Deranged epidermal differentiation in
               kl/kl
               mouse and the effects of βKlotho siRNA on the differentiation of HaCaT cells page 1
Deranged epidermal differentiation in
               kl/kl
               mouse and the effects of βKlotho siRNA on the differentiation of HaCaT cells page 2
Deranged epidermal differentiation in
               kl/kl
               mouse and the effects of βKlotho siRNA on the differentiation of HaCaT cells page 3

Deranged epidermal differentiation in kl/kl mouse and the effects of βKlotho siRNA on the differentiation of HaCaT cells

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  • DOI: 10.1111/exd.12258

    www.wileyonlinelibrary.com/journal/EXDLetter to the Editor

    Deranged epidermal differentiation in kl/kl mouse and the effectsof bKlotho siRNA on the differentiation of HaCaT cells

    Kozo Nakai1, Kozo Yoneda1, Reiji Haba2, Yoshio Kushida2, Naomi Katsuki2, Tetsuya Moriue1,Hiroaki Kosaka3, Yasuo Kubota1 and Shigeaki Inoue4

    1Department of Dermatology, Kagawa University, Kita-Gun, Japan; 2Department of Diagnostic Pathology, Kagawa University, Kita-Gun, Japan;3Department of Cardiovascular Physiology, Kagawa University, Kita-Gun, Japan; 4Institute of Innovative Science and technology, Tokai University,

    Isehara, Japan

    Correspondence: Kozo Nakai, Department of Dermatology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa

    761-0793, Japan, Tel.: +81-87-891-2162, Fax: +81-87-891-2163, e-mail: kozo@kms.ac.jp

    Abstract: Mice deficient in the klotho gene (kl/kl mice) display the

    phenotypes of human ageing. We found that the expression of

    epidermal differentiation-associated factors (keratin 1, keratin 10,

    filaggrin and loricrin) was lower in the skin of kl/kl mice than that

    of wild-type mice. In vitro experiments showed that the expression

    of bKlotho, a family of klotho gene-encoded protein, was inducedconcomitantly with the differentiation of an immortalized human

    epidermal keratinocyte cell line (HaCaT cells) when they were

    cultured in an airliquid interface. bKlotho knockdown by smallinterfering ribonucleic acid suppressed the expression of the above

    differentiation-associated factors in HaCaT cells. bKlotho small

    interfering ribonucleic acid increased the expression of keratin 14,

    which is expressed in mitotically active basal layer cells, and

    activated p44/p42 mitogen-activated protein kinase in the HaCaT

    cells grown in the airliquid interface. These findings suggest thatthe epidermal differentiation is deranged in kl/kl mice, and

    bKlotho is required for the differentiation of human epidermalkeratinocytes.

    Key words: epidermal differentiation Klotho

    Accepted for publication 1 October 2013

    BackgroundThe klotho gene was identified in 1997 as a gene mutated in the

    klotho (kl/kl) mouse, which displays the phenotypes of human age-

    ing (1). It has been reported that the skin of kl/kl mice demon-

    strates reductions in dermal and epidermal thicknesses and the

    number of hair follicles. The klotho gene encodes a putative type I

    membrane protein, Klotho. The Klotho protein functions as a

    humoral factor with pleiotropic activities (24). Klotho comprisesa family of proteins including aKlotho, bKlotho (5) and cKlotho(6). However, the details of these proteins in kl/kl mice were

    unknown.

    Question addressedAre bKlotho and differential markers suppressed in kl/kl miceand/or bKlotho knock-down human keratinocyte cells?Experimental designWe examined the expression of bKlotho and epidermal differenti-ation markers in the epidermis of kl/kl mice. We examined the

    expression of bKlotho in differentiating cells cultured in an airliquid interface and examined the effect of bKlotho knockdownsmall interfering ribonucleic acid (siRNA) on the differentiation of

    human epidermal keratinocytes. For details of the methods, see

    Data S1.

    ResultsThe skin of kl/kl mice showed an atrophic and scaly appearance

    (Fig. 1b) relative to wild-type (WT) mice (Fig. 1a). As previously

    reported, there were histological reductions in dermal and epider-

    mal thicknesses in kl/kl mice. We found that kl/kl mice had a

    wavy epidermis with basket-weave hyperkeratosis, implying the

    abnormal differentiation of the epidermis of kl/kl mice. Immuno-

    histological analysis revealed the relative absence of bKlotho in the

    epidermis of kl/kl mice (Fig. 1d) to that of WT mice (Fig. 1c). In

    WT mice, keratin 1, keratin 10, filaggrin and loricrin were

    expressed in the epidermis (Fig. 1e, g, i, k). However, the expres-

    sion of these differentiation-related proteins was suppressed in the

    epidermis of kl/kl mice (Fig. 1f, h, j, l). Western blotting revealed

    that the levels of protein expression of bKlotho, keratin 1, keratin10, filaggrin and loricrin were lower in the skin of kl/kl mice than

    those of WT mice (Figure S1).

    Next, we investigated the expression of bKlotho in human epi-dermal keratinocyte cells. bKlotho protein was barely detectable inmonolayer-cultured NHEK (Figure S2) and HaCaT cells (Fig. 2a).

    NHEK and HaCaT cells can differentiate and develop a multilay-

    ered epithelium when they are cultured in the airliquid interface.We detected the bKlotho protein in NHEK and HaCaT cellscultured in the airliquid interface (Figure S2 and 2a).

    To verify the relationship between bKlotho and epidermal differ-entiation, we knocked down bKlotho expression in HaCaT cellswith siRNA. bKlotho siRNA induced a cuboidal morphologicalchange in HaCaT cells grown in the airliquid interface (Fig. 2e).bKlotho siRNA inhibited the protein expression of bKlotho, keratin1, keratin 10 and loricrin in HaCaT cells grown in the airliquidinterface (Fig. 2f, g). bKlotho siRNA inhibited the mRNA expres-sion levels of filaggrin, loricrin, involucrin, keratin 1 and keratin 10

    (Figure S3). These results suggest that bKlotho is necessary for thenormal differentiation of epidermal keratinocytes.

    We also examined the effects of bKlotho siRNA on the mitoticactivity of HaCaT cells grown in the airliquid interface. Theexpression of keratin 14 and the activation of p44/p42 MAPK are

    reported in mitotically active layer cells of epidermis. bKlothosiRNA increased the expression levels of keratin 14 (Fig. 2h) and

    772 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

    Experimental Dermatology, 2013, 22, 748774

  • highly phosphorylated the p44/p42 MAPK protein in HaCaT cells

    grown in the airliquid interface (Fig. 2i). Moreover, bKlotho siR-NA enhanced the proliferation of HaCaT cells (Fig. 2j). These

    results suggest that bKlotho regulates the mitotic activity in differ-entiating epidermal keratinocytes.

    ConclusionThis study demonstrated a novel biological role of Klotho and/or

    bKlotho in skin. Keratinization of the epidermis is a well-definedprogramme of differentiation: keratinocytes progress vertically

    from basal cells into spinous and granular cells to flattened, dif-

    ferentiated squames in the stratum corneum. The suppression of

    differentiation markers suggests deranged keratinization of the

    epidermis, and it may partially explain the histological changes

    observed in the skin of kl/kl mice. We have shown abnormal

    differentiation of the epidermis in kl/kl mice. bKlotho was notexpressed in monolayer-cultured NHEK cells and HaCaT cells,

    but it was expressed when these cells were cultured in the airliquid interface. bKlotho knockdown by siRNA suppressed theexpression of these markers in HaCaT cells cultured in the airliquid interface. These data suggest that bKlotho may play a cru-cial role in the differentiation of epidermal keratinocytes. As the

    expression of aKlotho is also suppressed in kl/kl mice, anotherpossible mechanism of the deranged epidermal differentiation

    should be considered in the mice. Secreted Klotho has pleiotropic

    activities. It inhibits the insulin-like growth factor-1 receptor

    (IGF-1R) pathway (7). In skin, the IGF-1R pathway has been

    reported to interfere with keratinocyte differentiation (8) and

    H&

    E

    WT kl/kl

    WT kl/kl

    (a) (b)

    (c) (d)

    Klo

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    (e) (f)

    (g) (h)

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    (k) (l)

    Figure 1. The expression of bKlotho protein and epidermal differentiation-relatedprotein was suppressed in the skin of kl/kl mice. (a, b) Histology of the skin of kl/klmice and WT mice. Immunohistochemistry of bKlotho (c, d), keratin 1 (e, f),keratin 10 (g, h), filaggrin (i, j) and loricrin (k, l) in the skin of wild-type (WT) mice(a, c, e, g, i and k) and kl/kl mice (b, d, f, h, j and l).

    GAPDH

    (a) Monolayer Multilayer

    Klotho

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    CTRL KlothosiRNA

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    Day 1 Day 4

    CTRL siRNAKlotho siRNA

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    *

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    ****

    Figure 2. bKlotho siRNA suppressed the expression levels of differentiation-relatedfactors, increased keratin 14 expression and activated p44/p42 MAPK in HaCaTcells grown in the airliquid interface. HaCaT cells were grown in the airliquidinterface for 7 days to develop a multilayered epithelium. (a) bKlotho proteinexpression was analysed by Western blotting. (b) Histology of multilayer-culturedHaCaT cells. (c) Immunohistochemist