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

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<ul><li><p>DOI: 10.1111/exd.12258</p><p> to the Editor</p><p>Deranged epidermal differentiation in kl/kl mouse and the effectsof bKlotho siRNA on the differentiation of HaCaT cells</p><p>Kozo Nakai1, Kozo Yoneda1, Reiji Haba2, Yoshio Kushida2, Naomi Katsuki2, Tetsuya Moriue1,Hiroaki Kosaka3, Yasuo Kubota1 and Shigeaki Inoue4</p><p>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,</p><p>Isehara, Japan</p><p>Correspondence: Kozo Nakai, Department of Dermatology, Faculty of Medicine, Kagawa University, 1750-1 Ikenobe, Miki-cho, Kita-gun, Kagawa</p><p>761-0793, Japan, Tel.: +81-87-891-2162, Fax: +81-87-891-2163, e-mail:</p><p>Abstract: Mice deficient in the klotho gene (kl/kl mice) display the</p><p>phenotypes of human ageing. We found that the expression of</p><p>epidermal differentiation-associated factors (keratin 1, keratin 10,</p><p>filaggrin and loricrin) was lower in the skin of kl/kl mice than that</p><p>of wild-type mice. In vitro experiments showed that the expression</p><p>of bKlotho, a family of klotho gene-encoded protein, was inducedconcomitantly with the differentiation of an immortalized human</p><p>epidermal keratinocyte cell line (HaCaT cells) when they were</p><p>cultured in an airliquid interface. bKlotho knockdown by smallinterfering ribonucleic acid suppressed the expression of the above</p><p>differentiation-associated factors in HaCaT cells. bKlotho small</p><p>interfering ribonucleic acid increased the expression of keratin 14,</p><p>which is expressed in mitotically active basal layer cells, and</p><p>activated p44/p42 mitogen-activated protein kinase in the HaCaT</p><p>cells grown in the airliquid interface. These findings suggest thatthe epidermal differentiation is deranged in kl/kl mice, and</p><p>bKlotho is required for the differentiation of human epidermalkeratinocytes.</p><p>Key words: epidermal differentiation Klotho</p><p>Accepted for publication 1 October 2013</p><p>BackgroundThe klotho gene was identified in 1997 as a gene mutated in the</p><p>klotho (kl/kl) mouse, which displays the phenotypes of human age-</p><p>ing (1). It has been reported that the skin of kl/kl mice demon-</p><p>strates reductions in dermal and epidermal thicknesses and the</p><p>number of hair follicles. The klotho gene encodes a putative type I</p><p>membrane protein, Klotho. The Klotho protein functions as a</p><p>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</p><p>unknown.</p><p>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</p><p>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</p><p>human epidermal keratinocytes. For details of the methods, see</p><p>Data S1.</p><p>ResultsThe skin of kl/kl mice showed an atrophic and scaly appearance</p><p>(Fig. 1b) relative to wild-type (WT) mice (Fig. 1a). As previously</p><p>reported, there were histological reductions in dermal and epider-</p><p>mal thicknesses in kl/kl mice. We found that kl/kl mice had a</p><p>wavy epidermis with basket-weave hyperkeratosis, implying the</p><p>abnormal differentiation of the epidermis of kl/kl mice. Immuno-</p><p>histological analysis revealed the relative absence of bKlotho in the</p><p>epidermis of kl/kl mice (Fig. 1d) to that of WT mice (Fig. 1c). In</p><p>WT mice, keratin 1, keratin 10, filaggrin and loricrin were</p><p>expressed in the epidermis (Fig. 1e, g, i, k). However, the expres-</p><p>sion of these differentiation-related proteins was suppressed in the</p><p>epidermis of kl/kl mice (Fig. 1f, h, j, l). Western blotting revealed</p><p>that the levels of protein expression of bKlotho, keratin 1, keratin10, filaggrin and loricrin were lower in the skin of kl/kl mice than</p><p>those of WT mice (Figure S1).</p><p>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).</p><p>NHEK and HaCaT cells can differentiate and develop a multilay-</p><p>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).</p><p>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</p><p>(Figure S3). These results suggest that bKlotho is necessary for thenormal differentiation of epidermal keratinocytes.</p><p>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</p><p>reported in mitotically active layer cells of epidermis. bKlothosiRNA increased the expression levels of keratin 14 (Fig. 2h) and</p><p>772 2013 John Wiley &amp; Sons A/S. Published by John Wiley &amp; Sons Ltd</p><p>Experimental Dermatology, 2013, 22, 748774</p></li><li><p>highly phosphorylated the p44/p42 MAPK protein in HaCaT cells</p><p>grown in the airliquid interface (Fig. 2i). Moreover, bKlotho siR-NA enhanced the proliferation of HaCaT cells (Fig. 2j). These</p><p>results suggest that bKlotho regulates the mitotic activity in differ-entiating epidermal keratinocytes.</p><p>ConclusionThis study demonstrated a novel biological role of Klotho and/or</p><p>bKlotho in skin. Keratinization of the epidermis is a well-definedprogramme of differentiation: keratinocytes progress vertically</p><p>from basal cells into spinous and granular cells to flattened, dif-</p><p>ferentiated squames in the stratum corneum. The suppression of</p><p>differentiation markers suggests deranged keratinization of the</p><p>epidermis, and it may partially explain the histological changes</p><p>observed in the skin of kl/kl mice. We have shown abnormal</p><p>differentiation of the epidermis in kl/kl mice. bKlotho was notexpressed in monolayer-cultured NHEK cells and HaCaT cells,</p><p>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</p><p>expression of aKlotho is also suppressed in kl/kl mice, anotherpossible mechanism of the deranged epidermal differentiation</p><p>should be considered in the mice. Secreted Klotho has pleiotropic</p><p>activities. It inhibits the insulin-like growth factor-1 receptor</p><p>(IGF-1R) pathway (7). In skin, the IGF-1R pathway has been</p><p>reported to interfere with keratinocyte differentiation (8) and</p><p>H&amp;</p><p>E</p><p>WT kl/kl</p><p>WT kl/kl</p><p>(a) (b)</p><p>(c) (d)</p><p>Klo</p><p>tho</p><p>Fila</p><p>gg</p><p>rin</p><p>Lo</p><p>ricr</p><p>inK</p><p>erat</p><p>in 1</p><p>Ker</p><p>atin</p><p> 10</p><p>WT kl/kl</p><p>WT kl/kl</p><p>WT kl/kl</p><p>WT kl/kl</p><p>(e) (f)</p><p>(g) (h)</p><p>(i) (j)</p><p>(k) (l)</p><p>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).</p><p>GAPDH</p><p>(a) Monolayer Multilayer</p><p>Klotho</p><p>(f)</p><p>K1</p><p>Lor</p><p>GAPDH</p><p>CTRL KlothosiRNA</p><p>K10</p><p>Klotho</p><p>K14Phospho </p><p>p44/p42</p><p>p44/p42</p><p>Rel</p><p>ativ</p><p>e ar</p><p>bitr</p><p>ary </p><p>unit</p><p>(j)</p><p>Day 1 Day 4</p><p>CTRL siRNAKlotho siRNA</p><p>0</p><p>1</p><p>2</p><p>3</p><p>4</p><p>5</p><p>H&amp;</p><p>E</p><p>(b)</p><p>(c)</p><p>Klo</p><p>tho</p><p>0</p><p>0.2*</p><p>0.4</p><p>0.6</p><p>0.8</p><p>1.0</p><p>1.2</p><p>LorK1 K10Rel</p><p>ativ</p><p>e pr</p><p>otei</p><p>n ex</p><p>pres</p><p>sion</p><p>s</p><p>(g) CTRL siRNAKlotho siRNA</p><p>Klotho</p><p>0</p><p>0.4</p><p>0.8</p><p>1.2</p><p>Rel</p><p>ativ</p><p>e ex</p><p>pres</p><p>sion</p><p> leve</p><p>ls</p><p>(i)</p><p>0</p><p>1.5</p><p>3.0</p><p>4.5</p><p>6.0</p><p>CTRLsiRNA Klotho CTRLsiRNA Klotho</p><p>Rel</p><p>ativ</p><p>e ex</p><p>pres</p><p>sion</p><p> leve</p><p>ls(h)</p><p>GAPDH</p><p>Klotho siRNA</p><p>H&amp;</p><p>E</p><p>(d)</p><p>(e)</p><p>H&amp;</p><p>E</p><p>CTRL siRNA</p><p>*</p><p>*</p><p>*</p><p>****</p><p>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) Immunohistochemistry of bKlotho protein expression in multilayer-cultured HaCaT cells. (d, e) bKlotho siRNA induced a cuboidal morphologicalchange in HaCaT cells grown in the airliquid interface (f) HaCaT cells weretransfected with bKlotho or control (CTRL) siRNA and were grown in the airliquidinterface 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 &lt; 0.05; **P &lt; 0.005 versus the CTRLsiRNA group.</p><p>Letter to the Editor</p><p> 2013 John Wiley &amp; Sons A/S. Published by John Wiley &amp; Sons LtdExperimental Dermatology, 2013, 22, 748774 773</p></li><li><p>proliferation (9). Presumably, IGF-1R pathway might be activated</p><p>and the differentiation was suppressed in the skin of kl/kl mice.</p><p>Klotho inhibits the Wnt pathway. Increased Wnt activity was</p><p>shown by measuring b-galactosidase reporter activity in the skinof kl/kl mice crossed with the TOPGAL reporter strain, in</p><p>which the activity of the b-galactosidase reporter is under thecontrol of Wnt-responsive elements (10). The gene expression lev-</p><p>els of transcriptional targets of the Wnt pathway including cyclin</p><p>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-</p><p>way (11). The Wnt pathway is an important regulator of prolifer-</p><p>ation and differentiation in epidermal stem cell maintenance (12).</p><p>The activation of the Wnt pathway usually accompanies the</p><p>activation of p44/p42 MAPK (13, 14), which is another regula-</p><p>tor of the proliferation and differentiation of epidermal keratino-</p><p>cytes (15).</p><p>AcknowledgementsThis study was supported by the fund for Kagawa University Young Scien-</p><p>tists 2012 and Grants-in-Aid for scientific research to K. Nakai from the</p><p>Ministry of Education, Science, Sports, and Culture, Japan. We thank Ms.</p><p>Fumiko Nishiyama for her technical assistance and contribution of essential</p><p>reagents and tools.</p><p>Author contributionsKozo Nakai performed the research, designed the research study, analyzed</p><p>the data, wrote the paper; Kozo Yoneda, Reiji Haba, Yoshio Kushida,</p><p>Naomi Katsuki contributed essential reagents or tools; Tetsuya Moriue ana-</p><p>lysed the data; Hiroaki Kosaka and Yasuo Kubota analysed the data, wrote</p><p>the paper; Shigeaki Inoue performed the research, designed the research</p><p>study, contributed essential reagents or tools.</p><p>Conflict of interestThe authors have declared no conflicting interests. Animal care certification</p><p>was delivered.</p><p>References1 Kuro-o M, Matsumura Y, Aizawa H et al. Nat-</p><p>ure 1997: 390: 4551.2 Kuro-o M. Biochim Biophys Acta 2009: 1790:</p><p>10491058.3 Kuro-o M. Pflugers Arch 2010: 459: 333343.4 Kuro-o M. Korean J Intern Med 2011: 26:</p><p>113122.5 Ito S, Kinoshita S, Shiraishi N et al. Mech Dev</p><p>2000: 98: 115119.6 Ito S, Fujimori T, Hayashizaki Y et al. Biochim</p><p>Biophys Acta 2002: 1576: 341345.7 Kurosu H, Yamamoto M, Clark J D et al. Sci-</p><p>ence 2005: 309: 18291833.8 Sadagurski M, Yakar S, Weingarten G et al.</p><p>Mol Cell Biol 2006: 26: 26752687.</p><p>9 Isard O, Knol A C, Aries M F et al. J InvestDermatol 2011: 131: 5966.</p><p>10 Liu H, Fergusson M M, Castilho R M et al.Science 2007: 317: 803806.</p><p>11 Cadigan K M, Nusse R. Genes Dev 1997: 11:32863305.</p><p>12 Ito M, Yang Z, Andl T et al. Nature 2007: 447:316320.</p><p>13 Kim S E, Choi K Y. Cell Signal 2007: 19:15541564.</p><p>14 Yun M S, Kim S E, Jeon S H et al. J Cell Sci2005: 118: 313322.</p><p>15 Lin N, Moroi Y, Uchi H et al. J Dermatol Sci2007: 48: 7173.</p><p>Supporting InformationAdditional Supporting Information may be found inthe online version of this article:Data S1. Methods.Figure S1. The expression of bKlotho protein and</p><p>epidermal differentiation-related protein was suppressedin the skin of kl/kl mice.Figure S2. Normal human epidermal keratinocyte</p><p>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</p><p>levels of differentiation-related factors in HaCaT cellsgrown in the air-liquid interface.</p><p>Letter to the Editor</p><p>774 2013 John Wiley &amp; Sons A/S. Published by John Wiley &amp; Sons Ltd</p><p>Experimental Dermatology, 2013, 22, 748774</p></li></ul>


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