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1

Wnt Inhibitory Factor (WIF)-1 Promotes

Melanogenesis in Normal Human

Melanocytes

by

Hye Ran Kim

Major in Molecular Medicine

Department of Biomedical Sciences

The Graduate School, Ajou University

2

Wnt Inhibitory Factor (WIF)-1 Promotes

Melanogenesis in Normal Human Melanocytes

by

Hye Ran Kim

A Dissertation Submitted to The Graduate School of Ajou University

in Partial Fulfillment of the Requirements for the Degree of

MASTER OF BIOMEDICAL SCIENCES

Supervised by

Hee Young Kang, M.D., Ph.D.

Major in Molecular Medicine

Department of Biomedical Sciences

The Graduate School, Ajou University

August, 2011

3

4

감사

본 논 할 지 심 양면 도움 주시고 지도 조언 아끼지

않 셨 지도 이신 강희 님께 감사를 드립니다. 또한 좋

연구를 할 있도 많 조언과 격 를 주신 태 님과 강엽

님께도 감사 마 합니다.

그리고 연구 간 동안 도움 주신 피부과학 실 모든 생님들과 이

사님, 용 사님, 경이, 규 이, 조직 포 염색과 분 도 주신

생님께 감사 드립니다. 심 힘들 곁에 힘이 어 든든한

구 사, 사님과 연주 사, 권민 사 그리고 항상 격

해 주신 생님, 이명애 님께도 감사 드립니다.

언 나 아낌 없는 사랑 지원해 주시고 믿어주신 부모님과 큰엄마, 다른

가족들에게도 감사 마 합니다.

2011 6 월

자씀

5

- ABSTRACT -

Wnt Inhibitory Factor (WIF)-1 Promotes Melanogenesis in

Human Melanocytes

Background: Wnt inhibitory factor-1 (WIF-1) acts as a suppressor of Wnt antagonists.

There is no study available on melanogenesis effect of human melanocytes.

Objectives: In this study, we investigated the expression of WIF-1 in human melanocytes

and the effects of WIF-1 on melanogenesis.

Method: To determinutese whether WIF-1 is expressed in human melanocytes, we used

immunocytochemistry, immunhistochemistry, western blot analysis and reverse transcription

polymerase chain reaction. The cell proliferation was measured using the coulter counter.

The effects on pigmentation were investigated with measurement of melanin contents,

tyrosinase activity and its expression.

Result: WIF-1 was expressed in melanocytes. WIF-1 inhibited proliferation of human

melanocytes in a dose-dependent manner. WIF-1 also appeared to stimulate the melanin

synthesis of human melanocytes. This increase in pigmentation was due to stimulation of the

tyrosinase activity and increased expression of MITF and tyrosinase.

Conclusion: We demonstrated that WIF-1 was expressed in human melanocytes. Our

findings suggest that WIF-1 may play a role in melanogenesis.

Key Words: melanocyte, melanogenesis, WIF-1, pigmentation, Wnt antagonist

6

TABLE OF CONTENTS

ABSTRACT --------------------------------------------------------------------------------------------- 5

TABLE OF CONTENTS ------------------------------------------------------------------------------ 6

LIST OF FIGURES ------------------------------------------------------------------------------------- 8

LIST OF TABLES -------------------------------------------------------------------------------------- 9

I. INTRODUCTION ----------------------------------------------------------------------------------- 10

A. Human melanocyte development and pigmentati on ------------------------------------ 10

B. Wnt signaling pathway ----------------------------------------------------------------------- 12

C. The Wnt family -------------------------------------------------------------------------------- 14

D. The Wnt antagonist ---------------------------------------------------------------------------- 15

E. Melanocyte and Wnt signaling --------------------------------------------------------------- 18

II. MATERIALS AND METHODS ----------------------------------------------------------------- 19

A. Human melanocyte culture ------------------------------------------------------------------ 19

B. B16 melanoma cell lines culture ------------------------------------------------------------ 19

C. Human fibroblast culture --------------------------------------------------------------------- 20

D. Human keratinocyte culture ----------------------------------------------------------------- 20

E. Cell growth assay ------------------------------------------------------------------------------ 20

F. Melanin content determinutesation --------------------------------------------------------- 12

G. Tyrosinase activity assay --------------------------------------------------------------------- 21

H. Skin organ culture ----------------------------------------------------------------------------- 21

I. Pigmentation assay in cultured skin --------------------------------------------------------- 21

J. Immunocytochemistry ------------------------------------------------------------------------ 22

7

K. Western blottiong ----------------------------------------------------------------------------- 23

L. WIF-1 transfection ---------------------------------------------------------------------------- 23

M. Reverse transcriptase-polymerase chain reaction (RT-PCR) -------------------------- 24

N. Statistical analysis ----------------------------------------------------------------------------- 24

III. RESULTS ------------------------------------------------------------------------------------------- 25

A. WIF-1 is expressed in human melanocytes, fibroblasts and keratinocytes ---------- 25

B. WIF-1 increases pigmentation of cultured human skin --------------------------------- 27

C. WIF-1 treatment enhances pigmentation in B16 melanoma cells --------------------- 29

D. WIF-1 treatment enhances pigmentation in human melanocytes ---------------------- 32

E. WIF-1 transfection induces pigmentation in B16 melanoma cells -------------------- 35

F. WIF-1 transfection induces pigmentation in human melanocytes --------------------- 38

IV. DISCUSSION -------------------------------------------------------------------------------------- 41

V. CONCLUSION ------------------------------------------------------------------------------------- 42

REFERENCES ----------------------------------------------------------------------------------------- 43

국 요약 ------------------------------------------------------------------------------------------------ 49

8

LIST OF FIGURES

Fig. 1. Melanogenesis --------------------------------------------------------------------------------- 11

Fig. 2. Wnt signaling pathways ---------------------------------------------------------------------- 13

Fig. 3. WIF-1 is expressed in human melanocytes, fibroblasts and keratinocytes------------- 26

Fig. 4. WIF-1 increases pigmentation of cultured human skin ---------------------------------- 28

Fig. 5. WIF-1 treatment enhances pigmentation in B16 melanoma cells ---------------------- 30

Fig. 6. WIF-1 treatment enhances pigmentation in human melanocytes ---------------------- 33

Fig. 7. WIF-1 transfection induces pigmentation in B16 melanoma cells --------------------- 36

Fig. 8. WIF-1 transfection induces pigmentation in human melanocytes --------------------- 39

9

LIST OF TABLES

Table 1. Wnt antagonist family molecules --------------------------------------------------------- 17

Table 2. Effects of WIF-1 on proliferation, melanin contents and tyrosinase activity of

B16 melanoma cells -------------------------------------------------------------------------- 31

Table 3. Effects of WIF-1 on proliferation, melanin contents and tyrosinase activity of

human melanocytes -------------------------------------------------------------------------- 34

Table 4. Effects of WIF-1 transfection on proliferation, melanin contents and tyrosinase

activity of B16 melanoma cells ------------------------------------------------------------ 37

Table 5 . Effects of WIF-1 transfection on proliferation, melanin contents and tyrosinase

activity of human melanocytes ------------------------------------------------------------- 40

10

I. INTRODUCTION

A. Human melanocyte development and pigmentation

Human skin is made up of two main layers, the epidermis, which is described as a

stratified squamous epithelium mainly consisting of keratinocytes, and the dermis, an

underlying layer of vascularized connective tissue. Human melanocytes reside at the junction

of the dermis and the epidermis. Mature human melanocytes form long dendritic processes

that ramify among the neighboring keratinocytes. Human melanocytes are well known for

their role in skin pigmentation and their ability to produce and distribute melanin.

The human melanocyte transform the peptide tyrosinase into two different forms of

melanin, which then is spread throughout the dermal cells and the keratinocytes via

melanosomes to darken tissue. Figure 1 shows the chemical metabolism that occurs intra-

cellulary to produce melanin from the precursors phenylalanine and tyrosine. Eumelanin is

metabolized from DHICA and produces a brown color in hair in its intact form. Pheomelanin

is metabolized from 5,6-indolequione, which produces a red color in hair in its intact form.

From these two slightly different forms of pigment in various degrees of structural integrity

come all the differing shades of Caucasian hair (Prota, 2000). In addition to coloration,

melanin pigmentation in the skin also provides photoprotection from UV radiation to the

skin. However, the melanocytes themselves are not immune from radiation damage.

11

Fig. 1. Melanogenesis. L-Phenylalanine is metabolized in the presence of three major

enzymes, phenylalanine hydroxylase, tyrosine hydroxylase isoform I and tyrosinase, to

eumelanin and phaeomelanin. Alpha MSH, α-melanocyte stimulating hormone; UV,

ultraviolet. (Langan et al., 2010)

12

B. Wnt signaling pathway

Wnt proteins constitute a large family of 19 secreted glycoproteins that regulate many

aspects of cell physiology, including proliferation, migration and differentiation in

embryogenesis and adult tissue homeostasis (Cadigan and Nusse, 1997). The signaling

pathway mediated by Wingless-type (Wnt) proteins is highly conserved in evolution. This

pathway, initially described in Drosophila as critical for embryogenesis, has since been

shown to regulate cell fate decisions, cell proliferation, morphology, migration, apoptosis,

differentiation and stem cell self-renewal.

In the Wnt signaling pathway, ß-catenin is a key mediator. In the absence of Wnt

signals, cytosolic ß-catenin is recruited to a multi-protein ‘‘destruction complex’’ that

consists of casein kinase 1, adenomatous polyposis coli, Axin, and glycogen synthase kinase-

3ß (GSK-3ß) (Langan et al., 2010). In the resting state, ß-catenin is phosphorylated by GSK-

3ß and is subsequently targeted for degradation via the ubiquitin proteosome pathway (Batra

et al., 2006). Binding of Wnt ligands to their Frizzled receptors triggers the Wnt/ß-catenin

pathway, also termed the canonical Wnt pathway, through phosphorylation and increased

activity of Dishevelled (Dvl) (Itoh et al., 1998). Dvl activity alters the composition of the

‘‘destruction complex’’ with a net result of reduction. ß-catenin subsequently translocates to

the nucleus and interacts with TCF/LEF transcription factors (Behrens and Lustig, 2004;

Langan et al., 2010). TCF target genes include cyclin D1, VEGF, and Survivin, among

others (Shtutman et al., 1999). ß-catenin independent Wnt signaling, also termed non-

canonical signaling, may proceed through calcium flux, G proteins, and JNK (Veeman et al.,

2003).

13

Fig. 2. Wnt signaling pathways. In the absence of Wnt ligand, the "destruction complex"

composed of the core proteins Axin, adenomatous polyposis coli (APC), and glycogen

synthase kinase-3 (GSK3) rapidly phosphorylates (P) cytosolic ß-catenin, targeting it for

subsequent ubiquitination (Ub) and proteasome-mediated destruction. Binding of Wnt to

Frizzled (Fzd) and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) activates

the cytosolic protein Dishevelled (DVL), leading to inhibition of the destruction complex.

The resulting accumulated ß-catenin can then translocate to the nucleus to activate Wnt-

responsive target genes regulated by TCF and LEF family transcription factors, leading to

various cellular effects. Activation of the small GTPases Rho and Rac can result in

cytoskeletal rearrangements that affect cellular motility upon Wnt stimulation. Binding of

Wnt isoforms to either Fzd or receptors such as receptor tyrosine kinase-like orphan receptor

2 (Ror2, a receptor for Wnt-5a) can trigger ß-catenin-independent downstream sgnaling

14

events, including the inhibition of Wnt/ ß-catenin signaling. The mechanisms underlying ß-

catenin-independent Wnt signaling are not well defined and may be largely determinutese by

cellular context. The secreted inhibitor Dickkopf (DKK) can antagonize Wnt signaling by

competitively binding to LRP5/6. Secreted Fzd-related proteins (SFRPs) and Wnt inhibitory

factor (WIF) are thought to antagonize Wnt signaling by sequestering Wnt ligand in the

extracellular space (Chien et al., 2009).

C. The Wnt family

The Wnt gene family in mammalian systems is now known to consist of almost 20

members which generally fall into two classes: classical Wnts (Wnt1, Wnt3a, Wnt8, Wnt8b)

activate signaling through the canonical pathway and nonclassical Wnts (Wnt4, Wnt5a,

Wnt11) specifically activate noncanonical pathways. Wnt proteins are cysteine-rich secreted

glycoproteins and on the cell surface bind to co-receptors: Fz proteins which are members of

the frizzled (Fz) family of seven transmembrane receptors, and LRP-5 or LRP-6 which of

members of the family of low-density lipoprotein receptor-related protein (LRP) single

transmembrane receptors. In mammalian cells, there are 10 known Fz receptors which

contain seven transmembrane domains, an extracellular cysteine-rich domain (CRD) which

specifically binds to Wnt ligands, and an intracellular carboxyl tail. It has been difficult to

test which Wnts bind to which Fz receptors since Wnt proteins are hard to be purified.

15

D. The Wnt antagonist

According to the mechanisms of action, naturally endogenous Wnt antagonists can be

divided into two classes. The first class includes the secreted Frizzled-related protein (sFRP)

family, WIF-1, and Cerberus. These molecules exert inhibition through direct binding to Wnt

proteins. The second class including the Dickkof (DKK) family inhibits Wnt signaling by

binding to the Frizzled (Fz) coreceptor LRP5/LRP6 (Kawano and Kypta, 2003).

sFRPs are antagonists that directly bind to Wnts. They were initially given several names,

reflecting their simultaneous discovery by different approaches (Jones and Jomary, 2002).

There are presently eight known members of the family. A unifying nomenclature now

exists for five of these (sFRP1 to sFRP5) (Table 1.), although sFRP3 is still better known as

FrzB (for Frizzled motif associated with bone development). On the basis of sequence

homology, sFRP1, sFRP2, and sFRP5 form a subgroup as do sFRP3 and sFRP4, which are

quite distantly related to the other sFRPs. Sizzled, Sizzled2 and Crescent form a third

subgroup, but these have not been identified in mammals. There are conflicting reports on

the ability of Sizzled to inhibit Wnt signaling (Collavin and Kirschner, 2003; Veeman et al.,

2003). With one exception (Collavin and Kirschner, 2003), sFRPs (and the other Wnt

antagonists) have not been found in invertebrates. Despite this, many of them have been

demonstrated to inhibit the activity of the Drosophila Wnt homologue Wingless (Wg).

WIF is an evolutionary conserved protein of 379 aminuteso acid residues and

constitutes another member of the secretary Wnt modulators that directly bind to Wnt

proteins similar to sFRPs. WIF-1 was first identified as an expressed sequence tag from

human retina with highly conserved orthologues in xenopus and zebrafish (Collavin and

Kirschner, 2003).

16

The phenotype induced by injection of RNA encoding WIF-1 into early xenopus

embryos, namely induction of a partial secondary axis and abnormal somitogenesis,

suggested it played a role in the Wnt signaling pathway (Hsieh et al., 1999).

Cerberus is another Wnt antagonist that belongs to the same class as WIF-1 and sFRPs.

It was isolated from Xenopus as an abundant organizer-specific molecule (Bouwmeester et

al., 1996). It is expressed in the anterior endoderm including the Spemann's organizer and

has the unique property of inducing an ectopic head without trunk formation. Trunk

formation relies on Nodal and Wnt signaling, whereas head induction requires inhibition of

Wnt and bone morphogenetic protein (BMP) signaling. Cerberus, as a multivalent growth-

factor antagonist, inhibits all three signaling pathways, which leads to simultaneous head

formation and trunk inhibition.

The Dkk family comprises four members (Dkk-1 to Dkk-4) and a unique Dkk-3-related

protein named Soggy (Sgy). Dkks contain two characteristic cysteine-rich domains (Cys-1

and Cys-2) separated by a linker region of variable length (Glinka et al., 1998; Krupnik et al.,

1999). Cys-2, in particular, is highly conserved among all members of the family and

contains 10 conserved cysteine residues; this is similar to the proteins of the colipase family,

with which Dkk proteins share weak sequence similarity (Aravind and Koonin, 1998;

Krupnik et al., 1999). Detailed protein sequence and structural analysis suggested that Dkks

and colipase have the same disulfide-bonding pattern and a similar fold. Colipases are

essential for lipid hydrolysis by pancreatic lipases and interact with lipid (van Tilbeurgh et al.,

1999). It is not known whether the structural similarity between colipases and Dkks implies a

common function such as lipid interaction.

17

Table 1. Wnt antagonist family molecules (Kawano and Kypta, 2003).

18

E. Melanocyte and Wnt signaling

Melanocytes arise from neural crest cells that migrate from the lateral edge of the neural

plate to become a variety of cells including glial cells, smooth muscle cells, and neurons

(Dorsky et al., 1998). Wnt signaling plays a critical role in the development of the neural

crest, specifically Wnt1 and Wnt3A (Dorsky et al., 1998; Dunn et al., 2000). Canonical Wnt

signaling including Wnts such as Wnt6 (Schmidt et al., 2008) and Wnt8 is required both for

the induction of the neural crest, and for its expansion, and controls the expression of a

variety of key proteins including Slug and Snail (LaBonne and Bronner-Fraser, 1998; Sakai

et al., 2005). Wnt1 and Wnt3A also promote the development of neural crest cells into

pigment cells. When cells are depleted of these two proteins, they become neuronal rather

than pigmented cells (Dorsky et al., 1998). Wnt1 signals to melanoblasts in a paracrine

manner to increase melanocyte numbers, while Wnt3A and b-catenin can specify neural crest

cells to become melanocytes (Dunn et al., 2005). In mice, melanoblasts are concentrated in

the hair follicles where they differentiate into melanocytes, whereas in humans, melanocytes

interact with keratinocytes in the epidermis. This is controlled by DKK1 (Yamaguchi et al.,

2008) and Wnt signaling via Wnts 1, 3A, and 4 (Saitoh et al., 1998).

Wnt inhibitory factor-1 (WIF-1) acts as a suppressor of Wnt antagonists. However,

there is no study available on melanogenesis effect of human melanocytes. In this study, we

investigated the expression of WIF-1 in human melanocytes and the effects of WIF-1 on

melanogenesis.

19

II. MATERIALS AND METHODS

A. Human melanocyte culture

Normal human melanocytes were derived from the adult foreskin and maintained in a

F12 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1%

antibiotic/antimycotic solution (all from Gibco-BRL, Grand Island, NY, U.S.A.), 24 μg/, 3-

isobutyl-1-methylxanthine, 80 nM 12-O-tetradecanoyl phorbor-13-acetate, 1.2 ng/㎖ basic

fibroblast growth factor and 0.1 μg/㎖ cholera toxin (all from Sigma Chemical Co., St.

Louis, MO, U.S.A.). For experiments, melanocytes at passage 2 or 3 were used and

maintained in MCDB-153 containing 4% heat-inactivated fetal bovine serum (FBS), 0.6

ng/㎖ basic fibroblast growth factor, 5 μg/㎖ insulin, 1 μg/㎖ vitaminutes E, and 1

μg/㎖ transferrin (all from Sigma Chemical Co., St. Louis, MO, U.S.A.).

B. B16 melanoma cell lines culture

B16 melanoma cells was added in RPMI1640 (all from Gibco-BRL, Grand Island, NY,

U.S.A.) media containing 10% fetal bovine serum (FBS) and 10% penicillin-streptomycin

and incubated at 37°C, 5% co2. Media were changed every 3 days.

20

C. Human fibroblast culture

Normal human fibroblast were derived from the adult foreskin and maintained in a

RPMI1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 1%

antibiotic/antimycotic solution (all from Gibco-BRL, Grand Island, NY, U.S.A.). For

experiments, fibroblast at passage 2 or 3 were used and maintained in RPMI1640 medium

supplemented with 10% heat-inactivated fetal bovine serum and 1% antibiotic/antimycotic

solution (all from Gibco-BRL, Grand Island, NY, U.S.A.).

D. Human keratinocyte culture

Normal human keratinocyte were derived from the adult foreskin and maintained in a

Defined K-SFM medium supplemented with 5% heat-inactivated fetal bovine serum (FBS)

(all from Gibco-BRL, Grand Island, NY, U.S.A.). For experiments, fibroblast at passage 2

or 3 were used and maintained in RPMI1640 medium supplemented with 10% heat-

inactivated fetal bovine serum and 1% antibiotic/antimycotic solution (all from Gibco-BRL,

Grand Island, NY, U.S.A.).

E. Cell growth assay

Melanocytes or B16 melanoma cells line were plated at a density of 2×105 cells/㎠ in

60㎜ culture dishes. After 1 day of culture, the medium was changed to fresh medium

containing different concentrations of 0.5~5 ㎍/㎖ WIF-1 (R&D systems Inc.,

Minutesneapolis, MN, U.S.A.). After 5 days or 3 days of culture, the cells were counted with

Coulter counter (Coulter Electronics, Hialcah, FL). The cell cytotoxicity was examinutesed

using trypan blue exclusion assay.

21

F. Melanin content determinutesation

Melanocytes or B16 melanoma cells line were treated with different concentrations of

the 0.5-5 ㎍/㎖ WIF-1 for 5 days or 3 days. Melanin content was measured according to the

method of Tsuboi et al (Tsuboi et al., 1998). With a slight modification. pellets of 2X105

cells were solubilized in 1 M NaOH and the optical densities were measured at 490㎚ using

an enzyme-linked immunosorbent assay reader. The absorbance was compared with a

standard curve of synthetic melanin (Sigma Chemical Co., St. Louis, MO, U.S.A.).

G. Tyrosinase activity assay

Dopa oxidase activity was determinutesed by the method described by Tomita et al

(Tomita et al., 1992). With slight modification. Cells were solubilized with 1% Triton-X and

10 mmol/1 L-DOPA (Sigma Chemical Co., St. Louis, MO, U.S.A.). Following 90 minutes

incubation at 37 °C, the absorbance was measured at 490 ㎚.

H. Skin organ culture

Skin samples of human breast skin were obtained during breast surgery after receiving

consent and cultured as previously described (Jansen et al., 1998). A sterilized stainless steel

grid was placed on a 35-㎜ culture dish. Dulbecco’s modified Eagle’s minutesimal essential

medium supplemented with 10% FBS and 1% penicillin/streptomycin was filled up to the

stainless steel grid. The skin specimens were placed on the stainless steel grid and cultured in

incubator at 37°C with 5% CO2. The medium was exchanged every 3 days.

22

I. Pigmentation assay in cultured skin

The skin organ was treated with 1 mM WIF-1 (R&D systems Inc., Minutesneapolis,

MN, U.S.A.). After 3 days of culture, the specimens were fixed in 10% formalin, embedded

in paraffin section, and stained with hematoxylin and eosin for light microscope

examinutesation. Melanin pigments were visualized with Fontana–-Masson stain, performed

by usual methods without eosin background stain. The image analysis was evaluated using

Image Pro Plus Version 4.5 (Media Cybertics Co., MD, U.S.A.) and the pigmented area per

epidermal area (PA/EA) was measured.

J. Immunocytochemistry

Melanocytes, Fibroblastes, and keratinocytes (5×103) grown on Lab-Tek chambers

(Nalge Nunc International, Naperville, IL, U.S.A.) were fixed in 4% paraformaldehyde for

30 minutes at room temperature, and permeated in methanol followed by 0.1% Triton X-100

to achieve a nuclear permeance. Slides were placed in methanol containing 0.3% hydrogen

peroxide for 10 minutes, and the non-specific activity was blocked by normal goat serum for

10 minutes. They were incubated with rabbit polyclonal anti-WIF-1 (abcam, England,

Cambridge, UK) overnight at 4°C at 1:50 dilution. Biotinylated antibody against both rabbit

(Dako, Carpinteria, CA, U.S.A.) was incubated for 20 minutesutes at room temperature. The

substrate chromogen 3-aminuteso-9-ethyl-carbazol (Biomeda) was applied for 20 minutes.

Negative controls were made by applying normal rabbit serum (Vector Lab., Burlingame,

CA, U.S.A.) before the primary antibody.

23

K. Western blotting

Human melanocytes or B16 melanoma cell line were lysed in RIPA buffer (1% NP-40,

150㎜ NaCl, 10㎜ Tris–-HCl (pH 8.0), 1 mM EDTA) with 10 ㎍/㎖ aprotinin, 1 mM

sodium orthovandate and 100 ㎍/㎖ phenylmethylsulphonylfluoride, and separated with a

10% SDS-PAGE gel. The protein was then transferred onto a polyvinylidenedifluoride

membrane, and the membrane was probed with Tyrosinase, MITF (abcam, England,

Cambridge,UK), α-tubulin (Cell Signaling Boston Massachusetts, U.S.A.) antibodies. After

incubation with horseradish peroxidase-conjugated goat anti-rabbit (Tyrosinase and α-

tubulin, MITF) antibody, the membrane was developed with an enhanced

chemiluminutesescence detection kit (Amersham Int., Little Chalfont, UK).

L. WIF-1 transfection

Melanocytes or B16 melanoma cell line (2x105) were plated in 60㎜ plates 48 hours

before transfection. Cells were then transfected with a full-length WIF-1 construct in

pcDNA3.1 vector (FL) (Cell Signaling Boston Massachusetts, U.S.A.) or with an empty

pcDNA3.1 vector (EV), using Lipofectaminutese 2000 kit (Invitrogen, Carlsbad, CA, U.S.A.)

transfection reagent according to the manufacturer’s protocol. 48 hours after transfection,

cells were collected for western blot analysis.

24

M. Reverse transcriptase-polymerase chain reaction (RT-PCR)

Minutesi kit (Qiagen Inc., Valencia, CA, U.S.A.), and the cDNA was obtained using

SuperScriptTM III reverse transcriptase kit (Invitrogen, Carlsbad, CA, U.S.A.). Expression of

WIF-1 and ß-actin was measured using semiquantitative PCR. The cDNA was subjected to

PCR using accupower PCR premix kit (Bioneer, Daejeon, Korea). The oligonucleotide

primers for PCR were synthesized by bioneer and were as follows WIF-1, forward primer: 5’

– ATG GCA GAT CCA ACC GTC A, reverse primer: 3’ – AGG TTC CAT GTG CAC

CAC A and ß-actin, , forward primer : 5’ – GTG GGG CGC AGG CAC CA , reverse primer:

3’ – CTC CTT AAT GTC ACG CAC GAT TTC PCR amplification was performed with a

GeneAmp_PCR system 2700 (Applied Biosystems, Foster City, CA, U.S.A.) under the

following conditions: WIF-1 40 cycles at 94°C for 30 s (denaturation), 55°C for 30 seconds

(annealing) and 72°C for 45 seconds (elongation). The PCR products were electrophoresed

on 1.8% agarose gels. Each DNA band was visualized by staining with ethidium bromide.

N. Statistical analysis

All data were compared by one-way analysis of variance (ANOVA) (SPSS 12.0; SPSS

Inc., Chicago, U.S.A.). The results were expressed as mean±standard deviation (SD). All p

values were two tailed and p value less than 0.05 was considered to be of statistical

significance.

25

III. RESULTS

A. WIF-1 is expressed in human melanocytes, fibroblasts and keratinocytes

To investigate whether WIF-1 is expressed in cultured human melanocytes, fibroblasts

and keratinocytes, we first performed RT-PCR analysis. The RT-PCR studies indicated that

human melanocytes, fibroblasts and keratinocytes express WIF-1 mRNA (Fig. 3A.).

Immunocytochemical staining showed immunoreactivity for WIF-1 in human melanocytes,

fibroblasts and keratinocytes (Fig. 3B.).

26

(A)

(B)

Fig. 3. WIF-1 is expressed in human melanocytes, fibroblast and keratinocytes.

Expression of WIF-1 was assessed by RT-PCR (A). The protein expression was detected by

immunocytochemistry (x 400) (B).

27

B. WIF-1 increases pigmentation of cultured human skin

In cultured skin, 5 ㎍/㎖ WIF-1 induced pigmentation (Fig. 4A.). Fontana–Masson

staining showed increased epidermal pigmentation. Image analysis showed a significant

increase in the ratio of PA/EA in WIF-1-treated skin (0.228 ± 0.161), compared to control

skin (0.079 ± 0.056 ) (Fig. 4B.).

28

(A)

(B)

Fig. 4. Effect of WIF-1 on pigmentation in skin organ culture. Fontana–Masson stain. On

culture 3 days, melanin pigments were increased in WIF-1 (5 ㎍/㎖) treated human skin

compared to control (x 200) (A). An Image analysis. The values are the mean of three

independent experiments ± SD (B).

29

C. WIF-1 treatment enhances pigmentation in B16 melanoma cells

To explore the effect of WIF-1 in B16 melanoma cells, B16 melanoma cells were

incubated with different concentrations of WIF-1 (0.5 ㎍/㎖ and 5 ㎍/㎖). The WIF-1

dose-dependently inhibited proliferation of B16 melanoma cells at concentrations between 0

– 5 ㎍/㎖. At a concentration of 0.5 ㎍/㎖ or 5 ㎍/㎖, proliferation was inhibited by 95.8%

± 3.4% or 86.5% ± 7.63% (mean ± SD of DMSO control (100%), n=5) (Fig. 5A.). Next we

examinutesed whether WIF-1 affects pigmentation in B16 melanoma cells. The melanin

content was increased with treatment of WIF-1. WIF-1 (0.5 ㎍/㎖ or 5 ㎍/㎖) increased

melanin content to 117% ± 10.06% or 132.8% ± 17.86% (mean ± SD of DMSO control

(100%), *P <0.001) respectively (Fig. 3B.). Since tyrosinase is known for the key enzyme in

the process of melanin synthesis, dopa-oxidase activity and its expression were

examinutesed to study the action of WIF-1 on melanogenesis. As shown in Figure 5C, the

optical density in the presence of WF-1 (0.5 ㎍/㎖ or 5 ㎍/㎖) had significant increase by

138% ± 26.9% or 170.08% ± 49.6% (mean ± SD of DMSO control (100%), n=5, *P <0.001)

respectively. (Fig. 5C.). These results indicate that WIF-1 regulates tyrosinase and

subsequently induces melanin synthesis in B16 melanoma cells.

30

(A)

(B) (C)

Fig. 5. WIF-1 treatment enhances pigmentation in B16 melanoma cells. WIF-1

inhibits cell growth in B16 melanoma cells. Results were expressed as a percentage

proliferation of the respective controls. Values are the mean ± SD of five independent

experiments. *P < 0.001 using one-way ANOVA (A). Melanin content was

determinutesed by measuring the absorbance at 490 ㎚ of cell lysate. The values indicate

the mean of five independent experiments ± SD. *P < 0.001 using one-way ANOVA (B).

31

The cells were incubated with 10mM L−DOPA for 90 minutes and the absorbance was

measured at 490 ㎚. The values indicate the mean of five independent experiments ± SD.

*P < 0.001 using one-way ANOVA (C).

Table. 2. Effects of WIF-1 on proliferation, melanin contents and tyrosinase activity of

B16 melanoma cells.

32

D. WIF-1 treatment enhances pigmentation in human melanocytes

To explore the effect of WIF-1 in human melanocytes, human melanocytes were incubated

with different concentrations of WIF-1 (0.5㎍/㎖ and 5 ㎍/㎖). The WIF-1 dose-

dependently inhibited proliferation of human melanocytes at concentrations between 0 -

5㎍/㎖. At a concentration of 0.5㎍/㎖ or 5㎍/㎖, cell growth was inhibited by 93.7% ±

4.44% or 75.0% ± 17.68% (mean ± SD of DMSO control (100%), n=5, P <0.001) (Fig. 6A.).

Next we examinutesed whether WIF-1 affects pigmentation in human melanocytes. WIF-1

(0.5 ㎍/㎖ or 5 ㎍/㎖) increased melanin content to 113.3% ± 9.4% or 128.5% ± 20.1%

(mean ± SD of DMSO control (100%), P <0.001) respectively (Fig. 6B.). Dopa-oxidase

activity and its expression were examinutesed. As shown in Figure 6C, the optical density in

the presence of WF-1 (0.5 ㎍/㎖ or 5 ㎍/㎖) had significant increase by 111.6% ± 1.74%

or 151.3% ± 36.29% (mean ± SD of DMSO control (100%), n=5, P <0.001) respectively

(Fig. 6C.). WIF-1 treatment also increased the expression of tyrosinase and MITF. These

result suggesteds suggests that WIF-1 stimulates melanin synthesis through up-regulation of

MITF and tyrosinase expression in human melanocytes (Fig. 6D.).

33

(A) (B)

(C) (D)

Fig. 6. WIF-1 treatment enhanced pigmentation in human melanocytes. WIF-1

decreased cell growth in human melanocytes. Human melanocytes were incubated with

different concentrations of WIF-1 and cell proliferation was measured at 5 days using

the coulter counter. Values are the mean ± SD of five independent experiments. *P <

0.001 using one-way ANOVA (A). Human melanocytes were incubated with different

concentrations of WIF-1 for 5 days. Melanin content was determinutesed by measuring

34

the absorbance at 490 ㎚. The values indicate the mean of five independent experiments

± SD. *P < 0.001 using one-way ANOVA (B). The cells were incubated with 10 mM

L−DOPA for 90 minutes and the absorbance was measured at 490 ㎚. The values indicate

the mean of five independent experiments ± SD. *P < 0.001 using one-way ANOVA (C).

Cell lysates harvested after 5 days culture with MCDB153 medium were analyzed by

western blotting with tyrosinase antibody, MITF antibody (D).

Table. 3. Effects of WIF-1 on proliferation, melanin contents and tyrosinase activity of

human melanocytes.

35

E. WIF-1 transfection induces pigmentation in B16 melanoma cells

To further evaluate the effect of WIF-1 on B16 melanoma cell, we restored WIF-1

expression by gene transfection with a WIF-1-expressing pcDNA3.1 vector (FL). After WIF-

1 transfection for 48 hours, the WIF-1 dose-dependently inhibited proliferation of B16

melanoma cells. WIF-1 transfection also increased melanin content to 157.3% ± 40.6%

(mean ± SD of DMSO control (100%), P <0.001) (Fig. 7B.). WIF-1 transfection increased

expression of MITF and tyrosinase in B16 melanoma cells (Fig. 7C, D.).

36

(A) (B)

(C) (D)

Fig. 7. WIF-1 transfection induces pigmentation in B16 melanoma cells. WIF-1 inhibited

cell growth of B16 melanoma cells. B16 melanoma cells were transfected with WIF-1 vector

and cell proliferation was measured at 3 days using the Coulter counter. Results are

expressed as a percentage of proliferation to the respective controls (A). Melanin content

37

was determinutesed by measuring the absorbance at 490 ㎚ (B). The cells were incubated

with 10 mM DOPA for 90 minutes and the absorbance was measured at 490 ㎚ (C).

Western blot analysis. EV, empty pcDNA3.1 vector; WIF-1, WIF-1 construct in HA vector

(D). (*P < 0.001 using one-way ANOVA. Data are means ± SD.)

Table. 4. Effects of WIF-1 transfection on proliferation, melanin contents and

tyrosinase activity of B16 melanoma cells.

38

F. WIF-1 transfection induces pigmentation in human melanocytes

To further evaluate the effect of WIF-1 on human melanocytes, we restored WIF-1

expression by gene transfection with a WIF-1-expressing pcDNA3.1 vector (FL). After

transfection with WIF-1 for 48 hours, the WIF-1 dose-dependently inhibited proliferation of

human melanocytes. WIF-1 transfection also increased melanin content to 128.1% ± 19.8%

(mean ± SD of DMSO control (100%), *P <0.001 (Fig. 8B.). WIF-1 transfection also

increased expression of MITF and tyrosinase in human melanocytes (Fig. 8C, D.).

39

(A) (B)

(C) (D)

Fig. 8. WIF-1 transfection induces pigmentation in human melanocytes. WIF-1 inhibited

cell growth of human melanocytes. Human melanocytes were transfected with different

concentrations of WIF-1. The cell proliferation was measured at 5 days using the coulter

counter. Results were expressed as a percentage of proliferation to the respective controls

(A). Human melanocytes were transfected with different concentrations of WIF-1 for 5 days.

Melanin content was determinutesed by measuring the absorbance at 490 ㎚ (B). The cells

40

were incubated with 10 mM DOPA for 90 minutes and the absorbance was measured at 490

㎚ (C). Western blot analysis. EV, empty pcDNA3.1 vector; WIF-1, WIF-1 construct in HA

vector (D). (*P < 0.001 using one-way ANOVA. Data are means ± SD.)

Table. 5. Effects of WIF-1 transfection on proliferation, melanin contents and

tyrosinase activity of human melanocytes.

41

IV. DISCUSSION

This study presented for the first time that WIF-1 mRNA and protein are expressed in

human melanocytes. Also, this study clearly demonstrated that WIF-1 treatment or

transfection accelerated pigmentation of human melanocytes. The increase of pigmentation

was due to the stimulatory action of WIF-1 on dopa oxidase activity and MITF expression

which eventually led to the melanin biosynthesis. Wnt signaling is a major cell signal

transduction pathway and plays an important role in the proliferation and differentiation of

cells. Wnt-antagonists function as tumor suppressors and contribute to the pathogenesis of

several human malignancies (Ding et al., 2009). In the normal state, WIF-1 act as negative

regulators of Wnt signaling. Transfection with a WIF-1 gene construct had been reported to

result in significant tumor suppression in colorectal cancer cells (Taniguchi et al., 2005;

Yamaguchi et al., 2005) In this work, melanoma cell growth was suppressed with WIF-1.

It is generally known that inhibition of Wnt signaling pathway reduces pigmentation in

melanocytes. Therefore, the stimulatory effect of WIF-1 in melanocytes seems not to be

related to canonical Wnt pathway. WIF-1 may act as a stimulator of Wnt signaling pathway

in melanocytes. Further study is needed to investigate the exact role of WIF-1 in Wnt

signaling pathway in melanocytes.

42

V. CONCLUSION

1. WIF-1 mRNA and protein are expressed in normal human melanocytes, fibroblasts and

keratinocytes.

2. WIF-1 treatment and transfection induce pigmentation of cultured skin, B16 melanoma

cells and melanocytes.

3. WIF-1 treatment increased expression of MITF, and tyrosinase in B16 melanoma cells and

melanocytes.

In conclusion, this study suggests the regulatory role of the WIF-1 in melanogenesis of

human melanocytes.

43

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49

- 국 요약 -

인체 라닌 포 라닌 에 한 WIF-1 효과

아주 학 학원 생명과학과

란

(지도 : 강 희 )

경: Wnt inhibitory factor (WIF)-1 Wnt antagonist 억 알 있다.

아직 지 WIF-1이 인체 라닌 포 색소 과 에 미 는 향에 한

연구는 없다.

목 : 인체 라닌 포에 WIF-1 WIF-1 색소 과 에 미 는

효과를 알아보고자 하 다.

법: WIF-1 면역조직 학염색, 면역 포 학염색, 특 단 질 검출 검

사 역 사 합연쇄 이용하여 알아 보았다. 포증식 쿨 계 를

사용하여 하 다. 색소 라닌 양, 티 시나아 단 질

하 다.

결과: 라닌 포에 WIF-1 mRNA 단 질 인 하 다. WIF-1

라닌 포 증식 억 하고 라닌양과 티 시나 증가 시켰다.

WIF-1에 한 색소 증가를 티 시나 MITF 증가에 한 것

나타났다.

50

결 : WIF-1 라닌 포에 다. WIF-1 라닌 포 색소 도한

다.

핵심어 : 라닌 포, 색소 과 , WIF-2, 색소 , Wnt 억 자