Chen-yong Lin - Academia.edu (original) (raw)

Papers by Chen-yong Lin

Research paper thumbnail of Aberrant regulation favours matriptase proteolysis in neoplastic B-cells that co-express HAI-2

Journal of Enzyme Inhibition and Medicinal Chemistry, 2019

Matriptase is ectopically expressed in neoplastic B-cells, in which matriptase activity is enhanc... more Matriptase is ectopically expressed in neoplastic B-cells, in which matriptase activity is enhanced by negligible expression of its endogenous inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1. HAI-1, however, is also involved in matriptase synthesis and intracellular trafficking. The lack of HAI-1 indicates that other related inhibitor, such as HAI-2, might be expressed. Here, we show that HAI-2 is commonly co-expressed in matriptase-expressing neoplastic B-cells. The level of active matriptase shed after induction of matriptase zymogen activation in 7 different neoplastic B-cells was next determined and characterised. Our data reveal that active matriptase can only be generated and shed by those cells able to activate matriptase and in a rough correlation with the levels of matriptase protein. While HAI-2 can potently inhibit matriptase, the levels of active matriptase are not proportionally suppressed in those cells with high HAI-2. Our survey suggests that matriptase proteolysis might aberrantly remain high in neoplastic B-cells regardless of the levels of HAI-2.

Research paper thumbnail of Molecular Cloning of cDNA for Matriptase, a Matrix-degrading Serine Protease with Trypsin-like Activity

Journal of Biological Chemistry, 1999

A major protease from human breast cancer cells was previously detected by gelatin zymography and... more A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease. Elevated proteolytic activity has been implicated in neoplastic progression. Although the exact role(s) of proteolytic enzymes in the progression of tumor remains unclear, it seems that proteases may be involved in almost every step of the development and spread of cancer. A widely proposed view is that proteases contribute to the degradation of extracellular matrix and to tissue remodeling and are necessary for cancer invasion and metastasis. A wide array of extracellular matrixdegrading proteases have been discovered, the expression of some of which correlates with tumor progression, as reviewed by Magnatti and Rifkin (1). The plasmin/urokinase-type plas

Research paper thumbnail of Antimetastatic activity isolated from Colocasia esculenta (taro)

Anti-Cancer Drugs, 2012

Breast cancer mortality is primarily due to the occurrence of metastatic disease. We have identif... more Breast cancer mortality is primarily due to the occurrence of metastatic disease. We have identified a novel potential therapeutic agent derived from an edible root of the plant Colocasia esculenta, commonly known as taro, that has demonstrable activity in a preclinical model of metastatic breast cancer and that should have minimal toxicity. We have shown for the first time that a water-soluble extract of taro (TE) potently inhibits lung colonizing ability as well as spontaneous metastasis from mammary gland-implanted tumors, in a murine model of highly metastatic ER, PR and Her-2/neu negative breast cancer. TE modestly inhibits proliferation of some, but not all, breast and prostate cancer cell lines. Morphologic changes including cell rounding were observed. Tumor cell migration was completely blocked by TE. TE treatment also inhibited prostaglandin E 2 (PGE 2) synthesis and downregulated cyclooxygenase (COX) 1 and 2 mRNA expression. We purified the active compound(s) to near homogeneity with antimetastatic activity comparable to stock TE. The active compound with a native size of approximately 25 kD contains two fragments of nearly equal size. The N-terminal amino acid sequencing of both fragments reveals that the active compound is highly related to three taro proteins; 12 kD storage protein, tarin and lectin. All are similar in terms of amino acid sequence, post-translational processing and all contain a carbohydrate-binding domain. This is the first report describing a compound(s) derived from taro, that potently and specifically inhibits tumor metastasis.

Research paper thumbnail of Hepatocyte growth factor activator inhibitor-2 (HAI-2) is a favorable prognosis marker and inhibits cell growth through the apoptotic pathway in cervical cancer

Annals of Oncology, 2008

Background: In light of the poor prognosis for cervical cancer, research continues into the devel... more Background: In light of the poor prognosis for cervical cancer, research continues into the development of innovative and efficacious treatment modalities for this disease. We investigated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) and evaluated its clinical importance in cervical cancer. Patients and methods: HAI-2 expression was examined in cervical cancer specimens (n = 52) by immunohistochemistry. We further attempted to investigate the biological functions and inhibitory effects of HAI-2 using human papillomavirus (HPV) 16 type SiHa and HPV 18 type HeLa cervical cancer cell lines. Results: There were significant correlations between HAI-2 expression and stage (P = 0.017), lymph node metastasis (P = 0.005) and ovarian metastasis (P = 0.038). Low HAI-2 expression was a significant predictor for a poor prognosis compared with high HAI-2 expression (disease-free survival rate, P = 0.016; overall survival rate, P = 0.021). After transient transfection into the SiHa and HeLa cell lines, HAI-2 showed potential inhibitory effects mediated by reductions in hepsin and matriptase expression, which led to apoptosis by increasing the level of Bak and reducing the level of Bcl-2. Conclusions: The present findings indicate that low HAI-2 expression in cervical cancer may be associated with a poor prognosis. We propose that HAI-2 may represent a therapeutic target for the treatment of cervical cancer.

Research paper thumbnail of Mechanisms for the control of matriptase activity in the absence of sufficient HAI-1

American Journal of Physiology-Cell Physiology, 2011

Matriptase proteolytic activity must be tightly controlled for normal placental development, epid... more Matriptase proteolytic activity must be tightly controlled for normal placental development, epidermal function, and epithelial integrity. Although hepatocyte growth factor activator inhibitor-1 (HAI-1) represents the predominant endogenous inhibitor for matriptase and the protein molar ratio of HAI-1 to matriptase is determined to be >10 in epithelial cells and the majority of carcinoma cells, an inverse HAI-1-to-matriptase ratio is seen in some ovarian and hematopoietic cancer cells. In the current study, cells with insufficient HAI-1 are investigated for the mechanisms through which the activity of matriptase is regulated. When matriptase activation is robustly induced in these cells, activated matriptase rapidly forms two complexes of 100- and 140-kDa in addition to the canonical 120-kDa matriptase-HAI-1 complex already described. Both 100- and 140-kDa complexes contain two-chain, cleaved matriptase but are devoid of gelatinolytic activity. Further biochemical characterizatio...

Research paper thumbnail of Matriptase is inhibited by extravascular antithrombin in epithelial cells but not in most carcinoma cells

AJP: Cell Physiology, 2011

Antithrombin, a major anticoagulant, is robustly transported into extravascular compartments wher... more Antithrombin, a major anticoagulant, is robustly transported into extravascular compartments where its target proteases are largely unknown. This serpin was previously detected in human milk as complexes with matriptase, a membrane-bound serine protease broadly expressed in epithelial and carcinoma cells, and under tight regulation by hepatocyte growth factor activator inhibitor (HAI)-1, a transmembrane Kunitz-type serine protease inhibitor that forms heat-sensitive complexes with active matriptase. In the current study, we detect, in addition to matriptase-HAI-1 complexes, heat-resistant matriptase complexes generated by nontransformed mammary, prostate, and epidermal epithelial cells that we show to be matriptase-antithrombin complexes. These findings suggest that in addition to HAI-1, interstitial antithrombin participates in the regulation of matriptase activity in epithelial cells. This physiological mechanism appears, however, to largely be lost in cancer cells since matriptas...

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

American Journal of Physiology-Cell Physiology, 2005

Hepatocyte growth factor activator inhibitor-1 (HAI-1) was initially identified as cognate inhibi... more 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...

Research paper thumbnail of Increased matriptase zymogen activation in inflammatory skin disorders

American Journal of Physiology-Cell Physiology, 2010

Matriptase, a type 2 transmembrane serine protease, and its inhibitor hepatocyte growth factor ac... more 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 in...

Research paper thumbnail of Simultaneous activation and hepatocyte growth factor activator inhibitor 1-mediated inhibition of matriptase induced at activation foci in human mammary epithelial cells

American Journal of Physiology-Cell Physiology, 2004

Activation of single-chain, latent matriptase, a type II transmembrane serine protease, depends o... more Activation of single-chain, latent matriptase, a type II transmembrane serine protease, depends on the weak proteolytic activity of its own zymogen as well as its cognate inhibitor, hepatocyte growth factor activator inhibitor 1 (HAI-1). Oligomerization of matriptase zymogens and HAI-1, and probably its interaction with other proteins, has been proposed to occur during matriptase activation. In the present study, we examined the cellular events associated with matriptase activation triggered either by the physiological inducer sphingosine 1-phosphate (S1P) or by a chemical inducer, the polyanionic compound suramin. S1P-induced matriptase translocation to cell-cell contacts, where it is activated, is an F-actin polymerization-dependent process. Conversely, suramin-induced matriptase accumulation and activation at vesicle-like structures is an F-actin polymerization-independent process. While matriptase activation can occur at different subcellular locations, both S1P- and suramin-ind...

Research paper thumbnail of Matriptase Is Involved in ErbB-2-Induced Prostate Cancer Cell Invasion

The American Journal of Pathology, 2010

Deregulation of both ErbB-2 signaling and matriptase activity has been associated with human pros... more Deregulation of both ErbB-2 signaling and matriptase activity has been associated with human prostate cancer (PCa) progression. In this communication, we investigated the roles of both ErbB-2 signaling in matriptase zymogen activation and matriptase in ErbB-2-induced PCa malignancy. In a human PCa cell progression model, we observed that advanced PCa C-81 LNCaP cells exhibited an aggressive phenotype with increased cell migration and invasion capacity; these cells concurrently showed both enhanced ErbB-2 phosphorylation and increased matriptase zymogen activation compared with parental C-33 LNCaP cells. Moreover, ErbB2 activation , both ligand-dependent (eg , epidermal growth factor treatment) and ligand-independent (eg , overexpression) , was able to induce matriptase zymogen activation in this cell line. Inhibition of ErbB-2 activity by either the specific inhibitor, AG825 , in epidermal growth factor-treated C-33 LN-CaP cells or ErbB-2 knockdown in C-81 LNCaP cells, reduced matriptase activation. These observations were confirmed by similar studies using both DU145 and PC3 cells. Together , these data suggest that ErbB-2 signaling plays an important role in matriptase zymogen activation. ErbB-2-enhanced matriptase activation was suppressed by a phosphatidylinositol 3-kinase inhibitor (ie, LY294002) but not by a MEK inhibitor (ie, PD98059). Suppression of matriptase expression by small hairpin RNA knockdown in ErbB-2-overex-pressing LNCaP cells dramatically suppressed cancer cell invasion. In summary, our data indicate that ErbB-2 signaling via the phosphatidylinositol 3-kinase pathway results in up-regulated matriptase zymogen activity, which contributes to PCa cell invasion. (Am J

Research paper thumbnail of TMPRSS2, a Serine Protease Expressed in the Prostate on the Apical Surface of Luminal Epithelial Cells and Released into Semen in Prostasomes, Is Misregulated in Prostate Cancer Cells

The American Journal of Pathology, 2010

Research paper thumbnail of Polarized epithelial cells secrete matriptase as a consequence of zymogen activation and HAI-1-mediated inhibition

American Journal of Physiology-Cell Physiology, 2009

Matriptase, a transmembrane serine protease, is broadly expressed by, and crucial for the integri... more Matriptase, a transmembrane serine protease, is broadly expressed by, and crucial for the integrity of, the epithelium. Matriptase is synthesized as a zymogen and undergoes autoactivation to become an active protease that is immediately inhibited by, and forms complexes with, hepatocyte growth factor activator inhibitor (HAI-1). To investigate where matriptase is activated and how it is secreted in vivo, we determined the expression and activation status of matriptase in seminal fluid and urine and the distribution and subcellular localization of the protease in the prostate and kidney. The in vivo studies revealed that while the latent matriptase is localized at the basolateral surface of the ductal epithelial cells of both organs, only matriptase-HAI-1 complexes and not latent matriptase are detected in the body fluids, suggesting that activation, inhibition, and transcytosis of matriptase would have to occur for the secretion of matriptase. These complicated processes involved in...

Research paper thumbnail of Matriptase Expression and Zymogen Activation in Human Pilosebaceous Unit

Journal of Histochemistry & Cytochemistry, 2013

Studies of human genetic disorders and mouse models reveal the important roles of matriptase in h... more Studies of human genetic disorders and mouse models reveal the important roles of matriptase in hair growth. Here, we investigate matriptase expression and zymogen activation in hair follicles. We show: 1) layer-dependent distribution patterns, with much higher matriptase expression in cells of the outer root sheath and matrix cells of the hair bulb than in cells of the inner root sheath; 2) cycle-dependent expression patterns, with matriptase expressed in the anagen and catagen phases of the hair lifecycle, but not in the telogen phase; 3) reduced expression of the matriptase inhibitor, HAI-1, in the catagen phase, suggesting increased proteolytic activity in this phase; and 4) definitive matriptase zymogen activation patterns, with the highest matriptase activation observed in matrix cells and outer root sheath cells in the isthmus/bulge region. In sebaceous glands, matriptase is highly expressed in basal and ductal cells, with much lower expression in the differentiated, lipid-fi...

Research paper thumbnail of Purification from human milk of matriptase complexes with secreted serpins: mechanism for inhibition of matriptase other than HAI-1

American Journal of Physiology-Cell Physiology, 2008

Matriptase, a type 2 transmembrane serine protease, is predominately expressed by epithelial and ... more Matriptase, a type 2 transmembrane serine protease, is predominately expressed by epithelial and carcinoma cells in which hepatocyte growth factor activator inhibitor 1 (HAI-1), a membrane-bound, Kunitz-type serine protease inhibitor, is also expressed. HAI-1 plays dual roles in the regulation of matriptase, as a conventional protease inhibitor and as a factor required for zymogen activation of matriptase. As a consequence, activation of matriptase is immediately followed by HAI-1-mediated inhibition, with the activated matriptase being sequestered into HAI-1 complexes. Matriptase is also expressed by peripheral blood leukocytes, such as monocytes and macrophages; however, in contrast to epithelial cells, monocytes and macrophages were reported not to express HAI-1, suggesting that these leukocytes possess alternate, HAI-1-independent mechanisms regulating the zymogen activation and protease inhibition of matriptase. In the present study, we characterized matriptase complexes of 110...

Research paper thumbnail of Autoactivation of matriptase in vitro: requirement for biomembrane and LDL receptor domain

AJP: Cell Physiology, 2007

In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by ... more In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by lysophospholipids, androgens, and the polyanionic compound suramin. These structurally distinct chemicals induce different signaling pathways and cellular events that somehow, in a cell type-specific manner, lead to activation of matriptase immediately followed by inhibition of matriptase by hepatocyte growth factor activator inhibitor 1 (HAI-1). In the current study, we established an analogous matriptase autoactivation system in an in vitro cell-free setting and showed that a burst of matriptase activation and HAI-1-mediated inhibition spontaneously occurred in the insoluble fractions of cell homogenates and that this in vitro activation could be attenuated by a soluble suppressive factor(s) in cytosolic fractions. Immunofluorescence staining and subcellular fractionation studies revealed that matriptase activation occurred in the perinuclear regions. Solubilization of matriptase from ...

Research paper thumbnail of Matriptase and HAI-1 Are Expressed by Normal and Malignant Epithelial Cells in Vitro and in Vivo

The American Journal of Pathology, 2001

Matriptase and its cognate, Kunitz-type serine protease inhibitor, HAI-1, comprise a newly charac... more Matriptase and its cognate, Kunitz-type serine protease inhibitor, HAI-1, comprise a newly characterized extracellular matrix-degrading protease system that may function as an epithelial membrane activator for other proteases and latent growth factors. Both enzyme and inhibitor have been detected in breast cancer cells, immortalized mammary epithelial cells, and human milk, but not in cultured fibroblasts nor in fibrosarcoma cells. To test the hypothesis that this system is expressed by normal breast epithelium, invasive breast cancers, and other cancers of an epithelial origin (carcinomas) but not in cancers of a mesenchymal origin, we have expanded our expression analysis of matriptase and HAI-1 in vitro and in vivo. Matriptase and HAI-1 were detected at the protein and mRNA levels both in hormone-dependent and hormone-independent cultured breast cancer cells, and this expression correlated with the expression of the epithelial markers E-cadherin or ZO-1. However, none of the breast cancer cell lines tested that express the mesenchymal marker vimentin express matriptase or HAI-1, consistent with an epithelial-selective expression of this system. Expression of matriptase, as determined by Western blot analysis, was observed in primary human breast, gynecological, and colon carcinomas, but not in stromal-derived ovarian tumors and human sarcomas of various origins and histological grades. The epithelial-selective expression of matriptase and HAI-1 was further confirmed in human breast cancers by immunohistochemistry and in situ hybridization, where the expression of the protease and the inhibitor were found in the carcinoma cells and in surrounding normal breast epithelia. The expression of the matriptase/ HAI-1 system by malignant epithelial cells in vivo suggests a possible role for this protease in multiple aspects of the pathophysiology of epithelial malignancy, including invasion and metastasis.

Research paper thumbnail of Increased matriptase zymogen activation by UV irradiation protects keratinocyte from cell death

Journal of dermatological science, Jan 8, 2016

Overexposure to ultraviolet (UV) derived from solar light causes skin damage by causing DNA lesio... more Overexposure to ultraviolet (UV) derived from solar light causes skin damage by causing DNA lesions and the generation of reactive oxygen species (ROS) in keratinocytes and other epidermal cells. The type 2 transmembrane serine protease matriptase has characteristics that allow keratinocytes to respond to/recover from, environmental insults to the skin. This response may depend on its roles in epidermal proliferation and early differentiation, and its rapid activation in response to changes in the cellular chemical milieu, including increased oxidative stress. We investigate the regulation of matriptase activation and its role in the response of the skin to exposure to different parts of the UV spectrum including UVA UVB, and UVR. The activation state and distribution of matriptase in ex vivo UV exposed human skin specimens and sun damaged skin samples were analyzed by immunohistochemistry. HaCaT immortalized human keratinocytes were also used to investigate the mechanism of matript...

Research paper thumbnail of N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin

PloS one, 2015

The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor ... more The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB), is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa) due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with extensive terminal N-acetylglucosamine branching. The two different types of N-glycan differentially mask two epitopes on HAI-2 polypeptide, recognized by two different HAI-2 mAbs. The 30-40-kDa form may be mature HAI-2, and is primarily localized in vesicles/granules. The 25-kDa form is likely immature HAI-2, that remains in the endoplasmic reticulum (ER) in the perinuclear regions of mammary epithelial cells. The two different N-glycans could, therefore, represent different maturation stages of N...

Research paper thumbnail of An Epithelial-Derived Integral Membrane Kunitz-Type Serine Protease Inhibitor in Breast Cancer

Research paper thumbnail of Matriptase activation, an early cellular response to acidosis

Extracellular acidosis often rapidly causes intracellular acidification, alters ion channel activ... more Extracellular acidosis often rapidly causes intracellular acidification, alters ion channel activities, and activates G proteincoupled receptors. In this report, we demonstrated a novel cellular response to acidosis: induction of the zymogen activation of matriptase. Acid-induced matriptase activation is ubiquitous among epithelial and carcinoma cells and is characterized by rapid onset, fast kinetics, and the magnitude of activation seen. Trace amounts of activated matriptase can be detected 1 min after cells are exposed to pH 6.0 buffer, and the vast majority of latent matriptase within the cells is converted to activated matriptase within 20 min. Matriptase activation may be a direct response to proton exposure because acid-induced matriptase activation also occurs in an in vitro, cell-free setting in which intracellular signaling molecules and ion channel activities are largely absent. Acid-induced matriptase activation takes place both on the cell surface and inside the cells, likely due to the parallel intracellular acidification that activates intracellular matriptase. Following matriptase activation, the active enzyme is immediately inhibited by binding to hepatocyte growth factor activator inhibitor 1, resulting in stable matriptase-hepatocyte growth factor activator inhibitor 1 complexes that are rapidly secreted. As an early response to acidosis, matriptase activation can also be induced by perturbation of intracellular pH homeostasis by 5-(N-methyl-N-isobutyl)-amiloride and 5-(N-ethyl-Nisopropyl)-amiloride, both of which inhibit Na ؉ /H ؉ exchangers, and diisothiocyanostilbene-2,2-disulfonic acid, which can inhibit other acid-base ion channels. This study uncovers a novel mechanism regulating proteolysis in epithelial and carcinoma cells, and also demonstrates that a likely function of matriptase is as an early response to acidosis.

Research paper thumbnail of Aberrant regulation favours matriptase proteolysis in neoplastic B-cells that co-express HAI-2

Journal of Enzyme Inhibition and Medicinal Chemistry, 2019

Matriptase is ectopically expressed in neoplastic B-cells, in which matriptase activity is enhanc... more Matriptase is ectopically expressed in neoplastic B-cells, in which matriptase activity is enhanced by negligible expression of its endogenous inhibitor, hepatocyte growth factor activator inhibitor (HAI)-1. HAI-1, however, is also involved in matriptase synthesis and intracellular trafficking. The lack of HAI-1 indicates that other related inhibitor, such as HAI-2, might be expressed. Here, we show that HAI-2 is commonly co-expressed in matriptase-expressing neoplastic B-cells. The level of active matriptase shed after induction of matriptase zymogen activation in 7 different neoplastic B-cells was next determined and characterised. Our data reveal that active matriptase can only be generated and shed by those cells able to activate matriptase and in a rough correlation with the levels of matriptase protein. While HAI-2 can potently inhibit matriptase, the levels of active matriptase are not proportionally suppressed in those cells with high HAI-2. Our survey suggests that matriptase proteolysis might aberrantly remain high in neoplastic B-cells regardless of the levels of HAI-2.

Research paper thumbnail of Molecular Cloning of cDNA for Matriptase, a Matrix-degrading Serine Protease with Trypsin-like Activity

Journal of Biological Chemistry, 1999

A major protease from human breast cancer cells was previously detected by gelatin zymography and... more A major protease from human breast cancer cells was previously detected by gelatin zymography and proposed to play a role in breast cancer invasion and metastasis. To structurally characterize the enzyme, we isolated a cDNA encoding the protease. Analysis of the cDNA reveals three sequence motifs: a carboxyl-terminal region with similarity to the trypsin-like serine proteases, four tandem cysteine-rich repeats homologous to the low density lipoprotein receptor, and two copies of tandem repeats originally found in the complement subcomponents C1r and C1s. By comparison with other serine proteases, the active-site triad was identified as His-484, Asp-539, and Ser-633. The protease contains a characteristic Arg-Val-Val-Gly-Gly motif that may serve as a proteolytic activation site. The bottom of the substrate specificity pocket was identified to be Asp-627 by comparison with other trypsin-like serine proteases. In addition, this protease exhibits trypsin-like activity as defined by cleavage of synthetic substrates with Arg or Lys as the P1 site. Thus, the protease is a mosaic protein with broad spectrum cleavage activity and two potential regulatory modules. Given its ability to degrade extracellular matrix and its trypsin-like activity, the name matriptase is proposed for the protease. Elevated proteolytic activity has been implicated in neoplastic progression. Although the exact role(s) of proteolytic enzymes in the progression of tumor remains unclear, it seems that proteases may be involved in almost every step of the development and spread of cancer. A widely proposed view is that proteases contribute to the degradation of extracellular matrix and to tissue remodeling and are necessary for cancer invasion and metastasis. A wide array of extracellular matrixdegrading proteases have been discovered, the expression of some of which correlates with tumor progression, as reviewed by Magnatti and Rifkin (1). The plasmin/urokinase-type plas

Research paper thumbnail of Antimetastatic activity isolated from Colocasia esculenta (taro)

Anti-Cancer Drugs, 2012

Breast cancer mortality is primarily due to the occurrence of metastatic disease. We have identif... more Breast cancer mortality is primarily due to the occurrence of metastatic disease. We have identified a novel potential therapeutic agent derived from an edible root of the plant Colocasia esculenta, commonly known as taro, that has demonstrable activity in a preclinical model of metastatic breast cancer and that should have minimal toxicity. We have shown for the first time that a water-soluble extract of taro (TE) potently inhibits lung colonizing ability as well as spontaneous metastasis from mammary gland-implanted tumors, in a murine model of highly metastatic ER, PR and Her-2/neu negative breast cancer. TE modestly inhibits proliferation of some, but not all, breast and prostate cancer cell lines. Morphologic changes including cell rounding were observed. Tumor cell migration was completely blocked by TE. TE treatment also inhibited prostaglandin E 2 (PGE 2) synthesis and downregulated cyclooxygenase (COX) 1 and 2 mRNA expression. We purified the active compound(s) to near homogeneity with antimetastatic activity comparable to stock TE. The active compound with a native size of approximately 25 kD contains two fragments of nearly equal size. The N-terminal amino acid sequencing of both fragments reveals that the active compound is highly related to three taro proteins; 12 kD storage protein, tarin and lectin. All are similar in terms of amino acid sequence, post-translational processing and all contain a carbohydrate-binding domain. This is the first report describing a compound(s) derived from taro, that potently and specifically inhibits tumor metastasis.

Research paper thumbnail of Hepatocyte growth factor activator inhibitor-2 (HAI-2) is a favorable prognosis marker and inhibits cell growth through the apoptotic pathway in cervical cancer

Annals of Oncology, 2008

Background: In light of the poor prognosis for cervical cancer, research continues into the devel... more Background: In light of the poor prognosis for cervical cancer, research continues into the development of innovative and efficacious treatment modalities for this disease. We investigated the role of hepatocyte growth factor activator inhibitor-2 (HAI-2) and evaluated its clinical importance in cervical cancer. Patients and methods: HAI-2 expression was examined in cervical cancer specimens (n = 52) by immunohistochemistry. We further attempted to investigate the biological functions and inhibitory effects of HAI-2 using human papillomavirus (HPV) 16 type SiHa and HPV 18 type HeLa cervical cancer cell lines. Results: There were significant correlations between HAI-2 expression and stage (P = 0.017), lymph node metastasis (P = 0.005) and ovarian metastasis (P = 0.038). Low HAI-2 expression was a significant predictor for a poor prognosis compared with high HAI-2 expression (disease-free survival rate, P = 0.016; overall survival rate, P = 0.021). After transient transfection into the SiHa and HeLa cell lines, HAI-2 showed potential inhibitory effects mediated by reductions in hepsin and matriptase expression, which led to apoptosis by increasing the level of Bak and reducing the level of Bcl-2. Conclusions: The present findings indicate that low HAI-2 expression in cervical cancer may be associated with a poor prognosis. We propose that HAI-2 may represent a therapeutic target for the treatment of cervical cancer.

Research paper thumbnail of Mechanisms for the control of matriptase activity in the absence of sufficient HAI-1

American Journal of Physiology-Cell Physiology, 2011

Matriptase proteolytic activity must be tightly controlled for normal placental development, epid... more Matriptase proteolytic activity must be tightly controlled for normal placental development, epidermal function, and epithelial integrity. Although hepatocyte growth factor activator inhibitor-1 (HAI-1) represents the predominant endogenous inhibitor for matriptase and the protein molar ratio of HAI-1 to matriptase is determined to be >10 in epithelial cells and the majority of carcinoma cells, an inverse HAI-1-to-matriptase ratio is seen in some ovarian and hematopoietic cancer cells. In the current study, cells with insufficient HAI-1 are investigated for the mechanisms through which the activity of matriptase is regulated. When matriptase activation is robustly induced in these cells, activated matriptase rapidly forms two complexes of 100- and 140-kDa in addition to the canonical 120-kDa matriptase-HAI-1 complex already described. Both 100- and 140-kDa complexes contain two-chain, cleaved matriptase but are devoid of gelatinolytic activity. Further biochemical characterizatio...

Research paper thumbnail of Matriptase is inhibited by extravascular antithrombin in epithelial cells but not in most carcinoma cells

AJP: Cell Physiology, 2011

Antithrombin, a major anticoagulant, is robustly transported into extravascular compartments wher... more Antithrombin, a major anticoagulant, is robustly transported into extravascular compartments where its target proteases are largely unknown. This serpin was previously detected in human milk as complexes with matriptase, a membrane-bound serine protease broadly expressed in epithelial and carcinoma cells, and under tight regulation by hepatocyte growth factor activator inhibitor (HAI)-1, a transmembrane Kunitz-type serine protease inhibitor that forms heat-sensitive complexes with active matriptase. In the current study, we detect, in addition to matriptase-HAI-1 complexes, heat-resistant matriptase complexes generated by nontransformed mammary, prostate, and epidermal epithelial cells that we show to be matriptase-antithrombin complexes. These findings suggest that in addition to HAI-1, interstitial antithrombin participates in the regulation of matriptase activity in epithelial cells. This physiological mechanism appears, however, to largely be lost in cancer cells since matriptas...

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

American Journal of Physiology-Cell Physiology, 2005

Hepatocyte growth factor activator inhibitor-1 (HAI-1) was initially identified as cognate inhibi... more 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...

Research paper thumbnail of Increased matriptase zymogen activation in inflammatory skin disorders

American Journal of Physiology-Cell Physiology, 2010

Matriptase, a type 2 transmembrane serine protease, and its inhibitor hepatocyte growth factor ac... more 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 in...

Research paper thumbnail of Simultaneous activation and hepatocyte growth factor activator inhibitor 1-mediated inhibition of matriptase induced at activation foci in human mammary epithelial cells

American Journal of Physiology-Cell Physiology, 2004

Activation of single-chain, latent matriptase, a type II transmembrane serine protease, depends o... more Activation of single-chain, latent matriptase, a type II transmembrane serine protease, depends on the weak proteolytic activity of its own zymogen as well as its cognate inhibitor, hepatocyte growth factor activator inhibitor 1 (HAI-1). Oligomerization of matriptase zymogens and HAI-1, and probably its interaction with other proteins, has been proposed to occur during matriptase activation. In the present study, we examined the cellular events associated with matriptase activation triggered either by the physiological inducer sphingosine 1-phosphate (S1P) or by a chemical inducer, the polyanionic compound suramin. S1P-induced matriptase translocation to cell-cell contacts, where it is activated, is an F-actin polymerization-dependent process. Conversely, suramin-induced matriptase accumulation and activation at vesicle-like structures is an F-actin polymerization-independent process. While matriptase activation can occur at different subcellular locations, both S1P- and suramin-ind...

Research paper thumbnail of Matriptase Is Involved in ErbB-2-Induced Prostate Cancer Cell Invasion

The American Journal of Pathology, 2010

Deregulation of both ErbB-2 signaling and matriptase activity has been associated with human pros... more Deregulation of both ErbB-2 signaling and matriptase activity has been associated with human prostate cancer (PCa) progression. In this communication, we investigated the roles of both ErbB-2 signaling in matriptase zymogen activation and matriptase in ErbB-2-induced PCa malignancy. In a human PCa cell progression model, we observed that advanced PCa C-81 LNCaP cells exhibited an aggressive phenotype with increased cell migration and invasion capacity; these cells concurrently showed both enhanced ErbB-2 phosphorylation and increased matriptase zymogen activation compared with parental C-33 LNCaP cells. Moreover, ErbB2 activation , both ligand-dependent (eg , epidermal growth factor treatment) and ligand-independent (eg , overexpression) , was able to induce matriptase zymogen activation in this cell line. Inhibition of ErbB-2 activity by either the specific inhibitor, AG825 , in epidermal growth factor-treated C-33 LN-CaP cells or ErbB-2 knockdown in C-81 LNCaP cells, reduced matriptase activation. These observations were confirmed by similar studies using both DU145 and PC3 cells. Together , these data suggest that ErbB-2 signaling plays an important role in matriptase zymogen activation. ErbB-2-enhanced matriptase activation was suppressed by a phosphatidylinositol 3-kinase inhibitor (ie, LY294002) but not by a MEK inhibitor (ie, PD98059). Suppression of matriptase expression by small hairpin RNA knockdown in ErbB-2-overex-pressing LNCaP cells dramatically suppressed cancer cell invasion. In summary, our data indicate that ErbB-2 signaling via the phosphatidylinositol 3-kinase pathway results in up-regulated matriptase zymogen activity, which contributes to PCa cell invasion. (Am J

Research paper thumbnail of TMPRSS2, a Serine Protease Expressed in the Prostate on the Apical Surface of Luminal Epithelial Cells and Released into Semen in Prostasomes, Is Misregulated in Prostate Cancer Cells

The American Journal of Pathology, 2010

Research paper thumbnail of Polarized epithelial cells secrete matriptase as a consequence of zymogen activation and HAI-1-mediated inhibition

American Journal of Physiology-Cell Physiology, 2009

Matriptase, a transmembrane serine protease, is broadly expressed by, and crucial for the integri... more Matriptase, a transmembrane serine protease, is broadly expressed by, and crucial for the integrity of, the epithelium. Matriptase is synthesized as a zymogen and undergoes autoactivation to become an active protease that is immediately inhibited by, and forms complexes with, hepatocyte growth factor activator inhibitor (HAI-1). To investigate where matriptase is activated and how it is secreted in vivo, we determined the expression and activation status of matriptase in seminal fluid and urine and the distribution and subcellular localization of the protease in the prostate and kidney. The in vivo studies revealed that while the latent matriptase is localized at the basolateral surface of the ductal epithelial cells of both organs, only matriptase-HAI-1 complexes and not latent matriptase are detected in the body fluids, suggesting that activation, inhibition, and transcytosis of matriptase would have to occur for the secretion of matriptase. These complicated processes involved in...

Research paper thumbnail of Matriptase Expression and Zymogen Activation in Human Pilosebaceous Unit

Journal of Histochemistry & Cytochemistry, 2013

Studies of human genetic disorders and mouse models reveal the important roles of matriptase in h... more Studies of human genetic disorders and mouse models reveal the important roles of matriptase in hair growth. Here, we investigate matriptase expression and zymogen activation in hair follicles. We show: 1) layer-dependent distribution patterns, with much higher matriptase expression in cells of the outer root sheath and matrix cells of the hair bulb than in cells of the inner root sheath; 2) cycle-dependent expression patterns, with matriptase expressed in the anagen and catagen phases of the hair lifecycle, but not in the telogen phase; 3) reduced expression of the matriptase inhibitor, HAI-1, in the catagen phase, suggesting increased proteolytic activity in this phase; and 4) definitive matriptase zymogen activation patterns, with the highest matriptase activation observed in matrix cells and outer root sheath cells in the isthmus/bulge region. In sebaceous glands, matriptase is highly expressed in basal and ductal cells, with much lower expression in the differentiated, lipid-fi...

Research paper thumbnail of Purification from human milk of matriptase complexes with secreted serpins: mechanism for inhibition of matriptase other than HAI-1

American Journal of Physiology-Cell Physiology, 2008

Matriptase, a type 2 transmembrane serine protease, is predominately expressed by epithelial and ... more Matriptase, a type 2 transmembrane serine protease, is predominately expressed by epithelial and carcinoma cells in which hepatocyte growth factor activator inhibitor 1 (HAI-1), a membrane-bound, Kunitz-type serine protease inhibitor, is also expressed. HAI-1 plays dual roles in the regulation of matriptase, as a conventional protease inhibitor and as a factor required for zymogen activation of matriptase. As a consequence, activation of matriptase is immediately followed by HAI-1-mediated inhibition, with the activated matriptase being sequestered into HAI-1 complexes. Matriptase is also expressed by peripheral blood leukocytes, such as monocytes and macrophages; however, in contrast to epithelial cells, monocytes and macrophages were reported not to express HAI-1, suggesting that these leukocytes possess alternate, HAI-1-independent mechanisms regulating the zymogen activation and protease inhibition of matriptase. In the present study, we characterized matriptase complexes of 110...

Research paper thumbnail of Autoactivation of matriptase in vitro: requirement for biomembrane and LDL receptor domain

AJP: Cell Physiology, 2007

In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by ... more In live cells, autoactivation of matriptase, a membrane-bound serine protease, can be induced by lysophospholipids, androgens, and the polyanionic compound suramin. These structurally distinct chemicals induce different signaling pathways and cellular events that somehow, in a cell type-specific manner, lead to activation of matriptase immediately followed by inhibition of matriptase by hepatocyte growth factor activator inhibitor 1 (HAI-1). In the current study, we established an analogous matriptase autoactivation system in an in vitro cell-free setting and showed that a burst of matriptase activation and HAI-1-mediated inhibition spontaneously occurred in the insoluble fractions of cell homogenates and that this in vitro activation could be attenuated by a soluble suppressive factor(s) in cytosolic fractions. Immunofluorescence staining and subcellular fractionation studies revealed that matriptase activation occurred in the perinuclear regions. Solubilization of matriptase from ...

Research paper thumbnail of Matriptase and HAI-1 Are Expressed by Normal and Malignant Epithelial Cells in Vitro and in Vivo

The American Journal of Pathology, 2001

Matriptase and its cognate, Kunitz-type serine protease inhibitor, HAI-1, comprise a newly charac... more Matriptase and its cognate, Kunitz-type serine protease inhibitor, HAI-1, comprise a newly characterized extracellular matrix-degrading protease system that may function as an epithelial membrane activator for other proteases and latent growth factors. Both enzyme and inhibitor have been detected in breast cancer cells, immortalized mammary epithelial cells, and human milk, but not in cultured fibroblasts nor in fibrosarcoma cells. To test the hypothesis that this system is expressed by normal breast epithelium, invasive breast cancers, and other cancers of an epithelial origin (carcinomas) but not in cancers of a mesenchymal origin, we have expanded our expression analysis of matriptase and HAI-1 in vitro and in vivo. Matriptase and HAI-1 were detected at the protein and mRNA levels both in hormone-dependent and hormone-independent cultured breast cancer cells, and this expression correlated with the expression of the epithelial markers E-cadherin or ZO-1. However, none of the breast cancer cell lines tested that express the mesenchymal marker vimentin express matriptase or HAI-1, consistent with an epithelial-selective expression of this system. Expression of matriptase, as determined by Western blot analysis, was observed in primary human breast, gynecological, and colon carcinomas, but not in stromal-derived ovarian tumors and human sarcomas of various origins and histological grades. The epithelial-selective expression of matriptase and HAI-1 was further confirmed in human breast cancers by immunohistochemistry and in situ hybridization, where the expression of the protease and the inhibitor were found in the carcinoma cells and in surrounding normal breast epithelia. The expression of the matriptase/ HAI-1 system by malignant epithelial cells in vivo suggests a possible role for this protease in multiple aspects of the pathophysiology of epithelial malignancy, including invasion and metastasis.

Research paper thumbnail of Increased matriptase zymogen activation by UV irradiation protects keratinocyte from cell death

Journal of dermatological science, Jan 8, 2016

Overexposure to ultraviolet (UV) derived from solar light causes skin damage by causing DNA lesio... more Overexposure to ultraviolet (UV) derived from solar light causes skin damage by causing DNA lesions and the generation of reactive oxygen species (ROS) in keratinocytes and other epidermal cells. The type 2 transmembrane serine protease matriptase has characteristics that allow keratinocytes to respond to/recover from, environmental insults to the skin. This response may depend on its roles in epidermal proliferation and early differentiation, and its rapid activation in response to changes in the cellular chemical milieu, including increased oxidative stress. We investigate the regulation of matriptase activation and its role in the response of the skin to exposure to different parts of the UV spectrum including UVA UVB, and UVR. The activation state and distribution of matriptase in ex vivo UV exposed human skin specimens and sun damaged skin samples were analyzed by immunohistochemistry. HaCaT immortalized human keratinocytes were also used to investigate the mechanism of matript...

Research paper thumbnail of N-Glycan Branching Affects the Subcellular Distribution of and Inhibition of Matriptase by HAI-2/Placental Bikunin

PloS one, 2015

The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor ... more The gene product of SPINT 2, that encodes a transmembrane, Kunitz-type serine protease inhibitor independently designated as HAI-2 or placenta bikunin (PB), is involved in regulation of sodium absorption in human gastrointestinal track. Here, we show that SPINT 2 is expressed as two species of different size (30-40- versus 25-kDa) due to different N-glycans on Asn-57. The N-glycan on 25-kDa HAI-2 appears to be of the oligomannose type and that on 30-40-kDa HAI-2 to be of complex type with extensive terminal N-acetylglucosamine branching. The two different types of N-glycan differentially mask two epitopes on HAI-2 polypeptide, recognized by two different HAI-2 mAbs. The 30-40-kDa form may be mature HAI-2, and is primarily localized in vesicles/granules. The 25-kDa form is likely immature HAI-2, that remains in the endoplasmic reticulum (ER) in the perinuclear regions of mammary epithelial cells. The two different N-glycans could, therefore, represent different maturation stages of N...

Research paper thumbnail of An Epithelial-Derived Integral Membrane Kunitz-Type Serine Protease Inhibitor in Breast Cancer

Research paper thumbnail of Matriptase activation, an early cellular response to acidosis

Extracellular acidosis often rapidly causes intracellular acidification, alters ion channel activ... more Extracellular acidosis often rapidly causes intracellular acidification, alters ion channel activities, and activates G proteincoupled receptors. In this report, we demonstrated a novel cellular response to acidosis: induction of the zymogen activation of matriptase. Acid-induced matriptase activation is ubiquitous among epithelial and carcinoma cells and is characterized by rapid onset, fast kinetics, and the magnitude of activation seen. Trace amounts of activated matriptase can be detected 1 min after cells are exposed to pH 6.0 buffer, and the vast majority of latent matriptase within the cells is converted to activated matriptase within 20 min. Matriptase activation may be a direct response to proton exposure because acid-induced matriptase activation also occurs in an in vitro, cell-free setting in which intracellular signaling molecules and ion channel activities are largely absent. Acid-induced matriptase activation takes place both on the cell surface and inside the cells, likely due to the parallel intracellular acidification that activates intracellular matriptase. Following matriptase activation, the active enzyme is immediately inhibited by binding to hepatocyte growth factor activator inhibitor 1, resulting in stable matriptase-hepatocyte growth factor activator inhibitor 1 complexes that are rapidly secreted. As an early response to acidosis, matriptase activation can also be induced by perturbation of intracellular pH homeostasis by 5-(N-methyl-N-isobutyl)-amiloride and 5-(N-ethyl-Nisopropyl)-amiloride, both of which inhibit Na ؉ /H ؉ exchangers, and diisothiocyanostilbene-2,2-disulfonic acid, which can inhibit other acid-base ion channels. This study uncovers a novel mechanism regulating proteolysis in epithelial and carcinoma cells, and also demonstrates that a likely function of matriptase is as an early response to acidosis.