we analyzed its role in intestinal function using mice with intestinal epithelial cell-specific deletion of Spint1 generated by interbreeding mice carrying Spint1(LoxP) homozygous alleles with transgenic mice carrying the Cre recombinase gene controlled by the intestine-specific Villin promoter. Although the resulting mice had normal development and appearance, crypts in the proximal aspect of the colon, including the cecum, exhibited histologic abnormalities and increased apoptosis and epithelial cell turnover accompanied by increased intestinal permeability. Distended endoplasmic reticula were observed ultrastructurally in some crypt epithelial cells, indicative of endoplasmic reticular stress. To study the role of HAI-1/SPINT1 in mucosal injury, we induced colitis by adding dextran sodium sulfate to the drinking water. After dextran sodium sulfate treatment, intestine-specific HAI-1/SPINT1-deficient mice had more severe symptoms and a significantly lower survival rate relative to control mice. These results suggest that HAI-1/SPINT1 plays an important role in maintaining colonic epithelium integrity.