[Show abstract][Hide abstract] ABSTRACT: Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine.
[Show abstract][Hide abstract] ABSTRACT: The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) modulates the tight junction protein claudin and disrupts the tight junctional barrier. It also can enhance the effectiveness of anticancer agents. However, the detailed mechanisms of the effects of C-CPE remain unclear in both normal and cancerous cells. The C-CPE mutant called C-CPE 194 binds only to claudin-4, but the C-CPE 194 mutant called C-CPE m19 binds not only to claudin-4 but also to claudin-1. In the present study, to investigate the mechanisms of the effects of C-CPE on claudin expression, the tight junctional functions and the cytotoxicity of anticancer agents, human pancreatic cancer cells, and normal human pancreatic duct epithelial cells (HPDEs) were treated with C-CPE 194 and C-CPE m19. In well-differentiated cells of the pancreatic cancer cell line HPAC, C-CPE 194 and C-CPE m19 disrupted both the barrier and fence functions without changes in expression of claudin-1 and -4, together with an increase of MAPK phosphorylation. C-CPE 194, but not C-CPE m19, enhanced the cytotoxicity of the anticancer agents gemcitabine and S-1. In poorly differentiated pancreatic cancer cell line PANC-1, C-CPE 194, but not C-CPE m19, decreased claudin-4 expression and enhanced MAPK activity and the cytotoxicity of the anticancer agents. In normal HPDEs, C-CPE 194 and C-CPE m19 decreased claudin-4 expression and enhanced the MAPK activity, whereas they did not affect the cytotoxicity of the anticancer agents. Our findings suggest that the claudin-4 binder C-CPE 194 enhances effects of anticancer agents on pancreatic cancer cell lines via a MAPK pathway.
[Show abstract][Hide abstract] ABSTRACT: Tight junctions (TJs) are complex biochemical structures that seal the intercellular space and prevent the free movement of solutes across epithelial cell sheets. Modulating the TJ seal is a promising option for increasing the transdermal absorption of drugs. Within TJs, the binding of the claudin (CLDN) family of tetra-transmembrane proteins through cis- and trans-interactions is an integral part of seal formation. Because epidermal TJs contain CLDN-1 and -4, a binder for these CLDNs may be a useful modulator of the permeability of the epidermal barrier. Here we investigated whether m19, which can bind to CLDN-1/-4 （also CLDN-2/-5）, modulates the integrity of epidermal TJs and the permeability of cell sheets to solutes. Treatment of normal human epidermal keratinocytes (NHEKs) with the CLDNs binder reduced the integrity of TJs. A CLDN-1-specific binder (a monoclonal antibody, clone 7A5) also weakened the TJ seal in NHEKs. Although m19 attenuated the TJ barrier in human intestinal epithelial cells (Caco-2), 7A5 did not. Treatment of NHEKs with 7A5 enhanced permeation of a paracellular permeation marker. These findings indicate that CLDN-1 is a potential target for modulating the permeability of the epidermis and that our CLDN-1 binder is a promising candidate molecule for development as a dermal absorption enhancer.
The American Society for Pharmacology and Experimental Therapeutics.
No preview · Article · Jul 2015 · Journal of Pharmacology and Experimental Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Intranasal insulin administration has therapeutic potential for Alzheimer's disease and in intranasal administration across the nasal mucosa, the paracellular pathway regulated by tight junctions is important. The C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) binds the tight junction protein claudin and disrupts the tight junctional barrier without a cytotoxic effect. The C-CPE mutant called C-CPE 194 binds only to claudin-4, whereas the C-CPE 194 mutant called C-CPE m19 binds not only to claudin-4 but also to claudin-1.
In the present study, to investigate the effects of C-CPE mutants on the tight junctional functions of human nasal epithelial cells (HNECs) and on the permeability of human recombinant insulin across the cells, HNECs were treated with C-CPE 194 and C-CPE m19.
C-CPE 194 and C-CPE m19 disrupted the barrier and fence functions without changes in expression of claudin-1, -4, -7, and occludin or cytotoxicity, whereas they transiently increased the activity of ERK1/2 phosphorylation. The disruption of the barrier function caused by C-CPE 194 and C-CPE m19 was prevented by pretreatment with the MAPKK inhibitor U0126. Furthermore, C-CPE 194 and C-CPE m19 significantly enhanced the permeability of human recombinant insulin across HNECs and the permeability was also inhibited by U0126.
These findings suggest that C-CPE mutants 194 and m19 can regulate the permeability of insulin across HNECs via the MAPK pathway and may play a crucial role in therapy for the diseases such as Alzheimer's disease via the direct intranasal insulin administration.
[Show abstract][Hide abstract] ABSTRACT: Efficient vaccine delivery to mucosal tissues including mucosa-associated lymphoid tissues is essential for the development of mucosal vaccine. We previously reported that claudin-4 was highly expressed on the epithelium of nasopharynx-associated lymphoid tissue (NALT) and thus claudin-4-targeting using C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) effectively delivered fused antigen to NALT and consequently induced antigen-specific immune responses. In this study, we applied the C-CPE-based vaccine delivery system to develop a nasal pneumococcal vaccine. We fused C-CPE with pneumococcal surface protein A (PspA), an important antigen for the induction of protective immunity against Streptococcus pneumoniae infection, (PspA-C-CPE). PspA-C-CPE binds to claudin-4 and thus efficiently attaches to NALT epithelium, including antigen-sampling M cells. Nasal immunization with PspA-C-CPE induced PspA-specific IgG in the serum and bronchoalveolar lavage fluid (BALF) as well as IgA in the nasal wash and BALF. These immune responses were sufficient to protect against pneumococcal infection. These results suggest that C-CPE is an efficient vaccine delivery system for the development of nasal vaccines against pneumococcal infection.
[Show abstract][Hide abstract] ABSTRACT: Claudins constitute a family of at least 27 proteins with four transmembrane domains, and play a pivotal role in maintaining tight-junctions seals in diverse epithelial tissues. The expression of claudin-4 often changes in intestinal tissues of inflammatory bowel disease and various human cancers. Therefore, claudin-4 is a promising target for treatment of these diseases. In our previous study, we established a reporter cell line to monitor claudin-4 expression on the basis of a functional claudin-4 promoter. Using this cell line, we have performed a cell-based screen of a library containing 2642 biologically active small-molecule compounds to identify modulators of claudin-4 expression. The screen identified 24 potential modulators of the claudin-4 promoter activity. Fourteen of these compounds (12 of them novel) induced endogenous claudin-4 expression. The identified compounds might serve as lead compounds targeting aberrant gene expression in inflammatory bowel disease.
Full-text · Article · Feb 2015 · Biotechnology Letters
[Show abstract][Hide abstract] ABSTRACT: Claudin-1 (CLDN1), a known host factor for hepatitis C virus (HCV) entry and cell-to-cell transmission, is a target molecule for inhibiting HCV infection. We previously developed 4 clones of mouse anti-CLDN1 monoclonal antibody (mAb) that prevented HCV infection in vitro. Two of these mAbs showed the highest anti-viral activity. Here, we optimized the anti-CLDN1 mAbs as candidates for therapeutics by protein engineering. Although Fab fragments of the mAbs prevented in vitro HCV infection, their inhibitory effects were much weaker than those of the whole mAbs. In contrast, human chimeric IgG1 mAbs generated by grafting the variable domains of the mouse mAb light and heavy chains inhibited in vitro HCV infection as efficiently as the parental mouse mAbs. However, the chimeric IgG1 mAbs activated Fcγ receptor, suggesting that cytotoxicity against mAb-bound CLDN1-expressing cells occurred through the induction of antibody-dependent cellular cytotoxicity (ADCC). To avoid ADCC-induced side effects, we prepared human chimeric IgG4 mAbs. The chimeric IgG4 mAbs did not activate Fcγ receptor or induce ADCC, but they prevented in vitro HCV infection as efficiently as did the parental mouse mAbs. These findings indicate that IgG4 form of human chimeric anti-CLDN1 mAb may be a candidate molecule for clinically applicable HCV therapy.
The American Society for Pharmacology and Experimental Therapeutics.
No preview · Article · Jan 2015 · Journal of Pharmacology and Experimental Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Nanomaterials have been proposed as novel substrates for medical and commercial applications. However, such materials also may have novel toxicities, thus posing environmental and health concerns. We previously reported hepatic injury in mice following the intravenous administration of unmodified silica particles with diameters of 70 nm (SP70); this toxicity was not observed following administration by the same route of micro-size particles with diameters of 300 nm (SP300) or 1,000 nm (SP1000). In the present study, we used electron microscopy to investigate the dynamics of silica nanoparticles administered in mice. SP70 was observed in hepatocytes and in lung epithelial cells. Inclusion within hepatocytes was associated with accumulation of SP70 in the liver sinusoidal endothelial cells and passage through the space of Disse. In contrast, SP300 and SP1000 were not observed within the hepatocytes. To our knowledge, our report represents the first demonstration that silica nanoparticles accumulate in hepatocytes, liver sinusoidal endothelial cells, Kupffer cells, and lung tissue; accumulation of SP70 in liver sinusoidal endothelial cells correlated with the induction of liver injury.
[Show abstract][Hide abstract] ABSTRACT: Homoharringtonine (HHT), a natural alkaloid produced by various Cephalotaxus species, has antileukemic activity in acute and chronic myelogenous leukemia. However, HHT can also induce unanticipated effects in the gastrointestinal tract, such as diarrhea and nausea/vomiting, but the mechanism behind these adverse effects has not been clarified. In the present study, we show that HHT affects the epithelial permeability of intestinal Caco-2 cell monolayers. HHT reduced the transepithelial electrical resistance (TER) of Caco-2 cells in a dose- and time-dependent manner. The HHT effect was reversible and no cytotoxicity was observed at the concentrations used. HHT simultaneously increased the paracellular flux of the 4 kDa and 40 kDa FITC-dextrans associated with the TER reduction. Immunoblotting analysis revealed that HHT decreased the protein expression of TJ components such as claudin-3, -5, and -7. However, the transcription levels of these claudins were not repressed by HHT treatment. HHT also disturbed the cellular localization of claudin-1 and -4. These changes coincided with the reduced barrier function. Our findings suggest that HHT enhances the paracellular permeability of Caco-2 cell monolayers by modulating the protein expression and localization of claudin isoforms; these actions might be responsible for the gastrointestinal effects of HHT.
No preview · Article · Dec 2014 · European Journal of Pharmaceutics and Biopharmaceutics
[Show abstract][Hide abstract] ABSTRACT: Most malignant tumors are derived from epithelium, and claudin (CLDN)-3 and -4 are frequently overexpressed in such tumors. Although antibodies have potential in cancer diagnostics and therapy, development of antibodies against CLDNs has been difficult because the extracellular domains of CLDNs are too small and there is high homology among human, rat, and mouse sequences. Here, we created a monoclonal antibody that recognizes human CLDN-3 and -4 by immunizing rats with a plasmid vector encoding human CLDN-4. A hybridoma clone that produced a rat monoclonal antibody recognizing both CLDN-3 and -4 (clone 5A5) was obtained from a hybridoma screen by using CLDN-3- and -4-expressing cells; 5A5 did not bind to CLDN-1, -2, -5, -6, -7 or -9-expressing cells. Fluorescence-conjugated 5A5 injected into xenograft mice bearing human cancer MKN74 or LoVo cells could visualize the tumor cells. The human-rat chimeric IgG1 monoclonal antibody (xi5A5) activated FcgammaRIIIa in the presence of CLDN-3- or -4-expressing cells, indicating that xi5A5 may exert antibody-dependent cellular cytotoxicity. Administration of xi5A5 attenuated tumor growth in xenograft mice bearing MKN74 or LoVo cells. These results suggest that 5A5 shows promise in the development of a diagnostic and therapeutic antibody for cancers.
Preview · Article · Aug 2014 · Journal of Pharmacology and Experimental Therapeutics
[Show abstract][Hide abstract] ABSTRACT: Unlabelled:
Diacylglycerol acyltransferase-1 (DGAT1) is involved in the assembly of hepatitis C virus (HCV) by facilitating the trafficking of the HCV core protein to the lipid droplet. Here, we abrogated DGAT1 expression in Huh-7.5 cells by using either the transcription activator-like effector nuclease (TALEN) or lentivirus vector short hairpin RNA (shRNA) and achieved complete long-term silencing of DGAT1. HCV entry was severely impaired in DGAT1-silenced Huh-7.5 cell lines, which showed markedly diminished claudin-1 (CLDN1) expression. In DGAT1-silenced cell lines, the forced expression of CLDN1 restored HCV entry, implying that the downregulation of CLDN1 is a critical factor underlying defective HCV entry. The expression of the gene coding for hepatocyte nuclear factor 4α (HNF4α) and other hepatocyte-specific genes was also reduced in DGAT1-silenced cell lines. After DGAT1 gene rescue, CLDN1 expression was preserved, and HCV entry was restored. Strikingly, after DGAT1 silencing, CLDN1 expression and HCV entry were also restored by low-dose palmitic acid treatment, indicating that the downregulation of CLDN1 was associated with altered fatty acid homeostasis in the absence of DGAT1. Our findings provide novel insight into the role of DGAT1 in the life cycle of HCV.
In this study, we report the novel effect of complete silencing of DGAT1 on the entry of HCV. DGAT1 was recently reported as a host factor of HCV, involved in the assembly of HCV by facilitating the trafficking of the HCV core protein to lipid droplets. We achieved complete and long-term silencing of DGAT1 by either TALEN or repeated transduction of lentivirus shRNA. We found that HCV entry was severely impaired in DGAT1-silenced cell lines. The impairment of HCV entry was caused by CLDN1 downregulation, and the expression of HNF4α and other hepatocyte-specific genes was also downregulated in DGAT1-silenced cell lines. Our results suggest new roles of DGAT1 in human liver-derived cells: maintaining intracellular lipid homeostasis and affecting HCV entry by modulating CLDN1 expression.
Preview · Article · Jun 2014 · Journal of Virology
[Show abstract][Hide abstract] ABSTRACT: Objective:
The molecular basis of endothelial cell (EC)-specific gene expression is poorly understood. Roundabout 4 (Robo4) is expressed exclusively in ECs. We previously reported that the 3-kb 5'-flanking region of the human Robo4 gene contains information for lineage-specific expression in the ECs. Our studies implicated a critical role for GA-binding protein and specificity protein 1 (SP1) in mediating overall expression levels. However, these transcription factors are also expressed in non-ECs. In this study, we tested the hypothesis that epigenetic mechanisms contribute to EC-specific Robo4 gene expression.
Methods and results:
Bisulfite sequencing analysis indicated that the proximal promoter of Robo4 is methylated in non-ECs but not in ECs. Treatment with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine increased Robo4 gene expression in non-ECs but not in ECs. Proximal promoter methylation significantly decreased the promoter activity in ECs. Electrophoretic mobility shift assays showed that DNA methylation of the proximal promoter inhibited SP1 binding to the -42 SP1 site. In DNase hypersensitivity assays, chromatin condensation of the Robo4 promoter was observed in some but not all nonexpressing cell types. In Hprt (hypoxanthine phosphoribosyltransferase)-targeted mice, a 0.3-kb proximal promoter directed cell-type-specific expression in the endothelium. Bisulfite sequencing analysis using embryonic stem cell-derived mesodermal cells and ECs indicated that the EC-specific methylation pattern of the promoter is determined by demethylation during differentiation and that binding of GA-binding protein and SP1 to the proximal promoter is not essential for demethylation.
The EC-specific DNA methylation pattern of the Robo4 proximal promoter is determined during cell differentiation and contributes to regulation of EC-specific Robo4 gene expression.
Preview · Article · May 2014 · Arteriosclerosis Thrombosis and Vascular Biology
[Show abstract][Hide abstract] ABSTRACT: Most pathogens invade body through the mucosal epithelium, which is a primary target to prevent the infectious diseases. Mucosal vaccine has been considered to be an effective strategy to establish immunosurveillance against pathogens by the induction of antigen-specific immune responses at both mucosal and systemic immune compartments. The development of antigen delivery system and mucosal adjuvants are required for the sufficient induction of protective immunity in the development of mucosal vaccine. In this review, we shed light on the recent advances in the development of antigen delivery system using microbial functions for mucosal vaccines.
[Show abstract][Hide abstract] ABSTRACT: Epithelium plays pivotal roles in biological barrier separating the inside of body and the outside environment. Ninety percent of malignant tumors are derived from epithelium. Most pathological microorganisms invade into the body from mucosal epithelium. Thus, epithelium is potential targets for drug development. Claudins (CLs), a family of tetra-transmembrane protein consisting of over 20 members, are structural and functional components of tight junction-seals in epithelium. Modulation of CL-seals enhanced mucosal absorption of drugs. CLs are often over-expressed in malignant tumors. CL-4 expression is increased in the epithelial cells covering the mucosal immune tissues. Very recently, CLs are also expected to be targets for traumatic brain injury and regenerative therapy. In this review, we overview the past, the present and the future of CLs-targeted drug development.