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Non-Commercial Use Only Dermatology Reports 2009; volume 1:e1 Tight junctions in Hailey-Hailey rare blistering skin diseases inherited as an autosomal dominant trait. HHD results in Correspondence: Laura Raiko, Department of and Darier’s diseases mutations in the ATP2C1 gene, which Dermatology, University of Turku, 20520 Turku, encodes the Golgi secretory pathway Finland. E-mail: [email protected] 1 2,3 Laura Raiko, Pekka Leinonen, Ca2+/Mn2+ ATPase (hSPCA1).1,2 DD is caused Päivi M. Hägg,3 Juha Peltonen,4 by mutations in the ATP2A2 gene encoding Key words: adherens junction, claudin, Darier- Aarne Oikarinen,3 Sirkku Peltonen1 Ca2+-ATPase type 2 (SERCA2, sarcoplasm- White disease, tight junction, zonula occludens protein 1. 1Department of Dermatology, University ic/endoplasmic reticulum Ca2+-transport ATPase isoform 2b).3 The most prominent of Turku and Turku University Central Acknowledgments: we thank Dr Lauri Talve, Hospital, Turku, Finland; 2Department of common epidermal histological feature of DD Department of Pathology, Turku University Anatomy and Cell Biology and and HHD is suprabasal acantholysis, which Central Hospital for providing the archival tissue results from desmosomal disintegration. In 3Department of Dermatology, University material. This study was supported financially by addition DD and HHD epidermis show differ- grants from the Finnish Medical Foundation, of Oulu, Oulu, Finland; 4Institute of entiation and keratinization defects.4-6 Finnish Cultural Foundation, Academy of Finland, Biomedicine, Department of Anatomy, Specifically transition of keratin 14 to keratin the Turku University Foundation, the Southwest University of Turku, Turku, Finland 10 is abnormal as demonstrated by the pres- Finland Hospital District, and Northern ence of suprabasal keratinocytes expressing Ostrobothnia Hospital District, Finland. both cytokeratins that usually are exclusive in Contributions: LR, immunolabeling, confocal normal epidermis.6 Although the primary microscopy, writing the manuscript, and design- Abstract abnormalities in calcium metabolism in HHD ing the figures; PL, TEWL measurements, and DD are known, the sequence of events immunolabeling for frozen sections; PMH, obtain- Hailey-Hailey disease (HHD) and Darier’s leading to acantholysis is not understood fully 2+ ing patient samples in Oulu, TEWL measure- disease (DD) are caused by mutations in Ca - yet. ments; JP, writing the manuscript and supervising ATPases with the end result of desmosomal In normal human epidermis, tight junc- the work; AO, patient samples and supervising the disruption and suprabasal acantholysis. Tight tions (TJ) are located in the granular layer7,8 work in Oulu; SP, coordinating the work and par- junctions (TJ) are located in the granular cell where they contribute to the epidermal barri- onlyticipating in all parts except TEWL measurements. layer in normal skin and contribute to the epi- er function, especially the diffusion of water dermal barrier. Aberrations in the epidermal from inside out.9-11 The most important trans- Conflict of interest: the authors reported no con- differentiation, such as in psoriasis, have been membrane proteins of epidermal TJ are the flicts of interest. shown to lead to changes in the expression of members of the claudin family;use namely Received for publication: 1 October 2009. TJ components. Our aim was to elucidate the claudins-1 and -4, which are expressed in all Revision received: 2 November 2009. expression and dynamics of the TJ proteins 8,12 vital epidermal layers. Transmembrane pro- Accepted for publication: 3 November 2009. during the disruption of desmosomes in HHD tein occludin and intracellular linking mole- and DD lesions. Indirect immunofluorescence cule zonula occludens protein 1 (ZO-1) are This work is licensed under a Creative Commons and avidin-biotin labeling for TJ, desmosomal restricted to the granular layer normally.7 Attribution 3.0 License (by-nc 3.0). and adherens junction proteins, and subse- Previously we have shown that abnormal epi- ©Copyright L. Raiko et al., 2009 quent analyses with the confocal laser scan- dermal differentiation is associated with dis- Licensee PAGEPress, Italy ning microscope were carried out on 14 HHD 13 turbances in distribution of TJ proteins. Dermatology Reports 2009; 1:e1 and 14 DD skin samples. Transepidermal water Examples of aberrant differentiation include doi:10.4081/dr.2009.e1 loss (TEWL) was measured in normal and psoriatic epidermis and hypertrophic edges of lesional epidermis of nine HHD and eight DD healing blisters, which show spreading of ZO- patients to evaluate the function of the epider- 1 and occludin to the acanthotic spinous cell mal barrier in HHD and DD skin. The localiza- layers.7,13,14 Up-regulation of ZO-1 and occludin teins has not been studied in acantholysis. tion of TJ proteins claudin-1, claudin-4, ZO-1, is reversible and disappears during the heal- Studies on epidermal cadherin knockout mice and occludin in perilesional HHD and DD epi- ing of the psoriasis lesion.13 Regulation of TJ suggest the importance of cadherins in dermis was similar to that previously described proteins in epidermis is not well known but assembly and/or stability of TJ and desmo- in normal skin. In HHD lesions theNon-commercial tissue dis- the presence of TJ in the granular cell layers somes.17,18 Thus desmosomes and TJ seem to tribution of ZO-1 expanded to the acantholytic suggests that the prerequisite for the forma- be at least partly co-regulated. spinous cells. In agreement with previous find- tion of TJ is high extracellular calcium con- The aims of this study were: (1) to eluci- ings, desmoplakin was localized intracellularly. centration. In fact studies on cultured ker- date the role of TJ in the epidermal barrier in In contrast claudin-1 and ZO-1 persisted in the atinocytes have shown that development of TJ blistering disease; (2) to study the localiza- cell-cell contact sites of acantholytic cells. is calcium-inducible.7,15 We have demonstrat- tion of TJ in an abnormal epidermal calcium TEWL was increased in the lesional skin. The ed recently that in HHD and DD lesions the gradient; (3) to see whether the aberrant dif- current results suggest that TJ components fol- calcium concentration in the basal layer is ferentiation in DD has an effect on TJ in the low different dynamics in acantholysis of HHD lower than in normal skin.6 In addition to analogy of psoriatic skin and healing wounds; and DD compared to desmosomal and being decreased in the basal cells in the (4) to compare the dynamics of TJ compo- adherens junction proteins. lesional HH and DD epidermis, the calcium nents to that of desmosomal and adherens content was decreased in non-lesional DD junction proteins at the cellular level in the epidermis, probably linking to the keratiniza- acantholytic process. Specifically we investi- tion defect seen especially in DD.6 gated the localization of TJ components Introduction The localization of adherens junction and claudin-1, claudin-4, ZO-1, and occludin in desmosomal proteins in acantholytic lesions frozen and paraffin-embedded skin of 14 HHD Hailey-Hailey disease (HHD, OMIM 16960) of DD and HHD has been studied in detail and 14 DD patients using commercial anti- and Darier’s disease (DD, OMIM 124200) are previously.16 However the dynamics of TJ pro- bodies for indirect immunofluorescence and [Dermatology Reports 2009; 1:e1] [page 1] Article avidin-biotin immunolabeling. Co-localiza- Indirect immunofluorescence labeling confocal microscope. Confocal laser scanning tion of TJ proteins with adherens junction Punch biopsy samples were cut into 7 µm microscopy was carried out using a Zeiss LSM and desmosomal components was demon- cryosections, mounted on silanated glass 510 META confocal microscope equipped with strated using the confocal laser scanning slides, and fixed in 100% methyl alcohol at argon-ion and helium-neon lasers (Zeiss; microscope. In addition transepidermal water -20°C for 10 min. To prevent nonspecific bind- Jena, Germany) and LSM 3.0 software. The loss (TEWL) was elucidated by measuring ing, the samples were preincubated in 1% objectives were 40X (oil immersion, numeric TEWL in apparently normal and lesional HH bovine serum albumin (BSA) in phosphate aperture 1.3) and 63X (oil immersion, numer- and DD skin. The results showed that in both buffered saline (PBS) for 15 min. Antibodies ic aperture 1.4). For excitation, the 405-nm diseases the tissue localization of TJ proteins were diluted in 1% BSA-PBS, and incubated on line was used for Hoechst, the 488-nm line for TM remained apparently normal in the lesional the samples at 4°C for 20 h. Either primary Alexa Fluor 488, and the 543-nm line for TM and perilesional epidermis, while acantholy- antibodies were used alone or antibodies Alexa Fluor 568 and Cy3. The images were sis was associated with changes in the distri- raised in mouse and rabbit were mixed for saved in an LSM image browser program and bution of TJ proteins at the cellular level. double labeling. Following five 5-min washes exported to Adobe Photoshop in jpg format. in PBS, the samples were incubated with sec- ondary antibodies and Hoechst nuclear stain TEWL measurements (dilution 1:10,000) at 20°C for 1 h. Secondary The VapoMeter with a closed cylindrical Materials and Methods antibodies used were Alexa Fluor 568 conjugat- chamber (Delfin Technologies Ltd, Kuopio, ed goat anti-rabbit IgG (A11011) or Alexa Fluor Finland) was used for TEWL analyses. TEWL Biopsy samples 488 conjugated goat anti-mouse IgG (A11029) was measured from lesional skin of nine The skin biopsies were taken at the from Molecular Probes Inc. (Eugene, OR, patients with HHD and eight patients with DD. Department of Dermatology, Turku University USA). In double labeling the secondary anti- Measurements were obtained from lesional Central Hospital and the Department of bodies for mouse and rabbit were mixed. The and a corresponding healthy abdominal skin Dermatology, Oulu University Hospital, samples were washed in PBS, dipped in dou- area of each patient.
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