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• W2092976745 abstract "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. 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 (1Capozza R.F. Cure-Cure C. Cointry G.R. Meta M. Cure P. Rittweger J. Ferretti J.L. Association between low lean body mass and osteoporotic fractures after menopause.Menopause. 2008; 15: 905-913Crossref PubMed Scopus (32) Google Scholar, 2Khosla S. Atkinson E.J. Riggs B.L. Melton 3rd, L.J. Relationship between body composition and bone mass in women.J. Bone Miner Res. 1996; 11: 857-863Crossref PubMed Scopus (279) Google Scholar, 3Nakaoka D. Sugimoto T. Kaji H. Kanzawa M. Yano S. Yamauchi M. Sugishita T. Chihara K. Determinants of bone mineral density and spinal fracture risk in postmenopausal Japanese women.Osteoporos Int. 2001; 12: 548-554Crossref PubMed Scopus (56) Google Scholar). From these findings, we hypothesized that there might be some interactions between muscle tissues and bone metabolism. Bone morphogenetic proteins (BMPs) 2The abbreviations used are: BMPbone morphogenetic proteinALK2activin-like kinase 2OCNosteocalcinOGNosteoglycinALPalkaline phosphataseCol1type I collagenα-MEMα-minimal essential medium. are known to be important in fracture healing but have been disappointing in human trials (4Bragdon B. Moseychuk O. Saldanha S. King D. Julian J. Nohe A. Bone morphogenetic proteins. A critical review.Cell. Signal. 2011; 23: 609-620Crossref PubMed Scopus (488) Google Scholar). However, a recent study showed that fractures that are covered with relatively intact muscle improve more rapidly than fractures associated with more severe damage (5Utvåg S.E. Grundnes O. Rindal D.B. Reikerås O. Influence of extensive muscle injury on fracture healing in rat tibia.J. Orthop. Trauma. 2003; 17: 430-435Crossref PubMed Scopus (64) Google Scholar, 6Richards R.R. McKee M.D. Paitich C.B. Anderson G.I. Bertoia J.T. A comparison of the effects of skin coverage and muscle flap coverage on the early strength of union at the site of osteotomy after devascularization of a segment of canine tibia.J. Bone Joint Surg. Am. 1991; 73: 1323-1330Crossref PubMed Scopus (63) Google Scholar). 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-α (TNF-α), at the site of fracture induced the differentiation of stromal cells present in muscle into osteoprogenitor cells and promoted bone fracture healing (7Glass G.E. Chan J.K. Freidin A. Feldmann M. Horwood N.J. Nanchahal J. TNF-α promotes fracture repair by augmenting the recruitment and differentiation of muscle-derived stromal cells.Proc. Natl. Acad. Sci. U.S.A. 2011; 108: 1585-1590Crossref PubMed Scopus (272) Google Scholar). In that study muscle-derived mesenchymal cells were more effective as the source of cells that differentiate into osteoblastic cells than bone marrow-derived 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 (8Chan X.C. McDermott J.C. Siu K.W. Identification of secreted proteins during skeletal muscle development.J. Proteome Res. 2007; 6: 698-710Crossref PubMed Scopus (66) Google Scholar). 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. bone morphogenetic protein activin-like kinase 2 osteocalcin osteoglycin alkaline phosphatase type I collagen α-minimal essential medium. 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 (9Kaplan F.S. Le Merrer M. Glaser D.L. Pignolo R.J. Goldsby R.E. Kitterman J.A. Groppe J. Shore E.M. Fibrodysplasia ossificans progressiva.Best Pract. Res. Clin. Rheumatol. 2008; 22: 191-205Crossref PubMed Scopus (231) Google Scholar, 10Shore E.M. Xu M. Feldman G.J. Fenstermacher D.A. Cho T.J. Choi I.H. Connor J.M. Delai P. Glaser D.L. LeMerrer M. Morhart R. Rogers J.G. Smith R. Triffitt J.T. Urtizberea J.A. Zasloff M. Brown M.A. Kaplan F.S. A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva.Nat. Genet. 2006; 38: 525-527Crossref PubMed Scopus (866) Google Scholar, 11Fukuda T. Kohda M. Kanomata K. Nojima J. Nakamura A. Kamizono J. Noguchi Y. Iwakiri K. Kondo T. Kurose J. Endo K. Awakura T. Fukushi J. Nakashima Y. Chiyonobu T. Kawara A. Nishida Y. Wada I. Akita M. Komori T. Nakayama K. Nanba A. Maruki Y. Yoda T. Tomoda H. Yu P.B. Shore E.M. Kaplan F.S. Miyazono K. Matsuoka M. Ikebuchi K. Ohtake A. Oda H. Jimi E. Owan I. Okazaki Y. Katagiri T. Constitutively activated ALK2 and increased SMAD1/5 cooperatively induce bone morphogenetic protein signaling in fibrodysplasia ossificans progressiva.J. Biol. Chem. 2009; 284: 7149-7156Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). A mutation causing constitutive activation (617G→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 (10Shore E.M. Xu M. Feldman G.J. Fenstermacher D.A. Cho T.J. Choi I.H. Connor J.M. Delai P. Glaser D.L. LeMerrer M. Morhart R. Rogers J.G. Smith R. Triffitt J.T. Urtizberea J.A. Zasloff M. Brown M.A. Kaplan F.S. A recurrent mutation in the BMP type I receptor ACVR1 causes inherited and sporadic fibrodysplasia ossificans progressiva.Nat. Genet. 2006; 38: 525-527Crossref PubMed Scopus (866) Google Scholar). 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 <1/4 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. MC3T3-E1 cells were provided by Dr. H. Kodama (Ohu Dental College, Koriyama, Japan). Mouse calvarial osteoblasts from 2–4-day-old ICR mice were obtained from Primary Cell Co., Ltd., Sapporo, Japan. Human (h) recombinant BMP-2, anti-β-actin, PD98059, SB203580, curcumin, and recombinant human TGF-β1 were obtained from Sigma. Anti-alkaline phosphatase (ALP), anti-β-catenin, anti-phosphorylated Smad1/5/8, anti-Smad1, anti-Smad5, anti-TGF-β1, anti-OGN antibodies, OGN siRNA(m), and control siRNA were from Santa Cruz Biotechnology. Anti-extracellular signal-regulated kinase (ERK1/2) and anti-phospho ERK1/2 antibodies were from Cell Signaling Technology. Anti-The pcDEF3-ALK2 (R206H) V5-tagged construct was as previously described (11Fukuda T. Kohda M. Kanomata K. Nojima J. Nakamura A. Kamizono J. Noguchi Y. Iwakiri K. Kondo T. Kurose J. Endo K. Awakura T. Fukushi J. Nakashima Y. Chiyonobu T. Kawara A. Nishida Y. Wada I. Akita M. Komori T. Nakayama K. Nanba A. Maruki Y. Yoda T. Tomoda H. Yu P.B. Shore E.M. Kaplan F.S. Miyazono K. Matsuoka M. Ikebuchi K. Ohtake A. Oda H. Jimi E. Owan I. Okazaki Y. Katagiri T. Constitutively activated ALK2 and increased SMAD1/5 cooperatively induce bone morphogenetic protein signaling in fibrodysplasia ossificans progressiva.J. Biol. Chem. 2009; 284: 7149-7156Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). SB431542 was from Tocris Cookson Ltd. (Bristol, UK). Anti-Col1 antibody and human recombinant OGN were from Calbiochem and ADIPO BIOSCIENCE Inc. (Santa Clara, CA). The coding region of mouse OGN was amplified by reverse transcription PCR of total RNA from C2C12 cells using 5′-GCTGAAATGGAGACTGTGCACTCTA-3′ as the forward primer and 5-GGTTAGAAGTATGACCCTATGGGTA-3′ 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(−) (Invitrogen). All constructs were verified by restriction enzyme analysis and nucleotide sequencing. Mouse osteoblastic MC3T3-E1 and mouse calvarial osteoblastic cells were cultured in α-MEM (containing 50 μg/ml ascorbic acid) with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Invitrogen). Mouse myoblastic C2C12 cells (ATCC) were cultured in Dulbecco's modified Eagle's medium (Invitrogen) with 10% FBS and 1% penicillin-streptomycin. The medium was changed twice a week. Each vector was transfected into MC3T3-E1 or C2C12 cells with Lipofectamine (Invitrogen) as previously described (12Hisa I. Inoue Y. Hendy G.N. Canaff L. Kitazawa R. Kitazawa S. Komori T. Sugimoto T. Seino S. Kaji H. Parathyroid hormone-responsive Smad3-related factor, Tmem119, promotes osteoblast differentiation and interacts with the bone morphogenetic protein-Runx2 pathway.J. Biol. Chem. 2011; 286: 9787-9796Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Six hours later, the cells were supplied with fresh α-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 α-MEM or DMEM containing 10% FBS for 48 h, the cells were passaged, and clones were selected in α-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. 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. Total RNA was prepared from cells using TRIzol reagent. cDNA was synthesized using the Superscript TM First-Standard Synthesis System for RT-PCR (Invitrogen). Specific mRNA was quantified by real-time PCR using a 7500 Real-time PCR system (Applied Biosystems, Rotkreuz, Switzerland) with SYBR Premix Ex TaqTM Π (Perfect Real Time) kits (TaKaRa) according to the manufacturer's standard protocol. The mRNA value for each gene was normalized relative to the mouse GAPDH mRNA levels in RNA samples. Primer sequences (forward and reverse) were as follows: GAPDH, 5′-GTGTACATGGTTCCAGTATGAGTCC-3′ and 5′-AGTGAGTTGTCATATTTCTCGTGGT-3′; OGN, 5′-TGCTTTGTGGTCACATGGAT-3′ and 5′-GAAGCTGCACACAGCACAAT-3′; osteocalcin (OCN), 5′-CCTGAGTCTGACAAAGCCTTCA-3′ and 5′-GCCGGAGTCTGTTCACTACCTT-3′; Runx2, 5′-AAATGCCTCCGCTGTTATGAA-3′ and 5′-GCTCCGGCCCACAAATCT-3′; Osterix, 5′-AGCGACCACTTGAGCAAACAT-3′ and 5′-GCGGCTGATTGGCTTCTTCT-3′; ALP, 5′-ATCTTTGGTCTGGCTCCCATG-3′ and 5′-TTTCCCGTTCACCGTCCAC-3′; Col1, 5′-ATGCCTGGTGAACGTGGT-3′ and 5′-AGGAGAGCCATCAGCACCT-3′; MyoD, 5′-GACGGCTCTCTCTGCTCCTT-3′ and 5′-AGTAGAGAAGTGTGCGTGCT-3′; myogenin, 5′-GCTGCCTAAAGTGGAGATCCT-3′ and 5′-GCGCTGTGGGAGTTGCAT-3′; Myf-6, 5′-ATGGTACCCTATCCCCTTGC-3′ and 5′-TAGCTGCTTTCCGACGATCT-3′; TGF-β1, 5′-TTGCTTCAGCTCCACAGAGA-3′ and 5′-TGGTTGTAGAGGGCAAGGAC-3′. 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 A260/280>1.90. Double-stranded cDNA was synthesized using a T7-oligo(dT) primer with the 3′IVT 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 <0.005), decreased (signal log ratio change p value <0.005), or marginally increased or decreased. Mineralization of MC3T3-E1 cells was determined in 6-well plates using Alizarin Red staining. After confluent cells were grown in α-MEM supplemented with 10% FBS, 1% penicillin-streptomycin, and 10 mm β-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 (13Sowa H. Kaji H. Yamaguchi T. Sugimoto T. Chihara K. Activations of ERK1/2 and JNK by transforming growth factor β negatively regulate Smad3-induced alkaline phosphatase activity and mineralization in mouse osteoblastic cells.J. Biol. Chem. 2002; 277: 36024-36031Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Mouse OGN siRNA or control siRNA was transfected as recommended by the supplier (Santa Cruz Biotechnology) into cells seeded at 5 × 105 per well using LipofectamineTM RNAi MAX (Invitrogen). Cells were seeded at a density of 2 × 105 per 6-well plate. Twenty-four hours later, cells were transfected with 3 μg 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-β or 300 ng/ml BMP-2. Cells were lysed, and the luciferase activity was measured as previously described (14Sowa H. Kaji H. Hendy G.N. Canaff L. Komori T. Sugimoto T. Chihara K. Menin is required for bone morphogenetic protein 2- and transforming growth factor β-regulated osteoblastic differentiation through interaction with Smads and Runx2.J. Biol. Chem. 2004; 279: 40267-40275Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). All experiments were repeated at least three times. Data are expressed as the mean ± S.E. Statistical analysis was performed using analysis of variance. A p value <0.05 was taken to indicate a significance difference. 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 ∼45,000 genes on the Affymetrix gene chip, we identified 25 genes whose expression was decreased to <1/4 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 protein were suppressed in the stable ALK2 (R206H)-transfected C2C12 cells compared with those in the empty vector-transfected cells (Fig. 1B).TABLE 1Gene transcripts down-regulated in stable ALK-2(R206H)-transfected C2C12 cells versus vector-alone transfected cellsGene nameGenBankTM accession no.RatioB830045N13RikBM942851−6.3Trim44NM_020267−4.83110039M20RikAW494150−4.6Fndc1AK003938−4.5Ifi27AY090098−4.5Susd5AV226398−4.52810468N07RikAK013390−4.5Srpx2BC028307−4.4DecorinAV321547−4.3Auts2BB429147−4.0C1rBB558917−3.6Follistatin-like 1BI452727−3.3Del1AF031524−3.0Pros1Z25469−2.8NovX96585−2.5OsteoglycinBB542051−2.5Csrp2NM_007792−2.5Mospd2BB45345−2.42610301H18RikAK011956−2.4IL-33NM_133775−2.2LOC672215///Ntn1NM_008744−2.1Rnf157BB246182−2.1Samd9lBB145092−2.1TslpNM_021367−2.1HhipBB088162−2.0 Open table in a new tab 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 osteoblastic 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). We examined whether OGN would affect osteoblast phenotype 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 β-catenin protein in MC3T3-E1 cells (Fig. 3B). The optimal concentration of recombinant OGN protein in MC3T3-E1 cells was 10 μg/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 β-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 β-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 β-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 β-catenin protein in mouse primary osteoblasts (Fig. 3I). We examined the effects of OGN on BMP-2-induced differentiation of myoblasts into osteoblasts. Transient OGN overexpression significantly suppressed the levels of Runx2, Osterix, Col1, ALP, and OCN mRNA induced by BMP-2 in C2C12 cells (Fig. 4A). As shown in Figs. 4, B and C, transient OGN overexpression significantly antagonized BMP-2 and Smad5-induced transcriptional activity in C2C12 cells, although it did not affect the phosphorylation of Smad1/5/8. Moreover, OGN was not coimmunoprecipitated with Smad1 and Smad5 in these cells (data not shown), suggesting that OGN does not interact with Smad1 and Smad5 physically. 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 β-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 β-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). Because a previous study indicated that OGN induces bone formation in conjunction with TGF-β (15Tasheva E.S. Klocke B. Conrad G.W. Analysis of transcriptional regulation of the small leucine-rich proteoglycans.Mol. Vis. 2004; 10: 758-772PubMed Google Scholar, 16Hildebrand A. Romarís M. Rasmussen L.M. Heinegård D. Twardzik D.R. Border W.A. Ruoslahti E. Interaction of the small interstitial proteoglycans biglycan, decorin, and fibromodulin with transforming growth factor β.Biochem. J. 1994; 302: 527-534Crossref PubMed Scopus (858) Google Scholar), we examined the relationships between OGN and TGF-β 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-β-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-β-induced transcriptional activity. SB431542, an inhibitor of endogenous TGF-β signaling (ALK5 inhibitor), did not affect OGN-induced transcriptional activity, although it antagonized TGF-β effects (Fig. 6A). OGN significantly enhanced TGF-β-induced Col1 mRNA levels in MC3T3-E1 cells, and SB431542 did not affect OGN-induced Col1 mRNA levels, although it antagonized TGF-β effects (Fig. 6B). Next we examined whether OGN would affect the expression of TGF-β 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-β mRNA and protein compared with those in each control group in MC3T3-E1 cells. Moreover, OGN did not affect TGF-β-induced phosphorylation of Smad2/3 protein (Fig. 6E). Previous studies suggest that MAP kinases are related to Smad-independent signaling of TGF-β (13Sowa H. Kaji H. Yamaguchi T. Sugimoto T. Chihara K. Activations of ERK1/2 and JNK by transforming growth factor β negatively regulate Smad3-induced alkaline phosphatase activity and mineralization in mouse osteoblastic cells.J. Biol. Chem. 2002; 277: 36024-36031Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 40Janssens K. ten Dijke P. Janssens S. Van Hul W. Transforming growth factor-β1 to the bone.Endocr. Rev. 2005; 26: 743-774Crossref PubMed Scopus (562) Google Scholar). 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. 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 mus" @default.
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