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Osteoactivin (OA) is a novel osteogenic factor important for osteoblast differentiation and function. Previous studies showed that OA stimulates matrix ...
ORIGINAL RESEARCH ARTICLE

Journal of

Osteoactivin Induces Transdifferentiation of C2C12 Myoblasts Into Osteoblasts

Cellular Physiology

GREGORY R. SONDAG,1,2 SIBEL SALIHOGLU,1 SUZANNE L. LABABIDI,1 DOUGLAS C. CROWDER,1 FOUAD M. MOUSSA,1,2 SAMIR M. ABDELMAGID,1 1,2 AND FAYEZ F. SAFADI * 1

Department of Anatomy and Neurobiology, Northeast Ohio Medical University (NEOMED), Rootstown, Ohio

2

School of Biomedical Sciences, Kent State University, Kent, Ohio

Osteoactivin (OA) is a novel osteogenic factor important for osteoblast differentiation and function. Previous studies showed that OA stimulates matrix mineralization and transcription of osteoblast specific genes required for differentiation. OA plays a role in wound healing and its expression was shown to increase in post fracture calluses. OA expression was reported in muscle as OA is upregulated in cases of denervation and unloading stress. The regulatory mechanisms of OA in muscle and bone have not yet been determined. In this study, we examined whether OA plays a role in transdifferentiation of C2C12 myoblast into osteoblasts. Infected C2C12 with a retroviral vector overexpressing OA under the CMV promoter were able to transdifferentiate from myoblasts into osteoblasts. Immunofluorescence analysis showed that skeletal muscle marker MF-20 was severely downregulated in cells overexpressing OA and contained significantly less myotubes compared to uninfected control. C2C12 myoblasts overexpressing OA showed an increase in expression of bone specific markers such as alkaline phosphatase and alizarin red staining, and also showed an increase in Runx2 protein expression. We also detected increased levels of phosphorylated focal adhesion kinase (FAK) in C2C12 myoblasts overexpressing OA compared to control. Taken together, our results suggest that OA is able to induce transdifferentiation of myoblasts into osteoblasts through increasing levels of phosphorylated FAK. J. Cell. Physiol. 229: 955–966, 2014. ß 2013 Wiley Periodicals, Inc.

Mesenchymal Stem Cells (MSC’s) have the ability to differentiate into a variety of cell types. These cell types include chondrocytes, adipocytes, myoblasts, and osteoblasts (Grigoriadis et al., 1988; Yamaguchi et al., 1991). Osteoblasts are bone-forming cells that secrete mineralized extracellular matrix (ECM) (Franceschi, 1999). This ECM is composed of several proteins including glycosaminoglycans, collagen type I, and osteocalcin (Lian and Stein, 1992). MSC’s differentiation to osteoblasts undergo three stages of differentiation. The first stage is commitment of MSC’s into osteoblast precursors followed by a second stage of proliferation and the third stage is to differentiate into mature matrix secreting osteoblasts (Yamaguchi et al., 2000). These stages of osteoblast differentiation are controlled by local, systemic, and genetic factors. Runx2 and Osterix are transcription factors that are responsible for early commitment and differentiation of MSC’s into osteoblasts (Harada and Rodan, 2003). Myoblasts are also derived from MSC’s and differentiate into myocytes that fuse to form myotubes (Le Grand and Rudnicki, 2007; Yusuf and BrandSaberi, 2012). These differentiated myotubes form the contractile multinucleated myofibers of skeletal muscle (Sohn et al., 2009; Simionescu and Pavlath, 2011). Myogenic regulatory factors (MRFs) such as MyoD and Myogenin are transcription factors that bind upstream to muscle specific genes to promote the proliferation and differentiation of MSC’s into myoblasts (Dedkov et al., 2003). MyoD has been shown to be present not only during the proliferation and differentiation of myoblast but also in osteoblasts where it regulates their differentiation by Osterix. Myogenin is present as myoblasts withdraw from the cell cycle and begin to differentiate and fuse into myotubes (Amack and Mahadevan, 2004). Recent studies suggest the presence of specific factors secreted by myofibers that affect bone turnover. Bone morphogenic proteins (BMPs) are osteogenic growth factors that belong to the transforming growth factor (TGF)-b ß 2 0 1 3 W I L E Y P E R I O D I C A L S , I N C .

superfamily and are important regulators of morphogenesis during development (Wozney, 1998; Mu and Li, 2011). Previous group showed that BMPs can induce ectopic bone and cartilage formation in vivo (Urist, 1965). Other studies have shown that BMP-2 has the ability to stimulate transdifferentiation of C2C12 myoblasts into osteoblasts (Katagiri et al., 1994; Okubo et al., 1999). BMP-2 increased bone markers such as alkaline phosphatase and osteocalcin expression and inhibited myogenic markers such as MyoD and Myogenin. Another bone growth factor that is secreted by myofibers and stimulates bone remodeling is Osteoactivin (OA). OA is a type I transmembrane glycoprotein that is important for osteoblast and osteoclast differentiation (Owen et al., 2003; Abdelmagid et al., 2008; Sheng et al., 2008; Mendez-Ferrer et al., 2010; Singh et al., 2010). It was first characterized from studies using osteopetrotic mutant rats and has high homology to human glycoprotein (Gpnmb) and mouse DC-HIL (Safadi

The authors state they have no professional or financial affiliations that would bias this work. Contract grant sponsor: National Institutes of Health; Contract grant number: RO1AR048892-06. Contract grant sponsor: Ohio Department of Development. *Correspondence to: Fayez Safadi, Department of Anatomy and Neurobiology, Northeast Ohio Medical University, 4209 State Rt. 44, Building F143, Rootstown, OH 44224. E-mail: [email protected] Manuscript Received: 4 June 2013 Manuscript Accepted: 18 November 2013 Accepted manuscript online in Wiley Online Library (wileyonlinelibrary.com): 22 November 2013. DOI: 10.1002/jcp.24512

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et al., 2001). Our group was the first to report that OA has two isoforms, a mature glycosylated isoform with a MW of 115 kDa, and an immature non-glycosylated 65 kDa isoform. OA protein sequence has an N-terminal signal peptide, polycystic kidney disease domain (PKD), and a proline repeat rich domain (PRRD), di-leucine motif, and C-terminal containing RGD motif (Selim et al., 2007). We showed previously that OA stimulated markers of osteoblast differentiation and mineralization (Selim et al., 2003). We also showed that OA acts as a downstream mediator of BMP-2 function through the Smad1 signaling pathway (Abdelmagid et al., 2007). Moreover, our group also reported the temporal pattern of OA during fracture healing. We have previously shown that treatment of C3H10T1/2 pluripotent stem cells with recombinant OA upregulated bone formation markers with comparable response to BMP-2 when C3H10T1/2 cells were cultured on biodegradable hydrogels containing BMP-2 (Abdelmagid et al., 2010; Arosarena et al., 2011). OA has been found to have a protective function in skeletal muscle against long-term denervation. Previous study showed that OA is upregulated under muscle unloading conditions including denervation and space flight (Nikawa et al., 2004). OA also upregulated the expression of MMP-3 and MMP-9 in infiltrating fibroblasts of degenerated skeletal muscle fibers (Ogawa et al., 2005; Furochi et al., 2007a,b). With these data taken into consideration, OA serves as an interesting candidate for future musculoskeletal research and regenerative medicine. In this study, we determine the role of OA in transdifferentiating myoblasts into osteoblasts. Transfection of C2C12 myoblasts with a pBABE retroviral vector containing OA cDNA under the CMV promoter transdifferentiate C2C12 cells into osteoblasts. OA overexpression in C2C12 myoblasts downregulate myoblast markers; myotube formation, MyoD, and Myogenin production and upregulate osteoblast differentiation markers; alkaline phosphatase and Runx2. This cellular function was associated with increased FAK activation. OA overexpression in C2C12 myoblasts also decreases cell proliferation and survival. We propose that OA overexpression transdifferentiates C2C12 myoblasts into osteoblasts through phosphorylation of focal adhesion kinase (FAK).

medium (Invitrogen, Grand Island, NY) with 10% FBS and 1% penicillin–streptomycin and incubated at 37˚C with 5% CO2 (myoblast proliferation medium; MP). In order to induce myoblast differentiation (MD) into myotubes, cultures were switched to DMEM containing 2% horse serum. In some instances and depending on experimental procedures, C2C12 cells were differentiated with osteogenic media (OM) containing 10 mM bglycerophosphate and 50 mg/ml Ascorbic Acid. Expression of adenoviral vectors A pBABE viral vector containing OA cDNA was provided to us from Dr. Jeremy Rich (Cleveland Clinic, Cleveland OH). OApBABE viral vector construct was generated as previously described (Rich et al., 2003). For propagating the OA-viral vector, OA vector, or control (empty vector) was co-infected with a PCL10-A1 packaging vector, into subconfluent 293 HEK cell line using the calcium chloride method (Rich et al., 2003). Viruscontaining supernatant was harvested after 48 h and used to infect C2C12 cells. For generation of stable virus infected cell line, C2C12 cells were split and selected by replacing the medium with Bleomycin antibiotic (500 mg/ml) containing DMEM medium. The medium was changed after 72 h with continued selection. After 5 days, the antibiotic resistant colonies were selected and used for further culture. Production of the adenovirus expressing wild type FAK (WT-FAK) and dominant negative FAK (DN-FAK) is described elsewhere (Rafiq et al., 2006). FAK protein expression was co-infected with the Tet transactivator Ad-TA as described (Streblow et al., 1999). Cells were infected at 25 and 50 pfu/cell for Ad-WT-FAK or Ad-DN-FAK, respectively. The DN-FAK vector contains a mutation in the Tyr-397 domain which results in the reduction of FAK phosphorylation as previously described (Castillo et al., 2012). C2C12 cells were infected with either WTFAK or DN-FAK adenovirus in DMEM for 2 h, followed by the addition of 5% FBS in DMEM for 48 h. Stable infection was confirmed by quantitative (q)-PCR, ELISA, and Western blot analyses. Immunofluorescent staining

Anti-OA antibody was raised against osteoactivin peptide sequence within the c-terminal domain. Chickens were immunized, and the precipitated crude IgY was purified by affinity chromatography on Sepharose 4B derivatized with the immunizing OA peptide (Cambridge Research Biochemicals, Stockton-Tees, UK). The MF-20 monoclonal antibody developed by Dr. Donald Fischman was obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the NICHD and maintained by the University of Iowa, (Iowa City, IA). Primary antibodies against MyoD, Myogenin, and CyclinD1 were purchased from Santa Cruz Biotechnology (Cruz, CA). Primary antibodies against GAPDH, phosphorylated tyrosine, IgG, and HA were purchased from Cell Signaling (Danvers, MA). Primary antibodies against Caspase-12 were purchased from BD Biosciences (San Jose, CA), Desmin from Boster Biological Technology (Fremont, Ca), Runx2 from Sigma (St. Louis, MO), and FAK and phosphorylated FAK from Chemicon (Temecula, CA). AntiCaspase 3 was purchased from Bioss (Woburn, MA). Secondary HRP and fluorescent-conjugated antibodies were purchased from Jackson Immunolabs (West Grove, PA).

C2C12 myoblasts were cultured in chamber slides (BD Biosciences) at a density of 2,000 cells/chamber. C2C12 cells were infected with either an empty vector (pBABE-EV) or pBABE-OA construct or uninfected (control) and cultured for 24 h. Cells were fixed with 100% methanol for 5 min at room temperature. Cells were then washed three times with PBS. Cells were blocked using 2% donkey serum (Sigma) in PBS for 60 min at room temperature. Cells were incubated with primary antibodies against FAK or MF20 were incubated for 60 min at 4˚C and washed in PBS. Cells were incubated with secondary antibodies goat anti-mouse conjugated with cy2 (green) or cy3 (red) fluorescent dye and washed with PBS. Rhodamine phalloidin (Cytoskeleton, Denver, CO) and DAPI (Abcam, Cambride, MA) were used to stain the actin cytoskeleton and nucleus, respectively. Slides were then cover slipped and viewed with the Nikon Eclipse Ti-E inverted microscope with fluorescent cubes. Quantitation of the fusion index was adopted from the methods of (Sohn et al., 2009). The fusion index was calculated by determining the nuclei found within myotubes by the total number of nuclei in a microscopic field (three microscopic fields per sample; three independent experiments). Quantitation of desmin positive cells was modified from (Duprez et al., 1998) in which the number of desmin positive cells was counted in five randomly chosen fields of 0.135 mm2 in both uninfected control and infected OA overexpressing C2C12 cells.

Cell culture

Protein isolation

Mouse C2C12 myoblasts (American Type Culture Collection, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s

At the end of the culture period, the cells were washed with ice cold PBS, scraped, and centrifuged at 10,000 rpm. The PBS was

Materials and Methods Antibodies

JOURNAL OF CELLULAR PHYSIOLOGY

OSTEOACTIVIN INDUCES TRANSDIFFERENTIATION

removed and 500 ml of ice cold RIPA lysis buffer (Bio-Rad, Hercules, CA) was added to the cells. The cell lysate was transferred to a 1.5 ml tube and placed on an orbital rocker for 2 h at 4˚C. The protein concentration was determined using a BCA protein assay kit (Thermo Scientific, Rockford, IL).

buffer for 30 min. Aliquots were mixed with p-nitrophenol substrate in 10 triazene temolomide buffer. The ALP activity results were normalized to the total mg proteins.

Western blotting

C2C12 myoblast stable cell lines infected with pBABE OA or pBABE empty vector, or uninfected controls were stained on Day 14 of culture using an ALP staining kit (Sigma). Cells were briefly fixed with citrate–acetone–formaldehyde for 1 min and rinsed with dH2O. Alkaline dye mixture was added to the cells and incubated for 30 min in the dark. Cells were then rinsed with dH2O and evaluated with bright field E600 Nikon inverted microscope.

Protein was isolated and subjected to protein electrophoresis as described previously (Abdelmagid et al., 2007). Briefly, proteins isolated from different cultures were mixed with denature buffer and heated at 100˚C for 5 min. Proteins were subjected to SDSPAGE for 1 h. Proteins were then transferred to PVDF membrane by semi-dry transfer method (Bio-Rad) for 1 h at room temperature. The blot was incubated in blocking buffer for 1 h at room temperature. The appropriate primary antibodies were added to the blocking buffer at 4˚C overnight. The blot was then washed five times in TBST then incubated with HRP-conjugated secondary antibody, for 1 h at room temperature. The blot was washed again five times in TBST and the signals were developed using an ECL kit (Pierce) and detected on XL-exposure films (Kodack). Protein band intensities from Western blots were quantified via Image J (NIH).

Alkaline phosphatase histochemistry

Cell proliferation assay C2C12 myoblast stable cell lines infected with pBABE OA or pBABE EV, or uninfected control were plated in 96-well plates at a density of 1,000 cells per well and allowed to grow for 5 days. Cell proliferation was assessed using a CYQUANT kit (Invitrogen). A volume of 100 ml of CYQUANT dye binding solution was added into each well and incubated at 37˚C for 1 h. The fluorescence intensity of the sample was read at an excitation of 485 nm and emission of 530 nm, using a BioTek plate reader.

ELISA Total proteins were isolated from C2C12 myoblast stable cell lines infected with pBABE-EV or pBABE-OA or uninfected control and subject to ELISA using a DuoSet ELISA kit (R&D Systems, Minneapolis, MN). High affinity binding plates were coated with goat anti-mouse OA capture antibody and allowed to incubate overnight at 4˚C. Plates were then washed and blocked with 300 ml reagent diluent (R&D) for 1 h at room temperature. The plate was washed and 30 mg proteins from the appropriate sample were added to each well and allowed to incubate for 2 h at room temperature. The plates were washed and 100 ml of biotinylated goat anti-mouse OA was added for 2 h at room temperature. The wells were washed and 100 ml streptavidin-HRP was added to each well for 20 min at room temperature in the dark. The wells were washed and 100 ml of substrate solution was added to each well and allowed to incubate for 20 min at room temperature in the dark followed by 50 ml of stop solution. The sample color intensity was detected at an optical density of 450 nm using a BioTek plate reader. RNA isolation RNA was isolated as described previously (Abdelmagid et al., 2007). Cell layers were harvested and homogenized in Trizol, separated by chloroform, and RNA was recovered using isopropyl alcohol precipitation. Pellets were washed with 70% ethanol and RNA concentrations were determined using a spectrophotometer. RT and quantitative (q)PCR analysis RT-PCR analysis for OA and GAPDH was performed as described previously (Abdelmagid et al., 2007). Briefly, 2 mg of total RNA was reverse transcribed to cDNA. Two microliters of cDNA was amplified in 50 ml of qPCR reaction mixture. The primers for rat OA were forward; 50 -AAGGTACCTCACCTGCTAAGACTGC30 and reverse 50 -AAAGTCAGCATGGGTATGTTGGTGC-30 . qPCR reactions were performed in ABI PRISM 7700 using the SYBR Green method. OA values were calculated relative to G3PDH values using the equation (DDCT); CT ¼ threshold cycle. Alkaline phosphatase (ALP) activity measurement ALP activity was measured as described previously (Abdelmagid et al., 2007). Briefly, at day 14, cell layers were treated with TSM JOURNAL OF CELLULAR PHYSIOLOGY

Cell viability and mortality assays C2C12 myoblast stable cell lines infected with pBABE OA or pBABE EV, or uninfected cells were plated in 96-well plates at a density of 1,000 cells 24 h prior to termination. Cell viability was assessed using MTT (3-[4,5-dimethylthiazole-2-yl]-2,5diphenyltetrazolium bromide) assay (Sigma), or CellTiter-Glo luminescent assay (Promega, Madison, WI). For the MTT assay, substrate was added (100 ml/well) and cells were incubated at 37˚C for 4 h in the dark. After incubation solubilizer (20% SDS and 50% dimethyl formamide) was added (250 ml/well) and plates were rocked at room temperature overnight to solubilize the formazen crystals. Next day, 100 ml aliquots were mixed with 100 ml DMSO and transferred into a 96-well plate and sample color intensity were read at 570 nm, using a BioTek plate reader. For the CellTiter-Glo assay, a CellTiter reagent was added (100 ml/well) and the well-contents were mixed on an orbital shaker for 2 min. The plate was then incubated at room temperature for 10 min and luminescence was read using a Biotek microplate reader. In order to determine cell mortality rate, C2C12-pBABE-OA, -pBABE-EV, and uninfected control cells were cultured as above and after 24 h, cells were stained with trypan blue and counted using a hemocytometer. Flow cytometry C2C12 control, pBABE-EV, and pBABE-OA cells were cultured in DMEM with 10% FBS and 1% PSA for 5 days and then switched to serum free media for 0, 24, and 48 h. Samples were then stained with propidium iodide (PI) solution (50 mg/ml) for 30 min at room temperature. The samples were run on a BD LSR II flow cytometer (BD Biosciences) using FACSDiva version 6.0 software and analyzed by the ModFit LT DNA analysis software (Verity Software House, Topsham, ME; version 3.2.1; Model 1Dn0n-DSD). Immunoprecipitation Five hundred micrograms of total protein isolated from C2C12 control, pBABE-EV, pBABE-OA cultures were immuneprecipitated using an immuneprecipitation kit (Roche Diagnostic, Corp., Indianapolis, IN) to isolate FAK proteins. Twenty-five microliters of agarose A/G beads were added to each protein sample and incubated overnight at 4˚C. Beads were precipitated by

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centrifugation at 12,000g for 20 sec then the supernatant was transferred to a fresh tube for immuneprecipitation according to the manufacturer’s protocol. Twenty-five microliters of gel loading buffer was added to each sample; proteins were denatured by heating to 100˚C for 5 min and analyzed by SDS gel electrophoresis. Statistical analysis One-way analysis of variance (ANOVA) followed by Tukey’s post hoc test was used to determine whether there was a statistical significant difference between the means of control, pBABE-EV and pBABE-OA transfected C2C12 cells. The differences between the means were considered to be statistically significant if P-value is