β-catenin signaling activates nephronectin expression in osteoblasts

Accepted Manuscript Wnt/β-catenin signaling activates nephronectin expression in osteoblasts Mikiko Ikehata, Atsushi Yamada, Naoko Morimura, Masakatsu Itose, Tetsuo Suzawa, Tatsuo Shirota, Daichi Chikazu, Ryutaro Kamijo PII:

S0006-291X(17)30087-6

DOI:

10.1016/j.bbrc.2017.01.053

Reference:

YBBRC 37115

To appear in:

Biochemical and Biophysical Research Communications

Received Date: 28 December 2016 Accepted Date: 11 January 2017

Please cite this article as: M. Ikehata, A. Yamada, N. Morimura, M. Itose, T. Suzawa, T. Shirota, D. Chikazu, R. Kamijo, Wnt/β-catenin signaling activates nephronectin expression in osteoblasts, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.01.053. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Wnt/β β-catenin signaling activates nephronectin expression in osteoblasts

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Mikiko Ikehataa, b, Atsushi Yamadaa, Naoko Morimurac, Masakatsu Itosea, d, Tetsuo Suzawaa,

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Tatsuo Shirota d, Daichi Chikazub, Ryutaro Kamijoa

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Department of Oral and Maxillofacial Surgery, Tokyo Medical University, Tokyo, Japan;

Department of Integrative Physiology, Shiga University of Medical Science, Shiga, Japan; Department of Oral and Maxillofacial Surgery, School of Dentistry, Showa University, Tokyo,

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Department of Biochemistry, School of Dentistry, Showa University, Tokyo, Japan;

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Corresponding author: Atsushi Yamada, Ph.D.

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Department of Biochemistry, School of Dentistry, Showa University, 1-5-8 Hatanodai,

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Shinagawa, Tokyo 142-8555, Japan

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Tel: +81-3-3784-8163, Fax: +81-3-3784-5555, E-mail: [email protected]

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Abstract

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Nephronectin (Npnt), an extracellular matrix protein, is considered to play critical roles as an

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adhesion molecule in the development and functions of various organs and tissues, such as the

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kidneys and bone. In the present study, we found that Wnt3a strongly enhanced Npnt mRNA

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expression in osteoblast-like MC3T3-E1 cells, while it also induced an increase in Npnt gene

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expression in both time- and dose-dependent manners via the Wnt/β-catenin signaling pathway.

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These results suggest novel mechanisms for Wnt3a-induced osteoblast proliferation and cell

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survival via Npnt gene expression.

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Keywords

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nephronectin, Wnt3a, β-catenin

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1. Introduction

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Nephronectin (Npnt) is an extracellular matrix protein that was independently identified by two different groups. Brandenberger et al. reported Npnt as a ligand for integrin α8β1, which

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plays important roles in embryonic kidney development [1], while Morimura et al. identified

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Npnt among various epidermal growth factor (EGF)-like repeats containing proteins, such as

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fibrillin and laminin, from an osteoblast cell line [2]. Npnt has strong cell-adhesion properties,

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and is particularly involved in interactions between cells and the extracellular matrix during

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bone and kidney development [3, 4]. Furthermore, its expression is seen at a high level in

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osteoblasts. Thus, clarification of Npnt gene expression regulation is important for

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understanding the association between bone development and cell adhesion [2, 4, 5].

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Wnt proteins are secreted glycoproteins that have a variety of critical roles in early development and morphogenesis, as well as cell growth and differentiation, and are known to be

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essential elements for tissue regeneration [6]. It has also been shown that Wnt activates at least

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3 distinct intracellular signaling pathways, including Wnt/β-catenin, Wnt/Ca2+, and Wnt/polarity

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signaling [7, 8]. Wnt/β-catenin signaling is activated when Wnt binds to a Frizzled family receptor,

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which forms a complex with LRP5/6 [9], then intracellular β-catenin accumulates, with some

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β-catenin subsequently transported to the nucleus. Intranuclear β-catenin interacts with transcription

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factors to either increase or decrease the expression of specific target genes [10]. In contrast, when

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Wnt is absent, β-catenin is subjected to degradation by glycogen synthase kinase 3β (GSK3β), Axin,

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and the adenomatous polyposis coli (APC) protein complex [11]. Previous studies have suggested

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that Wnt3a induces Wnt/β-catenin signaling, which promotes proliferation and survival of

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pre-osteoblasts, and also has an important role in control of bone mass [12, 13].

We previously demonstrated that Npnt expression is down-regulated by cytokines, such as

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TGF-β, TNFα, and Oncostatin M, via the MAPK, NF-κβ, and JAK/STAT signaling pathways

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in MC3T3-E1 cells, a mouse calvaria-derived osteoblast-like cell line [14-16]. In the present

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study, we found that Wnt3a up-regulates the expression of Npnt in both time- and

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dose-dependent manners via the Wnt/β-catenin signaling pathway.

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2. Materials and Methods

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2.1. Cell culture

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MC3T3-E1 cells were maintained in MEM-α with L-glutamine and phenol red medium

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(Wako Pure Chemical Industries, Ltd., cat. # 135-15175), supplemented with 10% fetal bovine

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serum (FBS) (Life Technologies, cat. # 10437, or Biosera, cat. # FB-1360/500) and 1%

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penicillin-streptomycin at 37˚C in a CO2 incubator (5% CO2, 95% air). For the experiments,

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cells were plated at 1.0×105 cells/well in 6-well plates (Thermo Scientific Inc., cat. # 140675)

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and incubated until reaching 60-70% confluence.

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2.2. Reagents Mouse Wnt3a (cat. no. 1324-WN) and Wnt5a (cat. # 645-WN) were purchased from R&D

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Systems, Inc. The canonical Wnt signaling inhibitor XAV939 (cat. # 13596) was purchased

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from Cayman Chemical Company. Mouse Dkk-1 (cat. # 5897-DK) was purchased from R&D

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Systems, Inc.

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2.3. Quantitative real-time PCR

Total RNA was extracted using TRIzol reagent (Life Technologies, cat. # 15596018), then

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reverse transcribed using SuperScript III (Life Technologies, cat. # 18080-044). Quantitative

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real-time PCR was performed using Fast SYBR Green Master Mix (Applied Biosystems, cat. #

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4385612) with the following specific PCR primers: 5’-AAATGGTGAAGGTCGGTGTG-3’ and

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5’-TGAAGGGGTCGTTGATGG-3’ for glyceraldehyde3-phosphatedehydrogenase (Gapdh),

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and

for Npnt.

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2.4. Luciferase assay

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5’-CACGAGTAATTACGGTTGACAACAG-3’ and 5’-CTGCCGTGGAATGAACACAT-3’

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MC3T3-E1 cells were plated at 1.5×104 cells/well in 24-well plates and maintained

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overnight in 10% FBS containing MEMα. Adherent cells (250 ng/well) were co-transfected with

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a TOPFlash vector (Addgene, cat. # 12456), then incubated for 24 hours, followed by treatment

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with an inhibitor (XAV939 at 1 µM or Dkk-1 at 10 ng/mL) for 1 hour. Cells were harvested and

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luciferase activity was determined using the Dual Luciferase Reporter assay system (Promega

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cat. # E1910).

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2.5. Short interfering RNA (siRNA)-mediated knockdown of gene expression MC3T3-E1 cells were plated at 1.0×105 cells/ well in 6-well plates with 50 nM siRNA using Lipofectamine RNAi Max reagent (Life Technologies) in OPTI-MEM (Life Technologies, cat. #

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31985-070). The ID for β-catenin siRNAs was MSS202665. Stealth RNAi siRNA Negative

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Control Med GC Duplex #2 (ThermoFisher Scientific, cat. # 12935112) was used as the control.

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2.6. Western blot analysis

Protein samples were collected using Sample Buffer Solution with Reducing Reagent for

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SDS-PAGE (Nacalai Tesque, cat. # 09499-14) with a cell scraper (Corning Inc., cat. # 3010),

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then electrophoresed onto a 10% SDS polyacrylamide gel, which was then blotted onto a PVDF

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membrane. Npnt was detected using a primary antibody (1:2000) (R&D Systems Inc., cat. #

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AF4298) and then further probed with an anti-goat IgG horseradish peroxidase linked secondary

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antibody (1:2500) (Santa Cruz Biotechnology Inc., cat. # sc-2768). β-catenin was detected using

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a primary antibody (1:1000) (BD Transduction Laboratories, cat. # 61054) and then further

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probed with an anti-mouse IgG horseradish peroxidase linked secondary antibody (1:2500) (GE

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Healthcare, cat. # NA931VS). An anti-β-actin antibody (1:2000) (Sigma Aldrich, cat. # A5060)

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was used as the control antibody. To visualize the locations of the antigenic bands, peroxidase

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reactions were developed using ECL Prime Western Blotting Detection reagent (GE

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Healthcare).

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3. Results and Discussion In the present study, we initially examined regulation of Npnt expression in MC3T3-E1 cells by the Wnt family proteins Wnt3a and Wnt5a. Treatment of cells with 10 ng/mL of each for 24

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hours resulted in an obvious increase in expression of Npnt mRNA induced by Wnt3a (Suppl.

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Fig. S1). Next, we treated MC3T3-E1 cells with various concentrations of Wnt3a for 24 hours

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and noted an increase in Npnt mRNA level in cells treated with greater than 1 ng/mL Wnt3a as

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compared to untreated cells (Fig. 1A). The time-dependent effect on induction of Npnt

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expression was further examined using Wnt3a at 10 ng/mL and a significant increase in Npnt

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mRNA was detected at 12 hours after its addition (Fig. 1B). Together, these results suggest that

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Wnt3a induces up-regulation of Npnt gene expression in both time- and dose-dependent

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manners. The expression level of Npnt protein was also increased in MC3T3-E1 cells after 24 h

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of incubation with Wnt3a (Fig. 1c).

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Wnt3a is known to be a factor that activates the canonical Wnt signaling pathway [13, 17]. Binding of Wnts to Frizzled family receptors causes intracellular accumulation of β-catenin,

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which leads to translocation of β-catenin into the nucleus for target gene expression regulation

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[10]. To investigate the mechanism that governs regulation of Npnt expression by Wnt3a in

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osteoblasts, we performed several experiments. First, MC3T3-E1 cells were treated with

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DKK-1, which antagonizes Wnt by forming a complex with LRP5/6 [9, 18]. LRP5/6 is required

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for activating this pathway by the binding of Wnt to a receptor complex containing Frizzled [19].

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As shown in Figure 2A, the luciferase reporter activity of β-catenin-mediated transcriptional

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activation using a TOPFlash vector in MC3T3-E1 cells was increased by Wnt3a, while that

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increased activity was inhibited by DKK-1. When MC3T3-E1 cells were treated with both

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Wnt3a and DKK-1, the enhancement of Npnt mRNA expression by Wnt3a was inhibited by

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DKK-1 (Fig. 2B).

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XAV939 is known to increase the stability of the axin-GSK3β complex and promote

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β-catenin degradation [19]. As shown in Figure 3A, luciferase reporter activity induced by

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β-catenin-mediated transcriptional activation with a TOPFlash vector in MC3T3-E1 cells was

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increased by addition of Wnt3a, while that increase was inhibited by XAV939. When

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MC3T3-E1 cells were treated with both Wnt3a and XVA939, enhancement of Npnt mRNA

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expression by Wnt3a was again inhibited by XVA939 (Fig. 3B). These results suggest that Npnt

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gene expression is regulated by the Wnt/β-catenin signaling pathway.

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Finally, to investigate the mechanism that governs regulation of Npnt gene expression via

β-catenin in osteoblasts, MC3T3-E1 cells were treated with a small interfering RNA (siRNA)

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targeting β-catenin. We observed a significant decrease in the level of β-catenin protein in

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MC3T3-E1 cells following their treatment with β-catenin siRNA (Fig. 4A), which also inhibited

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Npnt gene induction by Wnt3a (Fig. 4B). These results indicate that Npnt gene expression is

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regulated by the Wnt/β-catenin signaling pathway (Suppl. Fig. S2).

In addition to our findings of regulation of Npnt gene expression by Wnt/β-catenin signaling, a previous study that used K14∆Nβ-cateninER mice, which express stabilized β-catenin fused

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with a mutant estrogen receptor under control of the K14 promoter, demonstrated that activation

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of Wnt/β-catenin signaling induced Npnt gene expression in the epidermis [20]. The present

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results shown in osteoblasts are consistent with their findings in epidermis samples. In the Npnt

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locus, several conserved putative binding sites for Lef/Tcfs, which are transcription factors in

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the Wnt/β-catenin signaling pathway, have been identified using the University of California

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Santa Cruz (UCSC) Genome Browser, with one of the conserved sites in Npnt gene introns

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showing consistent enrichment for Tcf4 in choromatin immunoprecipitation (ChIP) assays with

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an antibody to Tcf4 [20]. Nevertheless, additional studies are needed to fully reveal the

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responsible elements in the Wnt/β-catenin signaling pathway for Npnt gene promotion and

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enhancement.

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In conclusion, we report for the first time that Wnt3a promotes Npnt gene expression via the

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Wnt/β-catenin signaling pathway. Our results suggest novel mechanisms induced by Wnt3a that

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are involved in osteoblast proliferation and cell survival via Npnt gene expression.

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Acknowledgements This study was supported in part by the Project to Establish Strategic of the Research Center

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for Innovative Dentistry established by the Ministry of Education, Culture, Sports, Science and

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Technology of Japan, and Grants-in-Aid for Scientific Research from the Japan Society for the

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Promotion of Science (AY:15K11051, RK:15H05016,16K15782) .

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Figure legends

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Fig. 1. Dose- and time-dependent effects of Wnt3a on Npnt mRNA expression.

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(A) Dose-dependent effects of Wnt3a on Npnt mRNA expression. MC3T3-E1 cells were treated

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with 0, 0.01, 0.1, 1, or 10 ng/mL of Wnt3a for 24 hours. Results are shown as the mean ± SD of

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3 different samples relative to the level of untreated cells. **P <0.01, Student’s t-test. (B) Time

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course analysis of effect of Wnt3a on Npnt mRNA expression. MC3T3-E1 cells were treated

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with 10 ng/mL of Wnt3a for 3, 6, 12, or 24 hours. Total cellular RNA was extracted, and Npnt

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and Gapdh mRNA expressions were analyzed using real-time PCR. Results are shown as the

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mean ± SD of 3 different samples, and expressed relative to that of untreated cells at 0 hours.

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**P<0.01, Student’s t-test. (C) Western blotting analysis of Npnt protein levels in cells

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treated with and without Wnt3a. Cell lysates were collected after 24 hours of incubation

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with or without 10 ng/ml of Wnt3a.

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Fig. 2. Inhibition of Wnt3a-induced Npnt mRNA expression by DKK-1.

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(A) MC3T3-E1 cells were transfected with a TCF reporter gene (TOPFlash) vector for 4 hours.

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Following treatment with 100 ng/mL DKK-1 for 1 hour, Wnt3a at 10 ng/mL was added and

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incubation continued for another 24 hours, after which luciferase activity was detected in cell

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lysates. (B) Induction of Npnt gene expression by Wnt3a was inhibited by Dkk-1. MC3T3-E1

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cells were treated with 10 ng/mL of DKK-1 for 24 hours. Total cellular RNA was extracted, and

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Npnt and Gapdh mRNA expressions were analyzed by real-time PCR. Results are shown as the

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mean ± SD of 3 different samples and expressed relative to the level without Dkk-1 treatment.

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**P <0.01, Student’s t-test.

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Fig. 3. XAV939 inhibits Npnt gene expression induced byWnt3a.

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(A) MC3T3-E1 cells were transfected with a TCF reporter gene (TOPFlash) vector for 4 hours

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and treated with 1 µM XAV939 for 1 hour, followed by treatment with Wnt3a (10 ng/mL) for an

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additional 24 hours, after which luciferase activity in lysates was analyzed. (B) Expression of

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the Npnt gene induced by Wnt3a was inhibited by XAV939. MC3T3-E1 cells were treated with

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1 µM XAV939 for 24 hours. Total cellular RNA was extracted, and Npnt and Gapdh mRNA

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expressions were analyzed by real-time PCR. Results are shown as the mean ± SD of 3 different

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samples and are expressed relative to the untreated cells (control). **P <0.01, Student’s t-test.

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Fig. 4. siRNA mediated knockdown of β-catenin reduced the level of Npnt gene expression

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induced by Wnt3a

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(A) MC3T3-E1 cells were pretreated with 50 nM of β-catenin siRNA, then treated with Wnt3a

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(10 ng/mL) for 24 hours. Total cellular protein was extracted and the protein level of β-catenin

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was analyzed by western blotting. (B) Npnt gene expression induced by Wnt3a was inhibited by

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β-catenin siRNA. Total cellular RNA was extracted, and Npnt and Gapdh mRNA expressions

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were analyzed using real-time PCR. Results are shown as the mean ± SD of 3 different samples

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and are expressed relative to the untreated cells (control). **P <0.01, Student’s t-test.

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Supplementary Fig. S1. Enhancement of Npnt mRNA expression by Wnt3a.

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MC3T3-E1 cells were treated with 10 ng/mL of Wnt3a or Wnt5a for 24 hours. Total cellular

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RNA was extracted, and Npnt and Gapdh mRNA expressions were analyzed using real-time

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PCR. Results are shown as the mean ± SD of 3 different samples relative to untreated cells

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(control). **P <0.01, Student’s t-test.

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Supplementary Fig. S2. Model of regulation of Npnt gene expression via Wnt/β β -catenin

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signaling in osteoblasts.

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Wnt3a interacts with Frizzled and LRP5/6. β-catenin is stabilized and accumulated in cytoplasm,

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then transported to the nucleus. Intranuclear β-catenin interacts with the Npnt gene promoter to

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increase the expression of Npnt.

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Fig. 2. Ikehata, M. et al.

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Highlights Wnt3a enhances nephronectin gene expression. Nephronectin gene induction by Wnt3a is occurred by time- and dose- dependent

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manner.

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Expression of nephronectin is regulated via β-catenin activation.