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Published as doi: 10.1096/fj.07-9475com.
 (The FASEB Journal. 2008;22:1009-1020.)
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(The FASEB Journal. 2008;22:1009-1020.)
© 2008 FASEB

Dickkopf 1 (DKK1) regulates skin pigmentation and thickness by affecting Wnt/β-catenin signaling in keratinocytes

Yuji Yamaguchi*,{dagger},{ddagger}, Thierry Passeron*, Toshihiko Hoashi*, Hidenori Watabe*, François Rouzaud*, Ken-ichi Yasumoto*, Takahiko Hara*, Chiharu Tohyama{dagger}, Ichiro Katayama{dagger}, Toru Miki* and Vincent J. Hearing*

* Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA;

{dagger} Department of Dermatology, Osaka University Graduate School of Medicine, Osaka, Japan; and

{ddagger} Department of Geriatric and Environmental Dermatology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan

1Correspondence: Department of Geriatric and Environmental Dermatology, Nagoya City University Graduate School of Medical Sciences, 1-Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya, 467-8601 Japan. E-mail: yujin{at}med.nagoya-cu.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
The epidermis (containing primarily keratinocytes and melanocytes) overlies the dermis (containing primarily fibroblasts) of human skin. We previously reported that dickkopf 1 (DKK1) secreted by fibroblasts in the dermis elicits the hypopigmented phenotype of palmoplantar skin due to suppression of melanocyte function and growth via the regulation of two important signaling factors, microphthalmia-associated transcription factor (MITF) and β-catenin. We now report that treatment of keratinocytes with DKK1 increases their proliferation and decreases their uptake of melanin and that treatment of reconstructed skin with DKK1 induces a thicker and less pigmented epidermis. DNA microarray analysis revealed many genes regulated by DKK1, and several with critical expression patterns were validated by reverse transcriptase-polymerase chain reaction and Western blotting. DKK1 induced the expression of keratin 9 and {alpha}-Kelch-like ECT2 interacting protein ({alpha}KLEIP) but down-regulated the expression of β-catenin, glycogen synthase kinase 3β, protein kinase C, and proteinase-activated receptor-2 (PAR-2), which is consistent with the expression patterns of those proteins in human palmoplantar skin. Treatment of reconstructed skin with DKK1 reproduced the expression patterns of those key proteins observed in palmoplantar skin. These findings further elucidate why human skin is thicker and paler on the palms and soles than on the trunk through topographical and site-specific differences in the secretion of DKK1 by dermal fibroblasts that affects the overlying epidermis.—Yamaguchi, Y., Passeron, T., Hoashi, T., Watabe, H., Rouzaud, F., Yasumoto, K., Hara, T., Tohyama, C., Katayama, I., Miki, T., Hearing, V. J. Dickkopf 1 (DKK1) regulates skin pigmentation and thickness by affecting Wnt/β-catenin signaling in keratinocytes.


Key Words: keratin 9 • epithelial cell transforming sequence 2 • protease-activated receptor 2 • glucose synthase kinase • melanocyte


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
THE EPIDERMIS IN PALMOPLANTAR AREAS of the skin (palmoplantaris, the soles and palms) is thicker and less pigmented than in nonpalmoplantar areas (nonpalmoplantaris, the trunk). Gene profile analyses demonstrated that palmoplantar fibroblasts express higher levels of dickkopf 1 (DKK1), a canonical Wnt inhibitor (1) , than do nonpalmoplantar fibroblasts (2) . DKK1, which interacts with the Wnt receptor lipoprotein receptor-related protein 6 (LRP6; ref. 3 ), is a secreted antagonist of the canonical Wnt signaling pathway, which involves β-catenin and multiple protein complexes containing glycogen synthase kinase 3β (GSK3β), axin, adenomatous polyposis coli (APC), and Akt (4) . Keratin 14-DKK1 transgenic mice lack hair follicle development and pigmentation of their trunk skin because melanocytes do not exist in their interfollicular epidermis even in normal littermates (5) . A combined experimental and computerized modeling approach proves that mesenchymal DKK1 expression reduces overall appendage density, especially hair follicle development during embryogenesis (6) . We previously reported (2) that DKK1 inhibits melanocyte growth and function via the suppression of β-catenin and microphthalmia-associated transcription factor (MITF). Further study suggests that the rapid decrease in expression of MITF in melanocytes treated with DKK1 is concurrent with decreased activities of β-catenin and of GSK3β via phosphorylation at Ser-9 and with the up-regulated expression of protein kinase C{alpha} (7) . DNA microarray analyses (http://www.ncbi.nlm.nih.gov/geo/ with series GSE5515) also suggest various hypotheses to explain the mechanisms of hypopigmentation in palmoplantar skin: candidate receptors for DKK1 and the apoptosis pathway in human melanocytes (7) . Yasuda et al. (8) further reported that the decreased expression of fibronectin may account for the hypopigmentation in palmoplantar skin.

However, the mechanisms of thickened epidermis in palmoplantar skin have not been fully elucidated. Previously, we focused on site-specific (topographically or regionally different) interactions between fibroblasts and keratinocytes. Fibroblasts in palmoplantar dermis induce a thick epidermis and keratin 9 expression in keratinocytes through mesenchymal-epithelial interactions, whereas fibroblasts in nonpalmoplantar dermis do not (9) . Keratin 9 is exclusively observed in suprabasal palmoplantar epidermis and is only seen in acrosyringia (epidermal keratinocytes around sweat gland ducts) in nonpalmoplantar epidermis, which suggests that keratin 9 plays a role in the formation of the thick epidermis on the soles and palms (10) . This is further emphasized by the fact that mutations in the gene encoding keratin 9 result in genetic keratoderma (11) . Nonpalmoplantar epidermis (excluding dermal components) can be grafted to treat palmoplantar skin defects [e.g., caused by diabetes mellitus (12) and rheumatic diseases (13) ] because it can adopt a palmoplantar phenotype through mesenchymal-epithelial interactions (14 , 15) . These observations may result from expression differences of DKK1 between palmoplantar fibroblasts and nonpalmoplantar fibroblasts, which affect the adjacent epidermis in site-specific adult tissue.

Since keratinocytes play a major role in skin structure and pigmentation, we have now evaluated the effects of DKK1 on keratinocytes. We measured the effects of DKK1 on melanin uptake by keratinocytes and on keratinocyte gene expression profiles and Wnt signaling pathways to test the hypothesis that high levels of expression and secretion of DKK1 by palmoplantar fibroblasts account for the thick and hypopigmented phenotype of skin on the soles and the palms.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 CONCLUSIONS
 REFERENCES
 
Keratinocyte cultures and cell growth assay
Neonatal human foreskin keratinocytes were obtained from Cascade Biologics, Inc., (Portland, OR, USA). Keratinocyte cultures were grown in keratinocyte growth medium (KGM) consisting of Medium 154 and HKGS (both from Cascade Biologics). Keratinocytes from the third to fifth passage were used in these experiments. Keratinocytes were seeded on 24-well plates at a density of 1 x 104 cells/well and were incubated overnight to observe cell attachment and spreading. KGM medium was replenished with or without 50 ng/ml recombinant human DKK1 (rhDKK1; R&D Systems, Minneapolis, MN, USA) every 2 days, and cell growth was measured using the MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide] assay (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s instructions and/or cells were counted using a hemocytometer at 5 days.

Reconstructed skin
The epidermal equivalent MelanoDerm was obtained from MatTek Corp. (Ashland, MA, USA). Normal human keratinocytes and melanocytes were obtained from Asian neonatal foreskin tissues. MelanoDerms were grown at the air-liquid interface of the maintenance medium MEL-NHM-113 (MatTek), and the culture medium was renewed every 2 days. Where noted, the cultures were supplemented with 100 ng/ml rhDKK1 (R&D Systems) every 2 days for 4–10 days. rhDKK1 was dissolved in PBS with 0.1% BSA. The same concentrations of PBS and BSA were employed for mock-treated controls.

Histochemistry
Skin specimens were obtained from palmoplantar areas (palm and sole) and from nonpalmoplantar areas (trunk) and were taken from five adult Asian subjects (ages ranged from 31 to 47) during cutaneous surgery. Skin and epidermal equivalent samples were embedded in paraffin, and sections were cut, deparaffinized, and stained using standard techniques. The thickness of epidermis, and more specifically the thickness of the stratum corneum, was measured with Scion Image software (Scion Corp., Frederick, MD, USA). Melanin content was measured after Fontana-Masson staining and was also analyzed by Scion Image as described previously (16) . Indirect immunofluorescence primary antibodies used were as follows: mouse monoclonal antibody against keratin 9 (1:20, Abcam, Cambridge, MA, USA), rabbit polyclonal against β-catenin (1:50, Cell Signaling, Danvers, MA, USA), {alpha}-Kelch-like ECT2 interacting protein ({alpha}KLEIP; at 1:1,000) (17) , and proteinase-activated receptor-2 (PAR-2; 1:1,000, a kind gift of Dr. Glynis Scott, University of Rochester, Rochester, NY, USA) (18) . Secondary antibodies used were Alexa Fluor 594 goat anti-mouse IgG (H+L), Alexa Fluor 488 goat anti-mouse IgG (H+L), and Alexa Fluor 488 goat anti-rabbit IgG (H+L). DAPI (Vector, Burlingame, CA, USA) was used as a counterstain. Fluorescence was observed and captured using a Leica DMR B/D MLD fluorescence microscope (Leica, Wetzlar, Germany) and a Dage-MTI 3CCD 3-chip color video camera (Dage-MTI, Michigan City, IN, USA).

Transfection and melanin uptake assay
Transfection studies were performed using a DKK1 expressing plasmid, pcDNA3.1(–)-DKK1 (2) and the pcDNA3.1 vector alone as the control. Transfection was performed using lipofection for keratinocytes using lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Keratinocytes were seeded at 60% confluence 16 h before transfection in KGM. The amount of DNA used for each transfection was 2 µg per 1 x 106 cells. After 5 days, transfected cells were harvested for various analyses, including melanin uptake assay, immunocytochemistry, and Western blotting. The transfection efficiency was 70% as determined by the pEGFP-C1 vector (BD Biosciences Clontech, Palo Alto, CA, USA) and/or a β-Gal staining kit (Invitrogen). Melanin granules (2 µg/ml) purified from MNT1 melanoma cells (19) were added 1 days before the harvest. Bright field images were photographed (2) and were analyzed by Scion Image software as described previously (16) . Melanin content was also determined as described previously (2) by dissolving the cell pellets in 1 N NaOH and then measuring the absorbance at 405 nm in a SpectraMax 250 ELISA reader (Molecular Devices, Sunnyvale, CA, USA).

Microarray procedures
Modified oligo-DNA microarray analysis was performed as described previously (8) . Briefly, total RNA was prepared from cultured human keratinocytes treated with or without 50 ng/ml rhDKK1 (R&D Systems) for 2 h, using an RNeasy mini kit (Qiagen, Valencia, CA, USA). The quality (purity and integrity) of extracted total RNA was confirmed using an Agilent 2100 Bioanalyzer with an RNA 6000 Nano Assay (Agilent Technology, Palo Alto, CA, USA). Paired cDNA samples, labeled by cyanine 3- and cyanine 5-dUTP incorporation (Qiagen) during reverse transcription (Qiagen), were hybridized simultaneously with one oligo-DNA chip (Hs-Operon V2-vB2.2p13) as per National Cancer Institute (NCI) in-house protocol (available at http://mach1.nci.nih.gov/). Two fluorescent intensities of the oligo-DNA chip were scanned using a microarray scanner (GenePix 4000A; Axon Instruments, Inc., Sunnyvale, CA, USA). Differential gene expression was profiled with Genepix 3.0 software and was analyzed by NCI Center for Information Technology (CIT) programs and databases. All experiments were performed in triplicate independently.

Reverse transcriptase-polymerase chain reaction (RT-PCR)
To confirm the validity of oligo-DNA microarray results, RT-PCR was performed. The oligonucleotide primers for PCR were based on published mRNA sequences and were as follows: human {alpha}KLEIP sense primer 5'-tggctgtgttaggagggttc-3'; {alpha}KLEIP antisense primer 5'-ctactgccatgagctgtcca-3'; human GJB6 sense primer 5'-ggcgaggagagaagaggaat-3'; GJB6 antisense primer 5'-caactctgccacgttaagca-3'; human Snrpn sense primer 5'-gttttgggtctggtgttgct-3'; Snrpn antisense primer 5'-gacctctaatgcctggtgga-3'; human BMP2IK sense primer 5'-cacgccaactagcacaaaga-3'; BMP2IK antisense primer 5'-aattcgactggttgggactg-3'; MITF sense primer 5'-agagagcgagtgcccaggcatgaac-3'; MITF antisense primer 5'-tctttggccagtgctcttgcttcag-3'; human P4HA2 sense primer 5'-tgtcaaactgacaccccgta-3'; P4HA2 antisense primer 5'-atttactcgggccacaacag-3'; human Tulp3 sense primer 5'-acaccgtggatactgcttcc-3'; Tulp3 antisense primer 5'-ccgatccattccccttttat-3'; human PAR-2 sense primer 5'-tgctagcagcctctctctcc-3'; PAR-2 antisense primer 5'-cttcaaggggaaccagatga-3'; GAPDH sense primer 5'-accacagtccatgccatcac-3'; GAPDH antisense primer 5'-tccaccaccctgttgctgta-3'. After denaturation at 94°C for 2 min, PCR was performed for 30 cycles (30 s at 94°C, 1 min at 56°C, and 1 min at 72°C). All amplified products were sequence verified (8) . Control reactions were performed in the absence of reverse transcriptase and were negative. Each experiment was repeated at least in triplicate independently.

Immunoblotting
Cultures from 100 mm dishes were solubilized in 500 µl extraction buffer containing 1% Nonidet P 40 (Calbiochem, San Diego, CA, USA), 0.01% SDS, 0.1 M Tris:HCl, pH 7.2, and protease inhibitor cocktail (Roche, Mannheim, Germany). Protein concentrations of extracts were measured using the BCA protein assay kit (Pierce, Rockford, IL, USA). Cell extracts (1 µg) were separated on 8–14% gradient SDS-polyacrylamide gels (Invitrogen). After electrophoresis, proteins were transferred electrophoretically from the gels to Immobilon-P transfer membranes (Millipore, Bedford, MA). The filters were incubated in the presence of antibodies to PAR-2 (at 1:1000) (18) , {alpha}KLEIP (at 1:1000) (17) , keratin 9 (at 1:200, Abcam), β-catenin (at 1:1000, Cell Signaling Technology), GSK-3β (at 1:1000, Cell Signaling Technology), pGSK-3β (at 1:1000, Cell Signaling Technology), PKC{alpha} (at 1:10,000, Sigma, St. Louis, MO, USA), PKCβ2 (at 1:10,000, Sigma), extracellular-signal regulated kinase (ERK) 1/2 (p44/42 MAP kinase antibody) (at 1:1000, Cell Signaling Technology), pERK1/2 (at 1:1000, Cell Signaling Technology), or β-actin (at 1:3000, AC-15, Abcam) at 23°C for 1 h. They were then washed and incubated with horseradish peroxidase-linked anti-rabbit or anti-mouse whole antibodies (at 1:1000, Amersham, Piscataway, NJ, USA) at room temperature for 1 h. Antigens were detected using an ECL-plus Western blotting detection system (Amersham).

Immunocytochemical staining
Keratinocyte cultures in 2-well Lab-Tek chamber slides (Nalge Nunc International Corp., Naperville, IL, USA) were processed for indirect immunofluorescence to detect the expression of signal transduction proteins using primary antibodies to PAR-2 (at 1:1000) (18) ; {alpha}KLEIP (at 1:1000) (17) ; GSK-3β (1:50, Cell Signaling Technology); phospho-GSK-3β, which is specific for GSK-3β phosphorylated at Ser-9 (at 1:100, Cell Signaling Technology); β-catenin (at 1:50, Cell Signaling Technology and Santa Cruz Biotechnology, Santa Cruz, CA, USA); PKCβ1 (at 1:1000, Sigma); and PKC{alpha} (at 1:1000, Sigma).

Bound antibodies were visualized with appropriate secondary antibodies, Alexa Fluor 488 goat anti-rabbit IgG (H+L) (Molecular Probes, Inc., Eugene, OR, USA) and Alexa Fluor 594 mouse anti-rabbit IgG (H+L) (Molecular Probes) at 37°C for 30 min at 1:500 dilution with 5% goat serum. DAPI (Vector) was used as a counterstain. The fluorescence of green produced by Alexa 488, of red produced by Alexa 594, and of blue by DAPI was observed and captured using a Leica DMR B/D MLD fluorescence microscope and a Dage-MTI 3CCD 3-chip color video camera. Confocal microscopy was also used to investigate antibody localization, as detailed in Hoashi et al. (20) .

Statistical methods
Numbers reported in all figures are mean ± SD, and statistical analyses were performed using Student’s t test (NS=not significant).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 CONCLUSIONS
 REFERENCES
 
DKK1 decreases melanin uptake by keratinocytes and increases their proliferation in vitro
We initially investigated the effects of DKK1 on the proliferation of human keratinocytes and their uptake of melanin granules. Normal human keratinocytes (Fig. 1 ) were mock transfected or were transfected with DKK1, and their uptake of melanin granules was measured. After incubation for 24 h, keratinocytes transfected with DKK1 contained significantly less melanin than did those transfected with a mock control vector, as measured in bright field images analyzed by Scion Image (DKK1 vs. control; 14.0±4.0 vs. 44.0±9.0 arbitrary units; P=0.003; n=10) and by melanin content assay (DKK1 vs. control; 94.4±19.2 vs. 195.5±80.1 ng/ml; P=0.012; n=8). Further, transfection of DKK1 into normal human keratinocytes stimulated their growth and density (DKK1 vs. control; 29.3±1.9 vs. 24.0±1.2 cells/field; P=0.028; n=12), as recently reported for human mesenchymal stem cells (21) . The area of keratinocytes treated with 50 ng/ml rhDKK1 decreased significantly (P=0.006; n=8) in size (641±148 µm2) as compared with nontreated controls (1097±366 µm2). Interestingly, this tendency was also observed in skin in vivo (Supplemental Fig. 1), where the average area of palmoplantar epidermal keratinocytes (779±28 µm2) was significantly (P<0.001; n=3) lower than that of nonpalmoplantar keratinocytes (1425±97 µm2). Treatment of those keratinocytes with rhDKK1 significantly stimulated their growth, as measured by the MTT assay (DKK1 vs. control; 0.412±0.069 vs. 0.358±0.085 A405; P=0.012; n=6) and by cell counting (DKK1 vs. control; 5.41±0.91x104 vs. 3.99±0.86x104 cells/24-well plate; P=0.029; n=6).


Figure 1
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  		Figure 1. Melanin uptake of DKK1-transfected or mock-transfected keratinocytes in vitro using bright field (top) and phase contrast (bottom) microscopy. DKK1-transfected keratinocytes showed less melanin uptake than mock-transfected controls. Melanin content reflects analysis by Scion Image. Insets show higher-power images of areas determined in the box.

DKK1 decreases pigmentation and increases the thickness of reconstructed skin
To further examine whether the secretion of DKK1 by fibroblasts elicited the physiological differences observed between palmoplantar skin and nonpalmoplantar skin, we used reconstructed skin grown in the presence or absence of rhDKK1 (Fig. 2 ). Within 4 days, the rhDKK1-treated skin was already significantly less pigmented than the mock-treated control, and the difference was even clearer after 7 or 10 days of treatment (Fig. 2A ). This difference in pigmentation was confirmed by Fontana-Masson staining, which showed significant decreases in melanin content (P<0.002, P<0.002, and P=0.02 at 4, 7, and 10 days, respectively) in rhDKK1-treated skin (Fig. 2B, C ). The skin was also significantly thicker (P<0.05, P<0.02, and P<0.001 at 4, 7, and 10 days, respectively) after treatment by rhDKK1 (Fig. 2C ).


Figure 2
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  		Figure 2. Treatment of MelanoDerm skin reconstructs with or without rhDKK1. A) Macroscopic photo after 10 days of treatment with or without 100 ng/ml rhDKK1. The epidermis treated with rhDKK1 appeared thicker and less pigmented than the control. B) Fontana-Masson staining after 4, 7, or 10 days of treatment with or without rhDKK1. The untreated epidermis had more melanin than that treated with rhDKK1 at all time points. C) Graphs showing data from more than 10 independent experiments in terms of melanin content and thickness. White and gray bars indicate MelanoDerms treated with or without DKK1, respectively. All 6 comparisons of treated vs. control were statistically significant (P<0.05).

DKK1 regulates many genes critical to keratinocyte function
Although other cells and interactions no doubt also contribute to the regulation of skin morphology and pigmentation, the fact that DKK1 can recapitulate the palmoplantar phenotype in reconstituted skin prompted us to further study the mechanisms involved. For this purpose, we stimulated human keratinocytes with 50 ng/ml rhDKK1 for 2 h, harvested total RNA from those cells and from untreated controls, and then analyzed those transcripts using oligonucleotide-DNA microarray technology. Up-regulated or down-regulated genes are summarized in Supplemental Tables 1–5, and the entire dataset of DNA microarray data is deposited at http://www.ncbi.nlm.nih.gov/geo with series GSE9211.Treatment of keratinocytes with rhDKK1 had varied effects on genes related to cell contraction, to apoptosis [TNFRSF1A-associated via death domain (TRADD)], to noncanonical Wnt signaling pathways, to protein kinase Cβ1 (PKCβ1), to HOX transcription factors (HoxD8 and Hox12), and to keratins (keratin 9). We confirmed the differences in mRNA expression levels of interesting genes using RT-PCR, which validated that levels of some genes [such as {alpha}KLEIP and connexin 30 gap junction protein β6 (GJB6)] were significantly up-regulated in response to DKK1 treatment, whereas DKK1 significantly down-regulated the expression levels of other genes [such as MITF, procollagen-proline, 2-oxoglutarate 4-dioxygenase (P4HA2), Tubby-like protein 3 (Tulp3), and coagulation factor II receptor-like 1 or thrombin receptor-like 1 (PAR-2); Fig. 3 ].


Figure 3
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  		Figure 3. mRNA levels in human keratinocytes treated or untreated with 50 ng/ml rhDKK1 for 2 h, as analyzed by RT-PCR. The expression of {alpha}KLEIP, keratin 9, and GJB6 was up-regulated by rhDKK1, but PAR-2, P4HA2, Tulp3, and MITF were down-regulated; GAPDH is shown as a loading control. Densitometric analyses are reported at the bottom of each panel, with mock-treated normalized to 100.

DKK1 acts on {alpha}KLEIP, keratin 9, ERK, Wnt/β-catenin pathway, and PAR-2 to regulate skin thickness and melanin uptake
We further focused on {alpha}KLEIP, keratin 9, the Wnt/β-catenin pathway, and PAR-2, since the first three factors and the fourth factor may account for the thickened and for the hypopigmented epidermis in palmoplantar areas, respectively, via the effect of DKK1 on keratinocytes.

We first investigated the expression patterns of these proteins in skin in vivo. The expression of {alpha}KLEIP and keratin 9 (10) was up-regulated in palmoplantar skin, whereas the expression of PAR-2 and β-catenin (2) was down-regulated (Fig. 4 ). The expression patterns of GSK3β, Ser-9-phosphorylated GSK3β, and PKC{alpha} in palmoplantar skin was consistent with those obtained in our previous study (7) .


Figure 4
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  		Figure 4. Immunohistochemistry of plantar, transitional, and nonplantar skin. The expression of {alpha}KLEIP and keratin 9 was observed only in palmoplantar skin. In contrast, the expression of PAR-2 was decreased in palms and soles as compared to dorsal skin. {alpha}KLEIP and PAR-2 = green fluorescence; keratin 9 = red fluorescence; DAPI = blue fluorescence.

We next performed Western blotting to investigate the expression patterns of {alpha}KLEIP, keratin 9, and various proteins related with the Wnt/β-catenin signaling pathway in addition to PAR-2. We harvested extracts of keratinocytes after 2 h, 3 days of treatment with 50 ng/ml rhDKK1 and at 3 days after transfection with DKK1 and analyzed them (results for extracts at 3 days after treatment with rhDKK1 are shown in Fig. 5 ). The expression of DKK1 was confirmed in the DKK1-transfected cells. For {alpha}KLEIP, three nonspecific bands in keratinocytes were detected, as previously reported (17) , but the correct {alpha}KLEIP band (at 64 kDa) was also detected and its expression level in keratinocytes was increased in response to DKK1. In contrast to all other proteins examined in this study, treatment with KGM containing rhDKK1 did not induce keratin 9 expression in nonpalmoplantar keratinocytes, but those seeded onto the collagen gel embedded with nonpalmoplantar fibroblasts and treated at the air-liquid interface with 10% FBS/DMEM showed remarkable differences. Treatment with rhDKK1 resulted in the induction of keratin 9 compared with the control (Fig. 5) . The expression levels of nuclear and cytoplasmic β-catenin were down-regulated in response to DKK1. GSK3β is not only an enzyme involved in the control of glycogen metabolism; it also regulates a variety of cellular functions, including Wnt signaling pathways (22) . Inactivation of GSK3β via phosphorylation of Ser-9 is the major route by which insulin activates muscle glycogen synthase (23) . We investigated the expression of GSK3β phosphorylated at Ser-9 and found that it was up-regulated in response to DKK1. Although overall GSK3β expression was not significantly affected at 2 h, DKK1 reduced its expression level at 3 days. Since PKC{alpha} has been reported to inactivate GSK3β (24) , we examined the expression of PKC isoforms. DKK1 up-regulated the expression of PKC{alpha} and PKCβ2. These findings on Wnt/β-catenin signals were consistent with data obtained from melanocytes (7) . ERK 1/2 plays important roles in keratinocyte proliferation and migration (25 , 26) . Although levels of ERK were not changed by DKK1, expression of phosphorylated ERK1/2 (pERK, at Thr202/Tyr204) was up-regulated in response to DKK1. Finally, DKK1 treatment also resulted in decreased expression levels of PAR-2.


Figure 5
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  		Figure 5. Expression patterns of proteins expressed by keratinocytes treated or untreated with 50 ng/ml rhDKK1 for 3 days, as analyzed by Western blot. Expression of {alpha}KLEIP (arrow), keratin 9 (arrow), PAR-2, nuclear and cytoplasmic β-catenin, GSK3β, pGSK3β, PKC{alpha}, PKCβ2, ERK, and pERK are shown. β-actin is shown as a loading control. Densitometric analyses are reported at the bottom of each panel, with mock-treated normalized to 100.

We validated these expression patterns by immunocytochemistry (Fig. 6 ): DKK1 recapitulated the palmoplantar phenotype in terms of the expression patterns of {alpha}KLEIP, PAR-2, and β-catenin in keratinocytes (Fig. 6A ). Although induction of keratin 9 expression in keratinocytes did not occur at detectable levels (data not shown), we confirmed the expression patterns of Wnt/β-catenin signals including GSK3β, Ser-9-phosphorylated GSK3β (Fig. 6B ), PKC{alpha}, PKCβ (Fig. 6C ), and pERK (Supplemental Fig. 2) in response to DKK1. Confocal microscopy showed the colocalization of PKCβ and β-catenin in DKK1-treated keratinocytes, suggesting the membrane localization of PKCβ (Fig. 6C ).


Figure 6
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  		Figure 6. Immunocytochemistry of keratinocytes treated or untreated with 50 ng/ml rhDKK1 for 3 days. Immunocytochemistry recapitulates results obtained by Western blot. A) {alpha}KLEIP and PAR-2 = red fluorescence; β-catenin = green fluorescence; DAPI = blue fluorescence. B) Top panels: β-catenin = red and green fluorescence; DAPI = blue fluorescence. Middle and bottom panels: GSK3β and Ser-9-phosphorylated GSK3β = green fluorescence; β-catenin = red fluorescence; DAPI = blue fluorescence. C) Confocal images. PKC{alpha} and PKCβ = green fluorescence; β-catenin = red fluorescence; DAPI = blue fluorescence.

Finally, we confirmed the four key protein expression patterns in reconstructed skins grown either in the presence or absence of DKK1 (Fig. 7 ). Immunostaining of those reconstructed skins was consistent with human skin in situ (as noted above) and revealed increased expression of {alpha}KLEIP and decreased expression of PAR-2 and β-catenin after 10 days of treatment with DKK1. Interestingly, expression of keratin 9 appeared in several cells after treatment with DKK1.


Figure 7
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  		Figure 7. Immunohistochemistry of MelanoDerm skin reconstructs after 10 days of treatment with or without rhDKK1. The expression of {alpha}KLEIP was up-regulated while expression of PAR-2 and β-catenin was down-regulated by rhDKK1. In addition, some cells positive for keratin 9 were observed.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
CONCLUSIONS
 REFERENCES
 
We hypothesize that mesenchymal-epithelial interactions (such as dermal-epidermal interactions in the skin) play crucial roles in topographical and site-specific differentiation and regulation, not only during embryogenesis but also to maintain homeostasis in adults (27) .

Physiologically, keratinocytes in hypopigmented palmoplantar epidermis are more proliferative (generating a much thicker skin) but at the same time are less pigmented. In this study, we demonstrated that DKK1, which is secreted at high levels by fibroblasts in palmoplantar skin, decreases melanin uptake by keratinocytes and increases their proliferation. To elucidate the mechanisms underlying these effects, we further showed that DKK1 regulates the expression of {alpha}KLEIP, keratin 9, and Wnt/β-catenin signals to maintain the thickened characteristic of palmoplantar epidermis and that of PAR-2 for its hypopigmented characteristic.

Regulation of proliferation and cell density of keratinocytes via DKK1
{alpha}KLEIP is a protein that is involved in actin assembly at sites of cell-cell adhesion (17) and that associates with ECT2, a Rho nucleotide exchange factor involved in cytokinesis (28 , 29) . Enhanced mRNA and protein expression of {alpha}KLEIP by DKK1 may account, at least in part, for the enhanced actin assembly and cytokinesis of keratinocytes in palmoplantar skin, which is consistent with their increased proliferation and density in that tissue.

Keratin 9 may be expressed in keratinocytes that require strong support against mechanical stress since the expression of keratin 9 is observed only in suprabasal keratinocyte layers of palmoplantar skin and in acrosyringia (keratinocytes around sweat gland ducts) (10) . DKK1 enhanced the expression level of keratin 9, which can be induced in nonpalmoplantar keratinocytes by palmoplantar fibroblasts (9) , which suggests that DKK1 can alter keratin dimerization and patterning in keratinocytes. Although further investigation will be necessary to prove this, the altered keratin patterning seen in palmoplantar keratinocytes due to secreted DKK1 may also help account for the thickened phenotype of palmoplantar epidermis.

In addition to demonstrating the increased expression of {alpha}KLEIP and keratin 9 in response to DKK1 at the mRNA and protein levels, the decreased expression of cytoplasmic β-catenin we found (2) may result in decreased cell-cell contact, followed by the shrinkage (the decreased cytoplasmic occupation) of each cell. This decreased expression of β-catenin may account for the tiny cobblestoned appearance of keratinocytes in palmoplantar epidermis and those treated with DKK1 compared with those in nonpalmoplantar epidermis and those in the normal control group, as this concept is already published elsewhere (30) . Not only structural changes in keratinocytes by the decreased β-catenin expression in response to DKK1 but also numerous signals affecting proliferation are involved in this pathway. Although melanocytes and keratinocytes respond differently to DKK1 in terms of proliferation, the responses to Wnt/β-catenin signals were similar between the two cell types. Increased expression of PKC{alpha} and PKCβ may also account for the increased cell density since PKC isozymes regulate the proliferation and differentiation of keratinocytes (31) . ERK phosphorylation is activated at the healing wound edge. In addition, β2-adrenergic receptor, the activation of which delays wound healing, prevents localization of phospho-ERK to the lamellipodial edge (26) . ERK activation in response to DKK1 may also play a role in the stimulation of keratinocyte proliferation and the increased cellular density since ERK is also an important suppressive regulator of apoptosis (25) . Canonical Wnt signals activate β-catenin expression through the inhibition of β-catenin degradation by multiple protein complexes, including GSK3β, axin, and APC. However, Wnt-5a inhibits the canonical Wnt pathway by promoting GSK3β-independent β-catenin degradation (32) . GSK3β is a unique protein in that it is inactivated through phosphorylation (33) . Our finding of the elevated phosphorylation of GSK3β at Ser-9 in response to DKK1 may suggest a novel pathway for DKK1/Wnt/β-catenin signaling in addition to the decreased overall expression level of GSK3β.

Although further studies are necessary, including analyses of protein levels, we found several intriguing genes at the mRNA level that might explain the thickened epidermis elicited by DKK1.

Tulp3, which is ubiquitously expressed throughout embryonic development, belongs to the Tubby-like protein family. Tulp3-knockout mice exhibit embryonic lethality with a failure in neural tube closure characterized by neuroepithelial apoptosis (24) . The earliest phenotype of Tulp3-knockout mice is a significant reduction in the number of βIII-tubulin-positive neurons in the hindbrain, which suggests that Tulp3 maintains normally differentiating neuronal cell populations. Apoptosis is restricted to the ventral region of the neuroepithelium in the hindbrain of Tulp3-knockout mice, suggesting that Tulp3 is involved in selective cell death in specific types of cells (34 35 36) . DKK1 down-regulated the expression of Tulp3 in keratinocytes, which suggests that Tulp3 down-regulates the apoptosis of keratinocytes through βIII-tubulin interactions.

Further, we observed a slight up-regulation of GJB6 at the mRNA level, which suggests that altered cell-cell communications mediated by gap junctions may also play roles in regulating the proliferation, migration, and differentiation of keratinocytes (37 , 38) .

P4HA2, prolyl-4-hydroxylase {alpha}-subunit ({alpha}2), plays roles in collagen fiber formation, probably in response to hypoxia-inducible transcription factor 1 (39 40 41) . DKK1 down-regulates the expression of P4HA2 in keratinocytes, which suggests that the appearance of the dermis in palmoplantar areas might also differ from nonpalmoplantar areas due to the down-regulated expression of this hydroxylase. Since the extracellular matrix plays important roles in cell motility (42) , it will be intriguing to study specific enzymes related with collagen synthesis in the future.

Regulation of pigment transfer to keratinocytes via DKK1
We found that DKK1 significantly inhibits the uptake of melanin granules by keratinocytes, a process that is critical to the distribution of pigment in the epithelium. PAR-2 is expressed on keratinocytes and is involved in melanin uptake via phagocytosis (43) in a Rho-dependent fashion (44) . PAR-2 plays a significant role in modulating pigmentation in a skin type-dependent manner (45) and in response to ultraviolet (18) . Thus, the decreased melanin uptake elicited in keratinocytes by DKK1 may in large part be mediated through the suppressed expression of PAR-2. Since we found that DKK1 induces keratinocyte proliferation, it is possible that the down-regulation of PAR-2 in response to DKK1 is an epiphenomenon to proliferation, considering the fact that PAR-2 is expressed not in the basal layer but in the suprabasal layer of the epidermis (46 , 47) . Since PAR-2 plays important roles during cutaneous inflammation in vivo (48) , down-regulated expression of PAR-2 in palmoplantar skin may indicate antiinflammatory effects against friction and mechanical stress, although further studies will be necessary to elucidate this. Additionally, whether DKK1 might also decrease the secretion of melanosomes by melanocytes will be an important process to analyze in future studies.

Although we did not observe detectable levels of MITF protein in keratinocytes, differential display and RT-PCR showed that DKK1 suppresses the expression of MITF-V5 mRNA in those cells. We assume that keratinocytes express MITF-V5 at the mRNA level but do not make detectable levels of MITF-M protein. It should be noted that MITF can be expressed in many different isoforms (49) , one of which (MITF-M) is specific to melanocytes. Since the antibody we use is specific for the MITF-M protein, the lack of reactivity with keratinocytes may reflect this situation. However, this result indicates that not only does DKK1 suppress MITF expression in melanocytes (50) but also in keratinocytes. The decreased uptake of melanin granules by keratinocytes elicited by DKK1 is significant, and the microarray expression results may provide important clues into the regulation of this process in keratinocytes, a process that is currently not well understood. In addition to our previous studies that showed dramatic decreases in melanogenesis in melanocytes after DKK1 treatment through effects on MITF (2 , 6) , the decrease of melanin uptake by keratinocytes that we show here concur to explain the lighter pigmentation observed in palms and soles.


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 CONCLUSIONS
REFERENCES
 
Taken together, DKK1 has various effects on keratinocyte growth and function, including the up-regulation of cell density and the decreased melanin uptake, probably through numerous effects on gene expression patterns, suggesting the potential uses of DKK1 (or bioactive peptides) to regulate the palmoplantar properties of skin. The mechanism of action of DKK1 on keratinocytes is summarized in Fig. 8 .


Figure 8
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  		Figure 8. Schematic showing the effects of DKK1 produced by palmoplantar fibroblasts on keratinocytes and melanocytes in human skin to increase skin thickness and decrease visible pigmentation. Abbreviations are as defined in the text. T = suppression; {downarrow} = suppressive effect of DKK1 on that protein expression or function.

The combined results of the effects of DKK1 shown in this study, combined with the earlier observations of its involvement in determining the morphology of transplanted skin, demonstrate that DKK1 is involved in regulating pigmentation and thickness of skin not only during embryogenesis but also in adult skin. These results further support our hypothesis that mesenchymal-epithelial interactions play crucial roles in site-specific regulation in adults.

In addition to the genes that we have studied here, the DNA microarray data showed numerous up-regulated and down-regulated receptors and HOX-related genes that responded to DKK1 (as shown in the Supplemental Tables), suggesting that DKK1 can serve as a ligand (agonist) or an antagonist for those receptors and can affect the site-specific distribution of HOX. HOX gene family members are transcription factors regulating patterning in the primary and secondary axes of developing embryos that also control digit number and morphogenesis (51) . The collinear regulation of HOX genes during limb development is similar to that seen in the trunk: genes located in the middle of the HoxD complex (HoxD8) are expressed in proximal areas of the limb bud, whereas genes located upstream have a more distal expression (HoxD12) (52) . HOX genes are also known to direct topographical and site-specific differentiation of embryonic neurons in response to growth factors, especially those secreted by fibroblasts (53) . Gene profile analyses show that adult human fibroblasts obtained from different sites maintain key features of HOX gene expression patterns established during embryogenesis, suggesting that HOX genes may direct topographical and site-specific differentiation and underlie the detailed positional memory in adult fibroblasts (54) .

In conclusion, our study elucidates, at least in part, why palmoplantar epidermis is thicker and paler than nonpalmoplantar epidermis. In addition to dermal-epidermal interactions, which play important roles in regulating keratinocyte growth and differentiation through numerous growth factors (55) , we have proposed the necessity of those site-specific regulations (27) . We have shown that DKK1, a secretory protein highly expressed by palmoplantar fibroblasts (2) , has various effects on keratinocytes, the major type of cell in the epidermis. Enhanced {alpha}KLEIP expression in response to DKK1 may play a role in the enhanced cell contraction (compact cellular organization) in palmoplantar keratinocytes. Further, the decreased expression of β-catenin in the cytoplasm of keratinocytes elicited by DKK1 may result in the loose cell-cell contact and the increased cellular density seen in palmoplantar epidermis (10) . PKC isozymes and GSK3β may participate in DKK1/Wnt/β-catenin signaling pathways. DKK1 also induced palmoplantar specific keratin 9 in keratinocytes. The fact that DKK1 decreases melanocyte function and proliferation through MITF (2) and that melanosome transfer is decreased in keratinocytes in response to DKK1 (probably through PAR-2) may explain the hypopigmentation seen in the skin on palms and soles. However, the widespread effects of DKK1 observed in our DNA microarray data from noncanonical Wnt signaling pathways or HOX transcription factors to apoptosis strongly suggest the role of DKK1 in other processes in addition to pigmentation or skin thickness. Thus, some site specificities such as the higher occurrence of melanomas in palmoplantar areas in people with dark skin could find some explanation in the mesenchymal regulation. Further investigation will be directed at clarifying how to regenerate site specifically identical skin through the action of DKK1 and/or other growth factors.


   ACKNOWLEDGMENTS
 
This research was supported by the Intramural Research Program of the U.S. National Institutes of Health, the National Cancer Institute, and the Center for Cancer Research, and by a grant-in-aid from the Ministry of Education, Culture, Sports, and Technology (Japan; no. 18689028). We thank Dr. Akimichi Morita, a chairman of Department of Geriatric and Environmental Dermatology, Nagoya City University Graduate School of Medical Sciences (Nagoya, Japan), for critical review of this work.

Received for publication August 2, 2007. Accepted for publication September 27, 2007.


   REFERENCES
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 ABSTRACT
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 MATERIALS AND METHODS
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 DISCUSSION
 CONCLUSIONS
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