FEBS Letters 582 (2008) 3201–3205

Wnt4 and Wnt5a promote adipocyte differentiation

Makoto Nishizuka, Akiko Koyanagi, Shigehiro Osada, Masayoshi Imagawa*

Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University,3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan

Received 20 June 2008; revised 4 August 2008; accepted 11 August 2008

Available online 15 August 2008

Edited by Lukas Huber

Abstract The roles of the non-canonical Wnt pathway duringadipogenesis are not well known, though Wnt10b is known tofunction as a negative regulator for adipogenesis by activatingthe canonical Wnt pathway. We focused on the roles of Wnt4,Wnt5a and Wnt6, which are thought to be part of the non-canon-ical Wnt pathway. The expression of these genes changed dra-matically at the initial stage of adipogenesis. Furthermore, theinhibition of Wnt4 or Wnt5a expression prevented the accumula-tion of triacylglycerol and decreased the expression of adipogen-esis-related genes. Wnt4 and Wnt5a have crucial roles inadipogenesis as positive regulators.� 2008 Federation of European Biochemical Societies. Pub-lished by Elsevier B.V. All rights reserved.

Keywords: Adipocyte differentiation; Obesity; Wnt family;Gene expression; 3T3-L1 cells; Peroxisome proliferator-activated receptor c

1. Introduction

Peroxisome proliferator-activated receptor c (PPARc), the

CCAAT/enhancer-binding proteins (C/EBPs) and sterol regu-

latory element-binding protein 1 (SREBP1) were known to

be master regulators of adipogenesis [1]. On the other hand,

several inhibitors are also known. For example, preadipocyte

factor-1 (Pref-1) suppresses adipogenesis through the activa-

tion of MEK/extracellular signal-regulated kinase (ERK) sig-

naling [2] and transformation growth factor-b (TGF-b)

inhibits adipogenesis via SMAD3�s interaction with C/EBP

family [3]. In addition to these factors, recent reports have

demonstrated that Wnt10b is also a potent inhibitory factor

for adipocyte differentiation [4].

The Wnt family consists of at least 19 cysteine-rich, secreted

glycoproteins, which participate in a variety of developmental

processes such as cell proliferation, cell polarity, cell migration

and cell differentiation [5]. Members of the vertebrate Wnt

family can be divided into at least two functional classes

Abbreviations: PPARc, peroxisome proliferator-activated receptor c;C/EBP, CCAAT/enhancer-binding protein; SREBP1, sterol regulatoryelement-binding protein 1; Pref-1, preadipocyte factor-1; TGF-b,transforming growth factor-b; aP2, adipocyte fatty acid-bindingprotein 2

*Corresponding author. Fax: +81 52 836 3455.E-mail address: imagawa@phar.nagoya-cu.ac.jp (M. Imagawa).

0014-5793/$34.00 � 2008 Federation of European Biochemical Societies. Pu

doi:10.1016/j.febslet.2008.08.011

according to their biological activities: those of the Wnt/b-cate-

nin pathway (canonical Wnt pathway), which leads to the sta-

bilization of b-catenin and activates target genes in the

nucleus, and those of the non-canonical Wnt pathway, which

is independent of b-catenin signaling, and involves the stimula-

tion of intracellular Ca2+ release and the activation of phos-

pholipase C and protein kinase C (PKC) [6]. In the

adipocyte differentiation process, Wnt10b activates the Wnt/

b-catenin pathway and maintains preadipocytes in an undiffer-

entiation state by inhibiting expression of C/EBPa and PPARc[4]. These findings strongly indicate that the Wnt/b-catenin

pathway activated by the canonical Wnt family is crucial for

inhibiting adipocyte differentiation. However, the roles of the

non-canonical Wnt family during adipogenesis are not well

understood, even though non-canonical Wnts also have essen-

tial organizing roles in development.

In this study, we focused on the roles of Wnt4, Wnt5a and

Wnt6 during adipocyte differentiation. Since the ectopic

expression of Wnt4, Wnt5a and Wnt6 in C57 mammary epi-

thelial cells did not induce morphological transformation or

the accumulation of b-catenin, these proteins were thought

to be part of the non-canonical Wnt family [7,8]. In addition,

even though the functions of Wnt4, Wnt5a and Wnt6 were well

characterized in various cells, little was known about the bio-

logical roles of these proteins at the beginning of adipocyte dif-

ferentiation. We found that the expression of Wnt4, Wnt5a

and Wnt6 changed dramatically in the early stages of adipo-

genesis. The expression profiles of Wnt4 and Wnt5a were clo-

sely related to adipocyte differentiation. Furthermore, the

inhibition of Wnt4 and Wnt5a expression in 3T3-L1 cells pre-

vented the cytoplasmic accumulation of triacylglycerol and de-

creased the expression of adipogenesis-related genes. These

results strongly suggest that Wnt4 and Wnt5a have crucial

roles to promote adipocyte differentiation.

2. Materials and methods

2.1. Cell culture and differentiationThe mouse 3T3-L1 preadipocytes and mouse NIH-3T3 fibroblasts

were maintained in Dulbecco�s modified Eagle�s medium (DMEM)containing 10% calf serum. The differentiation experiment was per-formed as described previously [9]. In brief, the medium was changedto DMEM supplemented with 0.5 mM 3-isobutyl-1-methylxantine,10 lg/ml of insulin, 1 lM dexamethasone, and 10% fetal bovine serum(FBS) at 2 days postconfluence. After 2 days, the cells were transferredto DMEM containing 5 lg/ml insulin and 10% FBS. For Oil red Ostaining, 3T3-L1 adipocytes were fixed with 4% paraformaldehyde,then stained with 0.3% Oil red O (Amresco) solution for 60 min. Theamounts of triacylglycerol were measured using LIPIDOS LIQUID(Ono) according to the manufacturer�s instructions.

blished by Elsevier B.V. All rights reserved.

3202 M. Nishizuka et al. / FEBS Letters 582 (2008) 3201–3205

2.2. Real-time quantitative PCR (Q-PCR)An ABI PRISM 7000 sequence detection system (Applied Biosys-

tems) was used to perform Q-PCR. Pre-designed primers and probesets for Wnt4, Wnt5a, Wnt6, PPARc, C/EBPa, adipocyte fatty acid-binding protein 2 (aP2) and 18S rRNA were obtained from AppliedBiosystems. The reaction mixture was prepared using a TaqMan Uni-versal PCR Master mix according to the manufacturer�s instructions.Relative standard curves were generated in each experiment to calcu-late the inputted amounts of the unknown samples.

2.3. RNAi experimentFor the RNAi experiment, the two target regions (1: 374–394 bp and

2: 651–671 bp) for Wnt4 and the four target regions (1: 283–303 bp, 2:579–599 bp, 3: 602–622 bp and 4: 670–690 bp) for Wnt5a were selectedusing the QIAGEN siRNA online design tool (http://sir-na.quagen.com/). A 19-nt short hairpin RNA (shRNA)-coding frag-ment with a 5 0-TTCAAGAGA-3 0 loop was subcloned into the ApaI/EcoRI site of pSilencer 1.0-U6 (Ambion). As a negative control, thescrambled fragment 5 0-AAGAGGAGCATATTGGGAAGA-3 0,which does not have similarity with any mRNA listed in Genbank,was generated.

Transfection of shRNA-expressing plasmids into 3T3-L1 cells wasperformed with Nucleofector using Cell Line Nucleofector Kit V(Amaxa) according to the manufacturer�s instructions. The transfectedcells were subjected to differentiation experiments at 2 dayspostconfluence.

2.4. Western blot analysesProteins were separated using SDS/PAGE and transferred to the

polyvinylidene difluoride membrane. Rabbit polyclonal antibodiesagainst Wnt4 or Wnt5a (Santa Cruz) were used as primary antibody.

3. Results

3.1. Time course of Wnt4, Wnt5a and Wnt6 mRNA expression

during adipocyte differentiation

We first tested whether Wnt4, Wnt5a and Wnt6 were ex-

pressed during adipocyte differentiation of mouse 3T3-L1 cells.

At 2 days postconfluence, growth-arrested 3T3-L1 cells were

induced into adipocytes. The expression levels of these three

genes were investigated by Q-PCR (Fig. 1). The level of

Wnt4 mRNA increased after the treatment with inducers

and the peak of expression was at 6–12 h. The expression of

Wnt5a was slightly increased at 1 h and then rapidly decreased.

The expression of Wnt6 mRNA showed a biphasic pattern.

While the expression was decreased until 6 h after induction,

it was increased at 12 h and decreased at the middle and late

stages of adipocyte differentiation. These results indicated that

the levels of Wnt4, Wnt5a and Wnt6 changed dramatically

during adipocyte differentiation, especially early on.

3.2. Expression profiles of Wnt4, Wnt5a and Wnt6 in the

adipocyte differentiable state and the non-differentiable state

Next we examined whether the expression of Wnt4, Wnt5a

and Wnt6 at the beginning of adipocyte differentiation was

specific to adipogenesis. For the differentiation into adipo-

cytes, 3T3-L1 cells must first be grown to confluence and then

kept for 2 days. After that, the inducers are added to the med-

ium. Differentiation into adipocytes occurred only under the

growth-arrested condition. On the other hand, proliferating

3T3-L1 cells did not differentiate into adipocytes even in the

presence of inducers. Another mouse fibroblastic cell line,

NIH-3T3, does not differentiate into adipocytes in the

growth-arrested and proliferating states. Since both NIH-3T3

and 3T3-L1 cells were fibroblastic cell lines derived from

mouse embryo, we compared the expression profiles of these

three genes in the two cell lines under two conditions:

growth-arrested and proliferation (Fig. 2).

Although the expression of Wnt4 mRNA was slightly in-

creased in the proliferating state, that of Wnt4 was dramati-

cally induced in the growth-arrested state in 3T3-L1 cells

12 h after induction. In the NIH-3T3 cells, significant expres-

sion of Wnt4 was not observed in proliferating or growth-ar-

rested cells even after the induction. Although the Wnt5a

mRNA decreased in all cases, the expression of Wnt5a in

growth-arrested 3T3-L1 cells was drastically decreased. On

the other hand, the expression of Wnt 6 also decreased in all

four conditions, and the rates of decrease were almost the

same. These results indicate that the expression of Wnt4 and

Wnt5a, but not Wnt6, was drastically changed under differen-

tiable condition, suggesting that Wnt4 and Wnt5a are closely

related adipocyte differentiation.

3.3. The effect of knocking down the expression of Wnt4 or

Wnt5a on adipocyte conversion

To characterize the biological functions of Wnt4 and Wnt5a,

we blocked their expression at the early stage of adipocyte dif-

ferentiation using RNAi. Two shRNA expression plasmids for

Wnt4 and four plasmids for Wnt5a with different target re-

gions were introduced into 3T3-L1 cells. After that, the levels

of Wnt4 and Wnt5a were determined by semi-quantitative

PCR. Region 2 for Wnt4 and region 4 for Wnt5a showed

the greatest reduction (data not shown). For further analyses,

we therefore used the plasmids including region 2 and region 4

for the knockdown of Wnt4 and Wnt5a, respectively.

We first determined the expression levels of Wnt4 and

Wnt5a in 3T3-L1 cells transfected with each shRNA expres-

sion plasmid by Q-PCR and Western blot analyses. In

shRNA expression plasmid-transfected cells, the mRNA and

protein levels of Wnt4 (12 h after induction) or Wnt5a (before

induction) was reduced compared with that in the control

cells (Fig. 3A). Using these transfected cells, we performed

a differentiation experiment. After 8 days, these cells were

stained with Oil Red O to detect oil droplets and the amounts

of triacylglycerol were measured (Fig. 3B). The cell morphol-

ogy of the control cells altered from fibroblastic preadipocytes

to round adipocytes, and the cells stored the oil droplets well.

In contrast, the accumulation of oil droplets in the cells trans-

fected with the shRNA expression plasmids for Wnt4 was

clearly inhibited and the knockdown of Wnt5a expression

slightly decreased the accumulation of oil droplets. Further-

more, the Wnt4 RNAi-treatment more effectively blocked

the accumulation of triacylglycerol than the Wnt5a RNAi-

treatment. We next determined the level of aP2, an adipo-

genic marker. The expression of aP2 was also inhibited by

knockdown of the expression of Wnt4 and Wnt5a

(Fig. 3C). These results suggest that the reduction in Wnt4

and Wnt5a expression during adipogenesis impaired the adi-

pocyte differentiation.

Finally, we examined the expression levels of PPARcand C/EBPa during adipocyte differentiation (Fig. 4). The

expression of PPARc and C/EBPa was also found to be

inhibited by both the knockdown of Wnt4 and Wnt5a,

although the rates of inhibition were relatively low compared

with those observed for triacylglycerol accumulation and aP2

expression.

Fig. 1. Time course of mRNA expression of Wnt4, Wnt5a and Wnt6during adipocyte differentiation. Total RNA obtained from mouse3T3-L1 cells at various time points after treatment with inducers wassubjected to Q-PCR. The expression level of each gene was normalizedwith 18S rRNA expression. The bars indicate S.D. (n = 3).

Fig. 2. Expression of Wnt4, Wnt5a and Wnt6 in growth arrested orproliferating 3T3-L1 and NIH-3T3 cells. Total RNA obtained from3T3-L1 and NIH-3T3 cells at each time point was subjected to Q-PCR.The expression level of each gene was normalized with 18S rRNAexpression. Cells were treated with the inducers after growth arrest(growth arrested; the typical condition for adipocyte differentiation),or at the mid-exponential phase of growth (proliferating; not thetypical condition for adipocyte differentiation). The bars indicate S.D.(n = 3).

M. Nishizuka et al. / FEBS Letters 582 (2008) 3201–3205 3203

4. Discussion

Wnt10b seems to be a potent inhibitor for adipocyte differ-

entiation [4]. Kanazawa et al. indicated that Wnt5b promotes

adipogenesis by inhibiting the canonical Wnt/b-catenin signal-

ing pathway [10]. These reports strongly suggest that the Wnt/

b-catenin pathway plays a crucial role in the inhibition of adi-

pogenesis. On the other hand, non-canonical Wnt5a regulates

the growth, differentiation, apoptosis and insulin sensitivity of

the differentiating adipocytes, and induces osteoblastogenesis

by attenuating PPARc-induced adipogenesis in mesenchymal

stem cells of bone marrow [11,12]. However, little is known

about the roles of some Wnts family members involved in

the non-canonical Wnt signaling pathway at the early stage

of adipocyte differentiation. In the present study, we demon-

strated that Wnt4 and Wnt5a have an important role in the

promotion of adipocyte differentiation at the initial stage.

Wnt4 appears to have an important role in activating PKC

signaling, as shown by the report that PKCn is required for

Wnt4-stimulated outgrowth of commissural axons [13]. On

adipocyte conversion, PKC signaling is very important. Flem-

ing et al. indicated that PKCc, part of the PKC family, is

required for the mitotic clonal expansion, which occurs in

the early stages of adipogenesis [14]. Thus, our findings

Fig. 3. Effect of the knockdown of Wnt4 or Wnt5a on adipocyte conversion. (A) Knockdown of Wnt4 or Wnt5a gene expression by RNAi duringadipocyte differentiation. Total RNA and cell lysate obtained from 3T3-L1 cells transfected with the shRNA expression plasmid for Wnt4 (12 h afterinduction) or Wnt5a (before induction) were subjected to Q-PCR (left) and Western blot (right). 3T3-L1 cells transfected with a scrambled shRNAexpression plasmid was used as a control. On Q-PCR, the expression level of each gene was normalized with 18S rRNA expression. The bars indicateS.D. (n = 3). Cell lysates were separated by SDS–PAGE, and detected by Western blotting with antibodies against Wnt4 and Wnt5a. b-actin was usedas a loading control. (B) Differentiation of 3T3-L1 cells transfected with the shRNA expression plasmid for Wnt4 or Wnt5a. The cells after 8 days oftreatment were stained with Oil Red O (left). The measurement of triacylglycerol content was done on 24-well plates (right). The bars indicate S.D.(n = 5). *P < 0.01 vs. control. (C) The expression level of the aP2 gene on knockdown of Wnt4 or Wnt5a expression. The bars indicate S.D. (n = 3).*P < 0.01 vs. control.

3204 M. Nishizuka et al. / FEBS Letters 582 (2008) 3201–3205

indicate that Wnt4 may promote adipogenesis by regulating

PKC signaling and mitotic clonal expansion early on in the dif-

ferentiation process.

The endogenous expression of Wnt5a was abundant before

the induction and rapidly decreased after the induction. Inter-

estingly, even though this expression pattern was similar to

that of Wnt10b, the knockdown of Wnt5a expression led to

inhibition of adipogenesis. Wnt5a stimulates intracellular cal-

cium release and activation of PKC and calcium/calmodulin-

dependent kinase II [15]. On the other hand, Wnt5a has also

been suggested to antagonize canonical Wnt activity via inhi-

bition of b-catenin�s stabilization [16]. These reports imply that

Wnt5a acts in both the Wnt/b-catenin and non-canonical Wnt

signaling pathways. Further investigations are required to

solve which pathway is a main one at the early stage of adipo-

genesis.

Fig. 4. Effect of knockdown of Wnt4 or Wnt5a expression by RNAion the expression of master regulators of adipocyte differentiation.Total RNA obtained from the cells transfected with the shRNAexpression plasmid for Wnt4 or Wnt5a at each time point wassubjected to Q-PCR. The expression level of each gene was normalizedwith 18S rRNA expression. The bars indicate S.D. (n = 3). *P < 0.01;**P < 0.05 vs. control.

M. Nishizuka et al. / FEBS Letters 582 (2008) 3201–3205 3205

In Fig. 4, the expression of PPARc and C/EBPa was only

partly inhibited in the knockdown cells. It is possible that

the reduction of expression of PPARc and C/EBPa is caused

by either indirect effect or a complementary mechanism. To

dissolve the precise mechanisms on the reduction of these

genes, further analyses are definitely needed.

In summary, we showed that levels of Wnt4, Wnt5a and

Wnt6 changed dramatically at the beginning of adipocyte dif-

ferentiation. Furthermore, knockdown experiments using

RNAi demonstrated that both Wnt4 and Wnt5a function as

positive regulators of adipogenesis at the initial stage of the

differentiation process. These results may indicate that Wnts

family members mainly involved in the non-canonical Wnt sig-

naling pathway also regulates adipocyte differentiation.

Acknowledgements: This work was supported in part by grants fromthe Ministry of Education, Culture, Sports, Science and Technology(MEXT), Japan, and Japan Society for the Promotion of Science(JSPS), and by grants from the Takeda Science Foundation.

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