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Decorin expression in quiescent myogenic cells
Biochemical and Biophysical Research Communications 370 (2008) 383–387
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Decorin expression in quiescent myogenic cells Takanori Nishimura 1,*, Kenjiro Nozu 1, Yasuhiro Kishioka, Jun-ichi Wakamatsu, Akihito Hattori Meat Science Laboratory, Division of Bioresources and Product Science, Graduate School of Agriculture, Hokkaido University, Kita 9 Nishi 9, Kita-ku, Sapporo 060-8589, Japan
a r t i c l e
i n f o
Article history: Received 3 March 2008 Available online 17 March 2008
Keywords: Decorin Satellite cell Reserve cell Quiescence Myogenic cell Skeletal muscle
a b s t r a c t Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. When satellite cells are activated by myotrauma, they proliferate, migrate, differentiate, and ultimately fuse to existing myofibers. The remainder of these cells do not differentiate, but instead return to quiescence and remain in a quiescent state until activation begins the process again. This ability to maintain their own population is important for skeletal muscle to maintain the capability to repair during postnatal life. However, the mechanisms by which satellite cells return to quiescence and maintain the quiescent state are still unclear. Here, we demonstrated that decorin mRNA expression was high in cell cultures containing a higher ratio of quiescent satellite cells when satellite cells were stimulated with various concentrations of hepatocyte growth factor. This result suggests that quiescent satellite cells express decorin at a high level compared to activated satellite cells. Furthermore, we examined the expression of decorin in reserve cells, which were undifferentiated myoblasts remaining after induction of differentiation by serum-deprivation. Decorin mRNA levels in reserve cells were higher than those in differentiated myotubes and growing myoblasts. These results suggest that decorin participates in the quiescence of myogenic cells. Ó 2008 Elsevier Inc. All rights reserved.
Quiescent myogenic cells exist in postnatal skeletal muscle and are called satellite cells. Satellite cells are located between the basement membrane and the plasma membrane of muscle fibers. These cells play an important role in muscle hypertrophy during postnatal development of skeletal muscle and in the repair of muscle tissues in response to injury. Satellite cells are mitotically quiescent until activated by myotrauma such as stretch, exercise or injury, at which time they proliferate. A portion of the cells resulting from this proliferation migrate, differentiate, and ultimately fuse to existing myofibers or fuse with each other to produce new myofibers. The remainder of these cells do not differentiate, but instead return to quiescence and remain in a quiescent state until activation begins the process again [1]. This ability to maintain their own population, referred to as self-renewal, is important for skeletal muscle to maintain the capability to repair during postnatal life. However, the mechanisms by which satellite cells return to quiescence and maintain the quiescent state are still unclear. Like satellite cells in vivo, a subset of myoblasts remains quiescent and undifferentiated in vitro. These cells are called reserve
Abbreviations: TGF-b, transforming growth factor-b; GAPDH, glyceraldehydes-3phosphate dehydrogenase; PBS, phosphate buffered saline; HGF, hepatocyte growth factor. * Corresponding author. Fax: +81 11 716 0879. E-mail address: [email protected] (T. Nishimura). 1 These authors contributed equally to this study. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.03.025
cells and are predominantly quiescent while retaining their differentiation potential [2]. Reserve cells have been characterized by their high expression of p130 and myf5 and low expression of MyoD compared to myotubes, which express low levels of p130 and myf5 and high level of MyoD [3]. These results suggest that p130 could be a part of the specific pathway that blocks cell cycle progression and defines the pool of reserve cells during terminal differentiation. However, it still remains unclear why some myoblasts do not differentiate under the same culture conditions that cause others to differentiate to form myotubes. Decorin is a member of the small leucine-rich proteoglycan gene family and consists of a core protein and a dermatan/chondroitin sulfate chain [4]. Decorin binds several types of collagen [5,6] and regulates collagen fibril formation [7]. Decorin also plays an important role in cell growth through the modulation of growth factors, such as transforming growth factor-b (TGF-b) [8,9]. Forced expression of decorin in Chinese hamster ovary cells leads to the suppression of cell proliferation [10], which might result from inhibition of TGF-b activity [11]. Decorin also binds to epidermal growth factor receptor, activates the MAP-kinase pathway and up-regulates P21, an inhibitor of cyclin-dependent kinases, resulting in suppression of cell growth [12,13]. These results suggest that decorin participates in blocking cell cycle progression. Coppock et al. [14] have shown some genes that are expressed at a higher level in quiescent human lung fibroblasts than in logarithmically growing fibroblasts. These genes were named as quiecins and
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include decorin, several types of collagens and ribosomal protein S29, quiecin Q6. Furthermore, decorin expression in fibroblasts is highly stimulated by entrance into quiescence [15]. Given the role of decorin in quiescence of fibroblasts [14,15], we sought to investigate the involvement of decorin in the quiescence of myogenic cells. We here show that quiescent satellite cells express decorin at high levels compared to activated satellite cells in vitro. We also demonstrate that the expression level of decorin in reserve cells is higher than those in differentiated myotubes and actively growing myoblasts. These results suggest that decorin participate in the quiescence of myogenic cells. Materials and methods Satellite cell isolation and culture. Satellite cells were isolated from 5-weekold male Sprague–Dawley rats according to the method of Allen et al. [16]. Briefly, skeletal muscle (Musculus longissimus) was excised, hand minced with sterile scissors, and digested for 1 h at 37 °C with 1.25 mg/ml pronase. Cells were separated from muscle fiber fragments and tissue debris by differential centrifugation and plated on poly-L-lysine and fibronectin-coated dishes or 8well culture slides (Nunc, Roskilde, Denmark) in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Grand Island, NY) containing fetal bovine serum (FBS; 10% FBS–DMEM, Invitrogen). Cultures were maintained in a humidified atmosphere of 5% CO2 at 37 °C. In addition, companion satellite cell cultures were stained with the antibody against desmin (Sigma, St. Louis, MO), a specific protein expressed in muscle cells. Cultures with fewer than 97% of desmin-positive cells were not used for this study. After culturing in 10% FBS–DMEM for 72 h, differentiation was induced by substituting the media with DMEM containing 2% horse serum (HS; 2% HS–DMEM, Invitrogen), and the cultures were incubated thereafter for 48 h. All animal procedures were approved by the Experimental Animal Care and Use Committee at the Graduate School of Agriculture, Hokkaido University. Detection of quiescent satellite cells in vitro. To detect quiescent satellite cells, the cultures were incubated in 10% FBS–DMEM for 24 h, and then labeled for 16 h with 10 mM 5-bromo-2-deoxyuridine (BrdU; Amersham Biosciences, Piscataway, NJ) in 10% FBS–DMEM, followed by immunocytochemistry for detection of BrdU using a horseradish peroxidase-conjugated secondary antibody (Sigma). BrdU-negative cells were determined as quiescent satellite cells in vitro. Immunocytochemistry. Cultured cells were fixed at 24, 72, and 120 h with 2% formaldehyde in PBS (TPBS) for 10 min at room temperature. After washing three times with 0.5% Triton X-100 in PBS, specimens were blocked with 0.1% bovine serum albumin (Sigma) in PBS for 1 h at 37 °C, then incubated with anti-decorin antibody (1:100 dilution, Biogenesis, Kingston, NH) for 2 h at 37 °C, rinsed with TPBS
three times, and then treated with tetramethylrhodamine-50 -(and -6-)-isothiocyanate conjugated sheep IgG antibody (1:400 dilution, Chemicon International, Temecula, CA). After rinsing with TPBS, specimens were incubated with the antibody against myogenin (1:100 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 2 h at 37 °C, and then incubated with the secondary antibody conjugated with fluorescein isothiocyanate (1:500 dilution, Chemicon International) for 2 h at 37 °C. After rinsing, specimens were stained with DAPI (Dojindo, Kumamoto, Japan) and mounted in Aqua Poly Mount (PolyScience, Warrington, PA), and then observed using a microscope (DMI6000B, Leica Microsystems, Wetzlar, Germany). In vitro activation of satellite cells. Satellite cells isolated from adult rat skeletal muscle were cultured in 10% FBS–DMEM for 18 h, and then hepatocyte growth factor (HGF; R&D Systems, Minneapolis, MN) was added to the media to a final concentration of 5 or 10 ng/ml. After a further incubation for 22 h, cultures were pulse labeled for 2 h with 10 mM BrdU in 10% FBS–DMEM followed by immunocytochemistry for detection of BrdU using a horseradish peroxidase-conjugated secondary antibody. The percentage of BrdU labeled cells was used as an indicator of activation and entry into the cell cycle. RT-PCR. Total RNA was isolated from cell cultures using ISOGEN (Nippon gene, Tokyo, Japan), according to the manufacturer’s protocol. Decorin mRNA expression was analyzed by quantitative RT-PCR using a LightCycler instrument (Roche Diagnostics, Basel, Switzerland). The primers used to amplify the decorin fragment (250 bp, X59859) were 50 -TCGGATACATCCGCATCTCAGA-30 (forward) and 50 -G GCACTCTGAGGAGTTTGTTGT-30 (reverse). The primers for internal control glyceraldehydes-3-phosphate dehydrogenase (GAPDH) cDNA amplification (264 bp, AF106860) were 50 -TCACCACCATGGAGAAGGCT-30 (forward) and 50 -GCCATC CACAGTCTTCTGAG-30 (reverse). The real-time PCR conditions for amplification were as follows: denaturation at 95 °C for 30 s, annealing at 55 °C for 5 s, and extension at 72 °C for 10 s. Limited trypsinization to obtain myotubes and reserve cells. The method of Kitzmann et al. [17] was used to separate myotubes from reserve cells in differentiated cell cultures. Briefly, isolated satellite cells from adult rat skeletal muscle were seeded onto 10-cm culture dishes (Nunc) and cultured in 10% FBS–DMEM for 72 h and thereafter in 2% HS–DMEM for 48 h. Following incubation, a short trypsinization period (5 min, 0.01% trypsin–EDTA) and washing with PBS were used to preferentially detach myotubes. Reserve cells, which remained attached to the culture dish, were then detached by 10-min incubation with 0.25% trypsin. Statistical analysis. Data were analyzed using one-way analysis of variance (ANOVA) with Scheffe’s test. A probability of p < 0.05 was considered statistically significant.
Results First, we examined the expression of decorin during myogenesis by immunocytochemistry. After 24-h incubation in 10% FBS– DMEM, decorin staining was detectable in mononucleated myoblasts that were not stained with antibody against myogenin, a
Fig. 1. Expression of decorin during myogenesis in vitro. Satellite cells were isolated from 5-week-old Sprague–Dawley rats and cultured in 10% HS–DMEM for 72 h and thereafter in 2% HS–DMEM for 48 h. Cell cultures were fixed at 24 h, 72 h and 120 h and stained with anti-decorin and anti-myogenin antibodies.
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Relative abundance of decorin mRNA
marker of myogenic differentiation (Fig. 1). At 72 h, the decorin staining was observed in mononucleated myoblasts that were stained with antibody against myogenin and also detectable in differentiated myotubes with a few nuclei. After 48-h incubation in 2% HS–DMEM following the incubation in 10% FBS–DMEM for 72 h, the decorin staining was detectable in matured myotubes. These results demonstrate that decorin is expressed in proliferating myoblasts and differentiated myotubes. Next, we investigated if decorin was expressed in quiescent satellite cells by the BrdU-incorporation assay. Isolated satellite cells were incubated with BrdU for 16 h before staining with the antiBrdU antibody. Cells that were stained with anti-BrdU antibody (BrdU-positive cells) must be in the cell cycle and unstained cells (BrdU-negative cells) must be quiescent in the G0-phase. The decorin staining was observed in both BrdU-negative and -positive cells (Fig. 2). There was no difference in the staining pattern or strength between BrdU-positive and -negative cells. This result suggests that decorin is expressed in both quiescent and activated satellite cells. To compare the decorin expression level in quiescent satellite cells with that in activated satellite cells, we analyzed decorin mRNA expression in cell cultures having different ratios of activated satellite cells. Isolated satellite cells were cultured in 10% FBS–DMEM containing HGF (5 or 10 ng/ml) for 24 h, since HGF is known to activate satellite cells in vitro [18]. As shown in Fig. 3A, the ratio of activated satellite cells increased with the concentration HGF added. The ratio of activated satellite cells was 46% in cell cultures with 10 ng/ml of HGF added, whereas the ratio was 33% without HGF. The decorin mRNA level decreased with the concentration of HGF added. These results show that decorin mRNA expression in cell cultures with higher ratios of activated satellite cells is low, suggesting the possibility that decorin expression decreases when satellite cells are activated. In vitro there are myoblasts that remained undifferentiated when the cells are induced to differentiate with serum-deprivation, and they are termed as reserve cells [2]. Reserve cells are thought to be quiescent and similar to satellite cells in vivo. However, the mechanism by which reserve cells cannot commit to differentiation under the same conditions which cause others to differentiate is not known. In this study, we investigated the expression of decorin in reserve cells. First, we examined decorin expression in reserve cells by immunocytochemistry. Decorin staining was observed both reserve cells and differentiated myotu-
Raito of activated cells (%)
T. Nishimura et al. / Biochemical and Biophysical Research Communications 370 (2008) 383–387
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*
**
40
20
0
0 10 5 Concentration of HGF (ng/ml)
1.0 0.8 0.6 0.4
*
0.2 0 0 10 5 Concentration of HGF (ng/ml)
Fig. 3. Decorin mRNA expression in cell cultures having different ratios of activated satellite cells. (A) Ratio of activated satellite cells in response to hepatocyte growth factor (HGF). Isolated satellite cells were cultured in 10% FBS–DMEM for 18 h, and then HGF was added to the media to a final concentration of 5 or 10 ng/ml. After a further incubation for 22 h, cultures were pulse labeled for 2 h with 10 mM BrdU followed by immunocytochemistry for detection of BrdU. The percentage of BrdU labeled cells was used as an indicator of activation and entry into the cell cycle. The columns represent mean values, and bars indicate SE **p < 0.01, *p < 0.05. (B) Decorin mRNA expression in cells cultured with of HGF. Total RNA was isolated from cell cultures, and decorin mRNA expression was analyzed by quantitative RT-PCR. The columns represent relative values of decorin mRNA normalized to GAPDH and expressed as the ratio of decorin mRNA in control cultures without HGF. Bars indicate SE *p < 0.05.
bes (Fig. 4A). Myogenin staining, on the other hand, was not observed in reserve cells. Next, we investigated the expression level of decorin mRNA in reserve cells. We isolate reserve cells from differentiated myotubes in the same culture according to the method of Kitzmann et al. [17] and compared the mRNA expression level of decorin in isolated reserve cells with those in active growing myoblasts and differentiated myotubes. The level of decorin mRNA in reserve cells was 14-fold higher than that in active growing myoblasts and differentiated myotubes (Fig. 4B). Discussion
Fig. 2. Expression of decorin in quiescent and activated satellite cells. Isolated satellite cells were cultured in 10% FBS–DMEM for 24 h and then labeled for 16 h with 10 mM BrdU in 10% FBS–DMEM. Cultures were fixed and stained with antibodies against BrdU, decorin and desmin. In these views, two cells (arrows) are activated cells (BrdU-positive) and the others are quiescent cells (BrdU-negative).
Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. Although the mechanisms by which satellite cells are activated has been almost clarified [16,18,19], the mechanisms by which satellite cells return to quiescence and maintain the quiescent status are still unclear. The transition into quiescence in human fibroblasts is accompanied by the increased expression of a specific set of genes, which include decorin [14]. Furthermore, decorin expression in fibroblasts is highly stimulated by entrance into quiescence [15]. These results suggest that decorin plays an important role in induction and/or maintaining the quiescent status of fibroblasts. Thus, we sought to investigate the involvement of decorin in the quiescence of myogenic cells.
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Phase contrast
A
Decorin
DAPI
Myogenin
50 µm
Relative ab bundance of decorin mRNA
B
20
a
b
10
c
c 0 myoblasts
reserve cells & myotubes
reserve cells
myotubes
Fig. 4. Decorin expression in reserve cells. (A) Immunostaining of decorin and myogenin in reserve cells. Isolated satellite cells were cultured in 10% FBS–DMEM for 72 h and thereafter 2% HS–DMEM for 48 h. Cultures were fixed at 120 h and stained with antibodies against decorin and myogenin. (B) Decorin mRNA expression in reserve cells. After incubation with differentiation media (2% HS–DMEM) for 48 h, differentiated myotubes were detached by limited trypsinization (0.01% trypsin– EDTA) for 5 min, leaving a population of undifferentiated reserve cells. Total RNA was isolated from actively growing myoblasts cultured in 10% FBS–DMEM for 48 h, cell cultures (reserve cells and myotubes) before limited trypsinization, isolated reserve cells, and isolated myotubes. Decorin mRNA levels were detected by quantitative RT-PCR. The columns represent the relative values of decorin mRNA normalized to GAPDH and expressed as the ratio of decorin mRNA in active growing myoblasts. Different letters indicate significant differences p < 0.05.
Decorin was expressed in both activated satellite cells and quiescent satellite cells (Fig. 2). The expression level of decorin mRNA is lower in cell cultures that contained higher ratios of activated satellite cells (Fig. 3), suggesting that decorin expression must be high in quiescent satellite cells as compared with activated and proliferating cells. This is well consistence with the previously reported result that quiescent fibroblasts express decorin at high levels compared to proliferating fibroblasts [14]. Decorin may also play an important role in quiescence of satellite cells in skeletal muscle. At this time, it still remains unclear whether the reduction of decorin expression is needed to activate satellite cells, or whether activation induces the down-regulation of decorin expression. Activation stimulates quiescent satellite cells to exit quiescence and enter into the G1 phase of the cell cycle resulting in proliferation. Activation and proliferation appear to be separate but sequential events. Decorin over-expression inhibits proliferation of carcinoma cells via the up-regulation of p21, a cell cycle regulator [20,21]. Decorin might participate in proliferation of satellite cells after activation. An in vivo experiment has shown that progenies of a single satellite cell generate both differentiated myotubes and quies-
cent cells that are undifferentiated but still myogenic under differentiation-inducing conditions [22]. In vitro, a subset of myoblasts, termed reserve cell, remain quiescent and undifferentiated but retain their capacity for proliferation and myogenic differentiation [2]. We confirmed that reserve cells have the ability to differentiate when they were isolated from differentiating cultures and cultured again in 2% HS–DMEM (data not shown). Reserve cells have been characterized by their high expression of p130 and Myf5 and low expression of MyoD as compared to differentiated myotubes, which express low levels of p130 and Myf5 and high levels of MyoD [3]. Thus, the balance of these transcription factors could be important for the differentiation of myogenic cells. However, the mechanisms by which reserve cells cannot commit to differentiate under the same conditions as that induce other cells to differentiate is unknown. We sought to investigate if decorin participates in keeping reserve cells undifferentiated. Decorin mRNA expression in reserve cells was much higher than in differentiated myotubes and active growing myoblasts (Fig. 4). In our previous report, decorin over-expressing cells are not easily withdraw from the cell cycle and have delayed differentiation as compared with control cells, although decorin over-expressing cells eventually form multi-giant hypertrophic myotubes with larger size than control cells [23]. These results suggest that decorin negatively controls the initiation of differentiation. The high expression of decorin might suppress the exit from the cell cycle and maintain myogenic cells in an undifferentiated state. The total number of quiescent satellite cells remains constant over repeated cycles of degeneration and regeneration, suggesting that the steady state satellite cell population is maintained by self-renewal [24]. To keep the satellite cell pool ready for the next injury, some of the satellite cells that were activated and proliferated must reenter into the G0 phase and maintain quiescence. However, the induction and maintenance of the quiescent state in satellite cells remains unclear. Myostatin, a TGF-b super family member, has been shown to negatively regulate satellite cell activation to maintain quiescence [25]. Recently it has been shown that myostatin controls the process of self-renewal through regulation of Pax7 [26]. We have shown previously that decorin binds to myostatin and sequesters it within the extracellular matrix to regulate its activity [27] and that decorin attenuates myostatin signaling [23]. In this study, we have shown high expression of decorin in reserve cells, which are predominantly quiescent myoblasts, in response to a differentiation cue. Taken together, we speculate that decorin would play an important role in satellite cell self-renewal through attenuating myostatin signaling. Further study is needed to elucidate the role of decorin in satellite cell self-renewal. In summary, we here show that decorin is highly expressed in quiescent satellite cells in vitro and also that decorin expression is higher in reserve cells that do not commit to differentiation in vitro. These results suggest that decorin participates in keeping myogenic cells quiescent and undifferentiated. Acknowledgment We thank Dr. Shigeharu Fukunaga of the Biproduct Science Laboratory, Graduate School of Agriculture, Hokkaido University for technical assistance. References [1] T.J. Hawke, D.J. Garry, Myogenic satellite cells: physiology to molecular biology, J. Appl. Physiol. 91 (2001) 534–551. [2] N. Yoshida, S. Yoshida, K. Koishi, K. Masuda, Y. Nabeshima, Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’, J. Cell Sci. 111 (1998) 769–779.
T. Nishimura et al. / Biochemical and Biophysical Research Communications 370 (2008) 383–387 [3] G. Carnac, L.L. Fajas, A. L’honoré, C. Sardet, N.J.C. Lamb, A. Fernandez, The retinoblastoma-like protein p130 is involved in the determination of reserve cells in differentiating myoblasts, Curr. Biol. 10 (2000) 543–546. [4] R.V. Iozzo, The biology of the small leucine-rich proteoglycans, J. Biol. Chem. 274 (1999) 18843–18846. [5] E. Schönherr, H. Hausser, L. Beavan, H. Kresse, Decorin-type I collagen interaction. Presence of separate core protein-binding domains, J. Biol. Chem. 270 (1995) 8877–8883. [6] T. Nishimura, E. Futami, J. Wakamatsu, A. Hattori, Identification and characterization of proteoglycans in bovine neonatal skeletal muscle, Anim. Sci. J. 74 (2003) 407–416. [7] K.G. Danielson, H. Balibault, D.F. Holmes, H. Graham, K.E. Kadler, R.V. Iozzo, Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility, J. Cell Biol. 136 (1997) 729–743. [8] H. Hausser, A. Gröning, A. Hasilik, E. Schönherr, H. Kresse, Selective inactivity of TGF-beta/decorin complexes, FEBS Lett. 353 (1994) 243–245. [9] Y. Takeuchi, Y. Kodama, T. Matsumoto, Bone matrix decorin binds transforming growth factor-beta and enhances its bioactivity, J. Biol. Chem. 269 (1994) 32634–32638. [10] Y. Yamaguchi, E. Ruoslahti, Expression of human proteoglycan in Chinese hamster ovary cells inhibits cell proliferation, Nature 336 (1988) 244–246. [11] Y. Yamaguchi, D.M. Mann, E. Ruoslahti, Negative regulation of transforming growth factor-b by the proteoglycan decorin, Nature 346 (1990) 281–284. [12] S. Patel, M. Santra, D.J. McQuillan, R.V. Iozzo, A.P. Thomas, Decorin activates the epidermal growth factor receptor and elevates cytosolic Ca2+ in A431 carcinoma cells, J. Biol. Chem. 273 (1998) 3121–3124. [13] R.V. Iozzo, D.K. Moscatello, D.J. McQuillan, I. Eichstetter, Decorin is a biological ligand for the epidermal growth factor receptor, J. Biol. Chem. 274 (1999) 4489–4492. [14] D.L. Coppock, C. Kopman, S. Scandalis, S. Gilleran, Preferential gene expression in quiescent human lung fibroblasts, Cell Growth Differ. 4 (1993) 483–491. [15] D.L. Coppock, C. Kopman, J. Gudas, D.A. Cina-Poppe, Regulation of the quiescence-induced genes: Quiescin Q6, decorin, and ribosomal protein S29, Biochem. Biophys. Res. Commun. 269 (2000) 604–610. [16] R.E. Allen, C.J. Temm-Grove, S.M. Sheehan, G. Rice, Skeletal muscle satellite cell cultures, Methods Cell Biol. 52 (1997) 155–176.
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[17] M. Kitzmann, G. Carnac, M. Vandromme, M. Primig, N.J. Lamb, A. Fernandez, The muscle regulatory factors MyoD and Myf-5 undergo distinct cell cycle– specific expression in muscle cells, J. Cell Biol. 142 (1998) 1447–1459. [18] R. Tatsumi, J.E. Anderson, C.J. Nevoret, O. Halevy, R.E. Allen, HGF/SF is present in normal adult skeletal muscle and is capable of activating satellite cells, Dev. Biol. 194 (1998) 114–128. [19] R. Tatsumi, X. Liu, A. Pulido, M. Morales, T. Sakata, S. Dial, A. Hattori, Y. Ikeuchi, R.E. Allen, Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte growth factor, Am. J. Physiol. Cell Physiol. 290 (2006) C1487–C1494. [20] M. Santra, T. Skorski, B. Calabretta, E.C. Latrime, R.V. Iozzo, De novo decorin gene expression suppresses the malignant phenotype in human colon cancer cells, Proc. Natl. Acad. Sci. USA 92 (1995) 7016–7020. [21] M. Santra, D.M. Mann, E.W. Mercer, T. Skorski, B. Calabretta, R.V. Iozzo, Ectopic expression of decorin protein core causes a generalized growth suppression in neoplastic cells of various histogenetic origin and requires endogenous p21, an inhibitor of cyclin-dependent kinases, J. Clin. Invest. 100 (1997) 149–157. [22] A. Baroffio, M. Hamann, L. Bernheim, M.-L. Bochaton-Piallat, G. Gabbiani, C.R. Bader, Identification of self-renewing myoblasts in the progeny of single human muscle satellite cells, Differentiation 60 (1996) 47–57. [23] Y. Kishioka, M. Thomas, J. Wakamatsu, A. Hattori, M. Sharma, R. Kambadur, T. Nishimura, Decorin enhances the proliferation and differentiation of myogenic cells through suppressing myostatin activity, J. Cell. Physiol. (in press) . [24] H. Schmalbruch, D.M. Lewis, Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles, Muscle Nerve 23 (2000) 617–626. [25] S. McCroskery, M. Thomas, L. Maxwell, M. Sharma, R. Kambadur, Myostatin negatively regulates satellite cell activation and self-renewal, J. Cell Biol. 162 (2003) 1135–1147. [26] C. McFarlanea, A. Hennebry, M. Thomasa, E. Plummera, N. Ling, M. Sharma, R. Kambadur, Myostatin signals through Pax7 to regulate satellite cell selfrenewal, Exp. Cell Res. 314 (2008) 317–329. [27] T. Miura, Y. Kishioka, J. Wakamatsu, A. Hattori, A. Hennebry, C.J. Berry, M. Sharma, R. Kambadur, T. Nishimura, Decorin binds myostatin and modulates its activity to muscle cells, Biochem. Biophys. Res. Commun. 340 (2006) 675– 680.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Decorin expression in quiescent myogenic cells Takanori Nishimura 1,*, Kenjiro Nozu 1, Yasuhiro Kishioka, Jun-ichi Wakamatsu, Akihito Hattori Meat Science Laboratory, Division of Bioresources and Product Science, Graduate School of Agriculture, Hokkaido University, Kita 9 Nishi 9, Kita-ku, Sapporo 060-8589, Japan
a r t i c l e
i n f o
Article history: Received 3 March 2008 Available online 17 March 2008
Keywords: Decorin Satellite cell Reserve cell Quiescence Myogenic cell Skeletal muscle
a b s t r a c t Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. When satellite cells are activated by myotrauma, they proliferate, migrate, differentiate, and ultimately fuse to existing myofibers. The remainder of these cells do not differentiate, but instead return to quiescence and remain in a quiescent state until activation begins the process again. This ability to maintain their own population is important for skeletal muscle to maintain the capability to repair during postnatal life. However, the mechanisms by which satellite cells return to quiescence and maintain the quiescent state are still unclear. Here, we demonstrated that decorin mRNA expression was high in cell cultures containing a higher ratio of quiescent satellite cells when satellite cells were stimulated with various concentrations of hepatocyte growth factor. This result suggests that quiescent satellite cells express decorin at a high level compared to activated satellite cells. Furthermore, we examined the expression of decorin in reserve cells, which were undifferentiated myoblasts remaining after induction of differentiation by serum-deprivation. Decorin mRNA levels in reserve cells were higher than those in differentiated myotubes and growing myoblasts. These results suggest that decorin participates in the quiescence of myogenic cells. Ó 2008 Elsevier Inc. All rights reserved.
Quiescent myogenic cells exist in postnatal skeletal muscle and are called satellite cells. Satellite cells are located between the basement membrane and the plasma membrane of muscle fibers. These cells play an important role in muscle hypertrophy during postnatal development of skeletal muscle and in the repair of muscle tissues in response to injury. Satellite cells are mitotically quiescent until activated by myotrauma such as stretch, exercise or injury, at which time they proliferate. A portion of the cells resulting from this proliferation migrate, differentiate, and ultimately fuse to existing myofibers or fuse with each other to produce new myofibers. The remainder of these cells do not differentiate, but instead return to quiescence and remain in a quiescent state until activation begins the process again [1]. This ability to maintain their own population, referred to as self-renewal, is important for skeletal muscle to maintain the capability to repair during postnatal life. However, the mechanisms by which satellite cells return to quiescence and maintain the quiescent state are still unclear. Like satellite cells in vivo, a subset of myoblasts remains quiescent and undifferentiated in vitro. These cells are called reserve
Abbreviations: TGF-b, transforming growth factor-b; GAPDH, glyceraldehydes-3phosphate dehydrogenase; PBS, phosphate buffered saline; HGF, hepatocyte growth factor. * Corresponding author. Fax: +81 11 716 0879. E-mail address: [email protected] (T. Nishimura). 1 These authors contributed equally to this study. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.03.025
cells and are predominantly quiescent while retaining their differentiation potential [2]. Reserve cells have been characterized by their high expression of p130 and myf5 and low expression of MyoD compared to myotubes, which express low levels of p130 and myf5 and high level of MyoD [3]. These results suggest that p130 could be a part of the specific pathway that blocks cell cycle progression and defines the pool of reserve cells during terminal differentiation. However, it still remains unclear why some myoblasts do not differentiate under the same culture conditions that cause others to differentiate to form myotubes. Decorin is a member of the small leucine-rich proteoglycan gene family and consists of a core protein and a dermatan/chondroitin sulfate chain [4]. Decorin binds several types of collagen [5,6] and regulates collagen fibril formation [7]. Decorin also plays an important role in cell growth through the modulation of growth factors, such as transforming growth factor-b (TGF-b) [8,9]. Forced expression of decorin in Chinese hamster ovary cells leads to the suppression of cell proliferation [10], which might result from inhibition of TGF-b activity [11]. Decorin also binds to epidermal growth factor receptor, activates the MAP-kinase pathway and up-regulates P21, an inhibitor of cyclin-dependent kinases, resulting in suppression of cell growth [12,13]. These results suggest that decorin participates in blocking cell cycle progression. Coppock et al. [14] have shown some genes that are expressed at a higher level in quiescent human lung fibroblasts than in logarithmically growing fibroblasts. These genes were named as quiecins and
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include decorin, several types of collagens and ribosomal protein S29, quiecin Q6. Furthermore, decorin expression in fibroblasts is highly stimulated by entrance into quiescence [15]. Given the role of decorin in quiescence of fibroblasts [14,15], we sought to investigate the involvement of decorin in the quiescence of myogenic cells. We here show that quiescent satellite cells express decorin at high levels compared to activated satellite cells in vitro. We also demonstrate that the expression level of decorin in reserve cells is higher than those in differentiated myotubes and actively growing myoblasts. These results suggest that decorin participate in the quiescence of myogenic cells. Materials and methods Satellite cell isolation and culture. Satellite cells were isolated from 5-weekold male Sprague–Dawley rats according to the method of Allen et al. [16]. Briefly, skeletal muscle (Musculus longissimus) was excised, hand minced with sterile scissors, and digested for 1 h at 37 °C with 1.25 mg/ml pronase. Cells were separated from muscle fiber fragments and tissue debris by differential centrifugation and plated on poly-L-lysine and fibronectin-coated dishes or 8well culture slides (Nunc, Roskilde, Denmark) in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Grand Island, NY) containing fetal bovine serum (FBS; 10% FBS–DMEM, Invitrogen). Cultures were maintained in a humidified atmosphere of 5% CO2 at 37 °C. In addition, companion satellite cell cultures were stained with the antibody against desmin (Sigma, St. Louis, MO), a specific protein expressed in muscle cells. Cultures with fewer than 97% of desmin-positive cells were not used for this study. After culturing in 10% FBS–DMEM for 72 h, differentiation was induced by substituting the media with DMEM containing 2% horse serum (HS; 2% HS–DMEM, Invitrogen), and the cultures were incubated thereafter for 48 h. All animal procedures were approved by the Experimental Animal Care and Use Committee at the Graduate School of Agriculture, Hokkaido University. Detection of quiescent satellite cells in vitro. To detect quiescent satellite cells, the cultures were incubated in 10% FBS–DMEM for 24 h, and then labeled for 16 h with 10 mM 5-bromo-2-deoxyuridine (BrdU; Amersham Biosciences, Piscataway, NJ) in 10% FBS–DMEM, followed by immunocytochemistry for detection of BrdU using a horseradish peroxidase-conjugated secondary antibody (Sigma). BrdU-negative cells were determined as quiescent satellite cells in vitro. Immunocytochemistry. Cultured cells were fixed at 24, 72, and 120 h with 2% formaldehyde in PBS (TPBS) for 10 min at room temperature. After washing three times with 0.5% Triton X-100 in PBS, specimens were blocked with 0.1% bovine serum albumin (Sigma) in PBS for 1 h at 37 °C, then incubated with anti-decorin antibody (1:100 dilution, Biogenesis, Kingston, NH) for 2 h at 37 °C, rinsed with TPBS
three times, and then treated with tetramethylrhodamine-50 -(and -6-)-isothiocyanate conjugated sheep IgG antibody (1:400 dilution, Chemicon International, Temecula, CA). After rinsing with TPBS, specimens were incubated with the antibody against myogenin (1:100 dilution, Santa Cruz Biotechnology Inc., Santa Cruz, CA) for 2 h at 37 °C, and then incubated with the secondary antibody conjugated with fluorescein isothiocyanate (1:500 dilution, Chemicon International) for 2 h at 37 °C. After rinsing, specimens were stained with DAPI (Dojindo, Kumamoto, Japan) and mounted in Aqua Poly Mount (PolyScience, Warrington, PA), and then observed using a microscope (DMI6000B, Leica Microsystems, Wetzlar, Germany). In vitro activation of satellite cells. Satellite cells isolated from adult rat skeletal muscle were cultured in 10% FBS–DMEM for 18 h, and then hepatocyte growth factor (HGF; R&D Systems, Minneapolis, MN) was added to the media to a final concentration of 5 or 10 ng/ml. After a further incubation for 22 h, cultures were pulse labeled for 2 h with 10 mM BrdU in 10% FBS–DMEM followed by immunocytochemistry for detection of BrdU using a horseradish peroxidase-conjugated secondary antibody. The percentage of BrdU labeled cells was used as an indicator of activation and entry into the cell cycle. RT-PCR. Total RNA was isolated from cell cultures using ISOGEN (Nippon gene, Tokyo, Japan), according to the manufacturer’s protocol. Decorin mRNA expression was analyzed by quantitative RT-PCR using a LightCycler instrument (Roche Diagnostics, Basel, Switzerland). The primers used to amplify the decorin fragment (250 bp, X59859) were 50 -TCGGATACATCCGCATCTCAGA-30 (forward) and 50 -G GCACTCTGAGGAGTTTGTTGT-30 (reverse). The primers for internal control glyceraldehydes-3-phosphate dehydrogenase (GAPDH) cDNA amplification (264 bp, AF106860) were 50 -TCACCACCATGGAGAAGGCT-30 (forward) and 50 -GCCATC CACAGTCTTCTGAG-30 (reverse). The real-time PCR conditions for amplification were as follows: denaturation at 95 °C for 30 s, annealing at 55 °C for 5 s, and extension at 72 °C for 10 s. Limited trypsinization to obtain myotubes and reserve cells. The method of Kitzmann et al. [17] was used to separate myotubes from reserve cells in differentiated cell cultures. Briefly, isolated satellite cells from adult rat skeletal muscle were seeded onto 10-cm culture dishes (Nunc) and cultured in 10% FBS–DMEM for 72 h and thereafter in 2% HS–DMEM for 48 h. Following incubation, a short trypsinization period (5 min, 0.01% trypsin–EDTA) and washing with PBS were used to preferentially detach myotubes. Reserve cells, which remained attached to the culture dish, were then detached by 10-min incubation with 0.25% trypsin. Statistical analysis. Data were analyzed using one-way analysis of variance (ANOVA) with Scheffe’s test. A probability of p < 0.05 was considered statistically significant.
Results First, we examined the expression of decorin during myogenesis by immunocytochemistry. After 24-h incubation in 10% FBS– DMEM, decorin staining was detectable in mononucleated myoblasts that were not stained with antibody against myogenin, a
Fig. 1. Expression of decorin during myogenesis in vitro. Satellite cells were isolated from 5-week-old Sprague–Dawley rats and cultured in 10% HS–DMEM for 72 h and thereafter in 2% HS–DMEM for 48 h. Cell cultures were fixed at 24 h, 72 h and 120 h and stained with anti-decorin and anti-myogenin antibodies.
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Relative abundance of decorin mRNA
marker of myogenic differentiation (Fig. 1). At 72 h, the decorin staining was observed in mononucleated myoblasts that were stained with antibody against myogenin and also detectable in differentiated myotubes with a few nuclei. After 48-h incubation in 2% HS–DMEM following the incubation in 10% FBS–DMEM for 72 h, the decorin staining was detectable in matured myotubes. These results demonstrate that decorin is expressed in proliferating myoblasts and differentiated myotubes. Next, we investigated if decorin was expressed in quiescent satellite cells by the BrdU-incorporation assay. Isolated satellite cells were incubated with BrdU for 16 h before staining with the antiBrdU antibody. Cells that were stained with anti-BrdU antibody (BrdU-positive cells) must be in the cell cycle and unstained cells (BrdU-negative cells) must be quiescent in the G0-phase. The decorin staining was observed in both BrdU-negative and -positive cells (Fig. 2). There was no difference in the staining pattern or strength between BrdU-positive and -negative cells. This result suggests that decorin is expressed in both quiescent and activated satellite cells. To compare the decorin expression level in quiescent satellite cells with that in activated satellite cells, we analyzed decorin mRNA expression in cell cultures having different ratios of activated satellite cells. Isolated satellite cells were cultured in 10% FBS–DMEM containing HGF (5 or 10 ng/ml) for 24 h, since HGF is known to activate satellite cells in vitro [18]. As shown in Fig. 3A, the ratio of activated satellite cells increased with the concentration HGF added. The ratio of activated satellite cells was 46% in cell cultures with 10 ng/ml of HGF added, whereas the ratio was 33% without HGF. The decorin mRNA level decreased with the concentration of HGF added. These results show that decorin mRNA expression in cell cultures with higher ratios of activated satellite cells is low, suggesting the possibility that decorin expression decreases when satellite cells are activated. In vitro there are myoblasts that remained undifferentiated when the cells are induced to differentiate with serum-deprivation, and they are termed as reserve cells [2]. Reserve cells are thought to be quiescent and similar to satellite cells in vivo. However, the mechanism by which reserve cells cannot commit to differentiation under the same conditions which cause others to differentiate is not known. In this study, we investigated the expression of decorin in reserve cells. First, we examined decorin expression in reserve cells by immunocytochemistry. Decorin staining was observed both reserve cells and differentiated myotu-
Raito of activated cells (%)
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*
**
40
20
0
0 10 5 Concentration of HGF (ng/ml)
1.0 0.8 0.6 0.4
*
0.2 0 0 10 5 Concentration of HGF (ng/ml)
Fig. 3. Decorin mRNA expression in cell cultures having different ratios of activated satellite cells. (A) Ratio of activated satellite cells in response to hepatocyte growth factor (HGF). Isolated satellite cells were cultured in 10% FBS–DMEM for 18 h, and then HGF was added to the media to a final concentration of 5 or 10 ng/ml. After a further incubation for 22 h, cultures were pulse labeled for 2 h with 10 mM BrdU followed by immunocytochemistry for detection of BrdU. The percentage of BrdU labeled cells was used as an indicator of activation and entry into the cell cycle. The columns represent mean values, and bars indicate SE **p < 0.01, *p < 0.05. (B) Decorin mRNA expression in cells cultured with of HGF. Total RNA was isolated from cell cultures, and decorin mRNA expression was analyzed by quantitative RT-PCR. The columns represent relative values of decorin mRNA normalized to GAPDH and expressed as the ratio of decorin mRNA in control cultures without HGF. Bars indicate SE *p < 0.05.
bes (Fig. 4A). Myogenin staining, on the other hand, was not observed in reserve cells. Next, we investigated the expression level of decorin mRNA in reserve cells. We isolate reserve cells from differentiated myotubes in the same culture according to the method of Kitzmann et al. [17] and compared the mRNA expression level of decorin in isolated reserve cells with those in active growing myoblasts and differentiated myotubes. The level of decorin mRNA in reserve cells was 14-fold higher than that in active growing myoblasts and differentiated myotubes (Fig. 4B). Discussion
Fig. 2. Expression of decorin in quiescent and activated satellite cells. Isolated satellite cells were cultured in 10% FBS–DMEM for 24 h and then labeled for 16 h with 10 mM BrdU in 10% FBS–DMEM. Cultures were fixed and stained with antibodies against BrdU, decorin and desmin. In these views, two cells (arrows) are activated cells (BrdU-positive) and the others are quiescent cells (BrdU-negative).
Satellite cells are quiescent muscle stem cells that promote postnatal muscle growth and repair. Although the mechanisms by which satellite cells are activated has been almost clarified [16,18,19], the mechanisms by which satellite cells return to quiescence and maintain the quiescent status are still unclear. The transition into quiescence in human fibroblasts is accompanied by the increased expression of a specific set of genes, which include decorin [14]. Furthermore, decorin expression in fibroblasts is highly stimulated by entrance into quiescence [15]. These results suggest that decorin plays an important role in induction and/or maintaining the quiescent status of fibroblasts. Thus, we sought to investigate the involvement of decorin in the quiescence of myogenic cells.
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Phase contrast
A
Decorin
DAPI
Myogenin
50 µm
Relative ab bundance of decorin mRNA
B
20
a
b
10
c
c 0 myoblasts
reserve cells & myotubes
reserve cells
myotubes
Fig. 4. Decorin expression in reserve cells. (A) Immunostaining of decorin and myogenin in reserve cells. Isolated satellite cells were cultured in 10% FBS–DMEM for 72 h and thereafter 2% HS–DMEM for 48 h. Cultures were fixed at 120 h and stained with antibodies against decorin and myogenin. (B) Decorin mRNA expression in reserve cells. After incubation with differentiation media (2% HS–DMEM) for 48 h, differentiated myotubes were detached by limited trypsinization (0.01% trypsin– EDTA) for 5 min, leaving a population of undifferentiated reserve cells. Total RNA was isolated from actively growing myoblasts cultured in 10% FBS–DMEM for 48 h, cell cultures (reserve cells and myotubes) before limited trypsinization, isolated reserve cells, and isolated myotubes. Decorin mRNA levels were detected by quantitative RT-PCR. The columns represent the relative values of decorin mRNA normalized to GAPDH and expressed as the ratio of decorin mRNA in active growing myoblasts. Different letters indicate significant differences p < 0.05.
Decorin was expressed in both activated satellite cells and quiescent satellite cells (Fig. 2). The expression level of decorin mRNA is lower in cell cultures that contained higher ratios of activated satellite cells (Fig. 3), suggesting that decorin expression must be high in quiescent satellite cells as compared with activated and proliferating cells. This is well consistence with the previously reported result that quiescent fibroblasts express decorin at high levels compared to proliferating fibroblasts [14]. Decorin may also play an important role in quiescence of satellite cells in skeletal muscle. At this time, it still remains unclear whether the reduction of decorin expression is needed to activate satellite cells, or whether activation induces the down-regulation of decorin expression. Activation stimulates quiescent satellite cells to exit quiescence and enter into the G1 phase of the cell cycle resulting in proliferation. Activation and proliferation appear to be separate but sequential events. Decorin over-expression inhibits proliferation of carcinoma cells via the up-regulation of p21, a cell cycle regulator [20,21]. Decorin might participate in proliferation of satellite cells after activation. An in vivo experiment has shown that progenies of a single satellite cell generate both differentiated myotubes and quies-
cent cells that are undifferentiated but still myogenic under differentiation-inducing conditions [22]. In vitro, a subset of myoblasts, termed reserve cell, remain quiescent and undifferentiated but retain their capacity for proliferation and myogenic differentiation [2]. We confirmed that reserve cells have the ability to differentiate when they were isolated from differentiating cultures and cultured again in 2% HS–DMEM (data not shown). Reserve cells have been characterized by their high expression of p130 and Myf5 and low expression of MyoD as compared to differentiated myotubes, which express low levels of p130 and Myf5 and high levels of MyoD [3]. Thus, the balance of these transcription factors could be important for the differentiation of myogenic cells. However, the mechanisms by which reserve cells cannot commit to differentiate under the same conditions as that induce other cells to differentiate is unknown. We sought to investigate if decorin participates in keeping reserve cells undifferentiated. Decorin mRNA expression in reserve cells was much higher than in differentiated myotubes and active growing myoblasts (Fig. 4). In our previous report, decorin over-expressing cells are not easily withdraw from the cell cycle and have delayed differentiation as compared with control cells, although decorin over-expressing cells eventually form multi-giant hypertrophic myotubes with larger size than control cells [23]. These results suggest that decorin negatively controls the initiation of differentiation. The high expression of decorin might suppress the exit from the cell cycle and maintain myogenic cells in an undifferentiated state. The total number of quiescent satellite cells remains constant over repeated cycles of degeneration and regeneration, suggesting that the steady state satellite cell population is maintained by self-renewal [24]. To keep the satellite cell pool ready for the next injury, some of the satellite cells that were activated and proliferated must reenter into the G0 phase and maintain quiescence. However, the induction and maintenance of the quiescent state in satellite cells remains unclear. Myostatin, a TGF-b super family member, has been shown to negatively regulate satellite cell activation to maintain quiescence [25]. Recently it has been shown that myostatin controls the process of self-renewal through regulation of Pax7 [26]. We have shown previously that decorin binds to myostatin and sequesters it within the extracellular matrix to regulate its activity [27] and that decorin attenuates myostatin signaling [23]. In this study, we have shown high expression of decorin in reserve cells, which are predominantly quiescent myoblasts, in response to a differentiation cue. Taken together, we speculate that decorin would play an important role in satellite cell self-renewal through attenuating myostatin signaling. Further study is needed to elucidate the role of decorin in satellite cell self-renewal. In summary, we here show that decorin is highly expressed in quiescent satellite cells in vitro and also that decorin expression is higher in reserve cells that do not commit to differentiation in vitro. These results suggest that decorin participates in keeping myogenic cells quiescent and undifferentiated. Acknowledgment We thank Dr. Shigeharu Fukunaga of the Biproduct Science Laboratory, Graduate School of Agriculture, Hokkaido University for technical assistance. References [1] T.J. Hawke, D.J. Garry, Myogenic satellite cells: physiology to molecular biology, J. Appl. Physiol. 91 (2001) 534–551. [2] N. Yoshida, S. Yoshida, K. Koishi, K. Masuda, Y. Nabeshima, Cell heterogeneity upon myogenic differentiation: down-regulation of MyoD and Myf-5 generates ‘reserve cells’, J. Cell Sci. 111 (1998) 769–779.
T. Nishimura et al. / Biochemical and Biophysical Research Communications 370 (2008) 383–387 [3] G. Carnac, L.L. Fajas, A. L’honoré, C. Sardet, N.J.C. Lamb, A. Fernandez, The retinoblastoma-like protein p130 is involved in the determination of reserve cells in differentiating myoblasts, Curr. Biol. 10 (2000) 543–546. [4] R.V. Iozzo, The biology of the small leucine-rich proteoglycans, J. Biol. Chem. 274 (1999) 18843–18846. [5] E. Schönherr, H. Hausser, L. Beavan, H. Kresse, Decorin-type I collagen interaction. Presence of separate core protein-binding domains, J. Biol. Chem. 270 (1995) 8877–8883. [6] T. Nishimura, E. Futami, J. Wakamatsu, A. Hattori, Identification and characterization of proteoglycans in bovine neonatal skeletal muscle, Anim. Sci. J. 74 (2003) 407–416. [7] K.G. Danielson, H. Balibault, D.F. Holmes, H. Graham, K.E. Kadler, R.V. Iozzo, Targeted disruption of decorin leads to abnormal collagen fibril morphology and skin fragility, J. Cell Biol. 136 (1997) 729–743. [8] H. Hausser, A. Gröning, A. Hasilik, E. Schönherr, H. Kresse, Selective inactivity of TGF-beta/decorin complexes, FEBS Lett. 353 (1994) 243–245. [9] Y. Takeuchi, Y. Kodama, T. Matsumoto, Bone matrix decorin binds transforming growth factor-beta and enhances its bioactivity, J. Biol. Chem. 269 (1994) 32634–32638. [10] Y. Yamaguchi, E. Ruoslahti, Expression of human proteoglycan in Chinese hamster ovary cells inhibits cell proliferation, Nature 336 (1988) 244–246. [11] Y. Yamaguchi, D.M. Mann, E. Ruoslahti, Negative regulation of transforming growth factor-b by the proteoglycan decorin, Nature 346 (1990) 281–284. [12] S. Patel, M. Santra, D.J. McQuillan, R.V. Iozzo, A.P. Thomas, Decorin activates the epidermal growth factor receptor and elevates cytosolic Ca2+ in A431 carcinoma cells, J. Biol. Chem. 273 (1998) 3121–3124. [13] R.V. Iozzo, D.K. Moscatello, D.J. McQuillan, I. Eichstetter, Decorin is a biological ligand for the epidermal growth factor receptor, J. Biol. Chem. 274 (1999) 4489–4492. [14] D.L. Coppock, C. Kopman, S. Scandalis, S. Gilleran, Preferential gene expression in quiescent human lung fibroblasts, Cell Growth Differ. 4 (1993) 483–491. [15] D.L. Coppock, C. Kopman, J. Gudas, D.A. Cina-Poppe, Regulation of the quiescence-induced genes: Quiescin Q6, decorin, and ribosomal protein S29, Biochem. Biophys. Res. Commun. 269 (2000) 604–610. [16] R.E. Allen, C.J. Temm-Grove, S.M. Sheehan, G. Rice, Skeletal muscle satellite cell cultures, Methods Cell Biol. 52 (1997) 155–176.
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[17] M. Kitzmann, G. Carnac, M. Vandromme, M. Primig, N.J. Lamb, A. Fernandez, The muscle regulatory factors MyoD and Myf-5 undergo distinct cell cycle– specific expression in muscle cells, J. Cell Biol. 142 (1998) 1447–1459. [18] R. Tatsumi, J.E. Anderson, C.J. Nevoret, O. Halevy, R.E. Allen, HGF/SF is present in normal adult skeletal muscle and is capable of activating satellite cells, Dev. Biol. 194 (1998) 114–128. [19] R. Tatsumi, X. Liu, A. Pulido, M. Morales, T. Sakata, S. Dial, A. Hattori, Y. Ikeuchi, R.E. Allen, Satellite cell activation in stretched skeletal muscle and the role of nitric oxide and hepatocyte growth factor, Am. J. Physiol. Cell Physiol. 290 (2006) C1487–C1494. [20] M. Santra, T. Skorski, B. Calabretta, E.C. Latrime, R.V. Iozzo, De novo decorin gene expression suppresses the malignant phenotype in human colon cancer cells, Proc. Natl. Acad. Sci. USA 92 (1995) 7016–7020. [21] M. Santra, D.M. Mann, E.W. Mercer, T. Skorski, B. Calabretta, R.V. Iozzo, Ectopic expression of decorin protein core causes a generalized growth suppression in neoplastic cells of various histogenetic origin and requires endogenous p21, an inhibitor of cyclin-dependent kinases, J. Clin. Invest. 100 (1997) 149–157. [22] A. Baroffio, M. Hamann, L. Bernheim, M.-L. Bochaton-Piallat, G. Gabbiani, C.R. Bader, Identification of self-renewing myoblasts in the progeny of single human muscle satellite cells, Differentiation 60 (1996) 47–57. [23] Y. Kishioka, M. Thomas, J. Wakamatsu, A. Hattori, M. Sharma, R. Kambadur, T. Nishimura, Decorin enhances the proliferation and differentiation of myogenic cells through suppressing myostatin activity, J. Cell. Physiol. (in press) . [24] H. Schmalbruch, D.M. Lewis, Dynamics of nuclei of muscle fibers and connective tissue cells in normal and denervated rat muscles, Muscle Nerve 23 (2000) 617–626. [25] S. McCroskery, M. Thomas, L. Maxwell, M. Sharma, R. Kambadur, Myostatin negatively regulates satellite cell activation and self-renewal, J. Cell Biol. 162 (2003) 1135–1147. [26] C. McFarlanea, A. Hennebry, M. Thomasa, E. Plummera, N. Ling, M. Sharma, R. Kambadur, Myostatin signals through Pax7 to regulate satellite cell selfrenewal, Exp. Cell Res. 314 (2008) 317–329. [27] T. Miura, Y. Kishioka, J. Wakamatsu, A. Hattori, A. Hennebry, C.J. Berry, M. Sharma, R. Kambadur, T. Nishimura, Decorin binds myostatin and modulates its activity to muscle cells, Biochem. Biophys. Res. Commun. 340 (2006) 675– 680.