β-Catenin signaling

Journal Pre-proof Ectonucleotide pyrophosphatase 2 (ENPP2) plays a crucial role in myogenic differentiation through the regulation by WNT/␤-Catenin signaling Jay Prakash Sah (Conceptualization) (Investigation) (Writing original draft), Nguyen Thi Thu Hao (Investigation) (Validation), Xianghua Han (Investigation), Trinh Thi Tuyet Tran (Investigation), Sarah McCarthy (Investigation), Younjeong Oh (Investigation) (Visualization), Jeong Kyo Yoon (Conceptualization) (Writing review and editing) (Supervision) (Funding acquisition)

PII:

S1357-2725(19)30238-9

DOI:

https://doi.org/10.1016/j.biocel.2019.105661

Reference:

BC 105661

To appear in:

International Journal of Biochemistry and Cell Biology

Received Date:

12 August 2019

Revised Date:

28 November 2019

Accepted Date:

30 November 2019

Please cite this article as: Prakash Sah J, Hao NTT, Han X, Tran TTT, McCarthy S, Oh Y, Yoon JK, Ectonucleotide pyrophosphatase 2 (ENPP2) plays a crucial role in myogenic differentiation through the regulation by WNT/␤-Catenin signaling, International Journal of Biochemistry and Cell Biology (2019), doi: https://doi.org/10.1016/j.biocel.2019.105661

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier.

Ectonucleotide pyrophosphatase 2 (ENPP2) plays a crucial role in myogenic differentiation through the regulation by WNT/β-Catenin signaling

Jay Prakash Sah1,2,#, Nguyen Thi Thu Hao1,2, Xianghua Han3, Trinh Thi Tuyet Tran1,2, Sarah McCarthy3, Younjeong Oh1 and Jeong Kyo Yoon1,2*

Institute of Medi-Bio Science and 2Department of Integrated Biomedical Science, Soonchunhyang University, 25 Bongjeong-ro, Dongnam-gu, Cheonan-si 31151, South Korea

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1Soonchunhyang

3Center

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Running Title: Role of ENPP2 in skeletal myogenesis

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for Molecular Medicine, Maine Medical Center Research Institute, 81 Research Drive, Scarborough, ME 04074, USA

*Corresponding author (E-mail: [email protected]) # Present

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address: School of Health and Allied Science, Faculty of Health Science, Pokhara University, Lekhnath-12, Kaski, Nepal

ABSTRACT Ectonucleotide pyrophosphate phosphodiesterase type II (ENPP2), also known as Autotaxin (ATX), is an enzyme present in blood circulation that converts lysophosphatidyl choline (LPC) to lysophosphatidic acid (LPA). While LPA has been demonstrated to play diverse roles in skeletal myogenesis, mainly through in vitro studies, the role of ENPP2 in skeletal myogenesis has not been determined. We previously found that Enpp2 is induced by a positive WNT/β-Catenin

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signaling regulator, R-spondin2 (RSPO2), in C2C12 myoblast cells. As RSPO2 promotes myogenic differentiation via the WNT/β-Catenin signaling pathway, we hypothesized that ENPP2 may act as a key mediator for the crosstalk between WNT and LPA signaling during myogenic

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differentiation. Herein, we found that ENPP2 function is essential for myogenic differentiation in

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C2C12 cells. Pharmacological ENPP2 inhibitors or RNAi-mediated Enpp2 gene knockdown severely impaired the myogenic differentiation, including the cell fusion process, whereas

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administration of the recombinant ENPP2 protein enhanced myogenic differentiation. Consistent with the in vitro results, mice lacking the Enpp2 gene showed a disrupted muscle regeneration

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after acute muscle injury. The size of newly regenerated myofibers in Enpp2 mutant muscle was significantly reduced compared with wild-type regenerated muscle. Modified expression patterns of myogenic markers in Enpp2 mutant muscle further emphasized the impaired muscle regeneration process. Finally, we convincingly demonstrate that the Enpp2 gene is a direct

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transcriptional target for WNT/β-Catenin signaling. Functional TCF/LEF1 binding sites within the upstream region of Enpp2 gene were identified by chromatin immunoprecipitation using anti-βCatenin antibodies and reporter assay. Our study reveals that ENPP2 is regulated by WNT/βCatenin signaling and plays a key positive role in myogenic differentiation.

Key words: ENPP2, autotaxin, myogenic differentiation, LPA, WNT/β-Catenin, muscle regeneration

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Introduction

Lysophosphatic acid (LPA), the most abundant phospholipid in plasma, mediates many physiological and pathological processes through signaling-based activation of six distinct G

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protein-coupled receptors (GPCRs), named LPA receptor (LPAR)1–6 (Anliker and Chun, 2004;

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Yung et al., 2014). Previous studies have shown that LPA induces proliferation of myoblasts, allowing a continued expression of MYOD, but inhibits their differentiation (Xu et al., 2008;

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Yoshida et al., 1996). Another study demonstrated that LPA induces the activation of ERK1/2 MAP kinases and AKT/PKB in C2C12 cells, suggesting that LPA has mitogenic activity and

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induces C2C12 cell proliferation (Jean-Baptiste et al., 2005). Indeed, LPA treatment has been shown to induce a significant proliferation of C2C12 cells (Tsukahara and Haniu, 2012; Xu et al., 2008). More recently, it was reported that LPA acts as a downstream target of ceramide 1-phosphate (C1P), and transmits its signal through the LPAR1/LPAR3 receptors via AKT/ERK1/2 pathway to

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induce the proliferation of C2C12 cells (Bernacchioni et al., 2018). In contrast, LPA is also shown to induce the myogenic differentiation of C2C12 cells by activating the expression of protein kinase D2 (PKD2) through PI3K p110β and PKCδ pathways (Kleger et al., 2011; Lynch et al., 2013). In addition, LPA induces a reorientation of the nuclear centrosome towards the direction of migration, which, in turn, leads to fusion of myoblasts (Chang et al., 2015). Taken together, these

results indicate that LPA plays a key role in both proliferation and differentiation of myogenic cells. ENPP2 (ectonucleotide pyrophosphatase/phosphodiesterase 2; also known as autotaxin), originally isolated from melanoma cells as an autocrine motility stimulation factor, is a member of

the

ectonucleotide

pyrophosphatase/phosphodiesterase

family

that

hydrolyzes

phosphodiesterase (PDE) bonds of various nucleotides and their derivatives (Murata et al., 1994;

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Stefan et al., 2005). ENPP2 is produced as a secreted pre-proenzyme glycoprotein that becomes active after the proteolytic removal of its N-terminal signal peptide (Jansen et al., 2005). Unlike other members of the ENPP family, ENPP2 possesses lysophospholipase D (LysoD) activity that

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is mainly responsible for the catalysis of lysophosphocholine (LPC) into the most potent biolipid, LPA (van Meeteren and Moolenaar, 2007), thereby highlighting its central regulatory role in LPA

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production and signaling. Therefore, we reasoned that the elucidation of ENPP2 role in skeletal

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myogenesis would resolve the conflicting results from various earlier studies. We previously found that Enpp2 expression in C2C12 myoblast cells is induced by WNT/β-Catenin signaling regulator, R-spondin2 (RSPO2) (Han et al., 2014). As RSPO2 plays a

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positive role in myogenic differentiation, this result led us to hypothesize that ENPP2 is a key mediator for the crosstalk between LPA and WNT signaling during skeletal myogenesis. Until now, the role of ENPP2 in skeletal myogenesis has not been elucidated. In this study, we found

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that ENPP2 plays a critical role in myogenic differentiation, especially in cell fusion, in both primary myoblasts and the C2C12 myoblast cell line. We also show that loss of the Enpp2 gene disrupts skeletal muscle regeneration, via impaired myogenic marker expression and myofiber formation, after acute injury by BaCl2 injection in mice. Furthermore, we provide strong molecular evidence that Enpp2 is a direct transcriptional target of RSPO2-mediated WNT/β-Catenin

signaling. Our study convincingly demonstrates that ENPP2 is a critical regulator of skeletal muscle regeneration and myogenic differentiation, and a key mediator linking WNT/β-Catenin

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and LPA signaling in skeletal myogenesis.

Materials and Methods Cell culture The mouse myoblast cell line, C2C12, was obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in a growth medium (Dulbecco’s modified Eagle’s medium [DMEM, Corning Life Sciences, Oneonta NY, USA], supplemented with 10% fetal bovine serum [FBS, Corning Life Sciences] and 1% penicillin-streptomycin [Corning Life

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Sciences]) under 5% CO2 at 37 °C. For differentiation, 1 x 105 cells were seeded in each well of 24 well-plates and cultured until confluency, unless stated otherwise. Confluent C2C12 cells were cultured in a differentiation medium (DMEM containing 2% heat-inactivated horse serum

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[Corning Life Sciences] and 1% penicillin-streptomycin) for up to 3–5 days for myogenic differentiation. The differentiation media were replaced every 2 days with fresh differentiation

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medium.

Human recombinant ENPP2 protein (Cayman Chemicals, Ann Arbor, MI, USA), mouse

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RSPO2 protein (R&D Systems, St. Paul, MN, USA), mouse WNT3A protein (R&D Systems), HA-130 (Echelon Biosciences, Salt Lake City, UT, USA), PF-8380 (Echelon Biosciences), LPA lysophosphatidic

acid,

Tocris

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(1-Oleoyl

Biosciences,

Bristol,

UK),

LPC

(L-α-

Lysophosphatidylcholine, Sigma-Aldrich, St Louis, MO, USA), and Ki-16425 (Cayman Chemicals) were treated as indicated in the figure legends.

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Viral vector and transduction

Short hairpin RNAs (shRNAs) specific to the mouse Enpp2 or enhanced green fluorescent

protein (eGFP) genes were expressed in lentiviral vector, pLKO.1 (obtained from Thermo Scientific and Sigma-Aldrich, respectively). The lentiviruses were packaged in the recombinant

viral vector facility at Maine Medical Center Research Institute. C2C12 cells (2 x 105 cells) in 35 mm dishes were transduced with lentiviruses expressing control eGFP- or Enpp2-specific shRNA and selected in growth medium containing puromycin (2 µg/mL, Gibco) for 7 days, to establish cell pools with stable expression of specific shRNA. Approximately, over 90 % transduction efficiency was observed.

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DNA and siRNA transfection

Plasmid DNAs were transfected into the cells using Lipofectamine 2000 reagent (Invitrogen), according to the manufacturer’s instructions. In brief, DNA mixed with

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Lipofectamine 2000 reagent was treated into the growing cells and incubated for up to 12 h. After washing with 1x PBS, the cells were grown for 24 h in normal growth medium and then treated

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with RSPO2/WNT3A for another 24 h for luciferase activity measurement. Small interfering RNAs (siRNAs) specific to the mouse Enpp2 and Ctnnb1 genes, and control non-target siRNA

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were obtained from Thermo Scientific/Dharmacon (Lafayette, CO. USA). The siRNA was transfected into C2C12 cells using Lipofectamine RNAiMax reagent (Invitrogen) in a reverse-

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transfection mode, according to the manufacturer’s protocol.

Total RNA isolation and quantitative RT-PCR TRIzol reagent (Invitrogen) was used to isolate the total RNA from cultured cells or TA

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muscle. First-strand cDNA was synthesized from 1 µg of total RNA using a ProtoScript first-strand

cDNA synthesis kit (New England Bio-Labs, Ipswich, MA, USA). cDNA equivalent to 20 ng of total RNA was used for each qRT-PCR reactions that were performed in ABI-Step one plus qPCR machine housed at the Soonchunhyang Biomedical Science Core Facility Center of Korea Basic

Science Institute (KBSI). The PCR primer sequences used in this study are listed in Table S1 in the supplemental material.

Western blot analysis Cells were lysed with freshly prepared 1x Laemmli lysis buffer (60 mM Tris-HCl, pH-6.8; 10% W/V Glycerol; 2% SDS; 0.01% Bromophenol blue) mixed with 5% β-mercaptoethanol. The

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western blot analysis was performed as described previously (Han et al., 2011). Briefly, the cell lysates were separated by 8%–10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% skim milk in 1x phosphate-buffered

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saline containing 0.1% Tween 20 for 1 h at 22-24 °C with gentle rocking. The membranes were incubated overnight with primary antibodies at 4 °C with gentle rocking. Secondary antibodies

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conjugated with horseradish peroxidase were applied to the membranes, which were then incubated for 2 h at room temperature. After washing with 1x PBS-T, the membranes were

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incubated with ECLTM prime western blotting detection reagent (GE Healthcare, Buckinghamshire, UK) and the signal was recorded immediately using an Amersham Imager 600 (GE Healthcare).

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The images were quantified using the ImageJ software. Antibodies against ENPP2 (1:1,000 dilution, sc-66813, Santa Cruz Biotechnology, Santa Cruz, CA, USA), myogenin (MYOG) (F5D, 0.3 µg/mL; Developmental Studies Hybridoma Bank), PAX7 (0.3 µg/mL; Developmental Studies Hybridoma Bank), β-Tubulin (1:1,000 dilution, sc-5274, Santa Cruz Biotechnology), myosin

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heavy chain (MyHC) (MF20, 0.03 µg/mL; Developmental Studies Hybridoma Bank), MyoD (1:1000 dilution; sc-32758, Santa Cruz Biotechnology), β-Actin (1:5,000 dilution, A5441, Sigma), and GAPDH (1:5000 dilution, sc-25778; Santa Cruz Biotechnology) were used.

Construction of the Enpp2-luciferase reporter plasmids and luciferase assay Mouse Enpp2 genomic DNA spanning from bp -3860 to bp +213, containing the promoter and a portion of exon 1, was amplified by PCR. To create the Enpp2-Luc reporter construct, the amplified DNA fragment was inserted into the upstream of the luciferase gene in the pGL3 luciferase reporter plasmid (Promega, Madison, WI, USA). Other Enpp2-Luc derivatives were also constructed using PCR amplification. Promoter activity was studied by measuring the luciferase

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activity using the dual-luciferase assay kit (Promega), according to the manufacturer’s instructions. Renilla luciferase-thymidine kinase DNA (RL-TK) was used as a transfection control.

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Chromatin Immunoprecipitation Assay

C2C12 cells in two 100-mm dishes, incubated for 24 h in the absence or presence of

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RSPO2/WNT3A, were fixed with 2.7% formaldehyde for 10 min at room temperature, followed by 5 min of blocking in 125 mM glycine. Nuclei preparation was carried out by sonicating the

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cells with Covaris E220 Ultrasonicator for 12 pulses (each for 20 s), followed by resting for 30 s at 4 °C to obtain a sheared fragment size distribution of 100–800 bp. Each pulse was characterized

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by 70 W power, 20% duty factor, and 200 cycles/burst at 4 °C. Chromatin extraction and immunoprecipitation were performed according to the manufacturer’s protocol, using a SimpleChIP Enzymatic chromatin IP kit (magnetic beads) (Cell Signaling Technology, Danvers, MA, USA). Anti-β-Catenin antibody (D10A8, #8480, 1:25 dilution; Cell Signaling Technology),

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positive control Anti-Histone H3 antibody (D2B12XP, #4620, 1 µg/IP sample; Cell Signaling

Technology), and a negative-control normal rabbit antibody IgG (#2729, 1 µg/IP sample; Cell Signaling Technology) were used for chromatin immunoprecipitation. The primer sequences used

to amplify the Enpp2 genomic DNA isolated from the immunoprecipitated chromatin are listed in Table S2 in the supplemental materials. Mice and acute muscle injury Enpp2flox/flox mice (Tanaka et al., 2006) were kindly provided by Dr. Susan Smyth (University of Kentucky, USA) with permission from Dr. Wouter Moolenaar (Netherlands Cancer Institute, Netherland). CAG-Cre-ERT mice were obtained from the Jackson Laboratory (Bar

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Harbor, ME, USA). Enpp2flox/flox;CAG-Cre-ERT mice were produced by mating between Enpp2flox/flox and Enpp2flox/+;CAG-Cre-ERT mice. Genotyping for Enpp2flox and Enpp2∆Ex6-7 alleles was performed using the primers listed below. For wild type and Enpp2flox alleles, 5’(forward)

and

5'-ACAGACTTCTCTGAAGCTGAC-3'

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CATTTCCATTCCCTGCTCC-3’

(reverse) primers, which produced the 441 bp (wild type) and 540 bp (Enpp2flox) PCR products,

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were used. The Enpp2∆Ex6-7 allele was genotyped using 5'- GCACATACCTTTAATTCCAGCAC-

380 bp PCR product.

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3' (forward) and 5'-ACAGACTTCTCTGAAGCTGAC-3' (reverse) primers, which generated the

Enpp2flox/flox;CAG-Cre-ERT mice aged 12–13 weeks were used for muscle regeneration

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experiments. The mice were injected intraperitoneally with 75 mg/kg of tamoxifen (TMX, 20 mg/mL in corn oil, Sigma-Aldrich) or corn oil every 24 h for five consecutive days. At 3–4 days after the last TMX injection, 20 µL of 1.2% barium chloride (BaCl2, Sigma-Aldrich) was injected directly into the TA muscle of anesthetized mice. After 10 days of injury, mice were euthanized

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and their TA muscles were harvested for preparation of cryosections and RNA isolation. The mice were housed in a pathogen-free air-barrier facility. The handling and all

experimental protocols for the mice were approved by the Soonchunhyang University Animal Care

and Use Committee. The health status of all the mice used here was normal, and they were not involved in any previous experiments.

Hematoxylin and eosin staining and immunofluorescence staining Cryosections (15 µm thickness) of TA muscle were stained for 2 min in Gill’s No. 2 hematoxylin (Sigma-Aldrich), 8 min in eosin (Sigma-Aldrich), dehydrated with ethanol, and then

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cleared using Histo-clearing solution (Sigma-Aldrich). Images were taken using a Nikon digital SLR camera (DS-i2) attached to Nikon Eclipse Ti-U inverted microscope at the Soonchunhyang Biomedical Science Core Facility Center of Korea Basic Science Institute (KBSI).

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Immunofluorescence staining of the cultured cells and cryosections was performed as described previously (Han et al., 2011). The samples were fixed with 3.7% formaldehyde in 1x

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PBS for 15 min, and then washed and blocked with 1x PBS with 5% normal goat or donkey serum and 0.3% Triton X-100 for 1 h. Primary antibodies against myogenin (MYOG) (F5D, 3 µg/mL;

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Developmental Studies Hybridoma Bank, University of Iowa, IA, USA), sarcomeric myosin heavy chain (MyHC) (MF20, 0.3 µg/mL; Developmental Studies Hybridoma Bank), PAX7 (5 µg/mL;

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Developmental Studies Hybridoma Bank), and Laminin (1:50 dilution; Sigma-Aldrich) were used. The primary antibodies were incubated overnight at 4 °C. Secondary anti-mouse IgG antibody conjugated with Cy3 (1:400 dilution; Jackson Immuno-Research Laboratories, West Grove, PA, USA) and anti-rabbit IgG conjugated with Alexa Fluor 488 (1:400 dilution; Jackson Immuno-

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Research Laboratories) were used. Cell nuclei were stained with 4',6-diamidino-2-phenylindole

(DAPI; Invitrogen, Waltham, MA, USA). The cell images were obtained using a Nikon digital SLR camera (DS-i2) attached to Nikon eclipse Ti-U inverted microscope. The counting and measurements were carried out using ImageJ software.

Statistical analysis All in vivo experiments were performed with three biological replicates (as indicated in the figure legends) and the results are presented as mean ± SEM (standard error mean). The sample size for each experiment is indicated in the figure legends. The experimental groups were compared using two-tailed Student’s t-test. The graph and bar diagram were generated using Graphpad Prism software. P-values are indicated as *P ≤ 0.05, **P ≤ 0.01, and ***P ≤ 0.001, and

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P-values < 0.05 were considered statistically significant.

Results Inhibition of ENPP2 function prevents myogenic differentiation We previously reported that the Enpp2 gene is induced by WNT-signaling activator RSPO2 in the C2C12 myoblast cell line (Han et al., 2014). As RSPO2 enhances myogenic differentiation, there is a reasonable possibility that Enpp2 is a key mediator of RSPO2 activity and plays a positive role during myogenic differentiation. Therefore, we first examined ENPP2

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protein expression during myogenic differentiation in C2C12 cells. While ENPP2 was expressed at a very low level in undifferentiated cells, its expression increased robustly as soon as the differentiation process began and steadily increased during differentiation (Fig. 1A), suggesting

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that ENPP2 may play a role during myogenic differentiation.

To investigate whether ENPP2 function is essential for myogenic differentiation, we

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differentiated C2C12 cells in the presence of a pharmacological ENPP2 inhibitor, HA-130, up to day 5, and studied the onset and progression of myogenic differentiation by examining the

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expression of myogenic differentiation markers by western blot analysis (Fig. 1B). Reduced levels of an early differentiation marker, myogenin, were observed in HA-130-treated cells throughout

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the differentiation period. Consistent with the inhibition of early differentiation, the expression of a terminal differentiation marker, the sarcomeric Myosin heavy chain (MyHC), was also severely suppressed in HA-130 treated cells. However, MYOD expression was not significantly affected by the inhibitor. Interestingly, ENPP2 expression was significantly reduced by HA-130. This result

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suggests that ENPP2 enzymatic activity positively regulates ENPP2 expression. In contrast, as ENPP2 expression increased during myogenic differentiation, the reduced ENPP2 expression in HA-130 treated cells simply reflects poor myogenic differentiation. Next, we investigated the myogenic differentiation process by measuring the myogenic differentiation (the proportion of myogenin or MyHC-positive nuclei in total nuclei) and cell

fusion (the distribution of nuclei in MyHC-positive cells) indices by immunofluorescent staining of differentiating C2C12 cells treated with HA-130 or another pharmacological ENPP2 inhibitor, PF8380. Consistent with the results from western blot analysis, a reduced number of myogeninpositive cells was observed in HA-130 treated cultures at day 1 of differentiation (Fig. 1C, D). At day 4 of myogenic differentiation, the differentiation index was significantly lower in HA-130 treated cells than in control cells (Fig. 1C, E), suggesting that ENPP2 inhibition significantly

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diminished the onset and progression of myogenic differentiation. In addition, the process of cell fusion was also severely affected by HA-130 treatment (Fig. 1F). There was an accumulation of mononuclear cells, and simultaneously, the number of myofibers containing more than five nuclei

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was significantly decreased in the HA-130 treated cells. We observed a similar inhibitory effect of another specific pharmacological ENPP2 inhibitor, PF8380, on myogenic differentiation and cell

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fusion (Supplementary Fig. 1). Taken together, we concluded that ENPP2 function is required for myogenic differentiation.

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To investigate whether Enpp2 acts in a cell-autonomous manner during myogenic differentiation, we generated a C2C12 cell line expressing shRNA specific to the Enpp2 (Enpp2-

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KD) gene, by lentiviral transduction. qRT-PCR revealed a gene knockdown (KD) efficiency of over 75% in the Enpp2-KD cells, compared with the control cells expressing eGFP-specific shRNA (eGFP-KD) (Fig. 2A). Consistent to this result, ENPP2 protein expression was also severely reduced in Enpp2-KD cells compared to eGFP-KD cells (Fig. 2B). We determined the

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myogenic differentiation efficiency of Enpp2-KD cells in comparison with the eGFP-KD cells by

MyHC immunofluorescent staining. We observed a significant decrease in the level of differentiation in the Enpp2-KD cells (Fig. 2B, C). The cell fusion index in Enpp2-KD cells showed a considerable shift towards reduced nuclei number, compared with the eGFP-KD cells

(Fig. 2D). We further confirmed this observation of defective myogenic differentiation by examining the expression of several myogenic markers by western blot analysis (Fig. 2E). The expression of myogenin and MyHC were either severely reduced or abolished in the Enpp2-KD cells during myogenic differentiation, whereas MYOD expression remained unchanged. Collectively, these results indicate that Enpp2 function is required for myogenic differentiation in

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a cell-autonomous manner.

Recombinant ENPP2 protein mildly induces myogenic differentiation

To determine whether ENPP2 promotes myogenic differentiation, we measured the

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myogenic differentiation and cell fusion indices, as well as the expression of myogenic differentiation markers, in differentiated C2C12 cells treated with recombinant ENPP2 (rENPP2)

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protein (Fig. 3). It is known that LPA has a feedback inhibitory effect on enzymatic function of ENPP2 (Benesch et al., 2015), and the serum contains a significant amount of LPA(Hama et al.,

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2002). To prevent the feedback inhibition by serum LPA on the rENPP2 protein, we used charcoalstripped low lipid horse serum instead of normal horse serum as the differentiation medium. First,

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the expression of MyHC and myogenin proteins were analyzed in differentiating C2C12 cells treated rENPP2 by western blot. rENPP2 protein treatment did not change the level of myogenic marker expression (Fig. 3A). However, rENPP2 treatment mildly but significantly increased both myogenic differentiation and cell fusion indices compared with the control (Fig. 3B to D),

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indicating that rENPP2 can promote myogenic differentiation and cell fusion.

ENPP2 mediates the signal to induce myogenic differentiation through LPA

Next, we investigated whether impaired myogenic differentiation caused by ENPP2 inhibitor can be rescued by exogenous LPA. Compared with the control, myogenic differentiation and fusion indices were significantly reduced by HA-130 treatment, as shown above (Fig. 4). In contrast, these indices were significantly increased in LPA-treated cells. A robust enhancement in cell fusion was also noted, as the number of myofibers with more than five nuclei was considerably higher than that in control and HA-130 treated cells (Fig. 4C). More importantly, LPA treatment

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effectively rescued the differentiation defects produced by HA-130 treatment to a level comparable to untreated control cells. Differentiation index in LPA and HA-130 co-treated cells was significantly higher than in control and HA-130 treated cells (Fig. 4B). Reduced cell fusion index

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in HA-130 cells also recovered to the level of control cells upon LPA treatment (Fig. 4C).

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Loss of Enpp2 gene results in defective muscle regeneration in mice

To determine the role of Enpp2 gene during muscle regeneration, we first examined Enpp2

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expression during muscle regeneration. Enpp2 expression was transiently induced, with a peak at day 4, overlapping with the expression of myogenic commitment marker, myogenin, after injury

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during muscle regeneration (Fig. 5A). This stage of muscle regeneration typically involves a robust expansion of myogenic progenitors and initiation of myogenic differentiation (Musarò, 2014). We next analyzed muscle regeneration in the Enpp2 gene knockout mice. As conventional Enpp2 gene ablation results in embryonic lethality (Tanaka et al., 2006), we used the conditional gene knockout

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(CKO) approach to determine the Enpp2 gene function during muscle regeneration in adult mice. We deleted the Enpp2 gene in a whole-body format rather than skeletal muscle-specific manner, because we wanted to avoid any effects of the circulating ENPP2 protein derived from non-skeletal muscle tissues. We administered tamoxifen into Enpp2fl/fl;CAG-CreERT compound mice for five

consecutive days, as described in the materials and methods. No notable health issues and behavioral phenotypes were observed in mice after tamoxifen injection. Genomic DNA PCR and Enpp2 expression by qRT-PCR confirmed a successful CRE recombination of the Enpp2flox allele (Supplementary Fig. 2). Tibialis anterior (TA) muscles of Enpp2 CKO mice were injected with BaCl2 to induce acute muscle injury, and harvested at days 4 and 10 after injury (D4 and D10) (Fig. 5B). We

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analyzed muscle regeneration by measuring the cross-section area (CSA) of nascent myofibers (depicted by centered nuclei) at D10 after hematoxylin/eosin staining and immunofluorescence staining for laminin expression. Compared with the TA muscle isolated from control mice, the

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CSA of Enpp2 CKO TA myofibers was significantly reduced (Fig. 5C-E). Expression of myogenic genes was further assessed using qRT-PCR in the TA muscle at D4 after injury (Fig. 5F).

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Expression of most of the myogenic markers, including cell fusion markers, was severely disrupted, suggesting that the myogenic differentiation program was severely impaired. At D10,

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several myogenic markers, including myogenin, Myhc3, and Myomerger, remained reduced in the Enpp2 mutant muscle, whereas expression of other markers was restored to the level of wild-type

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muscle (Supplementary Fig. 3). Taken together, our results showed that Enpp2 gene ablation in mice significantly compromises the muscle regeneration process, confirming an essential role of Enpp2 during muscle regeneration.

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Enpp2 is a direct transcription target of WNT/β-Catenin signaling in myoblasts We previously reported that Enpp2 expression is increased upon treatment with WNT

signaling activator, RSPO2, in C2C12 cells (Han et al., 2014). In addition, enhanced myogenic

differentiation by RSPO2 was not observed in Enpp2-KD C2C12 cells, suggesting that Enpp2 is likely a functional mediator of RSPO2 function (Supplementary Fig. 4). To determine whether the Enpp2 is a direct target of WNT/β-Catenin signaling, we examined Enpp2 gene expression in C2C12 cells in which the β-Catenin-encoding Ctnnb1 gene was knocked-down by RNA interference. Ctnnb1 RNA expression, assessed by qRT-PCR, showed > 80% knockdown efficiency (Fig. 6A). A bona fide WNT/β-Catenin target, Axin2, was

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significantly down-regulated in Ctnnb1 knockdown cells (Fig. 6B), confirming the disruption of WNT/β-Catenin signaling. In these Ctnnb1 KD cells, RSPO2/WNT3A co-treatment was unable to

transfected with control non-target siRNA (Fig. 6C).

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induce Enpp2 expression, whereas its expression was increased by this treatment in C2C12 cells

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We next searched for potential TCF/LEF1 binding sites within the mouse Enpp2 gene region spanning from -3,866 bp of 5'-upstream sequences to +213 bp downstream sequences,

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including exon 1 (Fig. 6D). Seven highly conserved TCF/LEF1-binding elements (named TBE17) were identified within this region using the transcription factor binding site search program,

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Matinspector (Genomatix). Comparison of human and mouse genomic sequences by MUSSA (multi-species sequence analysis) program (available from Dr. Barbara Wold lab at Caltech) revealed that all these sites are well conserved in both human ENPP2 and mouse Enpp2 genes

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(Supplementary Fig. 5).

To determine whether the β-Catenin protein complexed with TCF/LEF1 transcription

factors binds to these TBE sites upon WNT signaling activation, we performed a ChIP assay using anti-β-Catenin antibody in C2C12 cells. To obtain a maximum activation of WNT/β-Catenin signaling, RSPO2 and WNT3A protein were cotreated. In C2C12 cells treated with

RSPO2/WNT3A for 24 h, specific binding of β-Catenin to the TBE1/2, TBE3, and TBE6 sites was detected from the chromatin immunoprecipitated with anti-β-Catenin antibody (Fig. 6E). In contrast, β-Catenin binding was not detected on the TBE4, TBE5, and TBE7 sites. The known βCatenin binding sites within the promoters of two WNT target genes, Axin2 and Follistatin, were also confirmed for β-Catenin binding in the same samples. None of the TBEs was detected in the chromatin immunoprecipitated with negative control IgG antibody from RSPO2/WNT3A-treated

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C2C12 cells or β-Catenin antibody from untreated cells (Fig. 6E). To further determine the function of these TBEs in Enpp2 expression, we created a series of mouse Enpp2 promoter-Luciferase reporter constructs, as shown in Fig. 7C. An Enpp2(-3.86)-

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Luc reporter containing all seven TBEs showed a prominent activation by RSPO2/WNT3A co-

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treatment (Fig. 6F). However, the Enpp2(-2.85)-Luc construct with deleted TBE5, TBE6, and TBE7 sites showed no significant activation upon RSPO2/WNT3A treatment, suggesting that the

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TBE6 site is functionally important for Enpp2 expression. Interestingly, Enpp2(-0.65)-Luc reporter containing the TBE1, TBE2, and TBE3 sites showed a reduced but significant response

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to RSPO2/WNT3A treatment. When the DNA region containing the TBE6 site (A region) was linked to the Enpp2(-0.65)-Luc reporter, this reporter (Enpp2(A+B)-Luc) was activated by RSPO2/WNT3A to the level equivalent to the Enpp2(-3.86)-Luc reporter. However, Enpp2(A)Luc did not show any response to RSPO2/WNT3A treatment. Collectively, these data strongly

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suggest that the Enpp2 gene is directly regulated by WNT/β-Catenin signaling through the multiple TBE sites identified by ChΙP within the Enpp2 promoter.

Discussion Essential role of ENPP2 in myogenic differentiation An active role of ENPP2, a key enzyme that produces the active biolipid LPA from LPC, has been identified in many developmental and physiological processes (Yuelling and Fuss, 2008). However, until now, no study had investigated the role of ENPP2 in skeletal myogenesis. In contrast, the functional roles of LPA, the product of ENPP2 enzyme, have been investigated in

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skeletal myogenesis. Interestingly, LPA appears to play complex roles during skeletal myogenesis. While several studies have demonstrated that LPA positively regulates myoblast proliferation (Bernacchioni et al., 2018; Jean-Baptiste et al., 2005; Tsukahara and Haniu, 2012; Xu et al., 2008;

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Yoshida et al., 1996), other studies have reported conflicting results, that LPA rather promotes

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myogenic differentiation without affecting cell proliferation (Kleger et al., 2011; Lynch et al., 2013). As LPA transmits its signal through different LPARs, these discrepancies may arise from

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the use of different myoblast cell types that may express different LPARs, eventually leading to different cellular outcomes. Differences in experimental conditions may also lead to the

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differences in outcomes.

In the present study, we found that ENPP2 plays a key role in myogenic differentiation processes, including myocyte fusion. Inhibition of ENPP2 by pharmacological inhibitors and RNA

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interference or treatment of recombinant ENPP2 protein convincingly showed that both myogenic differentiation initiation and myocyte fusion processes are negatively affected by ENPP2 inhibition in C2C12 cells. ENPP2 protein expression gradually increases during myogenic differentiation. Therefore, ENPP2 function may be associated with both myogenic differentiation initiation and myocyte fusion in C2C12 cells. Our observation is consistent with a previous report

that LPA increases C2C12-derived myocyte fusion by inducing the migration of cells accompanying nuclear centromere orientation towards the direction of migration (Chang et al., 2015). Consistent with the in vitro results, mice lacking the Enpp2 gene exhibited impaired muscle regeneration after acute muscle injury, displaying cellular and molecular signatures of disrupted myogenic differentiation, including myocyte fusion. Reduced expression of myogenic differentiation markers such as Pax7, MyoD, and Myogenin suggests that the initiation of

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myogenic differentiation was inhibited. Downregulation of Tmem8c and Myomerger at D4 and D10, and upregulation of Myhc3 at D10, indicate that the maturation of nascent myofibers may be

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delayed.

In some earlier studies, LPA is shown to stimulate the proliferation but not differentiation

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of myogenic cells (Xu et al., 2008; Yoshida et al., 1996). Since LPA is a major product of ENPP2 enzyme, our result is in contradiction with these earlier studies. As it has been previously

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demonstrated that WNT/β-Catenin signaling does not stimulate the proliferation of myogenic cells and rather enhances myogenic differentiation (Anakwe et al., 2003; Brack et al., 2008; Brack et

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al., 2009; Doi et al., 2014; Han et al., 2014; Huraskin et al., 2016; Kim et al., 2008; Tanaka et al., 2011; Yu et al., 2013), it is more plausible that WNT/β-Catenin signaling-induced Enpp2 enhances myogenic differentiation rather than proliferation.

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Enpp2 is a direct target of WNT/β-Catenin signaling We previously showed that Enpp2 gene expression is induced by a positive WNT

signaling regulator, RSPO2 (Han et al., 2014). A number of earlier studies also reported elevated levels of Enpp2 expression when β-Catenin signaling was activated or enhanced, especially in

different tumor cells (Cao et al., 2017; Chamorro et al., 2005; Richards et al., 2012; Tice et al., 2002; Wang et al., 2015; Zirn et al., 2006). For instance, Enpp2 expression was elevated approximately 4.3-fold in WNT1-treated C57MG mammary epithelial cells and 18-fold in ovaries expressing a stable form of β-Catenin (Cao et al., 2017; Chamorro et al., 2005; Richards et al., 2012; Tice et al., 2002; Wang et al., 2015; Zirn et al., 2006). However, none of earlier studies attempted to further determine whether Enpp2 is a direct target gene for activation of WNT/β-

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Catenin signaling. In this study, using a number of in vitro approaches, we confirm that Enpp2 is a direct target of WNT/β-Catenin signaling in myoblast cells. Myoblast cells in which the Ctnnb1 gene encoding β-Catenin was knocked-down by RNAi were no longer capable of inducing Enpp2

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expression by WNT signaling stimulation. Furthermore, using the ChIP and promoter reporter assays, we showed that the mouse Enpp2 gene region spanning from -650 bp of 5'-upstream to

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+213 bp downstream sequence are responsive to WNT3A/RSPO2 stimulation and contains three TCF/LEF1-binding sites complexed with the β-Catenin protein. These sites are highly conserved

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between the mouse and human Enpp2 gene sequences, suggesting that WNT/β-Catenin signaling

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regulates the Enpp2/ENPP2 gene expression in a similar fashion in mouse and human cells. While our study provides conclusive evidence linking WNT/β-Catenin signaling to the Enpp2 gene that regulates LPA production and signaling in myogenic cells, there is also mounting evidence suggesting that LPA regulates β-Catenin signaling in various non-myogenic cells (Badri

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and Lama, 2012; Burkhalter et al., 2015; Cao et al., 2017; Chiang et al., 2011; Guo et al., 2015; Yang et al., 2005). In human colon cancer cells, LPA stimulates cell proliferation by activation of β-Catenin via phosphorylation of Ser552 and Ser 675 (Guo et al., 2015), the phosphorylation sites unrelated to the WNT-stimulated phosphorylation sites clustered within the N-terminus of the βCatenin protein. β-Catenin carrying mutations on Ser552/675 responded correctly to WNT

stimulation, but failed to mediate LPA stimulation. Therefore, WNT and LPA stimulations converge to β-Catenin through these distinct phosphorylation events. Does LPA regulate β-Catenin in a similar manner in myogenic cells? We did not examine whether LPA regulates β-Catenin signaling in myogenic cells in this study. However, considering the positive roles of Enpp2 shown in this study and WNT/β-Catenin signaling (Anakwe et al., 2003; Brack et al., 2008; Brack et al., 2009; Doi et al., 2014; Han et al., 2011; Huraskin et al., 2016; Kim et al., 2008; Tanaka et al., 2011;

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Yu et al., 2013) in myogenic differentiation, the likelihood of positive feedback crosstalk between ENPP2/LPA and WNT/β-Catenin during myogenic differentiation is very high. This part of study

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is currently ongoing in our laboratory.

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Acknowledgements

This study was supported by a Global Research Development Program grant

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(2016K1A4A3914725) and a research grant (2016R1A2B4012956) from the National Research Foundation of Korea, as well as grants from the National Institute of Health of USA

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(5R01AR055278 and 8P20 GM103465) to JK Yoon. We thank to the members of Yoon

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laboratory for their assistance in the mice experiments.

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Figure legends Figure 1. Inhibition of Enpp2 function prevents myogenic differentiation. (A) ENPP2 protein expression in C2C12 cells during myogenic differentiation. Cell lysates prepared from exponentially growing, undifferentiated (Un) and differentiating C2C12 cells at different time points (D1–D4) cultured in duplicate in 12-well plates. Expression of the ENPP2 and a loading control β-actin protein was determined by western blot analysis. (B) Effect of ENPP2

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inhibition on the expression of myogenic protein markers in differentiating C2C12 cells. C2C12 cells were differentiated in the presence of an ENPP2 inhibitor, HA-130 (10 µM), or the vehicle (DMSO), for up to 5 days, and harvested at days 1, 3, and 5, respectively. ENPP2 and myogenic

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marker expression was determined by western blot analysis. Experiments were performed three

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times independently and similar results were obtained. (C–F) Immunofluorescence staining for myogenic markers in differentiating C2C12 cells treated with HA-130 (10 µM) or DMSO at days

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1 (D1) and 4 (D4). Cells were stained with anti-Myogenin (MYOG) and anti-MyHC primary antibodies, followed by Alexa Flour 488- and Cy3- conjugated secondary antibodies. Cell nuclei

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were counterstained with DAPI. Scale bars represent 100 µm. MYOG-positive cells (D) and MyHC-positive nuclei (E) are presented as the percentage of total nuclei number. Cell fusion index is presented as the distribution of MyHC-positive cells based on cell nuclei number: mono, 2–4, and ≥ 5 nuclei. More than 500 total nuclei were counted per well in triplicate wells of 24-well

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plates. Error bars indicate SEM. ***, P < 0.001; **, P < 0.01; *, P < 0.05.

Figure 2. Enpp2 acts in a cell-autonomous manner during myogenic differentiation.

(A) Enpp2 gene knockdown (KD) in C2C12 cells. Enpp2 gene knockdown efficiency was determined by qRT-PCR using RNA samples isolated from cells growing in 24-well plates in triplicate. Enpp2 RNA expression was normalized by Gapdh expression. C2C12 cells expressing eGFP-specific shRNA (eGFP KD) are used as control. (B-D) Immunofluorescence staining for MyHC expression in differentiated C2C12 cells in 24-well plates. Cell nuclei were counterstained with DAPI. Scale bars represent 100 µm. (C) Myogenic differentiation and (D) cell fusion indices

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are presented. More than 500 total nuclei were counted per well in triplicate. Error bars indicate SEM. ***, P < 0.001; **, P < 0.01; *, P < 0.05. (E) ENPP2 and myogenic marker expression in control eGFP and Enpp2 KD cells was determined by western blot analysis. * indicates non-

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specific signal. β-Tubulin expression was analyzed for the loading control. Experiments were

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performed two times independently and similar results were obtained.

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Figure 3. Recombinant ENPP2 mildly enhances myogenic differentiation. (A) C2C12 cells were differentiated in the presence of recombinant ENPP2 protein for up to 5 days, and harvested at days 1, 3, and 5, respectively. Expression of myogenic markers were

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analyzed by western blot assay in cell lysates prepared as described above. β-Tubulin expression was analyzed for the loading control. Experiments were performed two times independently and similar results were obtained. (B) Immunofluorescence staining for MyHC expression in

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differentiated C2C12 cells treated with recombinant human ENPP2 protein. C2C12 cells differentiate for 3 days in the presence of the ENPP2 protein (200 ng/mL) or BSA. Cells were stained with anti-MyHC (MF20) primary antibody and cell nuclei were counterstained with DAPI. Scale bar represents 100 µm. (C-D) Myogenic differentiation and cell fusion indices are presented.

At least 500 total nuclei were counted per well in triplicate wells. Error bars indicate SEM. *, P < 0.05.

Figure 4. LPA rescues ENPP2 inhibition during myogenic differentiation. (A) Immunofluorescence staining for MyHC expression in differentiated C2C12 cells treated with

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HA-130 (10 µM), LPA (20 µM), or both for 3 days in 24-well plates. Scale bar represents 100 µm. (B) Myogenic differentiation and (C) cell fusion indices were obtained from counting at least 500 total nuclei per well in triplicate. Error bars indicate SEM. ***, P < 0.001; **, P < 0.01; *, P <

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0.05.

Figure 5. Loss of the Enpp2 gene impairs muscle regeneration in mice.

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(A) Enpp2 mRNA expression during muscle regeneration. Total RNA isolated from normal (n=3) and regenerating (n=3) TA muscles after cardiotoxin-induced injury at days 2, 4, 7, and 15. Both

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Enpp2 and myogenin expression was normalized by Gapdh expression. (B) Schematic diagram of muscle regeneration in conditional Enpp2 gene knockout mice. Male mice were injected daily with tamoxifen (Enpp2 gene knockout, n=3) or corn oil (control, n=3) intraperitoneally for five consecutive days. No notable health issues and behavioral phenotypes were observed in mice after

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Enpp2 gene ablation. TA muscle was injured by injecting 20 µL of 2% barium chloride at day 3 after the last tamoxifen injection. At days 4 and 10 after injury, TA muscles were harvested and processed for cryosections and total RNA preparation. (C) Cryosections of the TA muscle (D10) were stained with hematoxylin and eosin (H&E) for histological analysis. Anti-Laminin antibody was also used for immunostaining the periphery of myofibers and the nuclei were counter-stained

with DAPI. Scale bars represent 100 µm. (D-E) Distribution of cross-sectional area (CSA) of regenerating myofibers and the mean of the CSA average obtained from three control and Enpp2 gene knockout mice, respectively. (G) Gene expression in regenerating TA muscle of D4 from control (n=3) and Enpp2KO (n=3) mice was analyzed by qRT-PCR. Gapdh expression was used as the normalization control. Error bars are SEM. ***, P < 0.001; **, P < 0.01; *, P < 0.05; NS,

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no statistical significance.

Figure 6. The Enpp2 gene is a direct transcription target of WNT/β-Catenin signaling in

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myoblasts.

(A) Effective knockdown of the Ctnnb1 gene encoding β-Catenin. C2C12 cells were transiently

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transfected with control non-targeting (nt) siRNA or siRNA specific to the Ctnnb1 gene in 24-well plates. Ctnnb1 RNA expression was determined by qRT-PCR and normalized by Gapdh gene

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expression. Samples were prepared in triplicate. (B-C) RNA expression in the Ctnnb1knockdowned C2C12 cells. C2C12 cells transiently transfected with siRNA were treated with

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recombinant RSPO2 protein (200 ng/mL) for one day. Axin2 (B) and Enpp2 (C) gene expression were analyzed by qRT-PCR. Gapdh gene expression was used for normalization. Samples were prepared in triplicate. (D) Schematic structures of various Enpp2-luciferase (Luc) reporter gene constructs. Mouse Enpp2 genomic DNA containing 3.8 kb 5’-upstream and 0.26 kb of a portion

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of exon 1 was cloned into pGL3 basic plasmid. Within 4.07 kb, seven conserved TCF/LEF1binding elements (TBEs) were identified. The TBE sites confirmed by β-Catenin antibody-

mediated chromatin immunoprecipitation are indicated as red-colored bars. (E) Detection of βCatenin binding to the TCF/LEF1 sites in the Enpp2 gene by chromatin immunoprecipitation assay. C2C12 cells were treated with recombinant RSPO2 (100 ng/mL) and WNT3A (20 ng/mL) proteins

before chromatin preparation. Chromatin was successively immunoprecipitated with anti-βCatenin, anti-Histone H3 (positive control), and normal rabbit IgG (negative control) antibodies. DNA extracted from the immunoprecipitated chromatins was amplified by PCR, using primer sets spanning the potential TBEs on genomic DNA of the mouse Enpp2 gene. PCR efficiency and accuracy were also determined using the genomic DNA isolated from the chromatin, prior to immunoprecipitation (input). Fst-T1/T2 and Axin2-T represent the TBEs identified in the previous

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studies and used for confirming the effectiveness of β-Catenin chromatin immunoprecipitation. Experiments were performed in triplicate and identical results were obtained. (F) Luciferase activities of the Enpp2-Luc reporter constructs. C2C12 cells were transfected with the various

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Enpp2-Luc reporter constructs (300 ng/well in a 24-well plate) and TK-Renilla luciferase plasmid (200 ng/well) as a normalization control. Transfected cells were treated with proteins RSPO2 (100

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ng/mL) and WNT3A (20 ng/mL) for 48 h before harvest. Firefly and Renilla luciferase activities were measured in triplicate samples. Error bars indicate SEM. ***, P < 0.001; **, P < 0.01; *, P <

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0.05; NS, no statistical significance.

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+

* **

Mono

+ +

Jo

ur na

lP

HA-130 LPA

Ctrl HA-130 LPA HA-130 + LPA

* **

re

***

*** Fusion index (%)

Differentiation index (%)

C

-p

B

2-4

≥5

Sah et.al., Fig. 5

B

CTX D0 D2 D4 D7 D15

Enpp2flox/flox;CAG-CreERT

Tamoxifen or corn oil Injection

-7

BaCl2

-3

Collect TA muscle

0

4

10 (day)

D M1

Wt

Enpp2 expression (fold)

Myogenin expression (fold)

A

M2

CTX D0 D2 D4 D7 D15

C Wt

Enpp2 KO

M1 M2

ro of

Enpp2 KO

M3

M3

F

Jo

ur na

lP

r ge er

m

yo

Tm

em

ck

8c

*

M

c3

*

M

yh

Wt KO

x7

0

*

*

M

0.5

-p

*

Pa

1.0

D yo g

1.5

M

2.0

Wt

Enpp2 KO

re

100μm

CSA (x 103 μm2)

LAMININ/DAPI

100μm

M

2.5

yo

E

RNA expression (fold)

H&E

CSA (μm2)

Sah et. al, Fig. 6

B

nt Ctnnb1

siRNA

T3 T2 T1 Exon 1

-3.86 kb

*

0.5 kb

+0.21 kb

A

B Luc

Enpp2(-3.86)-Luc

Luc

Enpp2(-2.85)-Luc

Luc

Enpp2(-0.65)-Luc

Luc

Enpp2(-0.05)-Luc

Luc

Enpp2(A+B)-Luc

Luc

Enpp2(A)-Luc

nt Ctnnb1

E

β-

T6 T7 Ctrl1 Ctrl2 Fst-T1/T2

***

Jo

ur na

lP

re

-p

ro of

Axin2-T

Relative luciferase activity (fold)

T4/5

***

6) -L uc p2 (- 2 .8 5) En -L pp uc 2( -0 .6 5) En -L pp uc 2( -0 .0 5) En -L pp uc 2( A+ B) -L En uc pp 2( A) -L uc

T3

Ctrl WNT3A+RSPO2

**

p2 (- 3 .8

T1/T2

En p

- + - + - + WNT3A+RSPO2

En p

t i-

An

pu

An

t

D In

Enpp2 RNA expression (fold)

Axin2 RNA expression (fold)

siRNA nt Ctnnb1 siRNA

***

T7 T6 T5 T4

Ctrl RSPO2

ca t C Hi Ab t rl st Ig H3 G Ab

**

D

C Ctrl RSPO2

t i-

Ctnnb1 RNA expression (fold)

A