Wnt4 and Wnt5a Promote Adipocyte Differentiation

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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

according to their biological activities: those of the Wnt/b-cate- Abstract The roles of the non-canonical Wnt pathway during adipogenesis are not well known, though Wnt10b is known to nin pathway (canonical Wnt pathway), which leads to the sta- function as a negative regulator for adipogenesis by activating bilization of b-catenin and activates target genes in the the canonical Wnt pathway. We focused on the roles of Wnt4, nucleus, and those of the non-canonical Wnt pathway, which Wnt5a and Wnt6, which are thought to be part of the non-canon- is independent of b-catenin signaling, and involves the stimula- ical Wnt pathway. The expression of these genes changed dra- tion of intracellular Ca2+ release and the activation of phos- matically at the initial stage of adipogenesis. Furthermore, the pholipase C and protein kinase C (PKC) [6]. In the inhibition of Wnt4 or Wnt5a expression prevented the accumula- adipocyte differentiation process, Wnt10b activates the Wnt/ tion of triacylglycerol and decreased the expression of adipogen- b-catenin pathway and maintains preadipocytes in an undiffer- esis-related genes. Wnt4 and Wnt5a have crucial roles in entiation state by inhibiting expression of C/EBPa and PPARc adipogenesis as positive regulators. [4]. These findings strongly indicate that the Wnt/b-catenin Ó 2008 Federation of European Biochemical Societies. Pub- lished by Elsevier B.V. All rights reserved. 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 Keywords: Adipocyte differentiation; Obesity; Wnt family; understood, even though non-canonical Wnts also have essen- Gene expression; 3T3-L1 cells; Peroxisome proliferator- tial organizing roles in development. activated receptor c 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 epithelial cells did not induce morphological transformation or the accumulation of b-catenin, these proteins were thought 1. Introduction to be part of the non-canonical Wnt family [7,8]. In addition, even though the functions of Wnt4, Wnt5a and Wnt6 were well Peroxisome proliferator-activated receptor c (PPARc), the characterized in various cells, little was known about the bio- CCAAT/enhancer-binding proteins (C/EBPs) and sterol regu- logical roles of these proteins at the beginning of adipocyte dif- latory element-binding protein 1 (SREBP1) were known to ferentiation. We found that the expression of Wnt4, Wnt5a be master regulators of adipogenesis [1]. On the other hand, and Wnt6 changed dramatically in the early stages of adipo- several inhibitors are also known. For example, preadipocyte genesis. The expression profiles of Wnt4 and Wnt5a were clo- factor-1 (Pref-1) suppresses adipogenesis through the activa- sely related to adipocyte differentiation. Furthermore, the tion of MEK/extracellular signal-regulated kinase (ERK) sig- inhibition of Wnt4 and Wnt5a expression in 3T3-L1 cells pre- naling [2] and transformation growth factor-b (TGF-b) vented the cytoplasmic accumulation of triacylglycerol and de- inhibits adipogenesis via SMAD3Õs interaction with C/EBP creased the expression of adipogenesis-related genes. These family [3]. In addition to these factors, recent reports have results strongly suggest that Wnt4 and Wnt5a have crucial demonstrated that Wnt10b is also a potent inhibitory factor roles to promote adipocyte differentiation. for adipocyte differentiation [4]. The Wnt family consists of at least 19 cysteine-rich, secreted glycoproteins, which participate in a variety of developmental 2. Materials and methods processes such as cell proliferation, cell polarity, cell migration and cell differentiation [5]. Members of the vertebrate Wnt 2.1. Cell culture and differentiation family can be divided into at least two functional classes The mouse 3T3-L1 preadipocytes and mouse NIH-3T3 fibroblasts were maintained in DulbeccoÕs modified EagleÕs medium (DMEM) containing 10% calf serum. The differentiation experiment was performed as described previously [9]. In brief, the medium was changed *Corresponding author. Fax: +81 52 836 3455. to DMEM supplemented with 0.5 mM 3-isobutyl-1-methylxantine, E-mail address: [email protected] (M. Imagawa). 10 lg/ml of insulin, 1 lM dexamethasone, and 10% fetal bovine serum (FBS) at 2 days postconfluence. After 2 days, the cells were transferred Abbreviations: PPARc, peroxisome proliferator-activated receptor c; to DMEM containing 5 lg/ml insulin and 10% FBS. For Oil red O C/EBP, CCAAT/enhancer-binding protein; SREBP1, sterol regulatory staining, 3T3-L1 adipocytes were fixed with 4% paraformaldehyde, element-binding protein 1; Pref-1, preadipocyte factor-1; TGF-b, then stained with 0.3% Oil red O (Amresco) solution for 60 min. The transforming growth factor-b; aP2, adipocyte fatty acid-binding amounts of triacylglycerol were measured using LIPIDOS LIQUID protein 2 (Ono) according to the manufacturerÕs instructions.

0014-5793/$34.00 Ó 2008 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2008.08.011 3202 M. Nishizuka et al. / FEBS Letters 582 (2008) 3201–3205

2.2. Real-time quantitative PCR (Q-PCR) mouse embryo, we compared the expression profiles of these An ABI PRISM 7000 sequence detection system (Applied Biosys- three genes in the two cell lines under two conditions: tems) was used to perform Q-PCR. Pre-designed primers and probe growth-arrested and proliferation (Fig. 2). sets for Wnt4, Wnt5a, Wnt6, PPARc, C/EBPa, adipocyte fatty acid- binding protein 2 (aP2) and 18S rRNA were obtained from Applied Although the expression of Wnt4 mRNA was slightly in- Biosystems. The reaction mixture was prepared using a TaqMan Uni- creased in the proliferating state, that of Wnt4 was dramati- versal PCR Master mix according to the manufacturerÕs instructions. cally induced in the growth-arrested state in 3T3-L1 cells Relative standard curves were generated in each experiment to calcu- 12 h after induction. In the NIH-3T3 cells, significant expres- late the inputted amounts of the unknown samples. sion of Wnt4 was not observed in proliferating or growth-arrested cells even after the induction. Although the Wnt5a 2.3. RNAi experiment For the RNAi experiment, the two target regions (1: 374–394 bp and mRNA decreased in all cases, the expression of Wnt5a in 2: 651–671 bp) for Wnt4 and the four target regions (1: 283–303 bp, 2: growth-arrested 3T3-L1 cells was drastically decreased. On 579–599 bp, 3: 602–622 bp and 4: 670–690 bp) for Wnt5a were selected the other hand, the expression of Wnt 6 also decreased in all using the QIAGEN siRNA online design tool (http://sir- four conditions, and the rates of decrease were almost the na.quagen.com/). A 19-nt short hairpin RNA (shRNA)-coding fragment with a 50-TTCAAGAGA-30 loop was subcloned into the ApaI/ same. These results indicate that the expression of Wnt4 and EcoRI site of pSilencer 1.0-U6 (Ambion). As a negative control, the Wnt5a, but not Wnt6, was drastically changed under differen- scrambled fragment 50-AAGAGGAGCATATTGGGAAGA-30, tiable condition, suggesting that Wnt4 and Wnt5a are closely which does not have similarity with any mRNA listed in Genbank, related adipocyte differentiation. was generated. Transfection of shRNA-expressing plasmids into 3T3-L1 cells was performed with Nucleofector using Cell Line Nucleofector Kit V (Amaxa) according to the manufacturerÕs instructions. The transfected 3.3. The effect of knocking down the expression of Wnt4 or cells were subjected to differentiation experiments at 2 days Wnt5a on adipocyte conversion postconfluence. To characterize the biological functions of Wnt4 and Wnt5a, we blocked their expression at the early stage of adipocyte dif- 2.4. Western blot analyses ferentiation using RNAi. Two shRNA expression plasmids for Proteins were separated using SDS/PAGE and transferred to the Wnt4 and four plasmids for Wnt5a with different target re- polyvinylidene difluoride membrane. Rabbit polyclonal antibodies against Wnt4 or Wnt5a (Santa Cruz) were used as primary antibody. 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 3. Results the greatest reduction (data not shown). For further analyses, we therefore used the plasmids including region 2 and region 4 3.1. Time course of Wnt4, Wnt5a and Wnt6 mRNA expression for the knockdown of Wnt4 and Wnt5a, respectively. during adipocyte differentiation We first determined the expression levels of Wnt4 and We first tested whether Wnt4, Wnt5a and Wnt6 were ex- Wnt5a in 3T3-L1 cells transfected with each shRNA expres- pressed during adipocyte differentiation of mouse 3T3-L1 cells. sion plasmid by Q-PCR and Western blot analyses. In At 2 days postconfluence, growth-arrested 3T3-L1 cells were shRNA expression plasmid-transfected cells, the mRNA and induced into adipocytes. The expression levels of these three protein levels of Wnt4 (12 h after induction) or Wnt5a (before genes were investigated by Q-PCR (Fig. 1). The level of induction) was reduced compared with that in the control Wnt4 mRNA increased after the treatment with inducers cells (Fig. 3A). Using these transfected cells, we performed and the peak of expression was at 6–12 h. The expression of a differentiation experiment. After 8 days, these cells were Wnt5a was slightly increased at 1 h and then rapidly decreased. stained with Oil Red O to detect oil droplets and the amounts The expression of Wnt6 mRNA showed a biphasic pattern. of triacylglycerol were measured (Fig. 3B). The cell morphol- While the expression was decreased until 6 h after induction, ogy of the control cells altered from fibroblastic preadipocytes it was increased at 12 h and decreased at the middle and late to round adipocytes, and the cells stored the oil droplets well. stages of adipocyte differentiation. These results indicated that In contrast, the accumulation of oil droplets in the cells trans- the levels of Wnt4, Wnt5a and Wnt6 changed dramatically fected with the shRNA expression plasmids for Wnt4 was during adipocyte differentiation, especially early on. clearly inhibited and the knockdown of Wnt5a expression slightly decreased the accumulation of oil droplets. Further- more, the Wnt4 RNAi-treatment more effectively blocked 3.2. Expression profiles of Wnt4, Wnt5a and Wnt6 in the the accumulation of triacylglycerol than the Wnt5a RNAi- adipocyte differentiable state and the non-differentiable state treatment. We next determined the level of aP2, an adipo- Next we examined whether the expression of Wnt4, Wnt5a genic marker. The expression of aP2 was also inhibited by and Wnt6 at the beginning of adipocyte differentiation was knockdown of the expression of Wnt4 and Wnt5a specific to adipogenesis. For the differentiation into adipo- (Fig. 3C). These results suggest that the reduction in Wnt4 cytes, 3T3-L1 cells must first be grown to confluence and then and Wnt5a expression during adipogenesis impaired the adi- kept for 2 days. After that, the inducers are added to the med- pocyte differentiation. ium. Differentiation into adipocytes occurred only under the Finally, we examined the expression levels of PPARc growth-arrested condition. On the other hand, proliferating and C/EBPa during adipocyte differentiation (Fig. 4). The 3T3-L1 cells did not differentiate into adipocytes even in the expression of PPARc and C/EBPa was also found to be presence of inducers. Another mouse fibroblastic cell line, inhibited by both the knockdown of Wnt4 and Wnt5a, NIH-3T3, does not differentiate into adipocytes in the although the rates of inhibition were relatively low compared growth-arrested and proliferating states. Since both NIH-3T3 with those observed for triacylglycerol accumulation and aP2 and 3T3-L1 cells were fibroblastic cell lines derived from expression. M. Nishizuka et al. / FEBS Letters 582 (2008) 3201–3205 3203

Fig. 1. Time course of mRNA expression of Wnt4, Wnt5a and Wnt6 during adipocyte diﬀerentiation. Total RNA obtained from mouse 3T3-L1 cells at various time points after treatment with inducers was subjected to Q-PCR. The expression level of each gene was normalized with 18S rRNA expression. The bars indicate S.D. (n = 3).

4. Discussion

Wnt10b seems to be a potent inhibitor for adipocyte differentiation [4]. Kanazawa et al. indicated that Wnt5b promotes adipogenesis by inhibiting the canonical Wnt/b-catenin signaling pathway [10]. These reports strongly suggest that the Wnt/ Fig. 2. Expression of Wnt4, Wnt5a and Wnt6 in growth arrested or proliferating 3T3-L1 and NIH-3T3 cells. Total RNA obtained from b-catenin pathway plays a crucial role in the inhibition of adi- 3T3-L1 and NIH-3T3 cells at each time point was subjected to Q-PCR. pogenesis. On the other hand, non-canonical Wnt5a regulates The expression level of each gene was normalized with 18S rRNA the growth, differentiation, apoptosis and insulin sensitivity of expression. Cells were treated with the inducers after growth arrest the differentiating adipocytes, and induces osteoblastogenesis (growth arrested; the typical condition for adipocyte differentiation), or at the mid-exponential phase of growth (proliferating; not the by attenuating PPARc-induced adipogenesis in mesenchymal typical condition for adipocyte differentiation). The bars indicate S.D. stem cells of bone marrow [11,12]. However, little is known (n = 3). 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- Wnt4-stimulated outgrowth of commissural axons [13].On strated that Wnt4 and Wnt5a have an important role in the adipocyte conversion, PKC signaling is very important. Flem- promotion of adipocyte differentiation at the initial stage. ing et al. indicated that PKCc, part of the PKC family, is Wnt4 appears to have an important role in activating PKC required for the mitotic clonal expansion, which occurs in signaling, as shown by the report that PKCn is required for the early stages of adipogenesis [14]. Thus, our findings 3204 M. Nishizuka et al. / FEBS Letters 582 (2008) 3201–3205

Fig. 3. Effect of the knockdown of Wnt4 or Wnt5a on adipocyte conversion. (A) Knockdown of Wnt4 or Wnt5a gene expression by RNAi during adipocyte differentiation. Total RNA and cell lysate obtained from 3T3-L1 cells transfected with the shRNA expression plasmid for Wnt4 (12 h after induction) or Wnt5a (before induction) were subjected to Q-PCR (left) and Western blot (right). 3T3-L1 cells transfected with a scrambled shRNA expression plasmid was used as a control. On Q-PCR, the expression level of each gene was normalized with 18S rRNA expression. The bars indicate S.D. (n = 3). Cell lysates were separated by SDS–PAGE, and detected by Western blotting with antibodies against Wnt4 and Wnt5a. b-actin was used as a loading control. (B) Differentiation of 3T3-L1 cells transfected with the shRNA expression plasmid for Wnt4 or Wnt5a. The cells after 8 days of treatment 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. indicate that Wnt4 may promote adipogenesis by regulating cium release and activation of PKC and calcium/calmodulin- PKC signaling and mitotic clonal expansion early on in the dif- dependent kinase II [15]. On the other hand, Wnt5a has also ferentiation process. been suggested to antagonize canonical Wnt activity via inhi- The endogenous expression of Wnt5a was abundant before bition of b-cateninÕs stabilization [16]. These reports imply that the induction and rapidly decreased after the induction. Inter- Wnt5a acts in both the Wnt/b-catenin and non-canonical Wnt estingly, even though this expression pattern was similar to signaling pathways. Further investigations are required to that of Wnt10b, the knockdown of Wnt5a expression led to solve which pathway is a main one at the early stage of adipo- inhibition of adipogenesis. Wnt5a stimulates intracellular cal- genesis. M. Nishizuka et al. / FEBS Letters 582 (2008) 3201–3205 3205

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