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Wen-tien Chen - One of the best experts on this subject based on the ideXlab platform.
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The propensity of invasive circulating tumor cells (iCTCs) in metastatic progression and therapeutic responsiveness.
Cancer medicine, 2019Co-Authors: Huan Dong, Donghai Chen, Qiang Zhao, Michael L. Pearl, Shaun Tulley, Leong Cho, Wen-tien ChenAbstract:Circulating tumor cells (CTCs) are important clinical indicators of metastatic progression and treatment efficacy. However, because of their low number and heterogeneity, reliable patient-derived CTC models are not readily available. We report here the isolation and characterization of the invasive population of CTCs, iCTCs, from blood of 10 patients with epithelial ovarian cancer (EOC) and one pancreatic cancer patient based on the avidity of tumor cells toward an artificial collagen-based adhesion matrix (CAM), in comparison with tumor progenitor (TP) cells isolated from tumor cell lines, tumors and ascites from EOC patients. CAM-avid cells identified to be iCTCs were indistinguishable with TP cells using either functional CAM uptake or surface markers (Seprase and CD44). In addition, iCTCs were characterized using peritoneal and spontaneous metastasis models in vivo to evaluate their metastatic propensity and therapeutic response. TP cells and iCTCs had a doubling time of about 34-42 hours. TP cells were rare (
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Transcriptional regulation of Seprase in invasive melanoma cells by transforming growth factor-β signaling.
The Journal of biological chemistry, 2014Co-Authors: Shaun Tulley, Wen-tien ChenAbstract:The tumor invasive phenotype driven by Seprase expression/activity has been widely examined in an array of malignant tumor cell types; however, very little is known about the transcriptional regulation of this critical protease. Seprase (also named fibroblast activation protein-α, antiplasmin-cleaving enzyme, and dipeptidyl prolyl peptidase 5) is expressed at high levels by stromal fibroblast, endothelial, and tumor cells in a variety of invasive tumors but is undetectable in the majority of normal adult tissues. To examine the transcriptional regulation of the gene, we cloned the human Seprase promoter and demonstrated that endogenous Seprase expression and exogenous Seprase promoter activity are high in invasive melanoma cells but not in non-invasive melanoma cells/primary melanocytes. In addition, we identified a crucial TGF-β-responsive cis-regulatory element in the proximal Seprase promoter region that enabled robust transcriptional activation of the gene. Treatment of metastatic but not normal/non-invasive cells with TGF-β1 caused a rapid and profound up-regulation of endogenous Seprase mRNA, which coincided with an abolishment of the negative regulator c-Ski, and an increase in binding of Smad3/4 to the Seprase promoter in vivo. Blocking TGF-β signaling in invasive melanoma cells through overexpression of c-Ski, chemically using SB-431542, or with a neutralizing antibody against TGF-β significantly reduced Seprase mRNA levels. Strikingly, RNAi of Seprase in invasive cells greatly diminished their invasive potential in vitro as did blocking TGF-β signaling using SB-431542. Altogether, we found that Seprase is transcriptionally up-regulated in invasive melanoma cells via the canonical TGF-β signaling pathway, supporting the roles of both TGF-β and Seprase in tumor invasion and metastasis.
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sphingosine 1 phosphate stimulates cell migration and active Seprase expression in human endothelial cells
山梨医科学雑誌, 2013Co-Authors: Hirotaka Okamoto, Wen-tien Chen, Masako Mitsumata, Tetsu Yamane, Yutaka Yatomi, Kaneo Satoh, Yukio Ozaki, Hideki FujiiAbstract:Objective: Sphingosine 1-phosphate (Sph-1-P), a bioactive lysophospholipid present in the plasma, is released from activated platelets. Our previous study dem- onstrated that Sph-1-P promoted the spreading on and migration of human umbilical vein endothelial cells (HUVEC) through the extracellular matrix (ECM), suggesting a possible induction of cell surface proteases in the Sph-1-P activated endothelial cells. In the present study, we examined whether Seprase, a type II transmembrane serine protease (TTSP) usually absent in tissue cells, can be induced in endothelial cells acti- vated by Sph-1-P. Methods: HUVEC migration through ECM was examined by modifi ed Boyden chamber assay. Western blotting and immunoprecipitation, using anti-Seprase monoclonal antibodies (mAbs), were used to confi rm the Seprase expression of HUVEC. Results: We show that Sph-1-P enhanced expression of active form Seprase in a time- and dose-dependent manner in HUVEC. The Sph-1-P inducible active form Seprase could be blocked by pertussis toxin and by C3 transferase, which inactivate Gi-type heterotrimetric G proteins and Rho, respectively. Conclusion: These results show that Sph-1-P can regulate migration of endothelial cells by inducing active form Seprase expression, which, in turn, is mediated through a Gi-coupled cell surface receptor and the Rho protein.
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Plasma Seprase and DPP4 Levels as Markers of Disease and Prognosis in Cancer
Disease markers, 2012Co-Authors: Mazyar Javidroozi, Stanley Zucker, Wen-tien ChenAbstract:Seprase (fibroblast activation protein α) has been examined as an invasion biomarker for various types of solid tumors. We studied whether plasma levels of Seprase and homologous protease, DPP4 in cancer might serve as tumor biomarkers. We developed sensitive and specific Enzyme-Linked Immunosorbent Assays (ELISAs) to measure these proteases. In 747 plasma samples (from 139 healthy volunteers and 561 cancer patients), mean Seprase and DPP4 levels were 0.51 ± 0.30 and 4.65 ± 6.37 μg/mL, respectively, and they were correlated with each other (R2 = 0.382). Plasma DPP4 and Seprase levels were significantly lower in cancer patients compared with healthy subjects (4.38 versus 5.65 μg/mL, p < 0.001 for DPP4; 0.46 versus 0.66 μg/mL, p < 0.001 for Seprase). Higher DPP4 was associated with better survival in all cancers combined (n = 346) as well as in head and neck malignancies (n = 38). Higher Seprase was associated with better survival in all non-metastatic cancers combined (n = 151) as well as head and neck malignancies, but worse survival in colorectal cancers (n = 47). This study demonstrates that in contrast to the high expression in solid tumors, plasma concentrations of Seprase and DPP4 are reduced and correlate inversely with survival in most types of cancer, suggesting that these circulating proteases represent useful tumor markers.
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Seprase, dipeptidyl peptidase IV and urokinase-type plasminogen activator expression in dysplasia and invasive squamous cell carcinoma of the esophagus. A study of 229 cases from Anyang Tumor Hospital, Henan Province, China.
Oncology, 2008Co-Authors: Mariusz Adam Goscinski, Wen-tien Chen, Zhenhe Suo, Jahn M. Nesland, Malgorzata Zakrzewska, Junsheng Wang, Shanshen Zhang, Vivi Ann Flørenes, Karl Erik GierckskyAbstract:Objective: Seprase, dipeptidyl peptidase IV (DPPIV) and urokinase-type plasminogen activator (uPA) play a crucial role in the degradation of the extracellular matrix and in the progression of various human tumors. However, their pathophysiologic significance in esophageal carcinoma has not yet been fully elucidated. Methods: The expression of Seprase, DPPIV and uPA in esophageal dysplasia, squamous cell carcinoma (SCC) and normal epithelium was examined by immunohistochemistry. Results: Seprase, DPPIV and uPA immunoreactivity was found in dysplastic and cancer cells as well as in stromal cells adjacent to dysplasia and cancer sites, but not in normal epithelium. We found a significant association between uPA expression and sex, tumor size and histological classification in carcinomas. High expression of DPPIV in cancer cells correlated with longer survival of the patients. No significant associations between Seprase and clinicopathological features either in dysplasia or in carcinomas were found. Finally, we demonstrated higher levels of Seprase, DPPIV and uPA in SCC cell lines than in normal esophageal epithelial cell lines. Conclusions: Our results showed that Seprase, DPPIV and uPA are expressed in both premalignant and malignant forms of SCC, but are lacking in normal esophageal epithelia, suggesting that they are involved in SCC neoplastic progression.
Thomas Kelly - One of the best experts on this subject based on the ideXlab platform.
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Seprase Promotes Rapid Tumor Growth and Increased Microvessel Density in a Mouse Model of Human Breast Cancer
Cancer research, 2004Co-Authors: Yan Huang, Sophie Wang, Thomas KellyAbstract:Seprase is a cell surface serine protease that is expressed to high levels by invading human breast carcinoma cells. To investigate the role of Seprase in breast cancer, MDA MB-231 human mammary adenocarcinoma cells were engineered to express active Seprase to high levels. All cells grow rapidly in cell culture. But differences are discovered when the cells are tested for tumorigenicity, growth, and microvessel density by implantation into the mammary fat pads of female severe combined immunodeficient mice. Control transfectants that do not express Seprase grow slowly whereas cells that express Seprase to high levels form fast-growing tumors that are highly vascular. Microvessel density is elevated in tumors of two different lines of Seprase transfectants to 146 ± 67.4 and 144 ± 33.42 vessels/mm 2 as compared with 50.5 ± 12.9 vessels/mm 2 for tumors of control-transfected cells that do not express Seprase. Seprase-expressing cells are better able to attract blood vessels and exhibit rapid tumor growth.
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Seprase, a membrane-bound protease, alleviates the serum growth requirement of human breast cancer cells
Clinical & Experimental Metastasis, 2003Co-Authors: Johnna D. Goodman, Tricia L. Rozypal, Thomas KellyAbstract:Seprase is a cell surface serine protease that is expressed to high levels by infiltrating ductal carcinomas of the breast but its function in malignancy is unknown. MDA-MB-435 (WT435) and MDA-MB-436 (WT436) human breast cancer cells express high levels of Seprase as do the carcinoma cells in tumors of human breast cancer patients. To investigate its role in the pathobiology of breast cancer, Seprase was specifically reduced in WT436 and WT435 cells by expression of antisense Seprase cDNA. Decreased expression of Seprase was confirmed in the antisense transfectants by zymography, immunoblotting, and fluorescence-activated cell sorting of cells labeled with antibody to Seprase. Control-transfectants continued to express high levels of Seprase. Seprase-deficient cells growing on type I collagen gels reveal a markedly different morphology than the parental or control-transfected cells that express high levels of Seprase. The Seprase-deficient cells grow in islands and aggregates of tightly attached cells while cells with high Seprase expression grow as groups of separate individual cells. Interestingly, the aggregated growth of the Seprase-deficient cells was not correlated with increased expression of E-cadherin. Seprase-deficient breast cancer cells also exhibit altered growth properties. Seprase-deficient cells and those with high Seprase levels proliferate in serum-containing media. However, in serum-free medium Seprase-deficient cells proliferate much more slowly than their Seprase-expressing counterparts. These findings indicate that Seprase promotes the aberrant growth of breast cancer cells by reducing their dependence on exogenous growth factors. Seprase may contribute to the pathogenesis of breast cancer by promoting growth of the primary tumor and by facilitating the growth of breast cancer cells in metastases at other sites of the body.
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Seprase complexes in cellular invasiveness
Cancer and Metastasis Reviews, 2003Co-Authors: Wen-tien Chen, Thomas KellyAbstract:A group of type II integral serine proteases, including dipeptidyl peptidase IV (DPP4/CD26), Seprase/fibroblast activation protein α (FAPα) and related type II transmembrane prolyl serine peptidases, exert their mechanisms of action on the cell surface. DPP4 and Seprase exhibit multiple functions due to their abilities to form complexes with each other and to interact with other membrane-associated molecules. Localization of the protease complexes at cell surface protrusions, called invadopodia, may have a prominent role in processing soluble factors (including chemokines and neuropeptide Y) and in degrading locally extracellular matrix components, that are essential to the cell migration and matrix invasion occurring during tumor invasion, angiogenesis and metastasis.
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DPPIV, Seprase, and related serine peptidases in multiple cellular functions
Current Topics in Developmental Biology, 2003Co-Authors: Wen-tien Chen, Thomas Kelly, Giulio GhersiAbstract:Publisher Summary Serine integral membrane peptidases (SIMP), including DPPIV, Seprase, and related prolyl peptidases, which are both Pro-Xaa cleaving enzymes and adhesion molecules, are likely to emerge as an important protease family. The main functions of SIMPs reside in their proteolytic and adhesive capacities, thus influencing cellular activities, migration, and invasion. These membrane proteases may form a physically and functionally linked complex at invadopodia during cellular invasion. The cysteine-rich domain of some peptidases exhibits the capability of binding to multiple molecules. This could allow not only activation of the peptidases themselves but also association with other membrane proteases to participate in cooperative extracellular matrix (ECM) protein degradation at invadopodia during cancer invasion. This chapter focuses on a small group of membrane serine peptidases, the SIMP, that are inducible, specific for proline-containing peptides and macromolecules, and active on the cell surface. Prototypes of SIMP members are DPPIV and Seprase. Other SIMP-related peptidases, including quiescent cell proline aminodipeptidase (QPP), prolyl carboxy-peptidase (PCP), prolyl endopeptidase (PEP), dipeptidyl peptidase 6 (DPP6), dipeptidyl peptidase 8 (DPP8), dipeptidyl peptidase 9 (DDP9), attractin, dipeptidyl peptidase II (DPPII), and dipeptidyl peptidase IV-β (DPPIV-β), have subtle structural and functional differences from DPPIV and Seprase, and they are also discussed in the chapter.
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Evaluation of Seprase activity
Clinical & Experimental Metastasis, 1999Co-Authors: Thomas KellyAbstract:Seprase is a serine protease that is integral to the plasma membrane and is overexpressed by invasive tumor cells (Piñeiro-Sánchez et al., J Biol Chem 1997; 272: 7595–601; Monsky et al., Cancer Res 1994; 54: 5702–10). Seprase activity is most often assessed by zymography, which is not a quantitative assay. This study establishes a relatively simple and quantitative method for determining Seprase activity. The degradation of a 3H-gelatin substrate is measured in the presence of 5 mM EDTA which inhibits matrix metalloproteinases but not Seprase. The quantitative character of the assay was demonstrated using partially purified Seprase from chicken embryos, a preparation that lacks detectable matrix metalloproteinase activity. In this assay, release of 3H-gelatin fragments is linear over time for 1.5 μg/assay Seprase concentration as well as for preparations concentrated or diluted by five fold (7.5 μg/assay and 0.3 μg/assay respectively). Additional experiments were performed to validate the quantification of Seprase activity using the radiographic assay by comparing the results to zymography. Exposure to 22 or 37 °C results in maximal Seprase activity while exposure to 80 or 100 °C completely abolishes Seprase activity in both zymography and the radiographic assay. Exposure to 60 °C abolished Seprase activity as judged by zymography, but about 50% gelatinase activity was observed using the 3H-gelatin substrate. Immunopreciptiation with Seprase-specific antibody specifically removed Seprase and lowered the Seprase activity remaining in the extracts as judged by both assays. Investigation of the Seprase that was partially purified from human breast cancer tissue revealed that its specific activity (cpm gelatin fragments released/ {mg protein×h}) is five times greater than that of Seprase purified from chicken embryos. This assay will be useful for determining the Seprase activity in extracts of tumor tissues and cells as well as for identifying inhibitors of Seprase.
Yunyun Yeh - One of the best experts on this subject based on the ideXlab platform.
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The protease complex consisting of dipeptidyl peptidase IV and Seprase plays a role in the migration and invasion of human endothelial cells in collagenous matrices.
Cancer research, 2006Co-Authors: Giulio Ghersi, Yunyun Yeh, Qiang Zhao, Stanley Zucker, Monica Salamone, Wen-tien ChenAbstract:Dipeptidyl peptidase IV (DPP4/CD26) and Seprase/fibroblast activation protein alpha are homologous type II transmembrane, homodimeric glycoproteins that exhibit unique prolyl peptidase activities. Human DPP4 is ubiquitously expressed in epithelial and endothelial cells and serves multiple functions in cleaving the penultimate positioned prolyl bonds at the NH(2) terminus of a variety of physiologically important peptides in the circulation. Recent studies showed a linkage between DPP4 and down-regulation of certain chemokines and mitogenic growth factors, and degradation of denatured collagens (gelatin), suggesting a role of DPP4 in the cell invasive phenotype. Here, we found the existence of a novel protease complex consisting of DPP4 and Seprase in human endothelial cells that were activated to migrate and invade in the extracellular matrix in vitro. DPP4 and Seprase were coexpressed with the three major protease systems (matrix metalloproteinase, plasminogen activator, and type II transmembrane serine protease) at the cell surface and organize as a complex at invadopodia-like protrusions. Both proteases were colocalized at the endothelial cells of capillaries, but not large blood vessels, in invasive breast ductal carcinoma in vivo. Importantly, monoclonal antibodies against the gelatin-binding domain of DPP4 blocked the local gelatin degradation by endothelial cells in the presence of the major metallo- and serine protease systems that modified pericellular collagenous matrices and subsequent cell migration and invasion. Thus, we have identified a novel mechanism involving the DPP4 gelatin-binding domain of the DPP4-Seprase complex that facilitates the local degradation of the extracellular matrix and the invasion of the endothelial cells into collagenous matrices.
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Regulation of Fibroblast Migration on Collagenous Matrix by a Cell Surface Peptidase Complex
The Journal of biological chemistry, 2002Co-Authors: Giulio Ghersi, Leslie A. Goldstein, Yunyun Yeh, Huan Dong, Lari Häkkinen, Hannu Larjava, Wen-tien ChenAbstract:The invasion of migratory cells through connective tissues involves metallo- and serine types of cell surface proteases. We show that formation of a novel protease complex, consisting of the membrane-bound prolyl peptidases Seprase and dipeptidyl peptidase IV (DPPIV), at invadopodia of migratory fibroblasts is a prerequisite for cell invasion and migration on a collagenous matrix. Seprase and DPPIV form a complex on the cell surface that elicits both gelatin binding and gelatinase activities localized at invadopodia of cells migrating on collagenous fibers. The protease complex participates in the binding to gelatin and localized gelatin degradation, cellular migration, and monolayer wound closure. Serine protease inhibitors can block the gelatinase activity and the localized gelatin degradation by cells. Antibodies to the gelatin-binding domain of DPPIV reduce the cellular abilities of the proteases to degrade gelatin but do not affect cellular adhesion or spreading on type I collagen. Furthermore, expression of the Seprase-DPPIV complex is restricted to migratory cells involved in wound closure in vitro and in connective tissue cells during closure of gingival wounds but not in differentiated tissue cells. Thus, we have identified cell surface proteolytic activities, which are non-metalloproteases, Seprase and DPPIV, that are responsible for the tissue-invasive phenotype.
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A Novel Protease-docking Function of Integrin at Invadopodia
The Journal of biological chemistry, 1999Co-Authors: Susette C. Mueller, Giulio Ghersi, Mayra L. Piñeiro-sánchez, Yunyun Yeh, Steven K. Akiyama, Linda Howard, Qing-xiang Amy Sang, Hirokazu Nakahara, Wen-tien ChenAbstract:Abstract Invadopodia are membrane extensions of aggressive tumor cells that function in the activation of membrane-bound proteases occurring during tumor cell invasion. We explore a novel and provocative activity of integrins in docking proteases to sites of invasion, termed invadopodia. In the absence of collagen, α3β1 integrin and the gelatinolytic enzyme, Seprase, exist as nonassociating membrane proteins. Type I collagen substratum induces the association of α3β1 integrin with Seprase as a complex on invadopodia. The results show that α3β1integrin is a docking protein for Seprase to form functional invadopodia. In addition, α5β1 integrin may participate in the adhesion process necessary for invadopodial formation. Thus, α3β1 and α5β1 integrins play major organizational roles in the adhesion and formation of invadopodia, promoting invasive cell behavior.
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Molecular cloning of Seprase: a serine integral membrane protease from human melanoma
Biochimica et biophysica acta, 1997Co-Authors: Leslie A. Goldstein, Giulio Ghersi, Mayra L. Piñeiro-sánchez, Salamone M, Yunyun Yeh, Denise Flessate, Wen-tien ChenAbstract:Seprase is a homodimeric 170 kDa integral membrane gelatinase whose expression correlates with the invasiveness of the human melanoma cell line LOX. Here, we report the molecular cloning of a cDNA that encodes the 97 kDa subunit of Seprase. Its deduced amino acid sequence predicts a type II integral membrane protein with a cytoplasmic tail of 6 amino acids, followed by a transmembrane domain of 20 amino acids and an extracellular domain of 734 amino acids. The carboxyl terminus contains a putative catalytic region (approximately 200 amino acids) which is homologous (68% identity) to that of the nonclassical serine protease dipeptidyl peptidase IV (DPPIV). The conserved serine protease motif G-X-S-X-G is present as G-W-S-Y-G. However, sequence analysis of Seprase cDNA from LOX and other cell lines strongly suggests that Seprase and human fibroblast activation protein alpha (FAP alpha) are products of the same gene. We propose that Seprase/FAP alpha and DPPIV represent a new subfamily of serine integral membrane proteases (SIMP).
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Identification of the 170-kDa Melanoma Membrane-bound Gelatinase (Seprase) as a Serine Integral Membrane Protease
The Journal of biological chemistry, 1997Co-Authors: Mayra L. Piñeiro-sánchez, Leslie A. Goldstein, Yunyun Yeh, Johannes Dodt, Linda Howard, Wen-tien ChenAbstract:Abstract The 170-kDa membrane-bound gelatinase, Seprase, is a cell surface protease, the expression of which correlates with the invasive phenotype of human melanoma and carcinoma cells. We have isolated Seprase from cell membranes and shed vesicles of LOX human melanoma cells. The active enzyme is a dimer of N-glycosylated 97-kDa subunits. Sequence analysis of three internal proteolytic fragments of the 97-kDa polypeptide revealed up to 87.5% identity to the 95-kDa fibroblast activation protein α (FAPα), the function of which is unknown. Thus, we used reverse transcription-polymerase chain reaction to generate a 2.4-kilobase cDNA from LOX mRNA with FAPα primers. COS-7 cells transfected with this cDNA expressed a 170-kDa gelatinase that is recognized by monoclonal antibodies directed against Seprase. Sequence analysis also showed similarities to the 110-kDa subunit of dipeptidyl peptidase IV (DPPIV). Like DPPIV, the gelatinase activity of Seprase was completely blocked by serine-protease inhibitors, including diisopropyl fluorophosphate. Seprase could be affinity-labeled by [3H]diisopropyl fluorophosphate, but the proteolytically inactive 97-kDa subunit could not, confirming the existence of a serine protease active site on the dimeric form. Proteolytic activity is lost upon dissociation into its 97-kDa subunit following treatment with acid, heat, or cysteine and histidine-modifying agents. We conclude that Seprase, FAPα, and DPPIV are related serine integral membrane proteases and that Seprase is similar to DPPIV, the proteolytic activities of which are dependent upon subunit association.
Giulio Ghersi - One of the best experts on this subject based on the ideXlab platform.
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Evaluation of shed membrane vesicles released from ECV-304 cultured to confluence and in migrating conditions; immunodetection of antigens by immunofluorescence staining.
2016Co-Authors: Monica Salamone, Francesco Carfì Pavia, Giulio GhersiAbstract:(A)—Shed membrane vesicles released from the same amount of ECV-304 cultured to confluence or in migrating conditions were compared to the amount of total proteins using Bradford colorimetric methods and reported as Arbitrary Units in the ordinate. (B)—Shed membrane vesicles obtained from confluent (a-i) or migrating (a’-i’) ECV-304 were coated on polylisine-treated cover glasses and immunostained with different primary antibodies against: (b-b’) mAb C27 against β1-integrin; (c-c’) mAb D28 against Seprase; (d-d’) mAb E34 against DPP4; (e-e’) anti MMP-2 (Sigma); (f-f’) mAb 13 against MT1-MMP; (g-g’) anti MMP-9 (Sigma); (h-h’) anti UpA (Santa Cruz Biotechnology); (i-i’) anti TIMP-2 (Sigma). The immunoreactions were detected using secondary antibodies against the primary animal species conjugated with FITC or TRITC fluorochrome; in (a-a’) vesicles were treated with a mixture of fluorescence conjugated secondary antibodies. The reported staining is not a quantitative evaluation of antigens. Bar = 2 μm.
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Immunostaining of endothelial cells cultured in 2D wounded and in 3D type-I collagen fibril gel systems.
2016Co-Authors: Monica Salamone, Francesco Carfì Pavia, Giulio GhersiAbstract:(A)—Endothelial cells were cultured to confluence and wounded. After 4 hours, cells were fixed and analyzed in immunofluorescence with antibodies against interest molecules; (a–f) cell staining of cells invading the wound healing area, (a’–f’) cell staining of corresponding unwounded areas, in which cells form a mono-layer. In (a–a’) anti MT1-MMP distribution; in (b–b’) anti MMP-2 distribution; in (c–c’) anti β-catenin distribution; in (d–d’) anti DPP-4 distribution; in (e–e’) anti Seprase distribution; in (f–f’) anti β1-integrin distribution. Yellow arrows show the wound area; while the blue arrows indicate immunostaining at the migration front of cells invading the wound healing area. Each squared area represents a 2X magnification to better mark, or not mark, immunolocalization of specific antigens in migrating and non-migrating cells. Barr = 50 μm. (B)–Endothelia cells were cultured inside to 3D type-I collagen fibril gels. (a–a’) phase contrast and anti DPP-4-FITC conjugate staining, respectively; (b–b’) phase contrast and anti MT1-MMP-TRITC directly conjugate staining, respectively; (c) anti Seprase staining; (d) anti β1-integrin staining; in c and d, the squared areas are 4X magnification of shed membrane vesicles released inside to type-I collagen fibril gel networks. In the images, the arrows indicate cell plasma membrane protrusions, and arrowed shed membrane vesicles released by cells in the extracellular matrix. Bar = 5 μm.
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The protease complex consisting of dipeptidyl peptidase IV and Seprase plays a role in the migration and invasion of human endothelial cells in collagenous matrices.
Cancer research, 2006Co-Authors: Giulio Ghersi, Yunyun Yeh, Qiang Zhao, Stanley Zucker, Monica Salamone, Wen-tien ChenAbstract:Dipeptidyl peptidase IV (DPP4/CD26) and Seprase/fibroblast activation protein alpha are homologous type II transmembrane, homodimeric glycoproteins that exhibit unique prolyl peptidase activities. Human DPP4 is ubiquitously expressed in epithelial and endothelial cells and serves multiple functions in cleaving the penultimate positioned prolyl bonds at the NH(2) terminus of a variety of physiologically important peptides in the circulation. Recent studies showed a linkage between DPP4 and down-regulation of certain chemokines and mitogenic growth factors, and degradation of denatured collagens (gelatin), suggesting a role of DPP4 in the cell invasive phenotype. Here, we found the existence of a novel protease complex consisting of DPP4 and Seprase in human endothelial cells that were activated to migrate and invade in the extracellular matrix in vitro. DPP4 and Seprase were coexpressed with the three major protease systems (matrix metalloproteinase, plasminogen activator, and type II transmembrane serine protease) at the cell surface and organize as a complex at invadopodia-like protrusions. Both proteases were colocalized at the endothelial cells of capillaries, but not large blood vessels, in invasive breast ductal carcinoma in vivo. Importantly, monoclonal antibodies against the gelatin-binding domain of DPP4 blocked the local gelatin degradation by endothelial cells in the presence of the major metallo- and serine protease systems that modified pericellular collagenous matrices and subsequent cell migration and invasion. Thus, we have identified a novel mechanism involving the DPP4 gelatin-binding domain of the DPP4-Seprase complex that facilitates the local degradation of the extracellular matrix and the invasion of the endothelial cells into collagenous matrices.
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DPPIV, Seprase, and related serine peptidases in multiple cellular functions
Current Topics in Developmental Biology, 2003Co-Authors: Wen-tien Chen, Thomas Kelly, Giulio GhersiAbstract:Publisher Summary Serine integral membrane peptidases (SIMP), including DPPIV, Seprase, and related prolyl peptidases, which are both Pro-Xaa cleaving enzymes and adhesion molecules, are likely to emerge as an important protease family. The main functions of SIMPs reside in their proteolytic and adhesive capacities, thus influencing cellular activities, migration, and invasion. These membrane proteases may form a physically and functionally linked complex at invadopodia during cellular invasion. The cysteine-rich domain of some peptidases exhibits the capability of binding to multiple molecules. This could allow not only activation of the peptidases themselves but also association with other membrane proteases to participate in cooperative extracellular matrix (ECM) protein degradation at invadopodia during cancer invasion. This chapter focuses on a small group of membrane serine peptidases, the SIMP, that are inducible, specific for proline-containing peptides and macromolecules, and active on the cell surface. Prototypes of SIMP members are DPPIV and Seprase. Other SIMP-related peptidases, including quiescent cell proline aminodipeptidase (QPP), prolyl carboxy-peptidase (PCP), prolyl endopeptidase (PEP), dipeptidyl peptidase 6 (DPP6), dipeptidyl peptidase 8 (DPP8), dipeptidyl peptidase 9 (DDP9), attractin, dipeptidyl peptidase II (DPPII), and dipeptidyl peptidase IV-β (DPPIV-β), have subtle structural and functional differences from DPPIV and Seprase, and they are also discussed in the chapter.
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Regulation of Fibroblast Migration on Collagenous Matrix by a Cell Surface Peptidase Complex
The Journal of biological chemistry, 2002Co-Authors: Giulio Ghersi, Leslie A. Goldstein, Yunyun Yeh, Huan Dong, Lari Häkkinen, Hannu Larjava, Wen-tien ChenAbstract:The invasion of migratory cells through connective tissues involves metallo- and serine types of cell surface proteases. We show that formation of a novel protease complex, consisting of the membrane-bound prolyl peptidases Seprase and dipeptidyl peptidase IV (DPPIV), at invadopodia of migratory fibroblasts is a prerequisite for cell invasion and migration on a collagenous matrix. Seprase and DPPIV form a complex on the cell surface that elicits both gelatin binding and gelatinase activities localized at invadopodia of cells migrating on collagenous fibers. The protease complex participates in the binding to gelatin and localized gelatin degradation, cellular migration, and monolayer wound closure. Serine protease inhibitors can block the gelatinase activity and the localized gelatin degradation by cells. Antibodies to the gelatin-binding domain of DPPIV reduce the cellular abilities of the proteases to degrade gelatin but do not affect cellular adhesion or spreading on type I collagen. Furthermore, expression of the Seprase-DPPIV complex is restricted to migratory cells involved in wound closure in vitro and in connective tissue cells during closure of gingival wounds but not in differentiated tissue cells. Thus, we have identified cell surface proteolytic activities, which are non-metalloproteases, Seprase and DPPIV, that are responsible for the tissue-invasive phenotype.
Masako Mitsumata - One of the best experts on this subject based on the ideXlab platform.
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sphingosine 1 phosphate stimulates cell migration and active Seprase expression in human endothelial cells
山梨医科学雑誌, 2013Co-Authors: Hirotaka Okamoto, Wen-tien Chen, Masako Mitsumata, Tetsu Yamane, Yutaka Yatomi, Kaneo Satoh, Yukio Ozaki, Hideki FujiiAbstract:Objective: Sphingosine 1-phosphate (Sph-1-P), a bioactive lysophospholipid present in the plasma, is released from activated platelets. Our previous study dem- onstrated that Sph-1-P promoted the spreading on and migration of human umbilical vein endothelial cells (HUVEC) through the extracellular matrix (ECM), suggesting a possible induction of cell surface proteases in the Sph-1-P activated endothelial cells. In the present study, we examined whether Seprase, a type II transmembrane serine protease (TTSP) usually absent in tissue cells, can be induced in endothelial cells acti- vated by Sph-1-P. Methods: HUVEC migration through ECM was examined by modifi ed Boyden chamber assay. Western blotting and immunoprecipitation, using anti-Seprase monoclonal antibodies (mAbs), were used to confi rm the Seprase expression of HUVEC. Results: We show that Sph-1-P enhanced expression of active form Seprase in a time- and dose-dependent manner in HUVEC. The Sph-1-P inducible active form Seprase could be blocked by pertussis toxin and by C3 transferase, which inactivate Gi-type heterotrimetric G proteins and Rho, respectively. Conclusion: These results show that Sph-1-P can regulate migration of endothelial cells by inducing active form Seprase expression, which, in turn, is mediated through a Gi-coupled cell surface receptor and the Rho protein.
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Sphingosine 1-phosphate induces extracellular matrix invasion and Seprase activity in human endothelial cells.
Cancer Research, 2008Co-Authors: Hirotaka Okamoto, Masako Mitsumata, Yoshiyuki Mori, Hideki Fujii, Tetsu Yamane, Yutaka Yatomi, Kaneo Satoh, Chen Wen-tien, Yukio OzakiAbstract:1151 Sphingosine 1-phosphate (Sph-1-P), a bioactive lysophospholipid present in the plasma, is released from activated platelets. Our previous study demonstrated that Sph-1-P promoted the spreading on and migration of human umbilical vein endothelial cells (HUVEC) through the extracellular matrix (ECM), suggesting a possible induction of cell surface proteases in the Sph-1-P activated endothelial cells. Here we provide the evidence that Seprase, a type II transmembrane serine protease (TTSP) usually absent in tissue cells, can be induced in endothelial cells activated by Sph-1-P. We show by immunoblot analysis using anti-Seprase monoclonal antibodies (mAbs) that Sph-1-P enhanced expression of Seprase in a time- and dose-dependent manner in HUVEC. The Sph-1-P inducible Seprase could be blocked by pertussis toxin and by C3 transferase, which inactivate Gi-type heterotrimetric G proteins and Rho, respectively. These results show that Sph-1-P can regulate migration of endothelial cells by inducing Seprase expression, which, in turn, is mediated through a Gi-coupled cell surface receptor and the Rho protein.
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The expression of a type II transmembrane serine protease (Seprase) in human gastric carcinoma
Oncology, 2004Co-Authors: Yoshiyuki Mori, Masako Mitsumata, Koji Kono, Yoshirou Matsumoto, Hideki Fujii, Tetsu Yamane, Wen-tien ChenAbstract:Objective: The invasion and metastasis of carcinoma cells require the proteolytic degradation of the extracellular matrix by various cell surface proteases. Among these, Seprase is a type II transmembrane serine protease absent in normal tissues and it has been implicated in the invasion of the extracellular matrix by both tumor and stromal cells in human breast carcinoma and melanoma. In the present study, the expression of Seprase mRNA, protein and its gelatin-degrading activity in human gastric carcinoma were examined to substantiate the potential role of Seprase in gastric carcinoma invasion. Methods: We have examined the Seprase expression in human gastric carcinoma (n = 34) by RT-PCR, Western immunoblotting analysis, immunohistochemistry, and gelatin zymography. Results: Immunoblotting analysis using mAb D8 directed against Seprase showed that the carcinoma tissues in 26 out of 34 cases of gastric cancer expressed a dimeric form of Seprase but their normal counterparts did not. Gelatin zymography confirmed that the isolated Seprase exhibited the gelatin-degrading activity and was active. Seprase-expressing carcinoma tissues were more often found in the scirrhous type than in other types of gastric carcinoma. RT-PCR analysis showed that Seprase mRNA was present in carcinoma tissues but not in normal tissues. Immunohistochemically, Seprase was mainly located in gastric carcinoma cells, weakly in stromal cells and microvessel endothelial cells in the tumor nest, and none in normal cells. Conclusions: Our studies showed the unique expression and localization of Seprase in the tumor and stromal cells within human gastric carcinoma but not in normal tissues, suggesting a role of Seprase in the invasive and metastatic progression of gastric carcinoma.
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Increased expression of Seprase, a membrane-type serine protease, is associated with lymph node metastasis in human colorectal cancer.
Cancer letters, 2003Co-Authors: Satoshi Iwasa, Xin Jin, Kyoko Okada, Masako Mitsumata, Akishi OoiAbstract:Seprase is a membrane-bound serine proteinase with gelatinase activity, which may be involved in cancer invasion and metastasis. We examined Seprase expression in colorectal cancer specimens obtained from 109 patients. Seprase immunoreactivity was found in cancer cells and adjacent stromal cells. Immunoblotting showed higher levels of Seprase protein in colorectal cancer tissue than in normal colorectal tissue. A semiquantitative assessment of the immunohistochemistry results revealed a significant correlation between Seprase expression and lymph node metastasis. These results suggested that an abundant expression of Seprase in colorectal cancer tissue is associated with lymph node metastasis.
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Expression patterns of Seprase, a membrane serine protease, in cervical carcinoma and cervical intraepithelial neoplasm.
Anticancer research, 2003Co-Authors: Xin Jin, Satoshi Iwasa, Kyoko Okada, Masako Mitsumata, Akishi OoiAbstract:Background: Seprase is a membrane-bound serine proteinase with gelatinase activity, which may be involved in cancer invasion and metastasis. Patients and Methods: We examined the Seprase expression patterns in invasive or preinvasive squamous epithelial lesions. Results and Conclusion: No Seprase immunoreactivity was found in the atypical epithelial cells and subepithelial stromal cells of CINI and 2 lesions. No Seprase expression could be detected even in CIN3 lesions. However, in contrast to CINI and 2, Seprase immunoreactivity was occasionally observed in the subepithelial stromal cells that were associated with moderate or severe inflammatory infiltrates. Some microinvasive carcinomas and all invasive carcinomas showed Seprase immunoreactivity in the cancer cells with various degrees of Seprase-positive stromal cells. Seprase may be an early marker of tumor invasion in squamous lesions of the uterine cervix.
