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Mechanical loading induces expression of osteo-anabolic

and osteo-catabolic factors by myotubes:

Role in myogenic adaptation

Petra Juffer1

Astrid D. Bakker1 Jenneke Klein-Nulend1

Richard T. Jaspers2

1 Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam and VU University Amsterdam, MOVE Research Institute Amsterdam,

Amsterdam, The Netherlands

2 MOVE Research Institute Amsterdam, Faculty of Human Movement Sciences, VU University Amsterdam, Amsterdam, The Netherlands

Submitted for publication

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ABSTRACT

Muscle and bone share numerous signaling pathways in the regulation of bone and muscle mass. However, of some crucial bone mass regulatory factors, such as RANKL, it is not known whether they are expressed in muscle, or whether they have an effect on muscle physiology. In addition, for some factors that are expressed in muscle and bone alike, i.e. COX-2, BMPs, Wnt signaling proteins, it is known that they play an important role in adaptation of bone mass to mechanical loading, but it is yet unknown whether the expression of these factors is affected by mechanical loading in muscle. Therefore we aimed to investigate 1) whether mechanical loading by cyclic uni-axial strain (CS) affects the expression of COX-2, BMP2, BMP6, and 7, Wnt signaling proteins, and RANKL by C2C12 myotubes, and 2) whether C2C12 myotube metabolism is affected by RANKL. C2C12 myotubes were subjected to CS (15% 1 Hz, 1 h), and gene expression of COX-2, BMP2, BMP6, BMP7, Wnt signaling proteins, and RANKL was analyzed immediately after 1 h CS using PCR. Prostaglandin E2 (PGE2) concentrations in the medium were measured at 3 h and 6 h post-CS. C2C12 myotubes were treated with rmRANKL (0, 0.2, 2, and 20 ng/mL). After 2h and 20h RANKL treatment, samples were collected for mRNA, DNA, and total protein analysis. After 20h RANKL treatment myotube thickness was measured. CS increased COX-2 mRNA levels (2-fold), but CS did not affect PGE2 production or mRNA levels of BMPs in myotubes. CS increased mRNA levels of Wnt1 (70-fold), and Wnt7a (200-fold), and decreased mRNA levels of Wnt4 (8-fold), and RANKL (1.8-fold). Addition of RANKL to myotubes increased total protein content and decreased myotube diameter, possibly as a result of changes in expression of the NFκB target genes c-fos and NFATC1. Our data show that cyclic uni-axial strain affects the expression of COX-2 and Wnts signaling proteins, as well as a novel factor RANKL. These typical osteo-anabolic and osteo-catabolic factors are produced by muscle cells in response to mechanical loading, and may play a role in the regulation of muscle fiber size, but may also affect bone remodeling via paracrine and endocrine signaling.

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INTRODUCTION

Physical activity prevents or retards loss of muscle and bone mass as occurs during aging and in many musculoskeletal disorders (36, 39, 47). In muscle, muscle fibers and their satellite cells transduce mechanical stimuli into biochemical signals that affect the rate of protein turnover, resulting in an increased muscle fiber size (9). In bone, the major mechanosensing bone cells (i.e. osteocytes) respond to mechanical loading by producing signals that affect the bone forming osteoblasts and/or to the bone resorbing osteoclasts (17). Despite the major differences in cellular and structural organization of muscle and bone, the mechanisms via which muscle cells and bone cells sense mechanical load and transduce these signals into biochemical responses are similar in many aspects. Important anabolic and catabolic signaling molecules such as prostaglandins, BMP2, BMP6, BMP7, Wnt signaling proteins, and RANKL have been established to play a role in bone adaptation. However, of some crucial bone mass regulatory factors, such as RANKL, it is not known whether they are expressed in muscle, or whether they have an effect on muscle physiology. In addition, for some factors that are expressed and/or produced in muscle and bone alike, i.e. prostaglandins, COX-2, BMPs, Wnt signaling proteins, it is known that they play an important role in muscle physiology, but it is yet unknown whether the expression of these factors is affected by mechanical loading in muscle. The aim of study was to assess whether mechanical loading affects the expression of key osteo-anabolic and/or osteo-catabolic factors in myotubes. Therefore we analyzed the effects of cyclic uni-axial strain (CS) on the production of prostaglandin E2 (PGE2), and mRNA expression levels of COX-2, BMP2, BMP6, BMP7, Wnt signaling proteins as well as the most studied factor involved in bone degradation, (i.e. RANKL), by C2C12 differentiated myotubes. We hypothesized that mechanical loading alters the expression of these known osteo-anabolic factors and/or osteo-catabolic factors that RANKL plays a role in the regulation of muscle fiber size. These results may reveal novel signaling pathways involved in the regulation of muscle fiber size by mechanical loading.

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MATERIALS AND METHODS

Myotube cultureC2C12 myoblasts were cultured in Dulbecco’s Modified Eagle Medium (D-MEM; Gibco, Paisly, UK) supplemented with 10 µg/ml penicillin (Sigma-Aldrich, St. Louis, MO, USA), 10 µg/ml streptomycin (Sigma-Aldrich), 50 µg/ml fungizone (Gibco), and 10% fetal bovine serum (FBS; Gibco), at 37°C in a humidified atmosphere of 5% CO

2 in air. This “growth medium” was

exchanged every 3 to 4 days. Upon 70% confluence, cells were harvested using 0.25% trypsin and 0.1% EDTA in phosphate-buffered saline. For CS treatment, cells were seeded at 2x104 cells per well of laminin-coated 6-well Bioflex® plates (Dunn Labortechnik GmbH, Asbach, Germany). For RANKL treatment, cells were seeded at 2x104 cells per well of 6-well tissue culture plastic plates. For CS treatment as well as RANKL treatment, cells were cultured in “growth medium”. After 3 days of culture, “growth medium” was replaced by medium consisting of D-MEM supplemented with 10 µg/mL penicillin, 10 µg/mL streptomycin, 50 µg/mL fungizone, and 2% horse serum (Gibco). This “differentiation medium” was refreshed daily. After 3 more days of culture the wells were covered with differentiated mature myotubes. Differentiated myotubes were subjected to mechanical loading by CS, or treated with RANKL as described below.

Application of CSOne hour before CS loading or static control culture, the “differentiation medium” of C2C12 myotube cultures was replaced by D-MEM containing 2% FBS, 10 µg/mL penicillin, 10 µg/mL streptomycin, and 50 µg/mL fungizone. During CS and static control culture, C2C12 myotubes were incubated at 37ºC in a humidified atmosphere of 5% CO2 in air. CS was applied using the FlexCell® FX4000™ Tension system according to the manufacturer’s instructions (FlexCell® Int Corp, Hillsborough, NC). Due to a vacuum force, the Flexcell® induces the flexible membranes of the Bioflex® plates to stretch. Briefly, C2C12 myotubes (n=3) were subjected to a CS in a sinusoidal pattern with a frequency of 1 Hz, and a maximum elongation of 15% for 1 h, and post-incubated up to 24 h without mechanical stimulation (12).

RANKL treatmentDifferentiated C2C12 myotubes were cultured in “differentiation medium” without or with 0.2, 2, or 20 ng/mL recombinant murine RANKL (R&D Systems, Minneapolis, MN) for 2 h (short treatment) or for 20 h (long treatment). Three independent experiments were performed. After 20 h RANKL treatment, micrographs were taken and used to measure the thickness of the myotubes at three different locations along the length of the myotubes. The myotube diameter was expressed as the mean of these three values. Per experiment, thirty myotubes were analyzed. After 2 h and 20 h RANKL treatment, cells were lysed in TriPure (RiboPure™ Kit; Applied Biosystems, Foster City, CA, USA) for isolation of RNA, DNA, and total protein.

RNA isolation and real-time PCR Total RNA was isolated using RiboPure™ Kit (Applied Biosystems). Total RNA concentration

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was measured using a BioTek Synergy™ microplate reader (BioTek Instruments, Inc., Vermont, USA). mRNA was reverse-transcribed to complementary DNA (cDNA) using a High Capacity RNA-to-cDNA Kit (Applied Biosystems). Expression of Wnt signaling genes was measured using RT2 mouse Wnt signaling pathway PCR Array (Qiagen Hamburg GmbH, Hamburg, Germany), and performed according to the manufacturer’s protocol on pooled RNA samples of myotubes treated with or without CS from 3 independent experiments. BMP2, BMP6, BMP7, COX-2, RANKL, and GAPDH mRNA levels were measured using Taqman® qPCR using inventoried? Taqman® gene expression assays (Applied Biosystems). GAPDH was used as housekeeping gene. RANK, c-Fos, NFATc1, and MuRF1 mRNA levels were measured using sybr green. HPRT was used as housekeeping gene. Primer sequences were as follows: c-Fos, 5’-TCACCCTGCCCCTTCTCA and 5’-CTGATGCTCTTGACTGGCTCC; RANK, 5’-TGGGCTTCTTCTCAGATGTCTTT and 5’-TGCAGTTGGTCCAAGGTTTG; NFATc1, 5’-CATGCGAGCCATCATCGA and 5’-TGGGATGTGAACTCGGAAGAC; MuRF1, 5’-GGGCTACCTTCCTCTCAAGTGC and 5’-CGTCCAGAGCGTGTCTCACTC; and HPRT, 5’-CCTAAGATGAGCGCAAGTTGAA and 5’-CCACAGGACTAGAACACCTGCTAA.

DNA isolationDNA was isolated by conventional phenol-chloroform extraction. Total DNA concentration was measured using a BioTek Synergy™ microplate reader (BioTek Instruments, Winooski, VT).

Total Protein isolationTotal protein was isolated by conventional phenol-chloroform extraction. Total protein was detected using Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific. Inc, Rockford, IL) and measured by BioTek Synergy™ microplate reader (BioTek Instruments).

Prostaglandin E2 AssayProstaglandin E2 (PGE2) release was measured in medium obtained from culture of C2C12 myotubes subjected to 1 h CS or static control culture and 3 h or 6 h post-incubation. PGE2 was measured using PGE2 Biotrak Enzyme immunoassay (EIA) System (GE Healthcare, Buckinghamshire, UK).

Statistical analysisStatistical analyses were performed using SPSS 20 (SPSS Inc., Chicago, IL). Groups were compared using one way ANOVAs with Bonferroni-adjusted t-test as post hoc test. Differences were considered significant if p<0.05. All data are expressed as mean ± SEM.

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50

RESULTS AND DISCUSSION

Comparison of the so far known mechanisms underlying adaptation of muscle and bone suggests that similar signaling pathways exist that are involved in adaptation of muscle fiber size and bone mass. In bone several signaling molecules are expressed which are known to have a strong effect on osteoblast and/or osteoclast activity, e.g. PGE2, Wnt signaling proteins, BMP2, BMP4, BMP7, and RANKL. Some of these molecules, such as PGE2 and Wnt signaling proteins, are also expressed in skeletal muscle, but only little is known whether the expression of these molecules is altered in muscle cells in response to mechanical loading. Here we suggest a role for some major osteo-anabolic and catabolic regulatory growth factors and cytokines, i.e. PGE2, COX-2, Wnts, BMP, and RANKL, in the adaptation of muscle fiber size to mechanical loading.

CS of C2C12 myotubes does not alter PGE2 production, but increases COX-2 mRNA levelsProstaglandins play a key role in the regulation of bone mass and structure (34). PGE2 is the most abundant prostaglandin synthesized (34). PGE2 has great impact in bone as it promotes both bone resorption (high concentrations of PGE2) and bone formation (low concentrations of PGE2) (3). PGE2 is synthesized after activation of the enzyme cyclo-oxygenase, which is expressed as two isoforms, COX-1 and COX-2. Mechanical loading of bone results in increased PGE2 synthesis and COX-2 gene expression by osteocytes and osteoblasts (10, 46). In skeletal muscle, PGE2 regulates protein turnover by modulating the rate of protein degradation (38). PGE2 enhances the regeneration of injured skeletal muscle by stimulating the fusion of myogenic precursor cells (43). To test whether mechanical loading increases PGE2 production by myotubes, and whether this is accompanied by an increase in COX-2 expression by myotubes, cells were subjected for 1 h to CS, and post-incubated for 3 h and 6 h to measure medium concentrations of PGE2 produced by the cells. COX-2 mRNA levels in myotubes were measured at several time points up to 24h post-CS. CS did not change PGE2 concentrations in the medium (fig.1A). However, mRNA levels of the rate-limiting enzyme of PGE2 production after mechanical loading, i.e. COX-2, were increased (2-fold) in response to mechanical strain by C2C12 myotubes (fig. 1B). These data agree with observations by others that COX-2 levels are increased in skeletal muscle in response to exercise in vivo (48). Although COX-2 mRNA levels were clearly increased in response to CS in myotubes, there was no concomitant stimulation of PGE2 production. Possibly the mechanical loading regime used was not optimal for the activation of COX-2, and therefore did not increase PGE2 production. Since PGE2 production increases during muscle contractions (44), our data suggest that the mechanical stimulus that triggers PGE2 production in skeletal muscle in vivo is likely not tensile strain, but a different type of mechanical stimulus, e.g. shear stress (chapter 5).

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Expression of BMPs in cyclically-strained myotubesBMPs are growth factors involved in many biological processes, such as cell differentiation and homeostasis of different cell types (35). Twenty BMPs have been identified, of which BMP2, 6, and 7 are known to induce (ectopic) bone formation (8, 26). BMP2 and 7 areused as therapeutics to treat bone defects as well as nonunions (27). Mechanical loadingupregulates BMP2, 6, and 7 gene expression in bone cells (28, 40). Since skeletal muscle expresses basal levels of BMP signaling proteins (11, 33), we questioned whether mechanical loading affects the expression of osteo-inductive BMPs, i.e. BMP2, 6, and 7 in myotubes. We tested the effect of CS on BMP2, 6, and 7 mRNA levels in C2C12 myotubes, and found that, in contrast to the effects of mechanical loading on bone cells, CS did not affect BMP2, 6, or 7 mRNA levels (fig. 2). This lack of effect in BMP expression in response to mechanical loading seems in line with lack of evidence that these osteo-inductive BMPs play a role in the adaptation of skeletal muscle. However, it is known that activated satellite cells respond to BMP-4, as they express BMP-receptor-1A (31), indicating that at least some of the BMP signaling pathways may be involved in muscle adaptation. However, whether this signaling is increased by mechanical loading remains to be determined. Note that in myotubes, some of the osteo-inductive BMPs were basally expressed, i.e. BMP2 and BMP6, which suggest that these may enable biochemical interactions between muscle and bone (15). Interestingly, such an interaction between vascular tissue and bone has been reported already (25).

Figure 1: CS did not affect PGE2 production, but increased COX-2 mRNA levels in C2C12 myotubes. Cells were subjected to CS (15% deformation, 1 Hz) for 1 h, and post-incubated without cyclic strain for 3, 6, and 24 h. (A) CS did not affect PGE2 production by myotubes. (B) CS increased COX-2 mRNA levels at all time points measured. CS, cyclic strain. Values are mean ± SEM (n=3). Significant effect of cyclic strain, *p < 0.05, ** p<0.01.

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CS alters the expression of Wnt-signaling genes by myotubesDownstream targets of BMP signaling are the Wnt family of signaling proteins, which are key regulatory proteins in the development of several tissues and in the maintenance of cellular homeostasis (13, 24, 30). Activation of the Wnt signaling pathway has an anabolic effect on bone mass during bone regeneration (16). In response to fluid flow-induced shear stress Wnt signaling pathways are activated in osteocytes (41), and therefore Wnt signaling likely plays an important role in mechanical loading-induced bone remodeling (22, 41). Wnts are also expressed in skeletal muscle in vivo, and it is known that Wnt signaling proteins are involved in skeletal muscle embryonic development (45), as well as muscle hypertrophy and muscle stem cell renewal (20, 45). Although these processes lead to changes in skeletal muscle mass, it is still unknown whether Wnts modulate the adaptation of skeletal muscle to mechanical loading. Here we show that the expression of several Wnt-signaling genes by myotubes in response to uni-axial CS is differentially changed (fig. 3). CS decreased mRNA levels of Wnt4 (8-fold) and Wnt8b (80-fold), but increased mRNA levels of SRY box containing gene 17 (Sox17) (9-fold), Wnt1 (70-fold), Wnt3 (6-fold), Wnt7b (10-fold), and Wnt7a (200-fold) in myotubes (fig. 3). Wnt1 and Wnt7a activate AKT-mTOR pathways that induce muscle hypertrophy, whereas Wnt4 negatively regulates muscle growth by inhibiting satellite cell proliferation (20, 21, 32, 37). However it is unknown whether Sox17, Wnt3, Wnt7b, and/or Wnt8b are associated with skeletal muscle adaptation. The present observations that Wnt1 and Wnt7a mRNA levels increased, while Wnt4 mRNA levels decreased in myotubes in response to CS, indicate that Wnts may play a role in mechanical loading-induced muscle adaptation.

Figure 2: Mechanical loading by cyclic uni-axial strain did not affect BMP gene expression in C2C12 myotubes. Cells were subjected to CS (15% deformation, 1Hz) for 1 h. CS did not affect BMP2 and BMP6 gene expression. BMP7 mRNA levels were not detectable. Values are mean ± SEM (n=3). N.D., not detectable; CS, cyclic strain.

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Figure 3 legend on next page

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Figure 3: CS alters expression of Wnt-signaling genes in C2C12 myotubes. Cells were subjected to CS (15% deformation, 1 Hz) for 1 h. A Wnt-signaling PCR array was performed on pooled cDNA of 3 independent experiments. Y-axis: Basal mRNA levels in unloaded myotubes. X-axis: Changes in mRNA levels as a result of CS. CS of myotubes increased mRNA levels of Wnt-1, Wnt-3, Wnt-7a, Wnt-7b, and Sox17. Cyclic strain decreased mRNA levels of Wnt-4 and Wnt-8b in myotubes. Dashed dotted line, no effect of CS; I, >300% decrease in gene expression in response to CS; II, no <300% decrease in gene expression in response to CS; III, <300% increase in gene expression in response to CS; IV, >300% increase in gene expression in response to CS.

RANKL, a novel factor in muscle fiber size adaptation?RANKL is recognized for its catabolic effect during bone remodeling, since it is a key factor for osteoclast differentiation (4). RANKL expression by osteoblasts and osteocytes in vitro decreases in response to mechanical loading (18, 52), suggesting that mechanical stimulation of bone mediates osteoclastogenesis via RANKL production by osteocytes and osteoblasts. However in vivo, it seems that the osteocytes are the major source of RANKL in bone remodeling (27, 50). Here we show that C2C12 myotubes also express RANKL, and that RANKL expression decreases in response to CS (fig. 4A). Thus, osteoclastogenesis may not only be regulated via osteocytes, but also by paracrine signaling from muscle cells (15). Besides affecting other cells than muscle fibers and their resident satellite cells, RANKL could also have an autocrine effect in muscle, since myotubes basally express RANK, the receptor for RANKL (data not shown). RANKL treatment of C2C12 myotubes resulted in increased total protein levels (fig. 4B). Treatment with 20 ng/mL RANKL decreased the diameter of myotubes (fig. 4C), suggesting that RANKL may attenuate the rate of protein synthesis and/or stimulated protein degradation resulting in myotube atrophy. In osteoclasts, RANKL activates NFATC1 and c-fos expression, primarily via the transcription factor NF-κB (51). In muscle the NF-κB signaling pathway is associated with skeletal muscle disorders, such as cachexia and disuse atrophy (1), whereas NFATC1 is associated with skeletal muscle hypertrophy (42). The transcription factor c-fos has distinct functions as it may activate or repress gene expression (7). C-fos expression is increased in response to exercise as well as in response to disuse (2, 29, 49). Since we observed that RANKL treatment increased total protein content in myotubes, and decreased myotube thickness, we questioned whether RANKL activates the expression of NF-κB target genes NFATC1, c-fos in C2C12 myotubes. Two hour RANKL treatment decreased c-fos mRNA, whereas 20 h treatment increased c-fos mRNA levels, dose dependently (fig. 4D), indicating that myotubes respond differently to a short 2 h RANKL treatment compared to a long 20 h RANKL treatment. Such differential effects were not observed for NFATC1 expression, since NFATC1 mRNA levels seemed to decrease in response to 2 h and 20 h RANKL treatment in myotubes (fig. 4E). As NFATC1 expression is associated with skeletal muscle hypertrophy (42), these results suggests that the rate of protein turnover decreases with increasing RANKL concentrations. The skeletal muscle atrophy marker muscle RING finger protein 1 (MuRF1) is a well known target gene of NF-κB (14). MuRF1 is involved in the ubiquitinaton and activity of the ubiquitin-proteasome system, and elevated MuRF1 expression levels in skeletal muscle are associated

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with an increased rate of protein degradation in vivo (6). mRNA levels of MuRF1 were only increased at 20 h treatment with 0.2 ng/mL RANKL, and not when RANKL was added at higher concentrations, suggesting that RANKL treatment would only induce skeletal muscle atrophy at lower RANKL concentrations, which is in contradiction with the observed decrease in myotubes thickness at high RANKL concentrations (fig 4C). Regarding these results, it seems that RANKL affects distinct signaling pathways in myotubes, and the net effect of RANKL on myotube metabolism is a result of multiple processes. More detailed mechanistic investigation is needed to unravel the mechanisms behind the effects of RANKL on muscle adaptation.

Figure 4: Effect of CS on RANKL mRNA levels, and effect of RANKL treatment on total protein, myotube diameter, and expression of c-fos, NFATC1, and Murf1 in C2C12 myotubes. (A) Cells were subjected to CS (15% deformation, 1 Hz) for 1 h and post-incubated without CS for 3, 6, and 24 h. Cyclic strain decreased RANKL mRNA levels at 6 h post-incubation. (B) Cells were treated without or with RANKL (0.2, 2, and 20 ng/mL) for 20 h. RANKL increased total protein in C2C12 myotubes. (C) RANKL (20 ng/mL) decreased myotube diameter. (D) Cells were treated without or with RANKL (0.2, 2, and 20 ng/mL) for 2 h and 20 h. RANKL decreased c-fos mRNA at 2 h, but increased c-fos mRNA levels at 20 h in a dose-dependent manner. (E) RANKL seemed to decrease NFATC1 mRNA levels dose-dependently. (F) Murf1 mRNA levels were not changed after 2 h RANKL treatment in C2C12 myotubes. RANKL (0.2 ng/mL) treatment for 20 h increased MuRF1 mRNA levels in myotubes. Values are mean ± SEM. * Significant effect of CS, p<0.05 (n=5), # Significant effect of RANKL, p<0.5 (n=3).

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SummaryThe present data shows that mechanical loading by CS alters the expression of several osteo-anabolic and osteo-catabolic factors, i.e. COX-2, Wnt1 , Wnt4, Wnt7a, and RANKL in C2C12 myotubes (5, 19, 23). Furthermore myotubes respond to RANKL treatment by decreasing their size, possibly via NF-κB signaling. These observations reveal new insights on COX-2 and Wnt signaling, and point to RANKL as a possible regulator of mechanical loading-induced muscle adaptation. Moreover, the expression of identical signaling proteins in response to mechanical loading by muscle cells and bone cells implies the possibility that bilateral biochemical communication exists between muscle and bone.

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Osteo-anabolic and osteo-catabolic factors in myotubes