Archives of Dermatological Research

, Volume 306, Issue 2, pp 131–141 | Cite as

p38δ mitogen-activated protein kinase regulates the expression of tight junction protein ZO-1 in differentiating human epidermal keratinocytes

  • Elina Siljamäki
  • Laura Raiko
  • Mervi Toriseva
  • Liisa Nissinen
  • Tuomas Näreoja
  • Juha Peltonen
  • Veli-Matti Kähäri
  • Sirkku Peltonen
Original Paper


Increasing evidence has recognized tight junctions (TJs) as the lower epidermal inside-out diffusion barrier located in granular cell layers of the epidermis. However, little is known about the regulation of TJ components in epidermis. p38 pathway is one of the mitogen-activated protein kinase pathways, which controls cell growth, differentiation, and apoptosis. We have investigated the role of p38 signaling pathway in the regulation of selected desmosomal, adherens and TJ components in human primary keratinocytes during Ca2+-induced differentiation, as well as in cultured squamous cell carcinoma cell lines. p38 signaling pathway was inhibited in cultured keratinocytes and cutaneous squamous cell carcinoma cells using recombinant adenoviruses, small inhibitory RNAs (siRNA) and chemical inhibitors. Expression of intercellular junction proteins was investigated using Western analysis and indirect immunofluorescence (IIF). The results showed that inhibition of p38δ function by siRNA or adenovirally delivered dominant negative mutant led to markedly decreased levels of Zonula occludens-1 (ZO-1) protein in keratinocytes, while the expression of other junctional proteins studied was not altered. Immunolocalization of ZO-1 revealed that intercellular junction areas were depleted from ZO-1. Inhibition of ZO-1 by siRNA silencing did not however result in an altered expression or subcellular localization of other TJ components studied. The expression of ZO-1 in carcinoma cells was also regulated by p38. The results indicate that ZO-1 is regulated by p38δ while the other junction proteins studied are not. Since ZO-1 is an integral component of functional TJs, various pathological processes affecting signaling via p38δ may also interfere with epithelial maturation and the formation and function of TJs.


Tight junction MAPK kinase Keratinocyte p38 ZO-1 Desmosome 



We thank Miso Immonen, Sari Pitkänen and Johanna Markola-Wärn for their skillful technical assistance. The authors thank Dr. Erkki Suominen from the Department of Surgery, Turku University Hospital for the tissue samples and Prof. Reidar Grénman from the Department of Otorhinolaryngology, Turku University Hospital for SCC cells. Markku Saari is acknowledged for his invaluable help with confocal imaging and image processing. Confocal imaging was performed at the Cell Imaging Core, Turku Centre for Biotechnology. This work was supported by grants from the Technology Development Center of Finland (TEKES), Academy of Finland (Projects 137687 and 251136), Turku University Hospital (Project 13336 and 13085), the Sigrid Jusélius Foundation, and the Cancer Research Foundation of Finland.

Conflict of interest

The authors have no conflict of interest to declare.

Supplementary material

403_2013_1391_MOESM1_ESM.pdf (78 kb)
Western analyses of five independent normal human keratinocyte experiments show biphasic activation of p38 cultured in high Ca2+ for various times. (PDF 77 kb)
403_2013_1391_MOESM2_ESM.pdf (154 kb)
Quantification of the Western analyses shown in the Fig. 2. Mean values of three independent western blot experiments were included in the diagram. The results demonstrate low expression of ZO-1 in cells with p38δ inhibition. (PDF 153 kb)
403_2013_1391_MOESM3_ESM.pdf (809 kb)
Inhibition of the activity of p38α and p38δ with BIRB796 does not affect the amount or localization of other junction proteins. (a–c) Keratinocyte cultures were untreated (control) or treated with BIRB796 (10 μM) or SB203580 (10 μM) and grown for 72 h in low or in high Ca2+ concentration. IIF demonstrates that the expression or localization of β-catenin, E-cadherin or desmoplakin was not affected. Bar 10 μm. (PDF 808 kb)


  1. 1.
    Boyce ST, Ham RG (1985) Cultivation, frozen storage, and clonal growth of normal human keratinocytes in serum free media. J Tissue Cult Methods 9:83–93CrossRefGoogle Scholar
  2. 2.
    Brandner JM, Kief S, Grund C, Rendl M, Houdek P et al (2002) Organization and formation of the TJ system in human epidermis and cultured keratinocytes. Eur J Cell Biol 81:253–263PubMedCrossRefGoogle Scholar
  3. 3.
    De Benedetto A, Rafaels NM, McGirt LY, Ivanov AI, Georas SN et al (2011) TJ defects in patients with atopic dermatitis. J Allergy Clin Immunol 127:773–786PubMedCentralPubMedCrossRefGoogle Scholar
  4. 4.
    Donnert G, Keller J, Medda R, Andrei MA, Rizzoli SO et al (2006) Macromolecular-scale resolution in biological fluorescence microscopy. Proc Natl Acad Sci USA 103:11440–11445PubMedCrossRefGoogle Scholar
  5. 5.
    D’Souza-Schorey C (2005) Disassembling adherens junctions: breaking up is hard to do. Trends Cell Biol 15:19–26PubMedCrossRefGoogle Scholar
  6. 6.
    Ebnet K (2008) Organization of multi protein complexes at cell–cell junctions. Histochem Cell Biol 130:1–20PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Eckert RL, Crish JF, Efimova T, Dashti SR, Deucher A et al (2004) Regulation of involucrin gene expression. J Invest Dermatol 123:13–22PubMedCrossRefGoogle Scholar
  8. 8.
    Furuse M, Hata M, Furuse K, Yoshida Y, Haratake A et al (2002) Claudin-based TJs are crucial for the mammalian epidermal barrier: a lesson from claudin-1-deficient mice. J Cell Biol 156:1099–1111PubMedCrossRefGoogle Scholar
  9. 9.
    Goldstein DM, Kuglstatter A, Lou Y, Soth MJ (2010) Selective p38alpha inhibitors clinically evaluated for the treatment of chronic inflammatory disorders. J Med Chem 53:2345–2353PubMedCrossRefGoogle Scholar
  10. 10.
    Gonzalez-Mariscal L, Betanzos A, Avila-Flores A (2000) MAGUK proteins: structure and role in the TJ. Semin Cell Dev Biol 11:315–324PubMedCrossRefGoogle Scholar
  11. 11.
    Gonzalez-Mariscal L, Betanzos A, Nava P, Jaramillo BE (2003) TJ proteins. Prog Biophys Mol Biol 81:1–44PubMedCrossRefGoogle Scholar
  12. 12.
    Gonzalez-Mariscal L, Tapia R, Chamorro D (2008) Crosstalk of TJ components with signaling pathways. Biochim Biophys Acta 1778:729–756PubMedCrossRefGoogle Scholar
  13. 13.
    Green KJ, Jones JC (1996) Desmosomes and hemidesmosomes: structure and function of molecular components. FASEB J 10:871–881PubMedGoogle Scholar
  14. 14.
    Green KJ, Simpson CL (2007) Desmosomes: new perspectives on a classic. J Invest Dermatol 127:2499–2515PubMedCrossRefGoogle Scholar
  15. 15.
    Gum RJ, McLaughlin MM, Kumar S, Wang Z, Bower MJ et al (1998) Acquisition of sensitivity of stress-activated protein kinases to the p38 inhibitor, SB 203580, by alteration of one or more amino acids within the ATP binding pocket. J Biol Chem 273:15605–15610PubMedCrossRefGoogle Scholar
  16. 16.
    Hell SW, Wichmann J (1994) Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. Opt Lett 19:780–782PubMedCrossRefGoogle Scholar
  17. 17.
    Howarth AG, Hughes MR, Stevenson BR (1992) Detection of the TJ-associated protein ZO-1 in astrocytes and other nonepithelial cell types. Am J Physiol 262:C461–C469PubMedGoogle Scholar
  18. 18.
    Itoh M, Nagafuchi A, Yonemura S, Kitani-Yasuda T, Tsukita S et al (1993) The 220 kD protein colocalizing with cadherins in non-epithelial cells is identical to ZO-1, a TJ-associated protein in epithelial cells: cDNA cloning and immunoelectron microscopy. J Cell Biol 121:491–502PubMedCrossRefGoogle Scholar
  19. 19.
    Johansen C, Vinter H, Soegaard-Madsen L, Olsen LR, Steiniche T et al (2010) Preferential inhibition of the mRNA expression of p38 mitogen-activated protein kinase regulated cytokines in psoriatic skin by anti-TNF-alpha therapy. Br J Dermatol 163:1194–1204PubMedCrossRefGoogle Scholar
  20. 20.
    Junttila MR, Ala-aho R, Jokilehto T, Peltonen J, Kallajoki M et al (2007) p38alpha and p38delta mitogen-activated protein kinase isoforms regulate invasion and growth of head and neck squamous carcinoma cells. Oncogene 26:5267–5279PubMedCrossRefGoogle Scholar
  21. 21.
    Kirschner N, Poetzl C, von den Driesch P, Wladykowski E, Moll I et al (2009) Alteration of TJ proteins is an early event in psoriasis: putative involvement of proinflammatory cytokines. Am J Pathol 175:1095–1106PubMedCrossRefGoogle Scholar
  22. 22.
    Kirschner N, Rosenthal R, Furuse M, Moll I, Fromm M, Brandner JM (2013) Contribution of tight junction proteins to ion, macromolecule, and water barrier in keratinocytes. J Invest Dermatol 133:1161–1169PubMedCrossRefGoogle Scholar
  23. 23.
    Kivisaari AK, Kallajoki M, Ala-aho R, McGrath JA, Bauer JW et al (2010) Matrix metalloproteinase-7 activates heparin-binding epidermal growth factor-like growth factor in cutaneous squamous cell carcinoma. Br J Dermatol 163:726–735PubMedCrossRefGoogle Scholar
  24. 24.
    Kraft CA, Efimova T, Eckert RL (2007) Activation of PKCdelta and p38delta MAPK during okadaic acid dependent keratinocyte apoptosis. Arch Dermatol Res 299:71–83PubMedCrossRefGoogle Scholar
  25. 25.
    Kuma Y, Sabio G, Bain J, Shpiro N, Márquez R et al (2005) BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo. J Biol Chem 280:19472–19479PubMedCrossRefGoogle Scholar
  26. 26.
    Leivonen SK, Ala-aho R, Koli K, Grénman R, Peltonen J et al (2006) Activation of Smad signaling enhances collagenase-3 (MMP-13) expression and invasion of head and neck squamous carcinoma cells. Oncogene 25:2588–2600PubMedCrossRefGoogle Scholar
  27. 27.
    Leivonen SK, Kähäri VM (2007) Transforming growth factor-β signaling in cancer invasion and metastasis. Int J Cancer 121:2119–2124PubMedCrossRefGoogle Scholar
  28. 28.
    Malminen M, Koivukangas V, Peltonen J, Karvonen SL, Oikarinen A et al (2003) Immunohistological distribution of the TJ components ZO-1 and occludin in regenerating human epidermis. Br J Dermatol 149:255–260PubMedCrossRefGoogle Scholar
  29. 29.
    Medicherla S, Ma JY, Reddy M, Esikova I, Kerr I et al (2010) Topical alpha-selective p38 MAP kinase inhibition reduces acute skin inflammation in guinea pig. J Inflamm Res 3:9–16PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Palatinus JA, O’Quinn MP, Barker RJ, Harris BS, Jourdan J et al (2011) ZO-1 determines adherens and gap junction localization at intercalated disks. Am J Physiol Heart Circ Physiol 300:H583–H594PubMedCrossRefGoogle Scholar
  31. 31.
    Peltonen S, Riehokainen J, Pummi K, Peltonen J (2007) TJ components occludin, ZO-1, and claudin-1, -4 and -5 in active and healing psoriasis. Br J Dermatol 156:466–472PubMedCrossRefGoogle Scholar
  32. 32.
    Pramanik R, Qi X, Borowicz S, Choubey D, Schultz RM et al (2003) p38 isoforms have opposite effects on AP-1-dependent transcription through regulation of c-Jun. The determinant roles of the isoforms in the p38 MAPK signal specificity. J Biol Chem 278:4831–4839PubMedCrossRefGoogle Scholar
  33. 33.
    Pummi K, Malminen M, Aho H, Karvonen SL, Peltonen J et al (2001) Epidermal TJs: ZO-1 and occludin are expressed in mature, developing, and affected skin and in vitro differentiating keratinocytes. J Invest Dermatol 117:1050–1058PubMedCrossRefGoogle Scholar
  34. 34.
    Raiko L, Leinonen P, Hägg PM, Peltonen J, Oikarinen A et al (2009) TJs in Hailey–Hailey and Darier’s diseases. Dermatol Rep, 1:e1.
  35. 35.
    Raman M, Chen W, Cobb MH (2007) Differential regulation and properties of MAPKs. Oncogene 26:3100–3112PubMedCrossRefGoogle Scholar
  36. 36.
    Schneeberger EE, Lynch RD (2004) The TJ: a multifunctional complex. Am J Physiol Cell Physiol 286:C1213–C1228PubMedCrossRefGoogle Scholar
  37. 37.
    Shin K, Fogg VC, Margolis B (2006) TJs and cell polarity. Annu Rev Cell Dev Biol 22:207–235PubMedCrossRefGoogle Scholar
  38. 38.
    Troy TC, Rahbar R, Arabzadeh A, Cheung RM, Turksen K (2005) Delayed epidermal permeability barrier formation and hair follicle aberrations in Inv-Cldn6 mice. Mech Dev 122:805–819PubMedCrossRefGoogle Scholar
  39. 39.
    Tsukita S, Furuse M, Itoh M (2001) Multifunctional strands in TJs. Nat Rev Mol Cell Biol 2:285–293PubMedCrossRefGoogle Scholar
  40. 40.
    Tuomi S, Mai A, Nevo J, Laine JO, Vilkki V et al (2009) PKCepsilon regulation of an alpha5 integrin-ZO-1 complex controls lamellae formation in migrating cancer cells. Sci Signal 2(77):ra32PubMedGoogle Scholar
  41. 41.
    Wang Y, Huang S, Sah VP, Ross J Jr, Brown JH et al (1998) Cardiac muscle cell hypertrophy and apoptosis induced by distinct members of the p38 mitogen-activated protein kinase family. J Biol Chem 273:2161–2168PubMedCrossRefGoogle Scholar
  42. 42.
    Wildanger D, Medda R, Kastrup L, Hell SW (2009) A compact STED microscope providing 3D nanoscale resolution. J Microsc 236:35–43PubMedCrossRefGoogle Scholar
  43. 43.
    Wilkinson GW, Akrigg A (1992) Constitutive and enhanced expression from the CMV major IE promoter in a defective adenovirus vector. Nucleic Acids Res 20:2233–2239PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Willig KI, Keller J, Bossi M, Hell SW (2006) STED microscopy resolves nanoparticles assemblies. New J Phys 8:106CrossRefGoogle Scholar
  45. 45.
    Yamamoto T, Saeki Y, Kurasawa M, Kuroda S, Arase S et al (2008) Effect of RNA interference of TJ-related molecules on intercellular barrier function in cultured human keratinocytes. Arch Dermatol Res 300:517–524PubMedCrossRefGoogle Scholar
  46. 46.
    Yoshida Y, Morita K, Mizoguchi A, Ide C, Miyachi Y (2001) Altered expression of occludin and TJ formation in psoriasis. Arch Dermatol Res 293:239–244PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Elina Siljamäki
    • 1
    • 2
  • Laura Raiko
    • 1
    • 3
  • Mervi Toriseva
    • 1
    • 2
  • Liisa Nissinen
    • 1
    • 2
  • Tuomas Näreoja
    • 3
    • 4
  • Juha Peltonen
    • 3
  • Veli-Matti Kähäri
    • 1
    • 2
  • Sirkku Peltonen
    • 1
  1. 1.Department of DermatologyUniversity of Turku and Turku University HospitalTurkuFinland
  2. 2.MediCity Research LaboratoryUniversity of TurkuTurkuFinland
  3. 3.Department of Cell Biology and AnatomyUniversity of TurkuTurkuFinland
  4. 4.Laboratory of Biophysics, Department of Cell Biology and AnatomyUniversity of TurkuTurkuFinland

Personalised recommendations