LETTER TO THE EDITOR

Journal of Dermatological Science (2007) 48, 233—236

www.intl.elsevierhealth.com/journals/jods

Human Fat2 is localized at immatureadherens junctions in epidermal kerati-nocytes

To the Editor,

KEYWORDSCadherin; Fat2; Keratinocyte; Adherens junction; Adhesionzipper

Fat cadherins are huge proteins that contain 34cadherin repeats and that form a distinct subfamilyof the cadherin superfamily. Although four kinds ofFat cadherin, Fat1, Fat2, Fat3 and Fat-J, have beenidentified, the role of Fat cadherins is not wellunderstood [1]. It has been reported that humanFat1 is expressed in the neuroepithelium, lung,kidney and skin [2], and Fat2 mRNA is expressedin cerebellar granule cells of the cerebellum andcutaneous squamous cell carcinoma (SCC) [3,4]. Inthis study, we found the expression of Fat2 in cul-tured keratinocytes and also in the epidermis at theprotein level for the first time.

We first performed RT-PCR analysis of normalhuman epidermal keratinocytes (NHEK, KURABO,Osaka, Japan) using degenerate primers that ampli-fied the mRNAs of multiple cadherins as describedpreviously [5]. Total RNA was extracted using Isogen(Nippon Gene, Tokyo, Japan), and first-strand cDNAswere generated using Super Script II (Invitrogen,Carlsbad, CA) with oligo dT primers. We obtaineda partial sequence of Fat2 cDNA (data not shown),and performed RT-PCR using specific primersfor Fat2 (forward: 50-cctctagttattccctgaagctccg-30,reverse: 50-tgaccgtgacgttgaacgagtagtg-30) as pre-viously described [6]. Amplification was carriedout with 30 cycles. Fat2 mRNAwas clearly expressedin NHEK cells and human cutaneous SCC cell lineHSC-1 cells [7], but not in melanoma cell line A375cells, normal human epidermal melanocytes or nor-mal human dermal fibroblasts (Fig. 1a).

To analyze the protein expression of Fat2, wenewly generated a polyclonal antibody for human

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Fat2 as outlined below. New Zealand rabbits wereimmunized by injection with purified glutathione S-transferase fusion protein containing the human Fat2cytoplasmic region (GST-Fat2, amino acid residues4092—4266). After the immunization, antisera werecollected and purified with an affinity column. Tocharacterize the reactivity of this antibody, weobtained a cloned cDNA encoding an NH2-teminal-truncated form of human Fat2 (Fat2C, amino acidresidues 2981—4349) by screening a lZAP cDNAlibrary with the PCR products of Fat2 amplified withthe degenerate primers. The Fat2C cDNA wasinserted into pFlag-CMV-1 (Sigma—Aldrich, St. Louis,MO), and was transiently transfected into mousefibroblast cell line L, which does not express anycadherin molecules. Then immunoblotting usingthe generated antibody (1:100 dilution) was per-formed as previously described [6]. A single bandwas detected in the Fat2C-transfected L cells butnot in parental L cells, indicating that the antibodyspecifically recognized Fat2 protein (Fig. 1b). Wefurther confirmed that a clear band was detectedin both NHEK and HSC-1 cells (Fig. 1c), and theposition of the band was roughly consistent withthe expected size of Fat2, approximately 480 kDa(calculated from the cDNA sequence). E-cadherinwas also detected using mouse anti-human E-cad-herin monoclonal antibody HECD-1 (a kind giftedfrom Dr. M. Takeichi), in accord with the result ofRT-PCR (Fig. 1a). Since keratinocytes are known toexpress Fat1 protein, it was important to excludecross-reactivity of the antibody with Fat1. We con-firmed by RT-PCR that both melanocytes and fibro-blasts expressedmRNA of Fat1 but not Fat2 (Fig. 1a).Unfortunately, we could not get sufficiently reactiveanti-Fat1 antibodies to detect human Fat1. The anti-Fat2 polyclonal antibody did not detect any band ineither of these types of cells (Fig. 1c), indicating thatthere was no cross-reactivity of the antibody withFat1. Therefore, we concluded that human kerati-nocytes express Fat2, in addition to various cadherinmolecules such as E-, P- and T-cadherins [6,7].

Next we examined the expression of Fat2 in epi-dermis. We obtained tissue from normal human skin

tology. Published by Elsevier Ireland Ltd. All rights reserved.

234 Letter to the Editor

Fig. 1 Keratinocytes express mRNA and protein of Fat2. (a) The mRNA expression of Fat2, Fat1 and/or E-cadherin wasanalyzed by RT-PCR in NHEK cells, HSC-1 cells, A375 cells, human normal epidermal melanocytes and human dermalfibroblasts. (b) The reactivity of the newly produced antibody for Fat2 protein was characterized by immunoblotting ofproteins from NH2-terminal-truncated Fat2-transfected L cells. The arrow indicates the truncated Fat2. Molecularweight markers are indicated on the left side. (c) The protein expression of Fat2 and E-cadherin was analyzed byimmunoblotting of NHEK cells, HSC-1 cells, human normal epidermal melanocytes and human dermal fibroblasts.

and SCC from separate patients with informed con-sent on the occasion of dermatological operations inKyoto University Hospital. Using the anti-Fat2 poly-clonal antibody (1:10 dilution), immunofluorescencestaining was performed as described previously [6].The results showed that Fat2 was expressed from thebasal layer to the granular layer of the normal epi-dermis (Fig. 2a). Although Fat2 was not detected indermal fibroblasts (Fig. 1a and c), weak signals wereobserved in the dermis (Fig. 2a). To examine thereason for this discrepancy, we examined the effectsof GST-Fat2-adsorption of the primary antibody. Thesignals in the epidermis almost disappeared,whereasthose in the dermis were still detected after suchadsorption (Fig. 2a). Similar results were obtained byomitting the primary antibody (data not shown).These results indicate that the signals in the dermiswere due to non-specific binding of the secondaryantibody. In addition, Fat2was detected in SCC tissue(Fig. 2b).

We further explored the cellular localization ofendogenous Fat2 in detail. Fat2 was located at cell—cell boundaries and in the nuclei of NHEK cells andHSC-1 cells (Fig. 2c and g), and this distribution wassimilar to that of Fat1, as shown in previous reports[2,8]. Interestingly, Fat2 was localized in a patternof two rows of dots at some cell—cell boundaries(Fig. 2c). In the early stage of adherens junction (AJ)formation, a structure formed by two rows ofpuncta, called the adhesion zipper, is detected.Adhesion zippers initiate contacts with neighboringcells, and also seal membranes into epithelial sheets

and finally become mature AJs. Many moleculessuch as E-cadherin, catenins and vinculin are clus-tered at adhesion zippers that play an importantrole in actin reorganization/polymerization [9]. Todetermine whether Fat2 is localized at adhesionzippers, a study of reconstitution of cell—cell con-tacts by calcium chelation was performed with HSC-1 cells [10]. We used mouse anti-human desmoglein1 and 2 monoclonal antibody DG3.10 (PROGEN,Heilberg, Germany) to detect desmosomes. Afterperturbing cell—cell junctions by the addition of2 mM EGTA, Fat2 was not colocalized with E-cad-herin (Fig. 2d). However, after replacement of theEGTA-containing medium with 1.8 mM Ca2+-contain-ing medium, Fat2 was colocalized with E-cadherinat adhesion zippers, but not with desmosomal cad-herins, during AJ formation (Fig. 2e and f). Wefurther analyzed the structural relationshipbetween Fat2 and actin filaments, which weredetected using Alexa fluor 488-conjugated phalloi-din (Invitrogen). Although Fat2 was present on actinbundles at cell—cell boundaries in non-treated cells(Fig. 2g), addition of 20 mM cytochalasin B (Sigma—Aldrich), a specific inhibitor of actin polymerization,affected the localization of Fat2, as well as that ofactin (Fig. 2h). Similar results were obtained bytreatment with HECD-1 to inhibit E-cadherin-depen-dent adhesion (data not shown).

In conclusion, we demonstrated that Fat2 isexpressed at adhesion zippers of keratinocytes.Our new findings will provide clues for clarifyingthe mechanism of formation of AJs.

Letter to the Editor 235

Fig. 2 Fat2 is a component of adhesion zippers. Immunofluorescence staining of Fat2 in normal human skin (a), SCC(b), NHEK cells (c) and HSC-1 cells (d—h) was carried out. (a) Normal human skin was stained using anti-Fat2 antibodywith (left panel) or without (right panel) adsorption by GST-Fat2. The indicated abbreviations are E-cad, E-cadherin;Dsg, Desmoglein 1 and 2. The area in the dotted white box was more highly magnified (c, e, f). Arrows denote adhesionzippers (c, e, f). HSC-1 cells were subjected to calcium chelation (d) or to replacement with 1.8 mM Ca2+-containingmedium (e, f). HSC-1 cells were treated with (h) or without (g) cytochalasin B and were double-stained with Fat2 and F-actin. Arrowheads show that actin depolymerization perturbs the distribution of Fat2 at cell—cell borders. Scale bar:20 mm.

References

[1] Tanoue T, Takeichi M. New insights into Fat cadherins. J CellSci 2005;118:2347—53.

[2] Tanoue T, Takeichi M. Mammalian Fat1 cadherin regulatesactin dynamics and cell—cell contact. J Cell Biol 2004;165:517—28.

[3] Nakayama M, Nakajima D, Yoshimura R, et al. MEGF1/fat2 proteins containing extraordinarily large extracellu-lar domains are localized to thin parallel fibers of

cerebellar granule cells. Mol Cell Neurosci 2002;20:563—78.

[4] Katoh Y, Katoh M. Comparative integromics on FAT1,FAT2, FAT3 and FAT4. Int J Mol Med 2006;18(3):523—8.

[5] Matsuyoshi N, Tanaka T, Toda K, et al. Identification of novelcadherins expressed in human melanoma cells. J InvestDermatol 1997;108(6):908—13.

[6] Zhou S, Matsuyoshi N, Liang SB, et al. Expression of T-cadherin in basal keratinocytes of skin. J Invest Dermatol2002;118:1080—4.

236 Letter to the Editor

[7] Furukawa F, Fujii K, Horiguchi Y. Roles of E- and P-cadherin inthe human skin. Microsc Res Tech 1997;38:343—52.

[8] Magg T, Schreiner D, Solis GP, et al. Processing of the humanprotocadherin Fat1 and translocation of its cytoplasmicdomain to the nucleus. Exp Cell Res 2005;307:100—8.

[9] Vasioukhin V, Bauer C, Yin M, et al. Directed actin polymer-ization is the driving force for epithelial cell—cell adhesion.Cell 2000;100:209—19.

[10] Rothen-Rutishauser B, Riesen FK, Braun A, et al. Dynamics oftight and adherens junctions under EGTA treatment. JMembr Biol 2002;188(2):151—62.

Seiya MatsuiAtsushi Utani

Kenzo TakahashiDepartment of Dermatology,

Graduate School of Medicine, Kyoto University,54 Kawahara-cho, Shogo-in, Sakyo-ku,

Kyoto 606-8317, Japan

Yohei MukoyamaDrug Discovery Research Laboratories,

Maruho Co., Ltd., Kyoto, Japan

Yoshiki MiyachiNorihisa Matsuyoshi*

Department of Dermatology,Graduate School of Medicine,

Kyoto University, 54 Kawahara-cho,Shogo-in, Sakyo-ku, Kyoto 606-8317, Japan

*Corresponding author. Tel.: +81 75 751 3311;fax: +81 75 751 4949

E-mail address: nori@kuhp.kyoto-u.ac.jp(N. Matsuyoshi)

23 January 2007