The transient receptor potential cation channel, subfamily V (TRPV), is expressed in the epidermis and considered to be a sensor of extrinsic stimuli such as temperature and other physical or chemical factors. In this study, we examined whether or not the activation of TRPVs by their agonists alters the epidermal tight junction (TJ) function in cultured human epidermal keratinocytes. Reverse transcription-polymerase chain reaction (RT-PCR) analyses showed that mRNA for TRPV1, 3 and 4 were expressed in differentiated keratinocytes in which TJs had formed. Stimulation of the keratinocytes with a TRPV4 agonist (4α-phorbol 12, 13-didecanoate, 4α-PDD) strengthened the TJ-associated barrier, analyzed by means of transepithelial electric resistance measurements and flux measurements of the paracellular tracer. Stimulation with TRPV1 and TRPV3 agonists did not have the same result. Simultaneously, the 4α-PDD-stimulated keratinocytes showed an upregulation of TJ structural proteins, occludin and claudin-4, and TJ regulatory factors, phospho-atypical PKCζ/ι. It was also observed that the amounts of occludin and phospho-atypical PKCζ/ι complex were higher in 4α-PDD stimulated keratinocytes. In conclusion, we demonstrated that the activation of TRPV4 strengthened the TJ-associated barrier of epidermal cells. It was also suggested that the upregulation of TJ structural proteins and/or the posttranslational modification of TJ structural proteins by phospho-atypical PKCζ/ι are responsible for the enhancement of TJ function. Our study supports the hypothesis that TJs change their function in response to a change in the external environment sensed through TRPVs.

Keywords:
Atypical PKCζ/ι, Cutaneous permeability barrier, Tight junction, Transient receptor potential cation channel, subfamily V (TRPV)
1.
Madison KC: Barrier function of the skin: ‘la raison d’être’ of the epidermis. J Invest Dermatol 2003;121:231–241.
2.
Tsukita S, Furuse M: Claudin-based barrier in simple and stratified cellular sheets. Curr Opin Cell Biol 2002;14:531–536.
3.
Brandner JM, Kief S, Grund C, Rendl M, Houdek P, Kuhn C, Tschachler E, Franke WW, Moll I: Organization and formation of the tight junction system in human epidermis and cultured keratinocytes. Eur J Cell Biol 2002;81:253–263.
4.
Furuse M, Hata M, Furuse K, Yoshida Y, Haratake A, Sugitani Y, Noda T, Kubo A, Tsukita S: Claudin-based tight junctions are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol 2002;156:1099–1111.
5.
Yuki T, Hachiya A, Kusaka A, Sriwiriyanont P, Visscher MO, Morita K, Muto M, Miyachi Y, Sugiyama Y, Inoue S: Characterization of tight junctions and their disruption by UVB in human epidermis and cultured keratinocytes. J Invest Dermatol 2011;131:744–752.
6.
Yuki T, Haratake A, Koishikawa H, Morita K, Miyachi Y, Inoue S: Tight junction proteins in keratinocytes: localization and contribution to barrier function. Exp Dermatol 2007;16:324–330.
7.
Kirschner N, Houdek P, Fromm M, Moll I, Brandner JM: Tight junctions form a barrier in human epidermis. Eur J Cell Biol 2010;89:839–842.
8.
Elias PM, Feingold KR: Coordinate regulation of epidermal differentiation and barrier homeostasis. Skin Pharmacol Appl Skin Physiol 2001;14(suppl 1):28–34.
9.
Denda M, Sato J, Tsuchiya T, Elias PM, Feingold KR: Low humidity stimulates epidermal DNA synthesis and amplifies the hyperproliferative response to barrier disruption: implication for seasonal exacerbations of inflammatory dermatoses. J Invest Dermatol 1998;111:873–878.
10.
Pensack RD, Michniak BB, Moore DJ, Mendelsohn R: Infrared kinetic/structural studies of barrier reformation in intact stratum corneum following thermal perturbation. Appl Spectrosc 2006;60:1399–1404.
11.
Tominaga M, Caterina MJ: Thermosensation and pain. J Neurobiol 2004;61:3–12.
12.
Chung MK, Lee H, Caterina MJ: Warm temperatures activate TRPV4 in mouse 308 keratinocytes. J Biol Chem 2003;278:32037–32046.
13.
Denda M, Fuziwara S, Inoue K, Denda S, Akamatsu H, Tomitaka A, Matsunaga K: Immunoreactivity of VR1 on epidermal keratinocyte of human skin. Biochem Biophys Res Commun 2001;285:1250–1252.
14.
Guler AD, Lee H, Iida T, Shimizu I, Tominaga M, Caterina M: Heat-evoked activation of the ion channel, TRPV4. J Neurosci 2002;22:6408–6414.
15.
Inoue K, Koizumi S, Fuziwara S, Denda S, Denda M: Functional vanilloid receptors in cultured normal human epidermal keratinocytes. Biochem Biophys Res Commun 2002;291:124–129.
16.
Denda M, Sokabe T, Fukumi-Tominaga T, Tominaga M: Effects of skin surface temperature on epidermal permeability barrier homeostasis. J Invest Dermatol 2007;127:654–659.
17.
Sokabe T, Fukumi-Tominaga T, Yonemura S, Mizuno A, Tominaga M: The TRPV4 channel contributes to intercellular junction formation in keratinocytes. J Biol Chem 2010;285:18749–18758.
18.
Watanabe H, Davis JB, Smart D, Jerman JC, Smith GD, Hayes P, Vriens J, Cairns W, Wissenbach U, Prenen J, Flockerzi V, Droogmans G, Benham CD, Nilius B: Activation of TRPV4 channels (hVRL-2/mTRP12) by phorbol derivatives. J Biol Chem 2002;277:13569–13577.
19.
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D: The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997;389:816–824.
20.
Moquich A, Hwang SW, Earley TJ, Petrus MJ, Murray AN, Spencer KS, Andahazy M, Story GM, Patapoutian A: Impaired thermosensation in mice lacking TRPV3, a heat and camphor sensor in the skin. Science 2005;307:1468–1472.
21.
Yuki T, Yoshida H, Akazawa Y, Komiya A, Sugiyama Y, Inoue S: Activation of TLR2 enhances tight junction barrier in epidermal keratinocytes. J Immunol 2011;187:3230–3237.
22.
Reiter B, Kraft R, Gunzel D, Zeissig S, Schulzke JD, Fromm M, Harteneck C: TRPV4-mediated regulation of epithelial permeability. FASEB J 2006;20:1802–1812.
23.
Helfrich I, Schmitz A, Zigrino P, Michels C, Haase I, le Bivic A, Leitges M, Niessen CM: Role of aPKC isoforms and their binding partners Par3 and Par6 in epidermal barrier formation. J Invest Dermatol 2007;127:782–791.
24.
Seth A, Sheth P, Elias BC, Rao R: Protein phosphatases 2A and 1 interact with occludin and negatively regulate the assembly of tight junctions in the CACO-2 cell monolayer. J Biol Chem 2007;282:11487–11498.
25.
Suzuki A, Ishiyama C, Hashiba K, Shimizu M, Ebnet K, Ohno S: aPKC kinase activity is required for the asymmetric differentiation of the premature junctional complex during epithelial cell polarization. J Cell Sci 2002;115:3565–3573.
26.
Lemini-Lopez A, Flores-Romo L, Arevalo-Lopez A, Meze I: Altered morphology and distribution of cellular junction protein in non-lesional psoriatic epidermis: an insight into disease severity. Arch Med Res 2006;37:36–44.
27.
Van Itallie C, Rahner C, Anderson JM: Regulated expression of claudin-4 decreases paracellular conductance through a selective decrease in sodium permeability. J Clin Invest 2001;107:1319–1327.
28.
Ebnet K, Suzuki A, Horikoshi Y, Hirose T, Meyer Zu Brickwedde MK, Ohno S, Vestweber D: The cell polarity protein ASIP/PAR-3 directly associates with junctional adhesion molecule (JAM). Embo J 2001;20:3738–3748.
29.
Hirase T, Kawashima S, Wong EY, Ueyama T, Rikitake Y, Tsukita S, Yokoyama M, Staddon JM: Regulation of tight junction permeability and occludin phosphorylation by Rhoa-p160ROCK-dependent and -independent mechanisms. J Biol Chem 2001;276:10423–10431.
30.
Hirose T, Izumi Y, Nagashima Y, Tamai-Nagai Y, Kurihara H, Sakai T, Suzuki Y, Yamanaka T, Suzuki A, Mizuno K, Ohno S: Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation. J Cell Sci 2002;115:2485–2495.
31.
Rao R: Occludin phosphorylation in regulation of epithelial tight junctions. Ann N Y Acad Sci 2009;1165:62–68.
32.
Sakakibara A, Furuse M, Saitou M, Ando-Akatsuka Y, Tsukita S: Possible involvement of phosphorylation of occludin in tight junction formation. J Cell Biol 1997;137:1393–1401.
33.
Wong V: Phosphorylation of occludin correlates with occludin localization and function at the tight junction. Am J Physiol 1997;273:C1859–C1867.
34.
Kida N, Sokabe T, Kashio M, Haruna K, Mizuno Y, Suga Y, Nishikawa K, Kanamaru A, Hongo M, Oba A, Tominaga M: Importance of transient receptor potential vanilloid 4 (TRPV4) in epidermal barrier function in human skin keratinocytes. Pflugers Arch 2012;463:715–725.
© 2012 S. Karger AG, Basel
2012
Copyright / Drug Dosage / Disclaimer
Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.
You do not currently have access to this content.