Adachi, M., Inoko, A., Hata, M., Furuse, K., Umeda, K., Itoh, M., Tsukita, S., 2006. Normal
Establishment of Epithelial Tight Junctions in Mice and Cultured Cells Lacking Expression of ZO-3,
a Tight-Junction MAGUK Protein. Mol. Cell. Biol. 26, 9003–9015.
https://doi.org/10.1128/MCB.01811-05
Aijaz, S., Balda, M.S., Matter, K., 2006. Tight Junctions: Molecular Architecture and Function, in:
International Review of Cytology. pp. 261–298. https://doi.org/10.1016/S0074-7696(06)48005-0
Amasheh, M., Schlichter, S., Amasheh, S., Mankertz, J., Zeitz, M., Fromm, M., Schulzke, J.D., 2008.
Quercetin enhances epithelial barrier function and increases claudin-4 expression in Caco-2 cells. J.
Nutr. 138, 1067–73. https://doi.org/10.1093/jn/138.6.1067
Appleton, B.A., Zhang, Y., Wu, P., Yin, J.P., Hunziker, W., Skelton, N.J., Sidhu, S.S., Wiesmann, C.,
2006. Comparative structural analysis of the Erbin PDZ domain and the first PDZ domain of ZO-1.
Insights into determinants of PDZ domain specificity. J. Biol. Chem. 281, 22312–22320.
https://doi.org/10.1074/jbc.M602901200
Balda, M.S., González-Mariscal, L., Contreras, R.G., Macias-Silva, M., Torres-Marquez, M.E., GarcíaSáinz, J.A., Cereijido, M., 1991. Assembly and sealing of tight junctions: possible participation of Gproteins, phospholipase C, protein kinase C and calmodulin. J. Membr. Biol. 122, 193–202.
Balda, M.S., Gonzalez-Mariscal, L., Matter, K., Cereijido, M., Anderson, J.M., 1993. Assembly of the
tight junction: the role of diacylglycerol. J. Cell Biol. 123, 293–302.
Cai, Y., Ma, W., Xiao, Y., Wu, B., Li, X., Liu, F., Qiu, J., Zhang, G., 2017. High doses of baicalin induces
kidney injury and fibrosis through regulating TGF-β/Smad signaling pathway. Toxicol. Appl.
Pharmacol. 333, 1–9. https://doi.org/10.1016/j.taap.2017.08.003
Chen, X.M., Kitts, D.D., 2017. Flavonoid composition of orange peel extract ameliorates alcohol-induced
tight junction dysfunction in Caco-2 monolayer. Food Chem. Toxicol. 105, 398–406.
https://doi.org/10.1016/j.fct.2017.04.009
Chuenkitiyanon, S., Pengsuparp, T., Jianmongkol, S., 2010. Protective effect of quercetin on hydrogen
peroxide-induced tight junction disruption. Int. J. Toxicol. 29, 418–24.
https://doi.org/10.1177/1091581810366487
Dhanasekaran, R., Owens, V., Sanchez, W., 2013. Chinese skullcap in move free arthritis supplement
causes drug induced liver injury and pulmonary infiltrates. Case reports Hepatol. 2013, 965092.
https://doi.org/10.1155/2013/965092
Fanning, A.S., Anderson, J.M., 1996. Protein–protein interactions: PDZ domain networks. Curr. Biol. 6,
1385–1388. https://doi.org/10.1016/S0960-9822(96)00737-3
19
Fanning, A.S., Mitic, L.L., Anderson, J.M., 1999. Transmembrane proteins in the tight junction barrier. J.
Am. Soc. Nephrol. 10, 1337–1345.
Förster, C., 2008. Tight junctions and the modulation of barrier function in disease. Histochem. Cell Biol.
130, 55–70. https://doi.org/10.1007/s00418-008-0424-9
Furuse, M., 2009. Knockout animals and natural mutations as experimental and diagnostic tool for
studying tight junction functions in vivo. Biochim. Biophys. Acta - Biomembr. 1788, 813–819.
https://doi.org/10.1016/j.bbamem.2008.07.017
Furuse, M., Fujita, K., Hiiragi, T., Fujimoto, K., Tsukita, S., 1998. Claudin-1 and -2: novel integral
membrane proteins localizing at tight junctions with no sequence similarity to occludin. J. Cell Biol.
141, 1539–50.
Furuse, M., Furuse, K., Sasaki, H., Tsukita, S., 2001. Conversion of zonulae occludentes from tight to
leaky strand type by introducing claudin-2 into Madin-Darby canine kidney I cells. J. Cell Biol. 153,
263–72. https://doi.org/10.1083/jcb.153.2.263
Furuse, M., Hata, M., Furuse, K., Yoshida, Y., Haratake, A., Sugitani, Y., Noda, T., Kubo, A., Tsukita, S.,
2002. Claudin-based tight junctions are crucial for the mammalian epidermal barrier. J. Cell Biol.
156, 1099–1111. https://doi.org/10.1083/jcb.200110122
González-Mariscal, L., Betanzos, A., Nava, P., Jaramillo, B.E., 2003. Tight junction proteins. Prog.
Biophys. Mol. Biol. 81, 1–44.
Harris, B.Z., Lim, W.A., 2001. Mechanism and role of PDZ domains in signaling complex assembly. J.
Cell Sci. 114, 3219–3231.
Hiroaki, H., Satomura, K., Goda, N., Nakakura, Y., Hiranuma, M., Tenno, T., Hamada, D., Ikegami, T.,
2018. Spatial Overlap of Claudin- and Phosphatidylinositol Phosphate-Binding Sites on the First
PDZ Domain of Zonula Occludens 1 Studied by NMR. Molecules 23, 2465.
https://doi.org/10.3390/molecules23102465
Hu, L., Wu, C., Zhang, Z., Liu, M., Maruthi Prasad, E., Chen, Y., Wang, K., 2019. Pinocembrin Protects
Against Dextran Sulfate Sodium-Induced Rats Colitis by Ameliorating Inflammation, Improving
Barrier Function and Modulating Gut Microbiota. Front. Physiol. 10, 908.
https://doi.org/10.3389/fphys.2019.00908
Ikenouchi, J., Umeda, K., Tsukita, Sachiko, Furuse, M., Tsukita, Shoichiro, 2007. Requirement of ZO-1
for the formation of belt-like adherens junctions during epithelial cell polarization. J. Cell Biol. 176,
779–86. https://doi.org/10.1083/jcb.200612080
Itoh, M., Furuse, M., Morita, K., Kubota, K., Saitou, M., Tsukita, S., 1999. Direct binding of three tight
junction-associated MAGUKs, ZO-1, ZO-2, and ZO-3, with the COOH termini of claudins. J. Cell
Biol. 147, 1351–1363.
20
Jiang, L., Liu, C., Leibly, D., Landau, M., Zhao, M., Hughes, M.P., Eisenberg, D.S., 2013. Structure-based
discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta. Elife 2, e00857.
https://doi.org/10.7554/eLife.00857
Jin, R., Song, Z., Yu, S., Piazza, A., Nanda, A., Penninger, J.M., Granger, D.N., Li, G., 2011.
Phosphatidylinositol-3-kinase gamma plays a central role in blood-brain barrier dysfunction in acute
experimental stroke. Stroke. 42, 2033–2044. https://doi.org/10.1161/STROKEAHA.110.601369
Li, X., Guo, L., Sun, Y., Zhou, J., Gu, Y., Li, Y., 2010. Baicalein inhibits melanogenesis through
activation of the ERK signaling pathway. Int. J. Mol. Med. 25, 923–927.
https://doi.org/10.3892/ijmm_00000423
Linnebur, S.A., Rapacchietta, O.C., Vejar, M., 2010. Hepatotoxicity associated with chinese skullcap
contained in Move Free Advanced dietary supplement: two case reports and review of the literature.
Pharmacotherapy 30, 750–750. https://doi.org/10.1592/phco.30.7.750
Liu, Y., 2010. New insights into epithelial-mesenchymal transition in kidney fibrosis. J. Am. Soc.
Nephrol. 21, 212–22. https://doi.org/10.1681/ASN.2008121226
Makino, T., Hishida, A., Goda, Y., Mizukami, H., 2008. Comparison of the major flavonoid content of S.
baicalensis, S. lateriflora, and their commercial products. J. Nat. Med. 62, 294–9.
https://doi.org/10.1007/s11418-008-0230-7
Nakahara, T., Nishitani, Y., Nishiumi, S., Yoshida, M., Azuma, T., 2017. Astilbin from Engelhardtia
chrysolepis enhances intestinal barrier functions in Caco-2 cell monolayers. Eur. J. Pharmacol. 804,
46–51. https://doi.org/10.1016/j.ejphar.2017.03.041
Noda, S., Tanabe, S., Suzuki, T., 2013. Naringenin enhances intestinal barrier function through the
expression and cytoskeletal association of tight junction proteins in Caco-2 cells. Mol. Nutr. Food
Res. 57, 2019–2028. https://doi.org/10.1002/mnfr.201300045
Nomme, J., Antanasijevic, A., Caffrey, M., Van Itallie, C.M., Anderson, J.M., Fanning, A.S., Lavie, A.,
2015. Structural Basis of a Key Factor Regulating the Affinity between the Zonula Occludens First
PDZ Domain and Claudins. J. Biol. Chem. 290, 16595–16606.
https://doi.org/10.1074/jbc.M115.646695
Paris, L., Tonutti, L., Vannini, C., Bazzoni, G., 2008. Structural organization of the tight junctions.
Biochim. Biophys. Acta 1778, 646–59. https://doi.org/10.1016/j.bbamem.2007.08.004
Pei, J., Grishin, N. V., 2014. PROMALS3D: Multiple Protein Sequence Alignment Enhanced with
Evolutionary and Three-Dimensional Structural Information, in: Multiple Sequence Alignment
Methods. pp. 263–271. https://doi.org/10.1007/978-1-62703-646-7_17
Piegholdt, S., Pallauf, K., Esatbeyoglu, T., Speck, N., Reiss, K., Ruddigkeit, L., Stocker, A., Huebbe, P.,
Rimbach, G., 2014. Biochanin A and prunetin improve epithelial barrier function in intestinal CaCo-
21
2 cells via downregulation of ERK, NF-κB, and tyrosine phosphorylation. Free Radic. Biol. Med. 70,
255–264. https://doi.org/10.1016/j.freeradbiomed.2014.02.025
Piotto, M., Saudek, V., Sklenár, V., 1992. Gradient-tailored excitation for single-quantum NMR
spectroscopy of aqueous solutions. J. Biomol. NMR 2, 661–665.
Schrödinger, LLC, 2015. The {PyMOL} Molecular Graphics System, Version~1.8.
Schumann, F.H., Riepl, H., Maurer, T., Gronwald, W., Neidig, K.-P., Kalbitzer, H.R., 2007. Combined
chemical shift changes and amino acid specific chemical shift mapping of protein-protein
interactions. J. Biomol. NMR 39, 275–89. https://doi.org/10.1007/s10858-007-9197-z
Sheng, M., 2001. Molecular organization of the postsynaptic specialization. Proc. Natl. Acad. Sci. U. S. A.
98, 7058–7061. https://doi.org/10.1073/pnas.111146298
Suzuki, T., Hara, H., 2011. Role of flavonoids in intestinal tight junction regulation. J. Nutr. Biochem. 22,
401–408. https://doi.org/10.1016/j.jnutbio.2010.08.001
Suzuki, T., Hara, H., 2009. Quercetin enhances intestinal barrier function through the assembly of zonula
occludens-2, occludin, and claudin-1 and the expression of claudin-4 in Caco-2 cells. J. Nutr. 139,
965–74. https://doi.org/10.3945/jn.108.100867
Suzuki, T., Tanabe, S., Hara, H., 2011. Kaempferol enhances intestinal barrier function through the
cytoskeletal association and expression of tight junction proteins in Caco-2 cells. J. Nutr. 141, 87–
94. https://doi.org/10.3945/jn.110.125633
Takeuchi, K., Satoh, H., 2015. NSAID-induced small intestinal damage - Roles of various pathogenic
factors. Digestion. https://doi.org/10.1159/000374106
Tang, W., Sun, X., Fang, J.S., Zhang, M., Sucher, N.J., 2004. Flavonoids from Radix Scutellariae as
potential stroke therapeutic agents by targeting the second postsynaptic density 95 (PSD-95)/disc
large/zonula occludens-1 (PDZ) domain of PSD-95. Phytomedicine 11, 277–284.
https://doi.org/10.1078/0944711041495173
Tenno, T., Goda, N., Umetsu, Y., Ota, M., Kinoshita, K., Hiroaki, H., 2013. Accidental interaction
between PDZ domains and diclofenac revealed by NMR-assisted virtual screening. Molecules 18,
9567–81. https://doi.org/10.3390/molecules18089567
Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F., Higgins, D.G., 1997. The CLUSTAL_X
windows interface: flexible strategies for multiple sequence alignment aided by quality analysis
tools. Nucleic Acids Res. 25, 4876–82.
Tsukita, S., Furuse, M., Itoh, M., 2001. Multifunctional strands in tight junctions. Nat. Rev. Mol. Cell
Biol. 2, 285–293. https://doi.org/10.1038/35067088
Umeda, K., Ikenouchi, J., Katahira-Tayama, S., Furuse, K., Sasaki, H., Nakayama, M., Matsui, T.,
Tsukita, Sachiko, Furuse, M., Tsukita, Shoichiro, 2006. ZO-1 and ZO-2 independently determine
22
where claudins are polymerized in tight-junction strand formation. Cell 126, 741–754.
https://doi.org/10.1016/j.cell.2006.06.043
Umetsu, Y., Goda, N., Taniguchi, R., Satomura, K., Ikegami, T., Furuse, M., Hiroaki, H., 2011. 1H, 13C,
and 15N resonance assignment of the first PDZ domain of mouse ZO-1. Biomol. NMR Assign. 5,
207–210. https://doi.org/10.1007/s12104-011-9301-x
Wang, X., Li, B., Zhao, W.-D., Liu, Y.-J., Shang, D.-S., Fang, W.-G., Chen, Y.-H., 2011. Perfluorooctane
sulfonate triggers tight junction “opening” in brain endothelial cells via phosphatidylinositol 3kinase. Biochem. Biophys. Res. Commun. 410, 258–263. https://doi.org/10.1016/j.bbrc.2011.05.128
Xie, L., Law, B.K., Chytil, A.M., Brown, K.A., Aakre, M.E., Moses, H.L., 2004. Activation of the Erk
Pathway Is Required for TGF-β1-Induced EMT In Vitro. Neoplasia 6, 603–610.
https://doi.org/10.1593/neo.04241
Yamashita, S., Saitoh, H., Nakanishi, K., Masada, M., Nadai, T., Kimura, T., 1985. Characterization of
enhanced intestinal permeability; electrophysiological study on the effects of diclofenac and
ethylenediaminetetraacetic acid. J. Pharm. Pharmacol. 37, 512–3. https://doi.org/10.1111/j.20427158.1985.tb03056.x
Zimmermann, P., 2006. The prevalence and significance of PDZ domain-phosphoinositide interactions.
Biochim. Biophys. Acta 1761, 947–56. https://doi.org/10.1016/j.bbalip.2006.04.003
Figure Legends
Fig. 1.
Multiple sequence alignment and structural comparison of the first PDZ domain of
ZO-1 and second PDZ domain of PSD-95. (A) Structure based multiple sequence alignment of
selected PDZ domains against crystal structure of mouse ZO-1-PDZ1 (PDB: 4yyx_A). Protein
names and accession numbers are as follows: ZO-1 mouse, P39447.2; rat, A0A0G2K2P5;
human, Q07157.3; PSD-95 mouse, Q62108.1; rat, P31016.1; and human P78352.3, respectively.
The secondary structure of ZO-1(PDZ1) was shown below the sequence. The residues
corresponding to the canonical binding pocket were boxed. The sequence alignment was
generated by PROMALS3D software (Pei and Grishin, 2014) and colored by ClustalX
23
(Thompson et al., 1997). Electrostatic surface potential diagrams of (B) ZO-1(PDZ1) and (C)
PSD-95(PDZ-2), with positive (blue) and negative (red) electrostatic potentials mapped onto a
van der Waals surface diagram of the canonical peptide binding site. Coordinates from PDB
codes 4YYX and 3GSL were used, respectively. The color scale ranges between +20 kBT (red)
and −20 kBT (blue), where kB is Boltzmann’s constant and T is temperature. The canonical
binding pockets were hexagonal boxed.
Fig 2.
Direct interaction between ZO-1(PDZ1) and the flavonoids from Radix
scutellariae. Overlaid HSQC spectra of 0.1 mM ZO-1(PDZ1) in the absence (black) and presence
of 0.2 mM of baicalin (A) and baicalein (B) (red), respectively. (C) Chemical structure of the
selected flavonoids from Radix scutellariae used in this study. Normalized chemical shift
changes of baicalin (C) and baicalein (D). The changes induced by baicalin and baicalein were
mapped to the structure (F, G). Resonances representing residues with larger chemical shift
changes than the threshold values are mapped onto the ribbon model of mouse ZO-1(PDZ1)
(PDB: 2RRM). The threshold values are indicated by dashed lines in the graphs (D, E). The
residues at the interface are displayed in the figure. Normalized chemical shift changes of
wogonin (H).
Fig. 3.
Effects of baicalin and baicalein on cell shape and tight junction integrity of
MDCK II cells. Immunofluorescence staining of CLD-2 (A-E) and bright-field differential
interference contrast (DIC) images (F-J) are arrayed. Continuous 96 h exposure by 100 µM
baicalin (B, G) and baicalein (D, I) and 48 h exposure by 100 µM baicalin (C, H) and baicalein
24
(E, J) followed by 48 h incubation in media with DMSO. Scale bar = 20 μm.
Fig. 4.
Changes in the amount of protein and mRNA of CLD-2 after 100 μM baicalin or
baicalein treatment in MDCK II cells. Western blotting analysis (A, B) and quantitative real-time
PCR analysis (C). (A) Reduction of CLD-2 by 48 h treatment with baicalin and baicalein. (B)
Restoration of CLD-2 by 48 h incubation without flavonoids after exposure to them. (C) Relative
mRNA expression level of CLD-2 after 48 h of baicalin or baicalein treatment.
Fig. 5.
DIC and immunofluorescence microscopy of baicalin or baialein-treated MDCK II
cells for 48 h. DIC images (A-C), immunofluorescence staining with anti-ZO-1 (D-F) and antiOCLN (G-I) and rhodamine-phalloidin staining (J-L). Cells were treated with 100 μM baicalin
(B, E, H, K) and baicalein (C, F, I, L) for 48 h. Scale bar = 20 μm.
Fig. 6.
Effect of ALK-5 inhibitor SB431542 and ERK/MEK inhibitor U0126 against the
TJ-mitigating activity of baicalin and baicalein. Immunofluorescence staining with anti-CLD-2
antibody (A) and bright-field DIC images (B) are depicted. Cells were exposed to 100 μM
baicalin or 50 μM baicalein for 48 h in the presence or absence of 10 μM SB431542. For U0126,
cells were treated flavonoids with above concentration for 45 h and followed by addition of the
inhibitor (5 µM) for 3 h. DSO control (a, g) with SB431542 (b) or U0126 (h), baicalin exposure
(c, i) with SB431542 (d) or U0126 (j), and baicalein exposure (e, k) with SB431542 (f) or U0126
(l) are arrayed.
25
Fig. 7.
Analysis of cell morphology of living MDCK II cells from DIC images. The
treatment for 48 h, continuous 96 h, and 48 h exposure followed by 48 h DMSO wash
experiments were analyzed (A, B). The effect of ALK-5 inhibitor SB431542 (10 µM) and
ERK/MEK inhibitor U0126 (5 µM) against flavonoids exposure (C, D) were examined. The
length of long axis (A, C) and short axis (B, D) are shown.
Fig. 8.
Variations of the major flavonoids’ content with the different extraction method
from the dried chopped Radix scutellariae root. Reversed-phase HPLC chart of the simple hotwater extract of Radix scutellariae root (A), extraction method A (B) and extraction method B
(C) (see text) are shown. Arrows 1–4 indicate baicalin, wogonoside, baicalein, and wogonin.
Bright-field DIC images (D-F) and immunofluorescence staining of CLD-2 (G-I) of MDCK II
cells are arrayed. Cells were treated with extract A (E, H) and extract B (F, I) for 48 h.
Fig. 9.
Paracellular flux of fluorescence-labeled insulin of Caco-2 monolayer cells treated
with 300 μM baicalin or baicalein for 24 h.
Fig. 10.
Potential molecular mechanisms of baicalin and baicalein with the downregulation
of the integrity of tight junctions. (A) Our primary working hypothesis based on the PDZdomain-derived competitive interaction between CLD-2 and ZO-1 or LNX1. When Radix
scutellariae flavonoids interfere with the contact of ZO-1 to CLD-2, LNX1 excessively promotes
ubiquitination and endocytosis of CLD-2 from the membrane; thus, tight junctions are
downregulated. (B) Potential contribution of the other known and unknown signaling pathways
that caused tight junction downregulation and partly irreversible cell-shape changes.
26
*Credit Author Statement
Author Contributions: Conceptualization, H.H., and T.T.; Data Analysis, M. Hisada and T.T.;
Investigation, M. Hisada., M.N., M.Hiranuma, N.G., and T.T.; Resources, T.T. and N.G.;
Writing-Original Draft Preparation, H.H.; Writing-Review & Editing, H.H.; Visualization, T.T.;
Manuscript revision, T.T.; Supervision, H.H.; Project Administration, H.H.; Funding
Acquisition, H.H.
SUPPORING INFORMATION
Flavonoids from Chinese skullcap can modulate tight junction integrity by partly targeting the first
PDZ domain of zonula occludens-1 (ZO-1)
Misaki Hisada1, Minami Hiranuma1, Mio Nakashima2, Natsuko Goda1, Takeshi Tenno1,3, and Hidekazu
Hiroaki1,2,3,*
1, Graduate School of Pharmaceutical Sciences, Nagoya University, Furocho, Chikusa, Nagoya,
Aichi, 464-8601, Japan
2, Department of Biological Sciences, Faculty of Science, Nagoya University
3. BeCerllBar, LLC., Nagoya, Aichi, Japan.
Supplementary Figure 1.
Chemical structure of the all selected flavonoids from used in this study.
...
論文の公開元へ
