ADAM10 Checks Myogenic Differentiation in Satellite Cells A Disintegrin and Metalloprotease 10 is Indispensable for Maintenance of the Muscle Satellite Cell Pool*

Satellite cells (SCs) are muscle-specific stem cells that are essential for the regeneration of damaged muscles. Although SCs have a robust capacity to regenerate myofibers, the number of SCs decreases with aging, leading to insufficient recovery after muscle injury. We herein show that ADAM10, a membrane-bound proteolytic enzyme with a critical role in Notch processing (S2 cleavage), is essential for the maintenance of SC quiescence. We generated mutant mice in which ADAM10 in SCs can be conditionally abrogated by tamoxifen injection. Tamoxifen-treated mutant mice did not show any apparent defects and grew normally under unchallenged conditions. However, these mice showed nearly complete loss of muscle regeneration after chemically induced muscle injury. In situ hybridization and flow cytometric analyses revealed that the mutant mice had significantly less SCs compared to wild type controls. Of note, we found that inactivation of ADAM10 in SCs severely compromised Notch 1 http://www.jbc.org/cgi/doi/10.1074/jbc.M115.653477 The latest version is at JBC Papers in Press. Published on October 9, 2015 as Manuscript M115.653477 Copyright 2015 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on O cber 7, 2015 hp://w w w .jb.org/ D ow nladed from ADAM10 Checks Myogenic Differentiation in Satellite Cells signaling and led to dysregulated myogenic differentiation, ultimately resulting in deprivation of the SC pool in vivo. Taken together, the present findings underscore the role of ADAM10 as an indispensable component of Notch signaling in SCs and for maintaining the SC pool. _______________________________________ Satellite cells (SCs) are muscle-specific stem cells that reside between myofibers and the basement membrane with essential roles in repairing damaged muscle (1-3). Under normal conditions, the majority of SCs exist in a quiescent state or G0 phase. However, in response to injury, SCs rapidly proliferate and differentiate to form myofibers and repair damaged muscles. The number of SCs and their regenerative potency decrease with age, leading to insufficient recovery after muscle trauma. Since loss of muscle strength and plasticity can compromise activity of daily living, especially in the elderly, it is mandatory to learn more about the mechanisms underlying the maintenance of the number and the regenerative capacity of SCs. Past studies have identified various molecules that are potentially involved in the maintenance and aging of SCs, including p38 MAP kinase (4,5), Wnt (6), TGF-β (7), Jak-Stat (8), p16 (9), and Notch (10-12). Notch receptors and their ligands are highly conserved gene families and are critically involved in various cellular functions, including cell fate decision, cell growth, and differentiation (13,14). There are four Notch receptors (Notch 1-4) and five ligands (Jagged 1 and 2, and DLL 1, 3, and 4) in mammalian. Notch signaling is also involved in the maintenance of stem cells in certain type of tissues, and loss of Notch signaling in the stem cells in these tissues often results in dysregulated differentiation. Accordingly, studies in the past few years have shown that Notch signaling has a crucial role in in the maintenance of SCs and that suppression of this signaling pathway results in dysregulated differentiation of SCs and a decrease in the SC population (10-12). The Notch signaling pathway has a complex and unique mode of action in transmitting cell signaling. Since the receptors and ligands are all membrane-bound, Notch signaling is initiated through cell-cell contact between a receptor expressing cell and a ligand expressing cell. Upon activation by ligand binding, Notch receptors undergo sequential proteolytic cleavage by a disintegrin and metalloprotease (ADAM) and the γ-secretase complex (each responsible for S2 and S3 cleavage, respectively), releasing the intracellular domain. The intracellular domain translocates into the nucleus to form a complex with a transcriptional cofactor Rbpj-1 and functions as a transcriptional activator (14,15). There are more than 20 ADAM genes in mammals. Among these, ADAM10 and ADAM17 (also known as TNFα-converting enzyme or TACE) are probably best characterized as enzymes involved in the proteolytic release of membranebound proteins, a process also referred to as ectodomain shedding (16,17). These two genes are closely related to one another and have distinct and overlapping substrates. The identity of the ADAM protease responsible for S2 cleavage of the Notch receptor remains somewhat controversial. Several studies have suggested the potential involvement of 2 by gest on O cber 7, 2015 hp://w w w .jb.org/ D ow nladed from ADAM10 Checks Myogenic Differentiation in Satellite Cells ADAM17 in S2 cleavage (18,19). However, results from studies of ADAM10 mutant mice, which often exhibit Notch-related defects (20-23), suggest that ADAM10 is the principal enzyme responsible for S2 cleavage in vivo. In the present study, we aimed to clarify the potential roles of ADAM10 in SCs and muscle regeneration. We generated a mutant mouse line in which Adam10 can be inactivated specifically in SCs upon tamoxifen injection. The mutant mice did not exhibit any apparent defects under unchallenged conditions; however, these mice almost completely lacked the capacity for muscle generation after muscle injury. Most importantly, we found that the mutant mice are nearly devoid of SCs in skeletal muscle due to defective Notch signaling. Collectively, our data show that ADAM10 is indispensable for maintaining the SC population and for Notch signaling in SCs, and further consolidate the notion that ADAM10 as the major sheddase for Notch in vivo. EXPERIMENTAL PROCEDURES Mice The generation of Adam10 mice was previously described (20). Adam10 mice were crossed with Pax7/J transgenic mice (24) to specifically abrogate the Adam10 allele from SCs (henceforth referred to as Adam10 mice). Conditional excision of the floxed allele was achieved by intraperitoneal injection of tamoxifen (75 μg/kg; Toronto Research Chemicals, Toronto, Canada) dissolved in corn oil (20 mg/ml). For fate-mapping experiments, we crossed Adam10mice with CAG-CAT-EGFP reporter mice (25) (referred to as Adam10 mice), by which deletion of the floxed Adam10 allele and transcriptional activation of EGFP in SCs can be simultaneously achieved. As a control, we also generated mice hemizygous for both the Pax7Cre and EGFP transgenes by crossing Pax7/J transgenic mice and CAGCAT-EGFP reporter mice (henceforth referred to as WT mice). SC-specific expression of Cre recombinase was confirmed by analyzing the expression of EGFP and immunostaining for PAX7 in the muscle fibers collected from the WT mice (data not shown). The Adam10 mice exhibited no apparent defects and were used as control animals in the present study (henceforth referred to as Ctrl mice) (20). All animal experiments were approved by the Institutional Animal Care and Use Committee of the Keio University School of Medicine. Reagents and antibodies All siRNAs were purchased from Sigma-Aldrich (St. Louis, MO). The following antibodies were used: antiPAX7 (1:100, ab34360; Abcam, Cambridge, England), anti-activated Notch1 (1:400, ab8925; Abcam), anti-MyoD (1:50, sc-32758; Santa Cruz, Dallas, TX), anti-GFP (1:500, GF090R; Nacalai Tesque, Kyoto, Japan), anti-Perilipin (1:500, D1D8; Cell Signaling Technology), anti-ADAM10 (1:2000, 422751; EMD Millipore, Germany), and anti-GAPDH (1:5000, G9545; Sigma Aldrich). Flow cytometry Skeletal muscle from 8to 12-week-old mice was harvested for flow cytometric analysis. After visible fat tissues, vessels, nerves, and tendons were removed, the muscles were thoroughly chopped and digested in a mixture of collagenase (Wako Pure Chemical Industries, Osaka, Japan), dispase (Life Technologies, Carlsbad, CA), and CaCl2. Digested 3 by gest on O cber 7, 2015 hp://w w w .jb.org/ D ow nladed from ADAM10 Checks Myogenic Differentiation in Satellite Cells samples were filtered through cell strainers to remove debris, and red blood cells were removed using Red Blood Cell Lysis Buffer (Roche Life Science, Indianapolis, IN) before antibody application. The following fluorochromeconjugated monoclonal antibodies were used: antiSca1 (1:200, D7), anti-CD31 (1:80, MEC13.3), and anti-CD45 (1:1333, 30-F11). These antibodies were purchased from Biolegend. The biotinylatedSM/C2.6 monoclonal antibody was generously provided by Dr. S. Fukada (26). The flow cytometric analysis was performed using a Gallios Flow Cytometer (Beckman Coulter, Brea, CA). In situ hybridization Paraffin-embedded sections of cardiotoxin-treated TA muscles were used for in situ hybridization. The sections were deparaffinized and then probed for Pax7 transcripts using an RNAscope Fluorescent Multiplex Reagent Kit (Probe-Mm-Pax7; 314181; Advanced Cell Diagnostics, Hayward, CA) as instructed by the manufacturer. The sections were counterstained with Mayer’s hematoxylin. Muscle-injury modelThe mice were anesthetized with a peritoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). Muscle injury was induced with an intramuscular injection of 50 μl cardiotoxin/PBS (10 μM) in the tibialis anterior (TA) muscle. The mice were closely monitored until fully recovering from the anesthesia and treatment. Isolation, culture, and staining of SCsSCs were isolated from myofibers of the extensor digitorum longus muscles digested with type 1 collagenase (Worthington, Lakewood, NJ). The isolated fibers were cultured in DMEM, high glucose, GlutaMax Supplement (Life Technologies), 20% FBS, 1% chicken embryo extract (USBiological, Pittsburgh, PA), and antibiotics on dishes coated with Matrigel (Corning, Corning, NY). When necessary, 4hydroxytamoxifen (4-OHT, Sigma-Aldrich) diluted in ethanol (10 μM) was added to the medium. The SCs that were detached from the fibers were cultured for 3 to 4 days. The cells were fixed with 4% paraformaldehyde, permeated wit