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 induced

concomitantly with the differentiation of an immortalized human

epidermal keratinocyte cell line (HaCaT cells) when they were

cultured in an air–liquid interface. bKlotho knockdown by small

interfering 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 air–liquid interface. These findings suggest that

the epidermal differentiation is deranged in kl/kl mice, and

bKlotho is required for the differentiation of human epidermal

keratinocytes.

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 (2–4). Klotho comprises

a 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 mice

and/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 air–liquid interface and examined the effect of bKlotho knockdown

small 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, keratin

10, 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 in

monolayer-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 air–liquid interface.

We detected the bKlotho protein in NHEK and HaCaT cells

cultured in the air–liquid interface (Figure S2 and 2a).

To verify the relationship between bKlotho and epidermal differ-

entiation, we knocked down bKlotho expression in HaCaT cells

with siRNA. bKlotho siRNA induced a cuboidal morphological

change in HaCaT cells grown in the air–liquid interface (Fig. 2e).

bKlotho siRNA inhibited the protein expression of bKlotho, keratin1, keratin 10 and loricrin in HaCaT cells grown in the air–liquidinterface (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 the

normal differentiation of epidermal keratinocytes.

We also examined the effects of bKlotho siRNA on the mitotic

activity of HaCaT cells grown in the air–liquid interface. The

expression 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

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

Experimental Dermatology, 2013, 22, 748–774

highly phosphorylated the p44/p42 MAPK protein in HaCaT cells

grown in the air–liquid 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-defined

programme 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 not

expressed in monolayer-cultured NHEK cells and HaCaT cells,

but it was expressed when these cells were cultured in the air–liquid interface. bKlotho knockdown by siRNA suppressed the

expression of these markers in HaCaT cells cultured in the air–liquid 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, another

possible 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)

βKlo

tho

Fila

gg

rin

Lo

ricr

inK

erat

in 1

Ker

atin

10

WT kl/kl

WT kl/kl

WT kl/kl

WT kl/kl

(e) (f)

(g) (h)

(i) (j)

(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

βKlotho

(f)

K1

Lor

GAPDH

CTRL βKlothosiRNA

K10

βKlotho

K14Phospho

p44/p42

p44/p42

Rel

ativ

e ar

bitr

ary

unit

(j)

Day 1 Day 4

CTRL siRNAβKlotho siRNA

0

1

2

3

4

5

H&

E

(b)

(c)

βKlo

tho

0

0.2*

0.4

0.6

0.8

1.0

1.2

LorK1 K10Rel

ativ

e pr

otei

n ex

pres

sion

s

(g) CTRL siRNAβKlotho siRNA

βKlotho

0

0.4

0.8

1.2

Rel

ativ

e ex

pres

sion

leve

ls

(i)

0

1.5

3.0

4.5

6.0

CTRLsiRNA βKlotho CTRLsiRNA βKlotho

Rel

ativ

e ex

pres

sion

leve

ls(h)

GAPDH

βKlotho siRNA

H&

E

(d)

(e)

H&

E

CTRL siRNA

*

*

*

****

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 air–liquid interface. HaCaT cells were grown in the air–liquidinterface for 7 days to develop a multilayered epithelium. (a) bKlotho proteinexpression was analysed by Western blotting. (b) Histology of multilayer-culturedHaCaT cells. (c) Immunohistochemistry of bKlotho protein expression in multilayer-cultured HaCaT cells. (d, e) bKlotho siRNA induced a cuboidal morphologicalchange in HaCaT cells grown in the air–liquid interface (f) HaCaT cells weretransfected with bKlotho or control (CTRL) siRNA and were grown in the air–liquidinterface for 7 days. Protein expression was analysed by Western blotting.Representative results of bKlotho, keratin 1 (K1), keratin 10 (K10) loricrin (Lor),keratin 14 (K14), p44/p42 and Phospho p44/p42 are shown. (g, h, i) Densitometricanalysis results were obtained from pooled data. (j) Cell number was assessedusing Cell Counting Kit-8. The results of relative levels of OD from pooled data.Values represent the mean � SE (n = 4). *P < 0.05; **P < 0.005 versus the CTRLsiRNA group.

Letter to the Editor

ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons LtdExperimental Dermatology, 2013, 22, 748–774 773

proliferation (9). Presumably, IGF-1R pathway might be activated

and the differentiation was suppressed in the skin of kl/kl mice.

Klotho inhibits the Wnt pathway. Increased Wnt activity was

shown by measuring b-galactosidase reporter activity in the skin

of kl/kl mice crossed with the TOPGAL reporter strain, in

which the activity of the b-galactosidase reporter is under the

control of Wnt-responsive elements (10). The gene expression lev-

els of transcriptional targets of the Wnt pathway including cyclin

D1 and c-myc are increased in the skin of kl/kl mice. b-Cateninhas been shown to be a key downstream effector in the Wnt path-

way (11). The Wnt pathway is an important regulator of prolifer-

ation and differentiation in epidermal stem cell maintenance (12).

The activation of the Wnt pathway usually accompanies the

activation of p44/p42 MAPK (13, 14), which is another regula-

tor of the proliferation and differentiation of epidermal keratino-

cytes (15).

AcknowledgementsThis study was supported by the fund for Kagawa University Young Scien-

tists 2012 and Grants-in-Aid for scientific research to K. Nakai from the

Ministry of Education, Science, Sports, and Culture, Japan. We thank Ms.

Fumiko Nishiyama for her technical assistance and contribution of essential

reagents and tools.

Author contributionsKozo Nakai performed the research, designed the research study, analyzed

the data, wrote the paper; Kozo Yoneda, Reiji Haba, Yoshio Kushida,

Naomi Katsuki contributed essential reagents or tools; Tetsuya Moriue ana-

lysed the data; Hiroaki Kosaka and Yasuo Kubota analysed the data, wrote

the paper; Shigeaki Inoue performed the research, designed the research

study, contributed essential reagents or tools.

Conflict of interestThe authors have declared no conflicting interests. Animal care certification

was delivered.

References1 Kuro-o M, Matsumura Y, Aizawa H et al. Nat-

ure 1997: 390: 45–51.2 Kuro-o M. Biochim Biophys Acta 2009: 1790:

1049–1058.3 Kuro-o M. Pflugers Arch 2010: 459: 333–343.4 Kuro-o M. Korean J Intern Med 2011: 26:

113–122.5 Ito S, Kinoshita S, Shiraishi N et al. Mech Dev

2000: 98: 115–119.6 Ito S, Fujimori T, Hayashizaki Y et al. Biochim

Biophys Acta 2002: 1576: 341–345.7 Kurosu H, Yamamoto M, Clark J D et al. Sci-

ence 2005: 309: 1829–1833.8 Sadagurski M, Yakar S, Weingarten G et al.

Mol Cell Biol 2006: 26: 2675–2687.

9 Isard O, Knol A C, Aries M F et al. J InvestDermatol 2011: 131: 59–66.

10 Liu H, Fergusson M M, Castilho R M et al.Science 2007: 317: 803–806.

11 Cadigan K M, Nusse R. Genes Dev 1997: 11:3286–3305.

12 Ito M, Yang Z, Andl T et al. Nature 2007: 447:316–320.

13 Kim S E, Choi K Y. Cell Signal 2007: 19:1554–1564.

14 Yun M S, Kim S E, Jeon S H et al. J Cell Sci2005: 118: 313–322.

15 Lin N, Moroi Y, Uchi H et al. J Dermatol Sci2007: 48: 71–73.

Supporting InformationAdditional Supporting Information may be found inthe online version of this article:Data S1. Methods.Figure S1. The expression of bKlotho protein and

epidermal differentiation-related protein was suppressedin the skin of kl/kl mice.Figure S2. Normal human epidermal keratinocyte

cells (NHEK cells) were grown in the air-liquid inter-face for 7 days to develop a multilayered epithelium.Figure S3. bKlotho siRNA suppressed the expression

levels of differentiation-related factors in HaCaT cellsgrown in the air-liquid interface.

Letter to the Editor

774ª 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd

Experimental Dermatology, 2013, 22, 748–774