Publications by authors named "Eisuke Mekada"

66 Publications

Identification of GII.14[P7] norovirus and its genomic mutations from a case of long-term infection in a post-symptomatic individual.

Infect Genet Evol 2020 12 1;86:104612. Epub 2020 Nov 1.

Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections, Nonthaburi, Thailand. Electronic address:

Norovirus is a leading cause of acute gastroenteritis worldwide. Norovirus shedding typically lasts one week to one month after the onset of diarrhea in immunocompetent hosts. The occurrence of mutations in the genome during infection has contributed to the evolution of norovirus. It has been suggested that genomic mutations in the P2-domain of capsid protein VP1, the major antigenic site for virus clearance, are involved in the evasion of host immunity and prolonged shedding of norovirus. In our previous study, we found a case of long-term shedding of GII.14 norovirus in a post-symptomatic immunocompetent individual that lasted about three months. In this study, we characterized the genomic sequence of the GII.14 strain to gain insight into the context of long-term shedding. By sequencing a 4.8 kb region of the genome corresponding to half of ORF1 and the entire ORF2 and ORF3, which encode several non-structural proteins and the structural proteins VP1 and VP2, the GII.14 strain was found to be classified as recombinant GII.14[P7]. Six point-mutations occurred during the three-month period of infection in a time-dependent manner in the genomic regions encoding RNA-dependent RNA polymerase, VP1, and VP2. Three of the six mutations were sense mutations, but no amino acid substitution was identified in the P2-domain of VP1. These results suggest that there is a mechanism by which long-term shedding of norovirus occurs in immunocompetent individuals independent of P2-domain mutations.
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http://dx.doi.org/10.1016/j.meegid.2020.104612DOI Listing
December 2020

Norovirus transmission mediated by asymptomatic family members in households.

PLoS One 2020 23;15(7):e0236502. Epub 2020 Jul 23.

Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections, Nonthaburi, Thailand.

The transmission of human norovirus excreted from infected persons occasionally causes sporadic infections and outbreaks. Both symptomatic patients and asymptomatic carriers have been reported to contribute to norovirus transmission, but little is known about the magnitude of the contribution of asymptomatic carriers. We carried out a 1-year survey of residents of a district of Bangkok, Thailand to determine the percentage of norovirus transmissions originating from asymptomatic individuals. We screened 38 individuals recruited from 16 families from May 2018 to April 2019 for GI and GII genotypes. Norovirus was detected every month, and 101 of 716 stool samples (14.1%) from individuals with no symptoms of acute gastroenteritis were norovirus-positive. The average infection frequency was 2.4 times per person per year. Fourteen genotypes were identified from the positive samples, with GII.4 being detected most frequently. Notably, 89.1% of the norovirus-positive samples were provided by individuals with no diarrhea episode. Similar to cases of symptomatic infections in Thailand, asymptomatic infections were observed most frequently in December. We detected 4 cases of NV infection caused by household transmission, and 3 of the 4 transmissions originated from asymptomatic individuals. We also identified a case in which norovirus derived from an asymptomatic individual caused diarrhea in a family member. These results suggest that asymptomatic individuals play a substantial role in both the maintenance and spreading of norovirus in a community through household transmission.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0236502PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377487PMC
September 2020

Isolation of intragenic suppressor of -splicing mutant in .

MicroPubl Biol 2019 Jul 23;2019. Epub 2019 Jul 23.

Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

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http://dx.doi.org/10.17912/micropub.biology.000126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7252243PMC
July 2019

The use of green fluorescent protein-tagged virus-like particles as a tracer in the early phase of chikungunya infection.

Virus Res 2019 10 21;272:197732. Epub 2019 Aug 21.

Thailand-Japan Research Collaboration Center on Emerging and Re-emerging Infections (RCC-ERI), Department of Medical Sciences, Ministry of Public Health, Nonthaburi, Thailand; Research Institute for Microbial Diseases (RIMD), Osaka University, Suita, Osaka, Japan. Electronic address:

To visually examine the early phase of chikungunya virus (CHIKV) infection in target cells, we constructed a virus-like particle (VLP) in which the envelope protein E1 is fused with green fluorescent protein (GFP). This chikungunya VLP-GFP (CHIK-VLP-EGFP), purified by density gradient fractionation, was observed as 60-70 nm-dia. particles and was detected as tiny puncta of fluorescence in the cells. CHIK-VLP-EGFP showed binding properties similar to those of the wild-type viruses. Most of the fluorescence signals that had bound on Vero cells disappeared within 30 min at 37 °C, but not in the presence of anti-CHIKV neutralizing serum or an endosomal acidification inhibitor (bafilomycin A1), suggesting that the loss of fluorescence signals is due to the disassembly of the viral envelope following the internalization of CHIK-VLP-EGFP. In addition to these results, the fluorescence signals disappeared in highly susceptible Vero and U251MG cells but not in poorly susceptible A549 cells. Thus, CHIK-VLP-EGFP is a useful tool to examine the effects of the CHIKV neutralizing antibodies and antiviral compounds that are effective in the entry phase of CHIKV.
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http://dx.doi.org/10.1016/j.virusres.2019.197732DOI Listing
October 2019

Anti-HB-EGF Antibody-Mediated Delivery of siRNA to Atherosclerotic Lesions in Mice.

Int Heart J 2018 Nov 5;59(6):1425-1431. Epub 2018 Nov 5.

Department of Cardiorenal and Cerebrovascular Medicine, Faculty of Medicine, Kagawa University.

For atherosclerotic cardiovascular diseases (ACD), gene therapy may be a potential therapeutic strategy; however, lack of effective and safe methods for gene delivery to atherosclerotic plaques have limited its potential therapeutic applications. To overcome this limitation, we developed a novel antibody-based gene delivery system (anti-HB-EGF/NA vector) by chemically crosslinking antibodies against human heparin-binding epidermal growth factor-like growth factor (HB-EGF). It has been shown to be excessively expressed in human atherosclerotic plaques and NeutrAvidin (NA) for conjugating biotinylated siRNA. Immunofluorescence staining and quantitative flow cytometry analysis using human HB-EGF-expressing cells showed both antibody-mediated selective cellular targeting and efficient intracellular delivery of conjugated biotin-fluorescence. Moreover, we demonstrated antibody-mediated significant and selective gene knockdown via conjugation with anti-HB-EGF/NA vector and biotinylated siRNA (anti-HB-EGF/NA/b-siRNA) in vitro. Furthermore, using high fat-fed human HB-EGF knock-in and apolipoprotein E-knockout (Hbegf hz/hz; Apoe-/-) mice, we demonstrated that the anti-HB-EGF/NA vector, conjugating biotin-fluorescence, increasingly accumulated within the atherosclerotic plaques of the ascending aorta in which human HB-EGF expression levels were highly elevated. Moreover, in response to a single intravenous injection of anti-HB-EGF/NA/b-siRNA in a dose-dependent manner, qPCR analysis of laser-dissected atherosclerotic plaques of the ascending aorta showed significant knockdown of the reporter gene expression. These results suggest that the anti-HB-EGF antibody-mediated siRNA delivery could be a promising delivery system for gene therapy of ACD.
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http://dx.doi.org/10.1536/ihj.17-644DOI Listing
November 2018

Double deletion of tetraspanins CD9 and CD81 in mice leads to a syndrome resembling accelerated aging.

Sci Rep 2018 03 23;8(1):5145. Epub 2018 Mar 23.

Department of Respiratory Medicine and Clinical Immunology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.

Chronic obstructive pulmonary disease (COPD) has been recently characterized as a disease of accelerated lung aging, but the mechanism remains unclear. Tetraspanins have emerged as key players in malignancy and inflammatory diseases. Here, we found that CD9/CD81 double knockout (DKO) mice with a COPD-like phenotype progressively developed a syndrome resembling human aging, including cataracts, hair loss, and atrophy of various organs, including thymus, muscle, and testis, resulting in shorter survival than wild-type (WT) mice. Consistent with this, DNA microarray analysis of DKO mouse lungs revealed differential expression of genes involved in cell death, inflammation, and the sirtuin-1 (SIRT1) pathway. Accordingly, expression of SIRT1 was reduced in DKO mouse lungs. Importantly, siRNA knockdown of CD9 and CD81 in lung epithelial cells additively decreased SIRT1 and Foxo3a expression, but reciprocally upregulated the expression of p21 and p53, leading to reduced cell proliferation and elevated apoptosis. Furthermore, deletion of these tetraspanins increased the expression of pro-inflammatory genes and IL-8. Hence, CD9 and CD81 might coordinately prevent senescence and inflammation, partly by maintaining SIRT1 expression. Altogether, CD9/CD81 DKO mice represent a novel model for both COPD and accelerated senescence.
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http://dx.doi.org/10.1038/s41598-018-23338-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865149PMC
March 2018

ErbB1 and ErbB4 generate opposing signals regulating mesenchymal cell proliferation during valvulogenesis.

J Cell Sci 2017 04 23;130(7):1321-1332. Epub 2017 Feb 23.

Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan

Heparin-binding EGF-like growth factor (HB-EGF) plays an indispensable role in suppression of cell proliferation during mouse valvulogenesis. However, ligands of the EGF receptor (EGFR/ErbB1), including HB-EGF, are generally considered as growth-promoting factors, as shown in cancers. HB-EGF binds to and activates ErbB1 and ErbB4. We investigated the role of ErbB receptors in valvulogenesis using ErbB1- and ErbB4-deficient mice, and an model of endocardial cushion explants. We show that HB-EGF suppresses valve mesenchymal cell proliferation through a heterodimer of ErbB1 and ErbB4, and an ErbB1 ligand (or ligands) promotes cell proliferation through a homodimer of ErbB1. Moreover, a rescue experiment with cleavable or uncleavable isoforms of ErbB4 in -null cells indicates that the cleavable JM-A, but not the uncleavable JM-B, splice variant of ErbB4 rescues the defect of the null cells. These data suggest that the cytoplasmic intracellular domain of ErbB4, rather than the membrane-anchored tyrosine kinase, achieves this suppression. Our study demonstrates that opposing signals generated by different ErbB dimer combinations function in the same cardiac cushion mesenchymal cells for proper cardiac valve formation.
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http://dx.doi.org/10.1242/jcs.196618DOI Listing
April 2017

BK-UM in patients with recurrent ovarian cancer or peritoneal cancer: a first-in-human phase-I study.

BMC Cancer 2017 01 31;17(1):89. Epub 2017 Jan 31.

Department of Cell Biology, Research Institute for Microbial Disease, Osaka University, 3-1 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Background: BK-UM (CRM197) is a mutant form of diphtheria toxin and a specific inhibitor of heparin-binding epidermal growth factor-like growth factor (HB-EGF). We assessed the safety, pharmacokinetics, recommended dose, and efficacy of BK-UM in patients with recurrent ovarian cancer (OC) or peritoneal cancer (PC), and measured HB-EGF levels in serum and abdominal fluid after BK-UM administration.

Methods: Eleven patients with advanced or recurrent OC or PC were enrolled and treated with BK-UM via the intraperitoneal route. The dose was escalated (1.0, 2.0, 3.3, and 5.0 mg/m) using a 3 + 3 design.

Results: Eight of 11 patients completed treatment. No dose-limiting toxicity (DLT) was experienced at dose levels 1 (1.0 mg/m) and 2 (2.0 mg/m). Grade 3 transient hypotension as an adverse event (defined as a DLT in the present study) was observed in two of four patients at dose level 3 (3.3 mg/m). Treatment with BK-UM was associated with decreases in HB-EGF levels in serum and abdominal fluid in seven of 11 patients and five of eight patients, respectively. Clinical outcomes included a partial response in one patient, stable disease in five patients, and progressive disease in five patients.

Conclusions: BK-UM was well tolerated at doses of 1.0 and 2.0 mg/m, with evidence for clinical efficacy in patients with recurrent OC or PC. A dose of 2.0 mg/m BK-UM is recommended for subsequent clinical trials.

Trial Registration: This trial was prospectively performed as an investigator-initiated clinical trial. The trial numbers are UMIN000001002 and UMIN000001001, with registration dates of 1/30/2008 and 2/4/2008, respectively. UMIN000001001 was registered as a trial for the continuous administration of BK-UM after UMIN000001002 .
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http://dx.doi.org/10.1186/s12885-017-3071-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286856PMC
January 2017

Reconstitution of a metastatic-resistant tumor microenvironment with cancer-associated fibroblasts enables metastasis.

Cell Cycle 2017 Mar 19;16(6):533-535. Epub 2017 Jan 19.

c AntiCancer, Inc. , San Diego , CA , USA.

The tumor microenvironment is critical for metastasis to occur. Subcutaneous xenografts of tumors in immunodeficient mice are usually encapsulated and rarely metastasize as opposed to orthotopic tumors which metastasize if the original tumor was metastatic. In the present report, we were able to reconstitute a metastatic tumor microenvironment by subcutaneously co-transplanting a human cervical cancer cell line and human cervical cancer-associated fibroblasts (CAFs), in athymic mice, which resulted in lymph node metastasis in 40% of the animals. In contrast, no metastasis occurred from the cervical cancer without CAFs. These results suggest that CAFs can overcome an anti-metastatic tumor environment and are a potential target to prevent metastasis.
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http://dx.doi.org/10.1080/15384101.2017.1281486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5384587PMC
March 2017

Characterization of a Novel Anti-Human HB-EGF Monoclonal Antibody Applicable for Paraffin-Embedded Tissues and Diagnosis of HB-EGF-Related Cancers.

Monoclon Antib Immunodiagn Immunother 2016 Apr 14;35(2):73-82. Epub 2016 Mar 14.

1 Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University , Osaka, Japan .

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor (EGFR/ErbB1) and ErbB4. HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Thus, monoclonal antibodies (mAbs) for HB-EGF detection could be an important tool in the therapeutic diagnosis of HB-EGF-related cancers and other diseases. However, few mAbs, especially those applicable for immunohistochemistry (IHC), have been established to date. In this study, we generated a clone of hybridoma-derived mAb 2-108 by immunizing mice with recombinant human HB-EGF protein expressed by human cells. The mAb 2-108 specifically bound to human HB-EGF but not to mouse HB-EGF and was successful in immunoblotting, even under reducing conditions, immunoprecipitation, and immunofluorescence for unfixed as well as paraformaldehyde-fixed cells. Notably, this mAb was effective in IHC of paraffin-embedded tumor specimens. Epitope mapping analysis showed that mAb 2-108 recognized the N-terminal prodomain in HB-EGF. These results indicate that this new anti-HB-EGF mAb 2-108 would be useful in the diagnosis of HB-EGF-related cancers and would be a strong tool in both basic and clinical research on HB-EGF.
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http://dx.doi.org/10.1089/mab.2015.0062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4845685PMC
April 2016

Identification of diphtheria toxin R domain mutants with enhanced inhibitory activity against HB-EGF.

J Biochem 2015 May 27;157(5):331-43. Epub 2014 Nov 27.

Department of Cell Biology and Department of Molecular Bacteriology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan; and Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan

Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a ligand of EGF receptor, is involved in the growth and malignant progression of cancers. Cross-reacting material 197, CRM197, a non-toxic mutant of diphtheria toxin (DT), specifically binds to the EGF-like domain of HB-EGF and inhibits its mitogenic activity, thus CRM197 is currently under evaluation in clinical trials for cancer therapy. To develop more potent DT mutants than CRM197, we screened various mutant proteins of R domain of DT, the binding site for HB-EGF. A variety of R-domain mutant proteins fused with maltose-binding protein were produced and their inhibitory activity was evaluated in vitro. We found four R domain mutants that showed much higher inhibitory activity against HB-EGF than wild-type (WT) R domain. These R domain mutants suppressed HB-EGF-dependent cell proliferation more effectively than WT R domain. Surface plasmon resonance revealed their higher affinity to HB-EGF than WT R domain. CRM197(R460H) carrying the newly identified mutation showed increased cell proliferation inhibitory activity and affinity to HB-EGF. These results suggest that CRM197(R460H) or other recombinant proteins carrying newly identified mutation(s) in the R domain are potential therapeutics targeting HB-EGF.
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http://dx.doi.org/10.1093/jb/mvu079DOI Listing
May 2015

Pre-clinical study of BK-UM, a novel inhibitor of HB-EGF, for ovarian cancer therapy.

Anticancer Res 2014 Aug;34(8):4615-20

Department of Obstetrics and Gynecology, Fukuoka University, Fukuoka, Japan Department of Central Research Institute for Advanced Molecular Medicine, Fukuoka University, Fukuoka, Japan

Background/aim: Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a member of the epidermal growth factor family, is a target for ovarian cancer therapy. The present study investigated the administration schedule of BK-UM, an anticancer agent targeting HB-EGF.

Materials And Methods: The ovarian cancer cell line, RMG-I, was injected subcutaneously into five-week-old female nude mice. The BK-UM was administered intraperitoneally, using three administration schedules with different doses. The tumor volume was calculated every week. Statistical significance was assessed using the Mann-Whitney U-test.

Results: At doses >0.1 mg/kg, BK-UM displayed significant antitumor effects, although the antitumor effects and body weights of mice did not significantly differ by dose or by three different administration schedules. At a dose <0.1 mg/kg, however, BK-UM had little inhibitory effect on tumor growth.

Conclusion: Daily administration of BK-UM, which has a potentially dose-dependent antitumor effect, may be the optimal schedule for clinical application.
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August 2014

Antibody-modified lipid nanoparticles for selective delivery of siRNA to tumors expressing membrane-anchored form of HB-EGF.

Biochem Biophys Res Commun 2014 Jul 20;449(4):460-5. Epub 2014 May 20.

Department of Medical Biochemistry, University of Shizuoka School of Pharmaceutical Sciences, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan. Electronic address:

An Fab' antibody against heparin-binding epidermal growth factor-like growth factor (HB-EGF) was applied to achieve advanced tumor-targeted delivery of siRNA. Lipid nanoparticles (LNP) encapsulating siRNA (LNP-siRNA) were prepared, pegylated, and surface modified with Fab' fragments of anti-HB-EGF antibody (αHB-EGF LNP-siRNA). αHB-EGF LNP-siRNA showed high-binding affinity to recombinant human HB-EGF in a Biacore assay. In addition, αHB-EGF LNP-siRNA selectively associated with cells expressing HB-EGF in vitro. Confocal microscopic images showed that siRNA formulated in αHB-EGF LNP-siRNA was efficiently internalized into MDA-MB-231 human breast cancer cells, on which HB-EGF is highly expressed. In addition, siRNA encapsulated in αHB-EGF LNP induced obvious suppression of both target mRNA and protein levels in MDA-MB-231 cells. These results indicate that αHB-EGF LNP have excellent potential to deliver siRNA to target cancer cells, resulting in effective gene silencing.
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http://dx.doi.org/10.1016/j.bbrc.2014.05.043DOI Listing
July 2014

Co-occurrence of tetraspanin and ROS generators: Conservation in protein cross-linking and other developmental processes.

Worm 2013 Apr;2(2):e23415

Department of Biology; Kurume University School of Medicine; Fukuoka, Japan.

The nematode exoskeleton, commonly called the cuticle, is a highly structured extracellular matrix mainly composed of collagen. Secreted collagen molecules from the underlying epidermal cells are cross-linked via their tyrosyl residues. Reactive oxygen species (ROS) are required for the cross-linking reaction to produce tyrosyl radicals. The conserved ROS generator enzyme in C. elegans, BLI-3/CeDUOX1, a homolog of dual oxidases (DUOXs), is responsible for production of hydrogen peroxide. The ROS generation system must be properly controlled since ROS are highly reactive molecules that irreversibly inhibit the functions of cellular components such as nucleic acids and proteins. We recently reported that the ROS generation system directed by BLI-3 requires the tetraspanin protein, TSP-15. Herein we outline the process of cuticle development with a focus on the molecular roles of TSP-15 in the BLI-3 system. We also propose the co-occurrence of tetraspanin and ROS generators by convergent evolution.
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http://dx.doi.org/10.4161/worm.23415DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3704445PMC
April 2013

Metastasis suppressor tetraspanin CD82/KAI1 regulates ubiquitylation of epidermal growth factor receptor.

J Biol Chem 2013 Sep 29;288(36):26323-26334. Epub 2013 Jul 29.

From the School of Cancer Sciences, College of Medical and Dental Sciences, University of Birmingham, Birmingham B15 2TT, United Kingdom.

Ligand-induced ubiquitylation of EGF receptor (EGFR) is an important regulatory mechanism that controls endocytic trafficking of the receptor and its signaling potential. Here we report that tetraspanin CD82/KAI1 specifically suppresses ubiquitylation of EGFR after stimulation with heparin-binding EGF or amphiregulin and alters the rate of recruitment of the activated receptor to EEA1-positive endosomes. The suppressive effect of CD82 is dependent on the heparin-binding domain of the ligand. Deletion of the C-terminal cytoplasmic domain of CD82 (CD82ΔC mutant) inhibits endocytic trafficking of the tetraspanin and compromises its activity toward heparin-binding EGF-activated EGFR. Reduced ubiquitylation of EGFR is accompanied by PKC-dependent increase in serine phosphorylation of c-Cbl in cells expressing elevated levels of CD82. Furthermore, phosphorylation of threonine 654 (PKC phosphorylation site) in the juxtamembrane domain of the receptor is considerably increased in CD82-expressing cells. These results describe previously unsuspected links between tetraspanin proteins and ubiquitylation of their molecular partners (e.g., EGFR). Our data identify CD82 as a new regulator of c-Cbl, which discriminatively controls the activity of this E3 ubiquitin ligase toward heparin-binding ligand-EGFR pairs. Taken together, these observations provide an important new insight into the modulatory role of CD82 in endocytic trafficking of EGF receptor.
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http://dx.doi.org/10.1074/jbc.M112.439380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3764837PMC
September 2013

Conditional loss of heparin-binding EGF-like growth factor results in enhanced liver fibrosis after bile duct ligation in mice.

Biochem Biophys Res Commun 2013 Jul 4;437(2):185-91. Epub 2013 Jun 4.

Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine, Osaka, Japan.

Our aims were to evaluate the involvement of heparin-binding EGF-like growth factor (HB-EGF) in liver fibrogenesis of humans and mice and to elucidate the effect of HB-EGF deficiency on cholestatic liver fibrosis using conditional HB-EGF knockout (KO) mice. We first demonstrated that gene expression of HB-EGF had a positive significant correlation with that of collagen in human fibrotic livers, and was increased in bile duct ligation (BDL)-induced fibrotic livers in mouse. We then generated conditional HB-EGF knockout (KO) mice using the interferon inducible Mx-1 promoter driven Cre recombinase transgene and wild type (WT) and KO mice were subjected to BDL. After BDL, KO mice exhibited enhanced liver fibrosis with increased expression of collagen, compared with WT mice. Finally, we used mouse hepatic stellate cells (HSCs) to examine the role of HB-EGF in the activation of these cells and showed that HB-EGF antagonized TGF-β-induced gene expression of collagen in mouse primary HSCs. Interestingly, HB-EGF did not prevent the TGF-β-induced nuclear accumulation of Smad3, but did lead to stabilization of the Smad transcriptional co-repressor TG-interacting factor. In conclusion, our data suggest a possible protective role of HB-EGF in cholestatic liver fibrosis.
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http://dx.doi.org/10.1016/j.bbrc.2013.05.097DOI Listing
July 2013

Soluble heparin-binding EGF-like growth factor (HB-EGF) detected by newly developed immuno-PCR method is a clear-cut serological biomarker for ovarian cancer.

Am J Transl Res 2012 10;4(4):415-21. Epub 2012 Oct 10.

Pharmacokinetic Research Laboratories, Kyowa Hakko Kirin Co. Ltd. 1188, Shimotogari, Nagaizumi-cho, Suntogun, Shizuoka, 411-8731, Japan.

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the EGF family of growth factors that bind to and activate the EGF receptor. HB-EGF is synthesized as a membrane-anchored protein (proHB-EGF), and then proteolytically cleaved, resulting in the mitogenically active soluble form. HB-EGF plays pivotal roles in pathophysiological processes such as development and cell proliferation. In this study, we developed an immuno-PCR system for the determination of soluble HB-EGF concentrations in human serum. Utilizing a monoclonal antibody with neutralizing activity against HB-EGF as a capture antibody resulted in increasing selectivity for an active form of HB-EGF in serum, and immuno-PCR system improved its sensitivity compared to the currently available methods. As a result of measurement of HB-EGF in 20 ovarian cancer patients and 20 healthy volunteers, ovarian cancer patients showed significantly higher concentrations than healthy volunteers (P< 0.05), which indicates that soluble HB-EGF detected by newly developed immuno-PCR system can be useful serological biomarkers such as a diagnostic biomarker for ovarian cancer, and a predictive and pharmacodynamic biomarker for anti-HB-EGF targeted therapies under development.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3493029PMC
November 2012

Tetraspanin is required for generation of reactive oxygen species by the dual oxidase system in Caenorhabditis elegans.

PLoS Genet 2012 Sep 20;8(9):e1002957. Epub 2012 Sep 20.

Department of Biology, Kurume University School of Medicine, Kurume, Fukuoka, Japan.

Reactive oxygen species (ROS) are toxic but essential molecules responsible for host defense and cellular signaling. Conserved NADPH oxidase (NOX) family enzymes direct the regulated production of ROS. Hydrogen peroxide (H(2)O(2)) generated by dual oxidases (DUOXs), a member of the NOX family, is crucial for innate mucosal immunity. In addition, H(2)O(2) is required for cellular signaling mediated by protein modifications, such as the thyroid hormone biosynthetic pathway in mammals. In contrast to other NOX isozymes, the regulatory mechanisms of DUOX activity are less understood. Using Caenorhabditis elegans as a model, we demonstrate that the tetraspanin protein is required for induction of the DUOX signaling pathway in conjunction with the dual oxidase maturation factor (DUOXA). In the current study, we show that genetic mutation of DUOX (bli-3), DUOXA (doxa-1), and peroxidase (mlt-7) in C. elegans causes the same defects as a tetraspanin tsp-15 mutant, represented by exoskeletal deficiencies due to the failure of tyrosine cross-linking of collagen. The deficiency in the tsp-15 mutant was restored by co-expression of bli-3 and doxa-1, indicating the involvement of tsp-15 in the generation of ROS. H(2)O(2) generation by BLI-3 was completely dependent on TSP-15 when reconstituted in mammalian cells. We also demonstrated that TSP-15, BLI-3, and DOXA-1 form complexes in vitro and in vivo. Cell-fusion-based analysis suggested that association with TSP-15 at the cell surface is crucial for BLI-3 activation to release H(2)O(2). This study provides the first evidence for an essential role of tetraspanin in ROS generation.
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http://dx.doi.org/10.1371/journal.pgen.1002957DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3447965PMC
September 2012

Conditional knockout of heparin-binding epidermal growth factor-like growth factor in the liver accelerates carbon tetrachloride-induced liver injury in mice.

Hepatol Res 2013 Apr 9;43(4):384-93. Epub 2012 Aug 9.

Department of Gastroenterology and Hepatology, Osaka University, Graduate School of Medicine Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, Osaka Department of Biochemistry and Molecular Genetics, Ehime University, Graduate School of Medicine Department of Cell Growth and Tumor Regulation, Proteo-Medicine Research Center (ProMRes), Ehime University, Ehime Kansai-Rosai Hospital, Hyogo, Japan.

Aim:   We previously demonstrated that heparin-binding epidermal growth factor-like growth factor (HB-EGF) is induced in response to several liver injuries. Because the HB-EGF knockout (KO) mice die in utero or immediately after birth due to cardiac defects, the loss of function study in vivo is limited. Here, we generated liver-specific HB-EGF conditional knockout mice using the interferon-inducible Mx-1 promoter driven cre recombinase transgene and investigated its role during acute liver injury.

Methods:   We induced acute liver injury by a single i.p. injection of carbon tetrachloride (CCl4 ) in HB-EGF KO mice and wild-type mice and liver damage was assessed by biochemical and immunohistochemical analysis. We also used AML12 mouse hepatocyte cell lines to examine the molecular mechanism of HB-EGF-dependent anti-apoptosis and wound-healing process of the liver in vitro.

Results:   HB-EGF KO mice exhibited a significant increase of alanine aminotransferase level and also showed a significant increase in the number of apoptotic hepatocytes assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling staining at 24 h after CCl4 injection. We also demonstrated that HB-EGF treatment inhibited tumor necrosis factor-α-induced apoptosis of AML12 mouse hepatocytes and promoted the wound-healing response of these cells.

Conclusion:   This study showed that HB-EGF plays a protective role during acute liver injury.
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http://dx.doi.org/10.1111/j.1872-034X.2012.01074.xDOI Listing
April 2013

MIG-13 controls anteroposterior cell migration by interacting with UNC-71/ADM-1 and SRC-1 in Caenorhabditis elegans.

FEBS Lett 2012 Mar 27;586(6):740-6. Epub 2012 Jan 27.

Department of Oncogene Research, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan.

The transmembrane protein MIG-13 is a key regulator required for anterior migration of neural cells in Caenorhabditis elegans, but the signaling mechanisms involved remain unknown. Here, we isolated a suppressor mutation in the unc-71/adm-1 gene, which rescued the AVM neuron migration defect in mig-13 mutants. Genetic analyses revealed that UNC-71 at least partly acts downstream of MIG-13 and has an inhibitory effect on the anterior cell migration. The unc-71 mutation also rescued the anterior migration defect of AVM neuron in src-1 mutants. These findings suggest that MIG-13 controls anteroposterior cell migration by interacting with UNC-71 and SRC-1 in C. elegans.
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http://dx.doi.org/10.1016/j.febslet.2012.01.031DOI Listing
March 2012

Development of anti-HB-EGF immunoliposomes for the treatment of breast cancer.

J Control Release 2012 Jun 14;160(2):274-80. Epub 2011 Oct 14.

Department of Medical Biochemistry and Global COE Program, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.

Increased expression of heparin-binding epidermal growth factor-like growth factor (HB-EGF) is frequently observed in certain cancers such as ovarian and breast cancers, and this protein is a desirable target for drug delivery by a drug delivery system (DDS). In the present study, we developed novel immunoliposomes targeting HB-EGF for cancer therapy. The immunoliposomes significantly associated with Vero-H cells overexpressing HB-EGF compared with their binding to wild-type Vero cells, whereas liposomes without modification by the antibody did not associate with either type of cells. Moreover, enhanced uptake of the immunoliposomes into Vero-H cells was observed as well as that into MDA-MB-231 human breast cancer cells, which are known to highly express HB-EGF. These results suggest that HB-EGF mediates the binding and uptake of the immunoliposomes in HB-EGF-expressing cells. Next, we determined the therapeutic effect of these immunoliposomes encapsulating an anticancer drug on tumor-bearing mice. For this purpose, we prepared doxorubicin (DOX)-encapsulated immunoliposomes and injected them intravenously into mice bearing MDA-MB-231 cancer cells. As a result, these DOX-encapsulated immunoliposomes suppressed not only tumor progression but also tumor regression. In conclusion, our results indicate that anti-HB-EGF antibody-modified liposomes could be a useful DDS carrier for the treatment of HB-EGF-expressing cancers.
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http://dx.doi.org/10.1016/j.jconrel.2011.10.010DOI Listing
June 2012

HB-EGF and PDGF mediate reciprocal interactions of carcinoma cells with cancer-associated fibroblasts to support progression of uterine cervical cancers.

Cancer Res 2011 Nov 18;71(21):6633-42. Epub 2011 Oct 18.

Obstetrics and Gynecology, Osaka-Saiseikai-Nakatsu Hospital, Japan.

Tumor stroma drives the growth and progression of cancers. A heparin-binding epidermal growth factor-like growth factor, HB-EGF, is an EGF receptor ligand that stimulates cell growth in an autocrine or paracrine fashion. While elevated expression of HB-EGF in cancer cells and its contribution to tumor progression are well documented, the effects of HB-EGF expression in the tumor stroma have not been clarified. Here, we show that HB-EGF is expressed in stromal fibroblasts where it promotes cancer cell proliferation. In uterine cervical cancers, HB-EGF was detected immunohistochemically in the stroma proximal to the cancer epithelium. Proliferation of cervical cancer cells in vitro was enhanced by coculture with fibroblasts isolated from tumor tissues of patients with cervical cancer. Inhibition of HB-EGF function or treatment with platelet-derived growth factor (PDGF) inhibitors abrogated cancer cell growth enhanced by cervical cancer-associated fibroblast (CCF) coculture. Furthermore, tumor formation in a mouse xenograft model was enhanced by cotransplantation of CCF or mouse embryonic fibroblasts, but not with embryonic fibroblasts from HB-EGF-deficient mice. Conversely, conditioned medium from cancer cells induced HB-EGF expression in CCF. Mechanistic investigations established that PDGF was the primary factor responsible. Together, our findings indicate that HB-EGF and PDGF reciprocally mediate the interaction of cancer cells with cancer-associated fibroblasts, promoting cancer cell proliferation in a paracrine manner that has implications for novel combinatorial cancer therapies.
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http://dx.doi.org/10.1158/0008-5472.CAN-11-0034DOI Listing
November 2011

A novel anti-human HB-EGF monoclonal antibody with multiple antitumor mechanisms against ovarian cancer cells.

Clin Cancer Res 2011 Nov 14;17(21):6733-41. Epub 2011 Sep 14.

Department of Obstetrics and Gynecology, Fukuoka University, Fukuoka, Japan.

Purpose: Heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) is a member of the EGF family and plays a pivotal role in tumor progression in ovarian cancer. We developed an anti-HB-EGF monoclonal antibody (mAb) and investigated its antitumor activities in vitro and in vivo to evaluate its potential as a therapeutic antibody against ovarian cancer.

Experimental Design: We prepared mAbs from HB-EGF null mice immunized with recombinant human soluble HB-EGF and evaluated their binding and neutralizing activity against HB-EGF. Next, we generated a mouse-human chimeric antibody and examined its in vitro and in vivo antitumor activities.

Results: Two murine anti-HB-EGF mAbs were developed, and one of them, KM3566, was revealed to have a high binding reactivity for membrane-anchored HB-EGF (pro-HB-EGF) expressed on the cell surface, as well as neutralizing activity against growth promoting activity of soluble HB-EGF. The mouse-human chimeric counterpart for KM3566 (cKM3566) induced dose-dependent antibody-dependent cellular cytotoxicity (ADCC) against cancer cells expressing HB-EGF in vitro, and significantly inhibited tumor growth in severe combined immunodeficient mice inoculated with MCAS or ES-2 human ovarian cancer cells.

Conclusions: A novel anti-HB-EGF chimeric antibody, cKM3566, with two antitumor mechanisms, neutralization and ADCC, exhibits potent in vivo antitumor activity. These results indicate that cKM3566 is a promising antiovarian cancer therapeutic antibody.
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http://dx.doi.org/10.1158/1078-0432.CCR-11-1029DOI Listing
November 2011

Assessment of HB-EGF levels in peritoneal fluid and serum of ovarian cancer patients using ELISA.

Anticancer Res 2011 Jul;31(7):2553-9

Department of Obstetrics and Gynecology, Faculty of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-0180, Japan.

Background: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a rational target for ovarian cancer therapy. The aim of this study was to examine HB-EGF levels in the peritoneal fluid and serum of ovarian cancer (OVCA) patients.

Patients And Methods: Samples were collected from six healthy women, 21 OVCA patients, and 21 ovarian cyst patients. HB-EGF levels were measured using a sandwich ELISA kit and calculated using a parallel line assay.

Results: No significant difference between the slopes of the standard and sample curves was observed at an anti-HB-EGF antibody concentration of 1.6 μg/ml. HB-EGF levels in the peritoneal fluid and serum of OVCA patients were significantly higher than those in patients with ovarian cysts or controls. Serum HB-EGF levels were also significantly correlated with levels in peritoneal fluid in OVCA patients.

Conclusion: We developed an assay for the exact measurement of HB-EGF levels in peritoneal fluid and serum.
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July 2011

Proteolytic activation of heparin-binding EGF-like growth factor by membrane-type matrix metalloproteinase-1 in ovarian carcinoma cells.

Cancer Sci 2011 Jan 14;102(1):111-6. Epub 2010 Oct 14.

Division of Cancer Cell Research, Institute of Medical Science, The University of Tokyo, Tokyo, Japan.

Increased expression of heparin-binding EGF-like growth factor (HB-EGF) and membrane-type matrix metalloproteinase-1 (MT1-MMP) is frequently associated with various types of malignant tumor. HB EGF-like growth factor has been reported to promote the malignant progression of ovarian carcinoma. Based on this finding, inhibition of HB-EGF activity with CRM197 is now under phase I clinical evaluation. On the other hand, MT1-MMP expressed in ovarian carcinoma cells is thought to promote invasion and growth of tumor cells by degrading the extracellular matrix. However, we recently demonstrated that co-expression of MT1-MMP and HB-EGF in gastric carcinoma cells leads to cleavage of HB-EGF within its N-terminal heparin-binding region, converting it into a potent heparin-independent growth factor. In this study, we evaluated the importance of regulation of HB-EGF by MT1-MMP in clinical samples of ovarian carcinoma. We detected co-expression of HB-EGF and MT1-MMP in clear cell ovarian carcinoma tissues, particularly at the invasion front and in tumor cells that had disseminated into the ascites, whereas HB-EGF alone was expressed in non-invasive borderline ovarian tumor tissue. Furthermore, a soluble HB-EGF fragment that corresponds to that processed by MT1-MMP was detected in malignant ascites obtained from patients with metastatic ovarian carcinoma. Ovarian carcinoma cells that express MT1-MMP and HB-EGF exhibited enhanced cell growth in a 3D-collagen matrix and anchorage-independent growth in suspension. These results indicate that MT1-MMP co-expressed with HB-EGF in ovarian carcinoma cells potentiates the activity of HB-EGF to promote invasive tumor growth and spreading in vivo.
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http://dx.doi.org/10.1111/j.1349-7006.2010.01748.xDOI Listing
January 2011

Membrane type 1-matrix metalloproteinase cleaves off the NH2-terminal portion of heparin-binding epidermal growth factor and converts it into a heparin-independent growth factor.

Cancer Res 2010 Jul 29;70(14):6093-103. Epub 2010 Jun 29.

Division of Cancer Cell Research, Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Epidermal growth factor (EGF) receptors (ErbB) and EGF family members represent promising targets for cancer therapy. Heparin-binding EGF (HB-EGF) is a member of the EGF family and is an important target for therapy in some types of human cancers. Processing of HB-EGF by proprotein convertases, and successively, by ADAM family proteases, generates a soluble growth factor that requires heparin as a cofactor. Although heparin potentiates HB-EGF activity in vitro, it is not clear how the heparin-binding activity of HB-EGF is regulated. Here, we show that membrane type 1-matrix metalloproteinase (MT1-MMP; MMP14), a potent invasion-promoting protease, markedly enhances HB-EGF-dependent tumor formation in mice. MT1-MMP additionally cleaves HB-EGF and removes the NH(2)-terminal 20 amino acids that are important for binding heparin. Consequently, the processing of HB-EGF by MT1-MMP converts HB-EGF into a heparin-independent growth factor with enhanced mitogenic activity, and thereby, expression of both proteins costimulates tumor cell growth in vitro and in vivo. The ErbB family of receptors expressed in human gastric carcinoma cells play a role in mediating enhanced HB-EGF activity by MT1-MMP during invasive cell growth in collagen. Thus, we shed light on a new mechanism whereby HB-EGF activity is regulated that should be considered when designing HB-EGF-targeted cancer therapy.
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http://dx.doi.org/10.1158/0008-5472.CAN-10-0346DOI Listing
July 2010

HB-EGF function in cardiac valve development requires interaction with heparan sulfate proteoglycans.

Development 2010 Jul;137(13):2205-14

Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan.

HB-EGF, a member of the EGF family of growth factors, plays an important role in cardiac valve development by suppressing mesenchymal cell proliferation. Here, we show that HB-EGF must interact with heparan sulfate proteoglycans (HSPGs) to properly function in this process. In developing valves, HB-EGF is synthesized in endocardial cells but accumulates in the mesenchyme by interacting with HSPGs. Disrupting the interaction between HB-EGF and HSPGs in an ex vivo model of endocardial cushion explants resulted in increased mesenchymal cell proliferation. Moreover, homozygous knock-in mice (HB(Delta)(hb/)(Delta)(hb)) expressing a mutant HB-EGF that cannot bind to HSPGs developed enlarged cardiac valves with hyperproliferation of mesenchymal cells; this resulted in a phenotype that resembled that of Hbegf-null mice. Interestingly, although Hbegf-null mice had abnormal heart chambers and lung alveoli, HB(Delta)(hb/)(Delta)(hb) mice did not exhibit these defects. These results indicate that interactions with HSPGs are essential for the function of HB-EGF, especially in cardiac valve development, in which HB-EGF suppresses mesenchymal cell proliferation.
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http://dx.doi.org/10.1242/dev.048926DOI Listing
July 2010

Anti-human HB-EGF monoclonal antibodies inhibiting ectodomain shedding of HB-EGF and diphtheria toxin binding.

J Biochem 2010 Jul 23;148(1):55-69. Epub 2010 Mar 23.

Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1, Yamadaoka, Suita, Osaka 565-0871, Japan.

HB-EGF is a member of the EGF family of growth factors that bind and activate the EGF receptor. HB-EGF is synthesized as a membrane-anchored protein (proHB-EGF), and then proteolytically cleaved, resulting in the mitogenically active soluble form. ProHB-EGF functions as the receptor for the diphtheria toxin (DT). HB-EGF plays pivotal roles in pathophysiological processes, including cancer. Monoclonal antibodies (mAbs) specific for HB-EGF could be an important tool in HB-EGF research. However, few such mAbs have been established to date. In this study, we newly generated seven clones of hybridoma-derived mAbs by immunizing HB-EGF null mice with recombinant human HB-EGF protein. All mAbs specifically bound to human HB-EGF but not to mouse HB-EGF. Epitope mapping analysis showed that most of the mAbs recognized the EGF-like domain. Although none of the newly isolated mAbs directly inhibited the mitogenic activity of HB-EGF for EGFR-expressing cells, some strongly inhibited DT-binding. Interestingly, some of the mAbs efficiently inhibited ectodomain shedding of proHB-EGF, and consequently prevented the cell growth of the EGFR-expressing cells in a co-culture system with proHB-EGF-expressing cells. Hence, these new anti-HB-EGF mAbs may advance clinical as well as basic research on HB-EGF.
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http://dx.doi.org/10.1093/jb/mvq033DOI Listing
July 2010

Humanized gene replacement in mice reveals the contribution of cancer stroma-derived HB-EGF to tumor growth.

Cell Struct Funct 2010 ;35(1):3-13

Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan.

Tumor progression is a complex process that involves the interaction of cancer cells with the cancer-surrounding stromal cells. The cancer stroma influences the cancer cell growth and metastatic potential. The EGF family growth factor HB-EGF is synthesized in cancer cells and plays pivotal roles in oncogenic transformation and tumor progression, but the contribution of HB-EGF expressed in tumor stromal cells to tumor growth remains unclear. In the present study, we found that HB-EGF was expressed in host-derived cancer stromal cells in xenograft and allograft mouse tumor models. CRM197 is a specific inhibitor of human HB-EGF that has no effect on mouse HB-EGF. To elucidate whether host-derived stromal HB-EGF contributes to tumor growth, we generated knock-in mice expressing a CRM197-inhibitable humanized mutant form of HB-EGF. Administration of CRM197 to humanized knock-in mice that were bearing tumors derived from human or mouse cancer cells revealed that inhibition of host-derived stromal HB-EGF by CRM197 significantly reduced tumor growth. These results suggest that HB-EGF in the cancer-associated stroma plays a significant role for tumor growth, and that the HB-EGF derived from the stroma, as well as that expressed by cancer cells, is a potential target for cancer therapy. The present results also suggest that the humanized HB-EGF knock-in mice could be utilized for pathophysiological studies of HB-EGF as well as the development of therapeutic strategies targeting HB-EGF.
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http://dx.doi.org/10.1247/csf.09025DOI Listing
May 2010

Integrin signal masks growth-promotion activity of HB-EGF in monolayer cell cultures.

J Cell Sci 2009 Dec 3;122(Pt 23):4277-86. Epub 2009 Nov 3.

Department of Cell Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871, Japan.

The extracellular environment and tissue architecture contribute to proper cell function and growth control. Cells growing in monolayers on standard polystyrene tissue culture plates differ in their shape, growth rate and response to external stimuli, compared with cells growing in vivo. Here, we showed that the EGFR (epidermal growth factor receptor) ligand heparin-binding EGF-like growth factor (HB-EGF) strongly stimulated cell growth in nude mice, but not in cells cultured in vitro. We explored the effects of HB-EGF on cell growth under various cell culture conditions and found that growth promotion by HB-EGF was needed in three-dimensional (3D) or two-dimensional (2D) culture systems in which cell-matrix adhesion was reduced. Under such conditions, cell growth was extremely suppressed in the absence of HB-EGF, but markedly potentiated in the presence of HB-EGF. When the integrin signal was reduced using antibodies or knockout of either integrin beta1 or focal adhesion kinase (FAK), cells showed HB-EGF-dependent growth. We also showed that EGF, transforming growth factor-alpha (TGFalpha) or ligands of other receptor tyrosine kinases (RTKs) stimulated cell growth in 3D culture, but not in tissue culture plates. These results indicate that the integrin signal was sufficient to support cell growth in 2D tissue culture plates without addition of the growth factor, whereas stimulation by growth factors was clearly demonstrated in culture systems in which integrin signals were attenuated.
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http://dx.doi.org/10.1242/jcs.054551DOI Listing
December 2009