Role of Osteoglycin in the Linkage between Muscle and Bone*

Background: The interaction between muscle tissues and bone metabolism has recently been noted. Results: Osteoglycin is produced in myoblastic cells and enhances bone formation parameters in osteoblasts. Conclusion: Osteoglycin may be a crucial humoral bone anabolic factor that is produced by muscle tissues. Significance: Osteoglycin may be the first potential humoral bone anabolic factor produced from muscle cells. The interaction between muscle tissues and bone metabolism is incompletely understood. We hypothesized that there might be some humoral factors that are produced in muscle tissues and exhibit bone anabolic activity. We, therefore, performed comparative DNA microarray analysis between mouse myoblastic C2C12 cells transfected with either stable empty vector or ALK2 (R206H), the mutation that constitutively activates the bone morphogenetic protein (BMP) receptor, to search for muscle-derived bone anabolic factors. Twenty-five genes whose expression was decreased to <1/4, were identified; these included osteoglycin (OGN). Stable overexpression of OGN significantly decreased the levels of Runx2 and Osterix mRNA compared with those in cells transfected with vector alone in MC3T3-E1 cells. On the other hand, it significantly enhanced the levels of alkaline phosphatase (ALP), type I collagen (Col1), and osteocalcin (OCN) mRNA as well as β-catenin and mineralization. A reduction in endogenous OGN level showed the opposite effects to those of OGN overexpression in MC3T3-E1 and mouse calvarial osteoblastic cells. Transient OGN overexpression significantly suppressed the levels of Runx2, Osterix, ALP, Col1, and OCN mRNA induced by BMP-2 in C2C12 cells. The conditioned medium from OGN-overexpressed and OGN-suppressed myoblastic cells enhanced and decreased, respectively, the levels of ALP, Col1, and β-catenin in MC3T3-E1 cells. Moreover, OGN increased Smad3/4-responsive transcriptional activity as well as Col1 mRNA levels independently of endogenous TGF-β in these cells. In conclusion, this study suggests that OGN may be a crucial humoral bone anabolic factor that is produced by muscle tissues.

Progress has recently been made in investigations of the relationships between bone metabolism and organ systems other than bone. For example, the linkages between bone and cardiovascular systems, nervous systems, adipose tissues, and pancreas have been noted. Previous studies indicate that muscle mass is closely related to high bone mass and a decrease in fracture risk in postmenopausal women (1)(2)(3). From these findings, we hypothesized that there might be some interactions between muscle tissues and bone metabolism.
Bone morphogenetic proteins (BMPs) 2 are known to be important in fracture healing but have been disappointing in human trials (4). However, a recent study showed that fractures that are covered with relatively intact muscle improve more rapidly than fractures associated with more severe damage (5,6). In that study muscle flaps applied to autogenous bone grafts improved healing, but coverage with skin did not. Moreover, proinflammatory cytokines, in particular tumor necrosis factor-␣ (TNF-␣), at the site of fracture induced the differentiation of stromal cells present in muscle into osteoprogenitor cells and promoted bone fracture healing (7). In that study muscle-derived mesenchymal cells were more effective as the source of cells that differentiate into osteoblastic cells than bone marrowderived mesenchymal cells. These findings suggest that muscle tissues play some important physiological and pathological roles through certain interactions between muscle tissues and bone metabolism. Muscle tissues can produce local growth factors, which have anabolic effects in bone tissues. For example, IGF-binding protein 5 (IGFBP5) is secreted from muscle tissues (8). We, therefore, hypothesized that there might be some humoral factors that are produced in muscle tissues and affect bone in an anabolic fashion, although there have been no reports about the relationships between the factors secreted from muscle tissues and bone metabolism.
Fibrodysplasia ossificans progressiva is a rare autosomal dominant disorder of skeletal malformations, which are characterized by postnatal progressive heterotopic ossification in soft tissues, especially skeletal muscle, and acute heterotopic ossification induced by muscle injury, such as accidental trauma or surgical operations (9 -11). A mutation causing constitutive activation (617G3 A; R206H) in a BMP type 1 receptor, the activin receptor type 1 (ACVR1)/activin-like kinase 2 (ALK2), is found in patients with the classic form of fibrodysplasia ossificans progressiva (10). This disease might provide some clue to link muscle tissues to bone.
In this study we performed comparative DNA microarray analysis between stable empty vector-and ALK2(R206H)transfected mouse myoblastic C2C12 cells to find novel factors that are produced in muscle tissues and exhibit bone anabolic activity. Osteoglycin (OGN) was identified as one of 25 genes whose expressions were decreased to Ͻ1/4 by ALK2 (R206H) overexpression in these cells. Our data suggest that OGN may be an important factor produced by muscle-derived cells and secreted into blood that exhibits bone anabolic effects.
The coding region of mouse OGN was amplified by reverse transcription PCR of total RNA from C2C12 cells using 5Ј-GCTGAAATGGAGACTGTGCACTCTA-3Ј as the forward primer and 5-GGTTAGAAGTATGACCCTATGGGTA-3Ј as the reverse primer. The cDNA was TA-cloned into the pCR2.1 vector (Invitrogen) according to the manufacturer's specifications. The OGN cDNA insert was cloned into the mammalian expression vector, pcDNA3.1(Ϫ) (Invitrogen). All constructs were verified by restriction enzyme analysis and nucleotide sequencing.
Transient and Stable Transfection-Each vector was transfected into MC3T3-E1 or C2C12 cells with Lipofectamine (Invitrogen) as previously described (12). Six hours later, the cells were supplied with fresh ␣-MEM or DMEM containing 10% FBS. Forty-eight hours later the transiently transfected cells were used for experiments. To generate stably transfected MC3T3-E1 or C2C12 cells, after incubation in ␣-MEM or DMEM containing 10% FBS for 48 h, the cells were passaged, and clones were selected in ␣-MEM or DMEM supplemented with G418 (0.3 mg/ml or 0.7 mg/ml, respectively; Invitrogen) and 10% FBS. Twenty-four clones were selected after 3 weeks of culture in G418. Several clones were selected after Western blotting with anti-OGN antibody. At least three independent clones for each stable transfection were characterized to rule out the possibility of clonal variation. Empty vector-transfected cell clones were used as control.
Protein Extraction and Western Blot Analysis-Cells were lysed with radioimmunoprecipitation buffer containing 0.5 mM phenylmethylsulfonyl fluoride, complete protease inhibitor mixture (Roche Applied Science), 1% Triton X-100, and 1 mM sodium orthovanadate. Proteins were transferred in 25 mM Tris, 192 mM glycine, and 20% methanol to polyvinylidene difluoride. Blots were blocked with 20 mM Tris-HCl (pH 7.5), 137 mM NaCl, 0.1% Tween 20 containing 3% dried milk powder. The membranes were immunoblotted with each primary antibody. The antigen-antibody complexes were visualized using the appropriate secondary antibodies (Sigma) and an enhanced chemiluminescence detection system as recommended by the manufacturer (Amersham Biosciences). The results depicted in each figure are representative of at least three independent cell preparations. Each experiment was repeated three times.
Microarray Analysis-Total RNA was extracted from C2C12 cells stably transfected with empty vector or ALK2 (R206H) using TRIzol reagent. Total RNA was purified using RNeasy MiniElute Clean Up kit (Qiagen, Tokyo, Japan) to yield an A 260/280 Ͼ1.90. Double-stranded cDNA was synthesized using a T7-oligo(dT) primer with the 3ЈIVT Express Kit (Affymetrix Inc., Santa Clara, CA). Hybridization samples were prepared and processed according to the GeneChip Expression Analysis Technical Manual. The Affymetrix Murine Genome 430 2.0 set was used to compare gene expression. Data were analyzed using the GeneChip Operating Software Version 1.4 (Affymetrix 690036) according to the GeneChip Expression Analysis Fundamentals, no.701190 Rev.4. Using DNA MicroArray Viewer (Kurabo, Osaka, Japan), the -fold changes in expression between two groups were calculated, log 2-transformed, and further classified as not changed, increased (signal log ratio change p value Ͻ0.005), decreased (signal log ratio change p value Ͻ0.005), or marginally increased or decreased.
Mineralization-Mineralization of MC3T3-E1 cells was determined in 6-well plates using Alizarin Red staining. After confluent cells were grown in ␣-MEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 10 mM ␤-glycerophosphate for 3 weeks, cells were fixed with ice-cold 70% ethanol and stained with Alizarin Red (Sigma) to detect calcification. For quantitation, cells stained with Alizarin Red were destained with ethylpyridinium chloride (Wako Pure Chemical Industries, Ltd.), the extracted stain was transferred to a 96-well plate, and absorbance at 562 nm was measured using a microplate reader as previously described (13).
Small Interfering RNA (siRNA)-Mouse OGN siRNA or control siRNA was transfected as recommended by the supplier (Santa Cruz Biotechnology) into cells seeded at 5 ϫ 10 5 per well using LipofectamineTM RNAi MAX (Invitrogen).
Luciferase Assay-Cells were seeded at a density of 2 ϫ 10 5 per 6-well plate. Twenty-four hours later, cells were transfected with 3 g of reporter plasmid (3GC2-Lux or 3TP-Lux) and empty vector only or OGN using Lipofectamine (Invitrogen). Fifteen hours later, the medium was changed to one containing 4% FBS. Thereafter, cells were cultured for 24 h in the presence or absence of 5 ng/ml TGF-␤ or 300 ng/ml BMP-2. Cells were lysed, and the luciferase activity was measured as previously described (14).
Statistics-All experiments were repeated at least three times. Data are expressed as the mean Ϯ S.E. Statistical analysis was performed using analysis of variance. A p value Ͻ0.05 was taken to indicate a significance difference.

RESULTS
Transcriptional Profiling-C2C12 cell clones stably expressing either empty vector or the V5-tagged-ALK2 (R206H) construct were prepared and screened by Western blot with an anti-V5 antibody. A clone with the highest level of expression was used for DNA microarray analysis in comparison with empty vector-transfected C2C12 cells. From ϳ45,000 genes on the Affymetrix gene chip, we identified 25 genes whose expression was decreased to Ͻ1/4 in the experimental versus the control group ( Table 1). The levels of OGN mRNA were significantly suppressed in the stable ALK2 R206H-transfected C2C12 cells compared with those in the empty vector-transfected cells and was further reduced by BMP-2 treatment (Fig. 1A). The levels of OGN FIGURE 1. The levels of OGN in myoblasts stably transfected with empty vector (V) and ALK2 (R206H). A, total RNA was extracted from C2C12 cells stably transfected with V or ALK2 (R206H) cultured in the presence or absence of 300 ng/ml BMP-2 for 48 h, and then real-time RT-PCR was performed. Data are expressed relative to the GAPDH mRNA value. *, p Ͻ 0.01 relative to V-transfected cells. **, p Ͻ 0.01 relative to ALK2 R206H-transfected C2C12 cells without BMP-2. B, total protein from C2C12 cells stably transfected with V or ALK2 (R206H) (#6, #8) was extracted, and Western blot analysis was performed with anti-OGN and ␤-actin antibodies.

Gene name
GenBank TM accession no. Ratio Ϫ2.0 protein were suppressed in the stable ALK2 (R206H)-transfected C2C12 cells compared with those in the empty vector-transfected cells (Fig. 1B).

OGN Expression during Osteoblast and Myotube
Differentiation-MC3T3-E1 cells in culture undergo osteoblastic differentiation with mineralization starting after 2-3 weeks. The levels of OGN mRNA increased for up to 3 weeks in MC3T3-E1 cells ( Fig. 2A). Myoblastic C2C12 cells differentiated into osteo-blastic cells when stimulated with BMP-2, and the levels of OGN mRNA increased with time (Fig. 2B). OGN levels did not change in the absence of BMP-2 (data not shown). C2C12 cells differentiated into myotube cells when stimulated with horse serum, and the levels of OGN mRNA increased with the myotube differentiation (Fig. 2C).
Effect of OGN on Osteoblast Phenotype and Mineralization-We examined whether OGN would affect osteoblast phenotype FIGURE 2. Expression of OGN increases during osteoblast and myotube differentiation. A, total RNA was extracted from wild-type MC3T3-E1 cells cultured for 7, 14, or 21 days, and real-time RT-PCR was performed. Data are expressed relative to the GAPDH mRNA value. *, p Ͻ 0.01 relative to wild-type MC3T3-E1 cells cultured for 7 days. B, total RNA was extracted from wild-type C2C12 cells treated with 300 ng/ml BMP-2 for 3, 10, or 17 days, and real-time RT-PCR was performed. Data are expressed relative to the GAPDH mRNA value. *, p Ͻ 0.01 relative to C2C12 cells treated for 3 days. C, total RNA was extracted from wild-type C2C12 cells cultured with or without horse serum for 6 days, and real-time RT-PCR was performed. Data are expressed relative to the GAPDH mRNA value. *, p Ͻ 0.01 relative to wild-type C2C12 cells cultured without horse serum.
in MC3T3-E1 cells. As shown in Fig. 3A, stable overexpression of OGN significantly suppressed the levels of Runx2 and Osterix mRNA compared with those of cells transfected only with vector in MC3T3-E1 cells. On the other hand, it significantly enhanced the levels of ALP, Col1, and OCN mRNA.
Moreover, stable OGN overexpression enhanced the levels of Col1 and ␤-catenin protein in MC3T3-E1 cells (Fig. 3B). The optimal concentration of recombinant OGN protein in MC3T3-E1 cells was 10 g/ml in our preliminary study (data not shown). As shown in Fig. 3C, recombinant OGN protein enhanced the levels of Col1, ALP, and OCN mRNA levels, although it significantly suppressed the levels of Runx2 and Osterix mRNA in MC3T3-E1 cells. Moreover, OGN enhanced the levels of Col1 and ␤-catenin protein in these cells (Fig. 3D). These results were consistent with the effects of OGN overexpression on osteoblast markers. Next, we examined the effects of OGN overexpression on mineralization in MC3T3-E1 cells. Stable OGN overexpression induced mineralization compared with empty vector-transfected cells in the presence of ascorbic acid and ␤-glycerophosphate. These findings were confirmed in quantitation analysis of Alizarin Red (Fig. 3E). Although a reduction in endogenous OGN levels by siRNA enhanced the levels of Runx2 and Osterix mRNA, it suppressed the levels of Col1, ALP, and OCN mRNA in MC3T3-E1 cells (Fig. 3F). A reduction in endogenous OGN level by siRNA suppressed the levels of Col1 and ␤-catenin protein in MC3T3-E1 cells (Fig.  3G). Moreover, we examined the effects of a reduction in endogenous OGN level by siRNA on the osteoblast phenotypes in mouse primary calvarial osteoblast cultures. As shown in Fig.  3H, a reduction in endogenous OGN levels by siRNA enhanced the level of Runx2 mRNA, and it suppressed the levels of Col1, ALP, and OCN mRNA in mouse primary osteoblasts. It suppressed the levels of Col1 and ␤-catenin protein in mouse primary osteoblasts (Fig. 3I). Role of Myoblastic Cell-derived OGN in Osteoblasts-We obtained the conditioned medium from OGN-overexpressed or -suppressed mouse myoblastic C2C12 cells. The levels of OGN in conditioned medium were shown in Fig. 5A. The levels of OGN were elevated and decreased in the conditioned medium from C2C12 cells, in which OGN was overexpressed by OGN transient transfection and decreased by siRNA transfection, respectively. Conditioned medium from transient OGN-overexpressing C2C12 cells decreased the levels of Runx2 and Osterix mRNA, and it enhanced the levels of Col1, ALP, and OCN mRNA in MC3T3-E1 cells compared with conditioned medium from empty vector-transfected C2C12 cells (Fig. 5B). Conditioned medium from stable OGN-overexpressing C2C12 cells enhanced the levels of Col1 and ␤-catenin protein in MC3T3-E1 cells (Fig. 5C). Moreover, although conditioned medium from endogenous C2C12 cells with OGN suppression by siRNA increased the levels of Runx2 and Osterix mRNA, it decreased the levels of Col1, ALP, and OCN mRNA in MC3T3-E1 cells compared with conditioned medium from control siRNA-transfected C2C12 cells (Fig. 5D). Moreover, it decreased the levels of Col1 and ␤-catenin protein in these cells (Fig. 5E). In addition, we examined the effects of conditioned medium from OGN-overexpressed or -suppressed C2C12 cells on the osteoblast phenotypes in mouse primary calvarial osteoblast cultures. Conditioned medium from transiently OGN-overexpressing mouse myoblastic C2C12 cells decreased the levels of Runx2 mRNA, and it enhanced the levels of Col1, ALP, and OCN mRNA in mouse primary osteoblasts (Fig. 5F). Moreover, conditioned medium from endogenous C2C12 cells with OGN suppression by siRNA increased the levels of Runx2 mRNA and decreased the levels of Col1, ALP, and OCN mRNA in mouse primary osteoblasts (Fig. 5G). Similar data were obtained from the experiments using the conditioned medium from stably OGN-overexpressing, horse serum-treated C2C12 cells (the putative myotube cells) (data not shown). Moreover, in our preliminary study the OGN protein was detected in human serum by Western blot analysis (data not shown).

Effects of OGN on BMP-2-induced Differentiation of Myoblasts into Osteoblasts-We
Role of TGF-␤ in Enhancement of Col1 Expression by OGN-Because a previous study indicated that OGN induces bone formation in conjunction with TGF-␤ (15, 16), we examined the relationships between OGN and TGF-␤ in the enhancement of Col1 expression by OGN in osteoblasts. We employed 3TP-Lux, a Smad3/4-responsive luciferase reporter, to examine the effects of OGN on TGF-␤-induced transcriptional activity in osteoblasts. As shown in Fig. 6A, OGN significantly increased Smad3/4-responsive transcriptional activity in MC3T3-E1 cells, and it additionally enhanced TGF-␤-induced transcriptional activity. SB431542, an inhibitor of endogenous TGF-␤ signaling (ALK5 inhibitor), did not affect OGN-induced transcriptional activity, although it antagonized TGF-␤ effects (Fig. 6A). OGN significantly enhanced TGF-␤-induced Col1 mRNA levels in MC3T3-E1 cells, and SB431542 did not affect OGN-induced Col1 mRNA levels, although it antagonized  OGN (#14, #17), and real-time RT-PCR for Runx2, Osterix, Col1, ALP, OCN, OGN, or GAPDH was performed. Data are expressed relative to the GAPDH mRNA value. *, p Ͻ 0.01 relative to V-transfected cells. B, total protein from MC3T3-E1 cells stably transfected with V or OGN was extracted, and Western blot analysis for anti-Col1, ␤-catenin, and ␤-actin antibodies was performed. C, total RNA was extracted from MC3T3-E1 cells treated with or without recombinant OGN (10 g/ml) for 48 h, and real-time RT-PCR for Runx2, Osterix, Col1, ALP, OCN, or GAPDH was performed. Data are expressed relative to the GAPDH mRNA value. *, p Ͻ 0.01 relative to OGN-untreated cells. D, total protein from MC3T3-E1 cells treated with or without recombinant OGN (10 g/ml) for 48 h was extracted, and Western blot analysis for anti-Col1, ␤-catenin, and ␤-actin antibodies was performed. E, MC3T3-E1 cells stably transfected with V, and OGN (#14, #17) were cultured for 21 days in the presence of 50 g/ml ascorbic acid and 10 mM ␤-glycerophosphate and Alizarin Red staining was performed. Mineralization was quantified by Alizarin Red staining as described under "Experimental Procedures." *, p Ͻ 0.01 relative to V-transfected cells. F, total RNA was extracted from MC3T3-E1 cells transfected with control siRNA or OGN siRNA, and real-time RT-PCR for Runx2, Osterix, Col1, ALP, OCN, OGN, or GAPDH was performed. Data are expressed relative to the GAPDH mRNA value. *, p Ͻ 0.01 relative to transient control siRNA-transfected cells. G, control siRNA-or OGN siRNA-transfected MC3T3-E1 cells were analyzed by Western blot with anti-Col1, ␤-catenin, and ␤-actin antibodies. H, total RNA was extracted from control siRNA-or OGN siRNA-transfected mouse calvarial bone cells, and real-time RT-PCR for Runx2, Col1, ALP, OCN, OGN, or GAPDH was performed. Data are expressed relative to the GAPDH mRNA value. *, p Ͻ 0.01 relative to control siRNA-transfected cells. I, control siRNA-or OGN siRNA-transfected mouse calvarial osteoblastic cells were analyzed by Western blot with anti-Col1, ␤-catenin, and ␤-actin antibodies.
TGF-␤ effects (Fig. 6B). Next we examined whether OGN would affect the expression of TGF-␤ in osteoblasts. As shown in Fig. 6, C and D, transient OGN overexpression or endogenous OGN suppression by siRNA did not affect the levels of TGF-␤ mRNA and protein compared with those in each control group in MC3T3-E1 cells. Moreover, OGN did not affect TGF-␤-induced phosphorylation of Smad2/3 protein (Fig.  6E). Previous studies suggest that MAP kinases are related to Smad-independent signaling of TGF-␤ (13,40). We, therefore, performed the additional experiments to examine the involvement of MAP kinases in OGN-induced Col1 mRNA levels using several MAP kinase inhibitors in MC3T3-E1 cells. PD98059, SB203580, and curcumin are inhibitors of ERK1/2, p38 MAP kinase, and c-Jun N-terminal kinase (JNK), respectively. As shown in Fig. 6F, inhibitors of p38 MAP kinase or JNK did not affect Col1 mRNA levels induced by OGN. However, OGN enhanced the levels of phosphorylated ERK1/2, and an inhibitor of ERK1/2 significantly antagonized the levels of Col1 mRNA enhanced by OGN in MC3T3-E1 cells (Fig. 6, F and G), suggesting that OGN seemed to enhance the levels of Col1 mRNA partly through ERK1/2 in osteoblasts.

DISCUSSION
Several lines of evidence suggest certain interactions between muscle tissues and bone metabolism. In this study we aimed to find some humoral factors that are produced in muscle tissues and exhibit bone anabolic activity. We hypothesized that the signal suppressed by the conversion of muscle tissues into bone might give us a clue to find out muscle-derived bone anabolic factors because those factors could be predominantly expressed in muscle tissues, compared with their expressions in
bone, and their systemic effects through blood could be more important than their local effects in muscle tissues. We, therefore, performed comparative DNA microarray analysis between stable empty vector-and ALK2(R206H)-transfected mouse myoblastic cells, as fibrodysplasia ossificans progressiva and its molecular pathogenesis could be a clue to help identify some muscle-derived humoral bone anabolic factors. Then we selected several factors that exhibited decreased expression levels upon ALK2(R206H) expression. Twenty-five genes showed expression that decreased to Ͻ1/4 in ALK2 (R206H)-transfected C2C12 cells compared with that in empty vector-transfected cells. Among them were several bone-related factors, such as decorin, follistatin, Nov, interleukin 33 (IL-33) and OGN. Decorin is a well characterized, secreted small leucinerich proteoglycan in bone, which binds to collagen and several bone matrix proteins (17). In a previous microarray analysis of C2C12 cells with and without BMP-2 treatment, several members of the leucine-rich-repeat family of proteoglycans, such as decorin, byglycan, osteomodulin, fibromodulin, and OGN, were down-regulated by BMP-2 treatment (18). Our preliminary study in MC3T3-E1 and C2C12 cells suggested that follistatin, Nov, and IL-33 do not enhance the osteoblast phenotype (data not shown). We, therefore, investigated the function of OGN because several studies have suggested its potential as a bone anabolic factor.
OGN is the seventh member of the small leucine-rich proteoglycans that was originally called osteoinductive factor. The small leucine-rich proteoglycans have core proteins ranging in size from 25 to 62 kDa and share a common protein structure that is composed of 6 -11 repeats of leucine-rich regions. OGN belongs to class III of the small leucine-rich proteoglycans, and it was initially isolated from bovine bone as an inducer of matrix mineralization (19). OGN cDNA is highly expressed in mouse hypertrophic chondrocytes as well as cochlea tissues (20,21). OGN down-regulation was found to be correlated with arteriogenesis in the adventitia of rabbit collateral arteries (22). High expression of OGN inhibits metastatic behavior and decreases gelatinase activity of murine hepatocarcinoma Hca-F cells (23,24). In this study the levels of OGN were elevated during osteoblast differentiation, which was consistent with a previous study (25). Moreover, some comparative microarray studies revealed that OGN is one of the mechanosensitive genes that mediates an anabolic response of mechanical loading in mice and in osteoblasts using a rotating wall vessel and a random positioning machine (26,27). These findings suggest that OGN might play an important role in bone formation by osteoblasts at the well differentiated stage. In our data the levels of OGN were decreased by activation of ALK2 signaling, which pathologically converts muscle tissues to bone in fibrodysplasia ossificans progressiva, and the levels of OGN increased when myoblastic cells differentiated into myotubes upon horse serum treatment. Moreover, Chan et al. (25) recently reported that OGN was included in differentially up-regulated secretome components during skeletal myogenesis in C2C12 cells in a recent study. These findings suggest that OGN might play some important role in muscle.
In this study stable OGN overexpression enhanced the levels of ALP, Col1, ␤-catenin, and OCN as well as mineralization in MC3T3-E1 cells, although it decreased the levels of Runx2 and Osterix. Moreover, endogenous reduction of OGN levels by siRNA decreased the levels of ALP, Col1, ␤-catenin, and OCN in these cells, although it elevated the levels of Runx2 and Osterix in these cells. In addition, OGN overexpression antagonized BMP-2-induced differentiation of myoblastic cells into osteoblasts.
A previous report showed that the osteoblast number increases at an early differentiation stage, but the osteoblast functions were impaired in osteoblast-specific Runx2-overexpressing transgenic mice (28), suggesting that Runx2 negatively acts on osteoblast differentiation of the osteoblastic cells at a later differentiation stage, whereas it induces osteoblast differentiation in immature osteoblasts at an earlier differentiation stage. From these findings we can speculate that OGN is a factor that suppresses osteoblastic differentiation of premature osteoblasts and enhances osteoblast phenotype and mineralization in well differentiated osteoblasts.
There are some humoral factors that are produced in nonendocrine organs and affect bone. Adiponectin is one of the adipokines that is produced in adipose tissue and regulates energy homeostasis and insulin sensitivity. Several studies indicate that adiponectin increases osteoclast apoptosis and decreases proliferation of osteoclast precursor cells (29,30). Leptin, a circulating hormone produced by adipose tissue, is a multifunctional hormone that plays important roles in body weight homeostasis, neuroendocrine function, fertility, immune function, and angiogenesis (31). A recent study showed FIGURE 5. Effects of conditioned medium from OGN-overexpressed or -suppressed myoblastic cells on osteoblast phenotype. A, conditioned medium was obtained from the cultures of C2C12 cells transiently transfected with empty vector (V) or OGN, or the cultures of C2C12 cells transfected with control siRNA-or OGN siRNA for 24 h. The conditioned medium was analyzed with Western blot analysis for anti-OGN and ␤-actin antibodies. B, conditioned medium was obtained from the cultures of C2C12 cells transiently transfected with empty vector (V) or OGN for 24 h. Wild-type MC3T3-E1 cells were cultured with 20% of each conditioned medium for 24 h. Total RNA was extracted from these cells, and real-time RT-PCR for Runx2, Osterix, Col1, ALP, OCN, or GAPDH was performed. Data are expressed relative to the GAPDH mRNA value. *, p Ͻ 0.01 relative to V-transfected groups. C, wild-type MC3T3-E1 cells were cultured with 20% of each conditioned medium for 48 h. Total protein from these cells was extracted, and Western blot analysis for anti-Col1, ␤-catenin, and ␤-actin antibodies was performed. D, conditioned medium was obtained from the cultures of control siRNA-or OGN siRNA-transfected C2C12 cells for 24 h. Wild-type MC3T3-E1 cells were cultured with 20% of each conditioned medium for 24 h. Total RNA was extracted from these cells, and real-time RT-PCR for Runx2, Osterix, Col1, ALP, OCN, or GAPDH was performed. Data are expressed relative to the GAPDH mRNA value. *, p Ͻ 0.01 relative to control siRNA-transfected groups. E, wild-type MC3T3-E1 cells were cultured with 20% of each conditioned medium for 48 h. Total protein from these cells was extracted, and Western blot analysis for anti-Col1 and ␤-catenin or ␤-actin antibodies was performed. F, conditioned medium was obtained from the cultures of transient V-or OGNtransfected C2C12 cells for 24 h. Mouse calvarial osteoblasts were cultured with 20% of each conditioned medium for 24 h. Total RNA was extracted from these cells, and real-time RT-PCR for Runx2, Col1, ALP, OCN, or GAPDH was performed. Data are expressed relative to the GAPDH mRNA value. *, p Ͻ 0.01 relative to V-transfected groups. G, conditioned medium was obtained from the cultures of control siRNA-or OGN siRNA-transfected C2C12 cells cultured for 24 h. Mouse calvarial osteoblasts were cultured with 20% of each conditioned medium for 24 h. Total RNA was extracted from these cells, and real-time RT-PCR for Runx2, Col1, ALP, OCN, or GAPDH was performed. Data are expressed relative to the GAPDH mRNA value. *, p Ͻ 0.01 relative to control siRNA-transfected groups. **, p Ͻ 0.05 relative to control siRNA-transfected groups.
that the role of leptin receptor signaling is as a negative modulator of bone mechanosensitivity (32). Moreover, leptin affects bone in a catabolic fashion through the central nervous system (33). On the other hand, IL-6, fibroblast growth factor 21, IL-8, and IL-15 are myokines that are produced by muscle tissues and affect other organs. IL-6 produced by type 1 and 2 muscle fibers increases hepatic glucose production during exercise and lipolysis in adipose tissues (34,35). Fibroblast growth factor 21, pro-duced from skeletal muscle, is an important endogenous regulator for systemic glucose and lipid metabolism (36,37). IL-8, produced by contracting myofibers, exerts its effect locally and plays a role in exercise-induced angiogenesis (38). IL-15 is produced in human skeletal muscle in vitro and in vivo, and it takes part in reducing adipose tissue mass (39). In this study conditioned medium from OGN-overexpressed and OGN-suppressed myoblastic cells enhanced and decreased the levels of FIGURE 6. Role of TGF-␤ in the enhancement of Col1 expression by OGN. A, MC3T3-E1 cells were transfected with 3 g of the Smad3/4 reporter construct, 3TP-Lux, and empty vector (V) or OGN. Cells were treated without (Ϫ) or with (ϩ) 5 ng/ml TGF-␤ for 24 h in the presence or absence of 5 M SB431542 pretreatment for 1 h. Cells were extracted, and relative luciferase activity was measured. Values are mean Ϯ S.E. of four experiments. *, p Ͻ 0.01 relative to V-transfected-TGF-␤ (Ϫ) SB431542 (Ϫ) group. **, p Ͻ 0.01 relative to V-transfected-TGF-␤ (ϩ) SB431542 (Ϫ) group. ***, p Ͻ 0.01 relative to OGN-transfected TGF-␤ (ϩ) SB431542 (Ϫ) group. B, MC3T3-E1 cells were transfected with 3 g of V or OGN. Cells were treated without (Ϫ) or with (ϩ) 5 ng/ml TGF-␤ for 24 h in the presence or absence of 5 M SB431542 pretreatment for 1 h. Total RNA was extracted, and real-time RT-PCR for Col1 or GAPDH was performed. Data are expressed relative to the GAPDH mRNA value. Values are the mean Ϯ S.E. *, p Ͻ 0.01 relative to V-transfected-TGF-␤ (Ϫ) SB431542 (Ϫ) group. **, p Ͻ 0.01 relative to V-transfected-TGF-␤ (ϩ) SB431542 (Ϫ) group. ***, p Ͻ 0.01 relative to OGN-transfected TGF-␤ (ϩ) SB431542 (Ϫ) group. C, total RNA was extracted from MC3T3-E1 cells transiently transfected with V or OGN (left) or control siRNA or OGN siRNA (right), and real-time RT-PCR for TGF-␤ or GAPDH was performed. Data are expressed relative to the GAPDH mRNA value. D, total protein was extracted from MC3T3-E1 cells transiently transfected with V or OGN (left) or control siRNA or OGN siRNA (right), and Western blot analysis for anti-TGF-␤ and ␤-actin antibodies was performed. E, MC3T3-E1 cells were transfected with 3 g of V or OGN. Cells were treated without (Ϫ) or with (ϩ) 5 ng/ml TGF-␤ for 24 h, and then total protein was extracted. Western blot analysis for anti-p-Smad2/3, Smad2, Smad3, or ␤-actin antibodies was performed. F, confluent MC3T3-E1 cells were treated in the presence or absence of 10 g/ml recombinant OGN without (Ϫ) or with (ϩ) PD98059, SB203580, or curcumin (each 10 M) for 48 h after pretreatment with these inhibitors for 1 h. Total RNA was extracted, and real-time RT-PCR for Col1 or GAPDH was performed. Data are expressed relative to the GAPDH mRNA value. Values are the mean Ϯ S.E. *, p Ͻ 0.01 relative to each inhibitor-untreated, recombinant OGN (Ϫ) group. **, p Ͻ 0.01 relative to each inhibitor-untreated, recombinant OGN (ϩ) group. G, MC3T3-E1 cells were treated for the indicated times with 10 g/ml recombinant OGN in serum free ␣-MEM. The cell extracts were then immunoblotted with anti-phosphorylated ERK1/2 and total ERK1/2 antibodies. ALP, Col1, and ␤-catenin in mouse osteoblastic cells, respectively. These effects were parallel to the effects of OGN overexpression and repression in osteoblasts. Moreover, OGN protein is detected in human serum and culture supernatant of mouse myoblastic cells. These findings indicate that OGN, produced in muscle, exhibits bone anabolic activity in osteoblasts. However, we cannot completely rule out the possibility that the conditioned media would contain changes besides just the enhanced production and secretion of OGN.
TGF-␤ is produced by osteoblasts and released from the bone matrix and regulates bone metabolism in various ways (40). TGF-␤ binds to its type ⌱ membrane receptor promoting dimerization with the type ⌸ receptor, stimulating its Ser/Thr kinase activity, and causing phosphorylation of Smad2 and Smad3 (p-Smad2/3); the activated Smads associate with Smad4 and translocate into the nucleus (41,42). Previous studies indicate that Smad3 is an important molecule for osteoblastic bone formation (13,(43)(44)(45). Several bioactive proteins, such as p53, basic fibroblast growth factor, interferon-␥, and BMP-1, were reported to interact with OGN (46 -50). Moreover, previous studies indicate that OGN interacts with TGF-␤ and induces bone formation in conjunction with TGF-␤ (15,16). In this study OGN induced Smad3/4-responsive transcriptional activity as well as Col1 mRNA levels independently of endogenous TGF-␤, and it augmented TGF-␤-induced transcriptional activity and collagen levels in mouse osteoblastic cells. Moreover, OGN did not affect the levels of TGF-␤ as well as the phosphorylation of Smad2/3 induced by TGF-␤. These findings suggest that OGN enhances TGF-␤-induced collagen expression through its interaction with the TGF-␤ pathway downstream rather than the phosphorylation of Smad2/3 by TGF-␤. This interaction between OGN and the TGF-␤ pathway might play some role in the bone anabolic activity by OGN. OGN plays some role in collagen fibrillogenesis in many tissues, such as eye and skin (50,51). OGN is one of the three major keratan sulfate-containing proteoglycans in the cornea, and its glycosaminoglycan side chains seem to affect fibril diameter, interfibrillar spacing, and normal tissue hydration (52). In an OGNdeficient mouse, the diameter of the corneal collagen fibrils was significantly increased by transmission electron microscopy and reduced tensile strength of the skin was observed (51,53). These data suggest that OGN might determine elasticity and tensile strength. Moreover, OGN might play a role in regulating the collagen structures of the auditory system and in hair cell development (20). Although further studies are necessary to clarify the details of the relationship between OGN and collagen in bone, OGN may enhance bone strength through alterations of the structure and quality of collagen matrix, other bone matrix proteins, and proteoglycans.
Exercise therapy and an increase in muscle mass are considered to be very effective for an increase in bone mineral density and a reduction in fracture risk in osteoporotic patients. However, therapy to improve these factors is clinically very difficult as the physical activity of osteoporotic patients is usually disturbed. Humoral bone anabolic factors, produced in muscle tissues, may be important as target molecules for the treatment and prevention of osteoporosis as well as bone metabolic indexrelated muscle and exercise therapy. Because OGN may be one of the humoral bone anabolic factors produced from muscle, a further clinical study using patient samples and an in vivo study using muscle-specific OGN-deleted or -transgenic mice are necessary.
In conclusion, OGN was selected as a molecule that is downregulated by ALK2 (R206H)-transfected myoblastic cells. The level of OGN as well as the effects of the conditioned medium from OGN-modulated myoblastic cells was positively correlated with osteoblast phenotype and mineralization in osteoblastic cells, although it seemed to reduce osteoblast differentiation in osteoblasts at the early differentiation stage and myoblasts. OGN may be a crucial humoral bone anabolic factor that is produced in muscle.