Msx2 Promotes Osteogenesis and Suppresses Adipogenic Differentiation of Multipotent Mesenchymal Progenitors
2003; Elsevier BV; Volume: 278; Issue: 46 Linguagem: Inglês
10.1074/jbc.m306972200
ISSN1083-351X
AutoresSu‐Li Cheng, Jian-Su Shao, Nichole Charlton-Kachigian, Arleen P. Loewy, Dwight A. Towler,
Tópico(s)Bone Metabolism and Diseases
ResumoIn the aorta, diabetes activates an osteogenic program that includes expression of bone morphogenetic protein-2 (BMP2) and the osteoblast homeoprotein Msx2. To evaluate BMP2-Msx2 signaling in vascular calcification, we studied primary aortic myofibroblasts. These cells express vascular smooth muscle cell (VSMC) markers, respond to BMP2 by up-regulating Msx2, and undergo osteogenic differentiation with BMP2 treatment or transduction with a virus encoding Msx2. The osteoblast factor osterix (Osx) is up-regulated 10-fold by Msx2, but Runx2 mRNA is unchanged; the early osteoblast marker alkaline phosphatase increases 50-fold with mineralized nodule formation enhanced 30-fold. Adipocyte markers are concomitantly suppressed. To better understand Msx2 actions on osteogenesis versus adipogenesis, mechanistic studies were extended to C3H10T1/2 mesenchymal cells. Msx2 enhances osteogenic differentiation in synergy with BMP2. Osteogenic actions depend upon intrinsic Msx2 DNA binding; the gain-of-function variant Msx2(P148H) directs enhanced mineralization, whereas the binding-deficient variant Msx2(T147A) is inactive. Adipogenesis (lipid accumulation, Pparg expression) is inhibited by Msx2. By contrast, suppression of adipogenesis does not require Msx2 DNA binding; inhibition occurs in part via protein-protein interactions with C/EBPα that control Pparg transcription. Thus, Msx2 regulates osteogenic versus adipogenic differentiation of aortic myofibroblasts. Myofibroblasts capable of both fates can be diverted to the osteogenic lineage by BMP2-Msx2 signaling and contribute to vascular calcification. In the aorta, diabetes activates an osteogenic program that includes expression of bone morphogenetic protein-2 (BMP2) and the osteoblast homeoprotein Msx2. To evaluate BMP2-Msx2 signaling in vascular calcification, we studied primary aortic myofibroblasts. These cells express vascular smooth muscle cell (VSMC) markers, respond to BMP2 by up-regulating Msx2, and undergo osteogenic differentiation with BMP2 treatment or transduction with a virus encoding Msx2. The osteoblast factor osterix (Osx) is up-regulated 10-fold by Msx2, but Runx2 mRNA is unchanged; the early osteoblast marker alkaline phosphatase increases 50-fold with mineralized nodule formation enhanced 30-fold. Adipocyte markers are concomitantly suppressed. To better understand Msx2 actions on osteogenesis versus adipogenesis, mechanistic studies were extended to C3H10T1/2 mesenchymal cells. Msx2 enhances osteogenic differentiation in synergy with BMP2. Osteogenic actions depend upon intrinsic Msx2 DNA binding; the gain-of-function variant Msx2(P148H) directs enhanced mineralization, whereas the binding-deficient variant Msx2(T147A) is inactive. Adipogenesis (lipid accumulation, Pparg expression) is inhibited by Msx2. By contrast, suppression of adipogenesis does not require Msx2 DNA binding; inhibition occurs in part via protein-protein interactions with C/EBPα that control Pparg transcription. Thus, Msx2 regulates osteogenic versus adipogenic differentiation of aortic myofibroblasts. Myofibroblasts capable of both fates can be diverted to the osteogenic lineage by BMP2-Msx2 signaling and contribute to vascular calcification. Mineral deposition in the skeleton is regulated by morphogenetic, metabolic, mechanical, inflammatory, and endocrine factors. With aging, abnormalities in orthotopic (e.g. bone formation) and heterotopic arterial vascular calcification are observed with very high prevalence (1Parhami F. Demer L.L. Curr. Opin. Lipidol. 1997; 8: 312-314Crossref PubMed Scopus (52) Google Scholar), the latter enhanced by hyperglycemia, hyperlipidemia, and chronic renal insufficiency (1Parhami F. Demer L.L. Curr. Opin. Lipidol. 1997; 8: 312-314Crossref PubMed Scopus (52) Google Scholar, 2Lehto S. Niskanen L. Suhonen M. Ronnemaa T. Laakso M. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 978-983Crossref PubMed Scopus (478) Google Scholar). At least three variants of vascular calcification have been described: (a) calcification of necrotic, intimal atherosclerotic plaques; (b) medial artery calcification; and (c) calcific sclerosis of the aortic valve. Vascular calcification is a highly significant complication of diabetes and has emerged as a powerful predictor of cardiovascular morbidity and mortality (2Lehto S. Niskanen L. Suhonen M. Ronnemaa T. Laakso M. Arterioscler. Thromb. Vasc. Biol. 1996; 16: 978-983Crossref PubMed Scopus (478) Google Scholar). The molecular mechanisms that perturb normal vascular calcium metabolism are only beginning to be understood (1Parhami F. Demer L.L. Curr. Opin. Lipidol. 1997; 8: 312-314Crossref PubMed Scopus (52) Google Scholar, 3Bostrom K. Demer L.L. Crit. Rev. Eukaryot. Gene Expr. 2000; 10: 151-158Crossref PubMed Google Scholar, 4Demer L.L. Tintut Y. Parhami F. Curr. Opin. Nephrol. Hypertens. 2002; 11: 437-443Crossref PubMed Scopus (53) Google Scholar). Demer et al. (5Bostrom K. Watson K.E. Horn S. Wortham C. Herman I.M. Demer L.L. J. Clin. Invest. 1993; 91: 1800-1809Crossref PubMed Scopus (891) Google Scholar) was the first to show that vascular calcification may progress via molecular processes similar to osteogenesis. This group showed that the powerful bone morphogen, bone morphogenetic protein 2 (BMP2) 1The abbreviations used are: BMP2, bone morphogenetic protein 2; ALP, alkaline phosphatase; C/EBP, CCAAT/enhancer-binding protein; Chol, cholesterol supplementation of a high fat diet; CMV, cytomegalovirus immediate early promoter; DII, dexamethasone/insulin/indomethacin; Fat, high fat diabetogenic diet; Gla, γ-carboxylated glutamic acid; GST, glutathione S-transferase; LacZ, SFG retrovirus expressing β-galactosidase; LEF1/TCF, lymphoid enhancer factor 1/T-cell factor; LDLR, low density lipoprotein receptor; LAP, liver activated protein; LIP, liver inhibitory protein; LRP, LDLR-related protein; LUC, luciferase gene; OPN, osteopontin; Osx, osterix; PPAR, peroxisome proliferator-activated receptor; RT, reverse transcription; VSMC, vascular smooth muscle cells.1The abbreviations used are: BMP2, bone morphogenetic protein 2; ALP, alkaline phosphatase; C/EBP, CCAAT/enhancer-binding protein; Chol, cholesterol supplementation of a high fat diet; CMV, cytomegalovirus immediate early promoter; DII, dexamethasone/insulin/indomethacin; Fat, high fat diabetogenic diet; Gla, γ-carboxylated glutamic acid; GST, glutathione S-transferase; LacZ, SFG retrovirus expressing β-galactosidase; LEF1/TCF, lymphoid enhancer factor 1/T-cell factor; LDLR, low density lipoprotein receptor; LAP, liver activated protein; LIP, liver inhibitory protein; LRP, LDLR-related protein; LUC, luciferase gene; OPN, osteopontin; Osx, osterix; PPAR, peroxisome proliferator-activated receptor; RT, reverse transcription; VSMC, vascular smooth muscle cells. is expressed in calcified atherosclerotic plaques of humans (5Bostrom K. Watson K.E. Horn S. Wortham C. Herman I.M. Demer L.L. J. Clin. Invest. 1993; 91: 1800-1809Crossref PubMed Scopus (891) Google Scholar). Bostrom et al. (6Zebboudj A.F. Imura M. Bostrom K. J. Biol. Chem. 2002; 277: 4388-4394Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar) further demonstrated that aortic calcification in response to matrix Gla protein deficiency was most likely via BMP2 signaling; matrix Gla protein can abrogate alkaline phosphatase (ALP) induction by inhibiting BMP2 association with the BMP receptor (6Zebboudj A.F. Imura M. Bostrom K. J. Biol. Chem. 2002; 277: 4388-4394Abstract Full Text Full Text PDF PubMed Scopus (299) Google Scholar). Thus, these studies point to a role for BMP2 in vascular calcification. Recently, we reported the use of the LDLR-/- mouse as a model of diet-induced vascular calcification in response to diabetes and dyslipidemia; mineral deposition is most notable early on in valve leaflets (7Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). RT-PCR analyses demonstrated diet-induced up-regulation of aortic Msx2, a homeodomain transcription factor that controls osteoblast differentiation and mineralization in the developing skull (8Satokata I. Ma L. Ohshima H. Bei M. Woo I. Nishizawa K. Maeda T. Takano Y. Uchiyama M. Heaney S. Peters H. Tang Z. Maxson R. Maas R. Nat. Genet. 2000; 24: 391-395Crossref PubMed Scopus (613) Google Scholar). In situ hybridization showed that Msx2 is expressed in a subset of aortic adventitial myofibroblasts and valve fibrosal cells (7Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Because the Msx2 gene is a direct gene target of BMP2 (9Sirard C. Kim S. Mirtsos C. Tadich P. Hoodless P.A. Itie A. Maxson R. Wrana J.L. Mak T.W. J. Biol. Chem. 2000; 275: 2063-2070Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar), we have studied the function of BMP2-Msx2 signaling in aortic osteogenesis. We show that aortic myofibroblasts are multipotent, capable of expressing VSMC, osteoblast, and adipocyte genes. Msx2 drives osteogenic differentiation of myofibroblasts without hindering VSMC phenotype but suppresses adipogenesis. Unlike osteogenic activation, suppression of adipogenesis is independent of intrinsic Msx2 DNA binding, occurring in part via antagonistic protein-protein interactions with C/EBPα that precludes transactivation of key target genes such as Pparg. Thus, BMP2-Msx2 signaling promotes osteogenic differentiation of aortic myofibroblasts. Msx2 can regulate the lineage fate of multipotent myofibroblasts via distinct cell autonomous mechanisms that are either dependent (osteogenesis) or independent (adipogenesis) of intrinsic Msx2 DNA-binding activity. Animals, Cell Culture, and Retroviruses—LDLR-/- mice and C57BL/6 mice were maintained as detailed previously (10Bidder M. Shao J.S. Charlton-Kachigian N. Loewy A.P. Semenkovich C.F. Towler D.A. J. Biol. Chem. 2002; 277: 44485-44496Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), following procedures approved by the Washington University Animal Studies Committee. Aortic RNA was isolated from 10-week-old LDLR-/- mice (four per group) maintained on mouse chow, a diabetogenic high fat diet with cholesterol (Fat plus Chol), or a high fat diabetogenic diet without cholesterol (Fat) for 5 weeks as previously detailed (7Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Primary mouse aortic myofibroblasts (adventitial cells) were isolated from 6-week-old male C57BL/6 mice as previously described (10Bidder M. Shao J.S. Charlton-Kachigian N. Loewy A.P. Semenkovich C.F. Towler D.A. J. Biol. Chem. 2002; 277: 44485-44496Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). C3H10T1/2 cells (11Hirschi K.K. Rohovsky S.A. D'Amore P.A. J. Cell Biol. 1998; 141: 805-814Crossref PubMed Scopus (689) Google Scholar, 12Ahrens M. Ankenbauer T. Schroder D. Hollnagel A. Mayer H. Gross G. DNA Cell Biol. 1993; 12: 871-880Crossref PubMed Scopus (307) Google Scholar) and CV1 cells were obtained from the American Type Cell Culture and cultured in Basal Eagle's medium containing 10% serum. Our N-terminal Met-FLAG-tagged wild type Msx2, Msx2(P148H), or Msx2(T147A) cDNAs (13Newberry E.P. Latifi T. Battaile J.T. Towler D.A. Biochemistry. 1997; 36: 10451-10462Crossref PubMed Scopus (70) Google Scholar) were subcloned into the NcoI and BamHI sites of SFG retroviral vector (14Ory D.S. Neugeboren B.A. Mulligan R.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11400-11406Crossref PubMed Scopus (794) Google Scholar). As a negative control, SFG-LacZ encoding β-galactosidase was generated in the same fashion (15Lai C.F. Chaudhary L. Fausto A. Halstead L.R. Ory D.S. Avioli L.V. Cheng S.L. J. Biol. Chem. 2001; 276: 14443-14450Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 16Kalajzic I. Stover M.L. Liu P. Kalajzic Z. Rowe D.W. Lichtler A.C. Virology. 2001; 284: 37-45Crossref PubMed Scopus (39) Google Scholar). The SFG retrovirus has been shown to have no effect on osteoblast differentiation (15Lai C.F. Chaudhary L. Fausto A. Halstead L.R. Ory D.S. Avioli L.V. Cheng S.L. J. Biol. Chem. 2001; 276: 14443-14450Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 16Kalajzic I. Stover M.L. Liu P. Kalajzic Z. Rowe D.W. Lichtler A.C. Virology. 2001; 284: 37-45Crossref PubMed Scopus (39) Google Scholar). Pseudotyped retroviral particles were prepared as previously detailed (14Ory D.S. Neugeboren B.A. Mulligan R.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11400-11406Crossref PubMed Scopus (794) Google Scholar, 15Lai C.F. Chaudhary L. Fausto A. Halstead L.R. Ory D.S. Avioli L.V. Cheng S.L. J. Biol. Chem. 2001; 276: 14443-14450Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). First passage-transduced cells were used for all the assays. Osteogenic Gene Expression and Mineralization Assays—RNA extraction and RT-PCR was performed as detailed previously (7Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 17Willis D.M. Loewy A.P. Charlton-Kachigian N. Shao J.S. Ornitz D.M. Towler D.A. J. Biol. Chem. 2002; 277: 37280-37291Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). The primers for Msx2, OPN, and glyceraldehyde-3-phosphate dehydrogenase were reported previously (7Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar, 10Bidder M. Shao J.S. Charlton-Kachigian N. Loewy A.P. Semenkovich C.F. Towler D.A. J. Biol. Chem. 2002; 277: 44485-44496Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). For real-time fluorescence RT-PCR, relative mRNA levels were expressed as percentage of 18 S ribosomal RNA levels. Amplimers used and designed with Primer Express Software V1.0 (ABI PE Biosystems, Palo Alto, CA) were: Msx2, 5′-ACC ACG TCC CAG CTT CTA GC-3′ and 5′-GCT CTG CGA TGG AGA GGT ACT G-3′; BMP2, 5′-CAC CGT GCT CAG CTT CCA-3′ and 5′-TCG GGA AGT TTT CCC ACT CA-3′; Osx, 5′-CCC TTC TCA AGC ACC AAT GG-3′ and 5′-AAG GGT GGG TAG TCA TTT GCA TA-3′; ALP, 5′-ACA CCA ATG TAG CCA AGA ATG TCA-3′ and 5′-GAT TCG GGC AGC GGT TAC T-3′; Runx2 5′-CCG TGG CCT TCA AGG TTG T-3′ and 5′-TTC ATA ACA GCG GAG GCA TTT-3′; PPARγ2, 5′-ACC ACT CGC ATT CCT TTG AC-3′ and 5′-TGG GTC AGC TCT TGT GAA TG-3′; adipsin, 5′-TGC ATC AAC TCA GAG GTG TCA ATC A-3′ and 5′-TGC GCA GAT TGC AGG TTG T-3′; VSMC α-actin, 5′-CGG GAG AAA ATG ACC CAG ATT AT-3′ and 5′-GGA CAG CAC AGC CTG AAT AGC-3′; SM22α, 5′-GAG GGA TCG AAG CCA GTG AA-3′ and 5′-TGA GCC ACC TGT TCC ATC TG-3′; and 18 S, 5′-CGG CTA CCA CAT CCA AGG AA-3′ and 5′-GCT GGA ATT ACC GCG GCT-3′. Western blot analyses, ALP activity assays, and Alizarin red staining of calcium deposition were performed as described previously (18Lecanda F. Avioli L.V. Cheng S.L. J. Cell Biochem. 1997; 67: 386-396Crossref PubMed Scopus (246) Google Scholar). The PPARγ antibody H-100 was purchased from Santa Cruz Biotechnology (catalog #7196, Santa Cruz, CA). The tubulin antibody TU-01 was purchased from Zymed Laboratories Inc. (South San Francisco, CA). For mineralized nodule assays, myofibroblasts were maintained in growth medium supplemented with 50 μg/ml ascorbic acid, and 10 mm β-glycerol phosphate for 25 days. At the end of the culture period, cultures were stained for mineral deposition using the von Kossa method as previously described (7Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Digital photomicrographs were captured with a Spot Enhanced camera mounted on a Zeiss Axiovert S100 microscope, and von Kossa-stained osteogenic colonies were quantified using Kodak 1D automated image analysis software version 3.5.3. Eukaryotic Expression Constructs, PPARγ LUC Promoter-Reporter Constructs, Transient Transfection Assays, and GST Pull-down Assays—Expression constructs for Msx2, Msx2(T147A), Msx2(P148H), and Msx2(Δ132-148) have been previously described and characterized (13Newberry E.P. Latifi T. Battaile J.T. Towler D.A. Biochemistry. 1997; 36: 10451-10462Crossref PubMed Scopus (70) Google Scholar, 17Willis D.M. Loewy A.P. Charlton-Kachigian N. Shao J.S. Ornitz D.M. Towler D.A. J. Biol. Chem. 2002; 277: 37280-37291Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Mouse C/EBPα cDNA was obtained by RT-PCR amplification (which also introduced convenient 5′- and 3′-linkers) of the mRNA obtained from adipocytes generated from C3H10T1/2 cells (see below) and subcloned into the KpnI-BamHI sites of pcDNA3 (Invitrogen, Carlsbad, CA) using techniques previously detailed (13Newberry E.P. Latifi T. Battaile J.T. Towler D.A. Biochemistry. 1997; 36: 10451-10462Crossref PubMed Scopus (70) Google Scholar). Amplimers used were 5′-GAT GGT ACC ATG GAG TCG GCC GAC TTC TAC-3′ and 5′-GAC GGA TCC CTA GTC CTG GCT TGC GCG-3′. The LIP and LAP forms of C/EBP-β (19Rosen E.D. Walkey C.J. Puigserver P. Spiegelman B.M. Genes Dev. 2000; 14: 1293-1307PubMed Google Scholar) were the kind gift of Dr. Linda Sandell (Washington University, St. Louis, MO). The synthesis of 700 PPARγ LUC (luciferase reporter in KpnI-MluI sites of pGL2 Basic (Promega, Madison, WI; mouse 0.7-kb PPARγ promoter fragment -615 to +66 (20Shi X.M. Blair H.C. Yang X. McDonald J.M. Cao X. J. Cell Biochem. 2000; 76: 518-527Crossref PubMed Scopus (101) Google Scholar)) was obtained by PCR using mouse genomic DNA as a template, applying techniques previously described (10Bidder M. Shao J.S. Charlton-Kachigian N. Loewy A.P. Semenkovich C.F. Towler D.A. J. Biol. Chem. 2002; 277: 44485-44496Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Amplimers used were 5′-GAC GGT ACC TTT ATA GAA TTT GGA TAG CAG-3′ and 5′-GAT CAC GCG TAA CAG CAT AAA ACA GAG ATT TG-3′. All constructs were sequenced to verify fidelity (ABI Prism Dye Terminator kit, Foster City, CA). For transfection, either C3H10T1/2 mural pleuripotent mesenchymal cells (11Hirschi K.K. Rohovsky S.A. D'Amore P.A. J. Cell Biol. 1998; 141: 805-814Crossref PubMed Scopus (689) Google Scholar, 12Ahrens M. Ankenbauer T. Schroder D. Hollnagel A. Mayer H. Gross G. DNA Cell Biol. 1993; 12: 871-880Crossref PubMed Scopus (307) Google Scholar, 20Shi X.M. Blair H.C. Yang X. McDonald J.M. Cao X. J. Cell Biochem. 2000; 76: 518-527Crossref PubMed Scopus (101) Google Scholar) or CV1 fibroblastic cells were transfected using LipofectAMINE (Invitrogen) as outlined in the figure legends. A CMV β-galactosidase plasmid was included as an internal control for transfection efficiency. The ratio of C/EBP to Msx2 expression construct was systematically varied as outlined in the figure legends. Empty pcDNA3 expression vector was used to maintain constant DNA concentrations in all transient transfections. For experiments designed to demonstrate specificity for particular C/EBP family members, the C/EBP to Msx2 expression plasmid ratio was 10:1. One day following transfection, cultures were re-fed with growth medium and cellular luciferase, and β-galactosidase activities were measured 24-48 h later as previously described (13Newberry E.P. Latifi T. Battaile J.T. Towler D.A. Biochemistry. 1997; 36: 10451-10462Crossref PubMed Scopus (70) Google Scholar, 21Newberry E.P. Boudreaux J.M. Towler D.A. J. Biol. Chem. 1997; 272: 29607-29613Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The purification of GST fusion proteins expressed in Escherichia coli and coupled in vitro transcription/translation (Promega) of [35S]Met-radiolabeled transcription factors has been previously detailed (13Newberry E.P. Latifi T. Battaile J.T. Towler D.A. Biochemistry. 1997; 36: 10451-10462Crossref PubMed Scopus (70) Google Scholar, 21Newberry E.P. Boudreaux J.M. Towler D.A. J. Biol. Chem. 1997; 272: 29607-29613Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). Pull-down assays, SDS-PAGE, and autoradiography were performed as recently detailed (17Willis D.M. Loewy A.P. Charlton-Kachigian N. Shao J.S. Ornitz D.M. Towler D.A. J. Biol. Chem. 2002; 277: 37280-37291Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Adipogenesis Assays—Confluent cultures of transduced C3H10T1/2 cells were subjected to adipogenic medium containing 0.1 μm dexamethasone, 50 μm indomethacin, and 5 μg/ml insulin (DII medium) for 14 days. The progression of adipogenesis was monitored under light microscope. At the end of culture period, cells were stained for lipid droplets using Oil Red-O stain as described (12Ahrens M. Ankenbauer T. Schroder D. Hollnagel A. Mayer H. Gross G. DNA Cell Biol. 1993; 12: 871-880Crossref PubMed Scopus (307) Google Scholar). Statistics—Statistical analyses were performed using Student's unpaired t test or one-way analysis of variance as previously detailed (22Rifas L. Towler D.A. Avioli L.V. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7549-7554Crossref PubMed Scopus (41) Google Scholar). Each experiment was performed at least twice, and the representative data were presented as mean ± S.E. of independent replicates (n ≥ 3). BMP2-Msx2 Signaling Promotes Osteogenic Differentiation of Vascular Myofibroblasts—We previously demonstrated that high fat diabetogenic diets induce calcific vasculopathy and aortic Msx2 and osteopontin expression in LDLR-/- mice (7Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). To understand the mechanisms leading to aortic calcification, we analyzed effects of high fat diets on the expression of BMP2, a potent osteogenic agent (23Katagiri T. Akiyama S. Namiki M. Komaki M. Yamaguchi A. Rosen V. Wozney J.M. Fujisawa-Sehara A. Suda T. Exp. Cell Res. 1997; 230: 342-351Crossref PubMed Scopus (119) Google Scholar) that activates Msx2 expression (9Sirard C. Kim S. Mirtsos C. Tadich P. Hoodless P.A. Itie A. Maxson R. Wrana J.L. Mak T.W. J. Biol. Chem. 2000; 275: 2063-2070Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar) and has been identified in human atherosclerotic plaques (5Bostrom K. Watson K.E. Horn S. Wortham C. Herman I.M. Demer L.L. J. Clin. Invest. 1993; 91: 1800-1809Crossref PubMed Scopus (891) Google Scholar). As compared with mouse chow, high fat diabetogenic diets, either with (Fat plus Chol) or without (Fat) cholesterol supplementation, concomitantly up-regulated aortic BMP2 and Msx2 mRNA accumulation (Fig. 1A; also see Ref. 7Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Msx2 expression in aorta is primarily localized to a sub-population of adventitial and valvular fibrosal cells that also expressed VSMC α-actin (7Towler D.A. Bidder M. Latifi T. Coleman T. Semenkovich C.F. J. Biol. Chem. 1998; 273: 30427-30434Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). To confirm that BMP2-Msx2 signaling can facilitate aortic calcification, we studied effects of BMP2 and Msx2 on the commitment of aortic myofibroblasts to osteogenic differentiation. Primary aortic myofibroblasts (VSMC α-actin+) were prepared as previously detailed (10Bidder M. Shao J.S. Charlton-Kachigian N. Loewy A.P. Semenkovich C.F. Towler D.A. J. Biol. Chem. 2002; 277: 44485-44496Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). As shown in Fig. 1B, BMP2 treatment of myofibroblasts for 8 days up-regulated ALP activity, an early mineralization marker indicating commitment to osteogenic differentiation (16Kalajzic I. Stover M.L. Liu P. Kalajzic Z. Rowe D.W. Lichtler A.C. Virology. 2001; 284: 37-45Crossref PubMed Scopus (39) Google Scholar, 18Lecanda F. Avioli L.V. Cheng S.L. J. Cell Biochem. 1997; 67: 386-396Crossref PubMed Scopus (246) Google Scholar). Concomitantly, BMP2 dose-dependently up-regulated Msx2 mRNA in myofibroblasts, quantified by real-time fluorescence RT-PCR (Fig. 1C). To evaluate the role of Msx2 in myofibroblast function, we used the pantropic vesicular stomatitis virus-G protein pseudotyped retrovirus (14Ory D.S. Neugeboren B.A. Mulligan R.C. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11400-11406Crossref PubMed Scopus (794) Google Scholar, 15Lai C.F. Chaudhary L. Fausto A. Halstead L.R. Ory D.S. Avioli L.V. Cheng S.L. J. Biol. Chem. 2001; 276: 14443-14450Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar, 16Kalajzic I. Stover M.L. Liu P. Kalajzic Z. Rowe D.W. Lichtler A.C. Virology. 2001; 284: 37-45Crossref PubMed Scopus (39) Google Scholar) to transduce myofibroblasts with either the LacZ control virus or virus expressing FLAG-tagged Msx2. Staining for β-galactosidase activity (Fig. 2A), analysis of Msx2 mRNA accumulation (Fig. 2B) and Western blot for FLAG-Msx2 (Fig. 2C) confirmed the efficient transduction of >90% of cells and robust expression of Msx2. Like BMP2 treatment, Msx2 transduction up-regulated mRNA accumulation (Fig. 2D; 50-fold) and ALP activity (Fig. 2E; 10-fold). Thus, components of the BMP2-Msx2 signaling cascade necessary for the initiation of osteoblast lineage differentiation are present in vascular myofibroblasts.Fig. 2Transduction of Msx2 into primary aortic myofibroblasts up-regulates expression and activity of alkaline phosphatase, an early osteoblast phenotypic marker. A, aortic myofibroblasts are efficiently transduced by pseudotyped SFG retrovirus as shown by >90% cells exhibiting β-galactosidase (LacZ) activity by histochemical stain. Msx2 mRNA (B) and Msx2 protein (C) are expressed by myofibroblasts transduced with SFG-Msx2 virus as demonstrated by Western blot for FLAG-tagged Msx2 (C). D, SFG-Msx2-transduced cells express higher levels of ALP mRNA accumulation (D) and ALP activity (E). *, p < 0.001 versus LacZ control cells. See text for details.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Msx2 Expression Stimulates Osteogenesis of Myofibroblasts—Osx is a BMP2-induced transcription factor that directs osteoblast-specific differentiation, up-regulates ALP expression, and is necessary for mineralization (24Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2700) Google Scholar). To show that Msx2 enhances osteogenesis in myofibroblasts, we evaluated effects of Msx2 on Osx expression and mineralized nodule formation. As shown in Fig. 3A, Msx2 up-regulated Osx 10-fold; by contrast, expression of Runx2, a transcription factor that demarcates the bipotential osteoblast and chrondrocyte progenitor (24Nakashima K. Zhou X. Kunkel G. Zhang Z. Deng J.M. Behringer R.R. de Crombrugghe B. Cell. 2002; 108: 17-29Abstract Full Text Full Text PDF PubMed Scopus (2700) Google Scholar), was unchanged (Fig. 3B). Mineralized nodule formation was also increased 30-fold by Msx2 (Fig. 3, C and D). VSMC phenotypic markers SM22α and VSMC α-actin were not suppressed (in fact they increased ∼3- to 4-fold; Fig. 3E). By contrast, the adipogenic marker, PPARγ, was suppressed >90% by Msx2 (Fig. 3F). Thus, Msx2 enhances osteogenesis of aortic myofibroblasts as evidenced by Osx expression, ALP activation, and enhanced mineralized nodule formation. Adipogenic markers such as Pparg are concomitantly suppressed (see below). Msx2 Enhances Osteogenic Differentiation of C3H10T1/2 Cells via Mechanisms Dependent upon Intrinsic Msx2 DNA-binding Activity—C3H10T1/2 is a well-characterized multipotential cell line for studying mesenchymal cell differentiation (12Ahrens M. Ankenbauer T. Schroder D. Hollnagel A. Mayer H. Gross G. DNA Cell Biol. 1993; 12: 871-880Crossref PubMed Scopus (307) Google Scholar, 20Shi X.M. Blair H.C. Yang X. McDonald J.M. Cao X. J. Cell Biochem. 2000; 76: 518-527Crossref PubMed Scopus (101) Google Scholar). To better understand the molecular mechanisms whereby Msx2 regulates osteogenic versus adipogenic differentiation, we therefore evaluated the effects of Msx2 on C3H10T1/2 lineage potential. C3H10T1/2 cells were transduced with either SFG-LacZ (control) or SFG-Msx2, and osteogenic differentiation stimulated with BMP2 (100 ng/ml) as detailed under “Experimental Procedures.” Consistent with our results in transduced primary aortic myofibroblasts, Msx2 markedly up-regulated ALP activity (Fig. 4A), and mRNA accumulation (Fig. 4B), and increased culture mineralization (Fig. 4C; Alizarin red stain for calcium) in C3H10T1/2 cells. Previously, we generated and biochemically characterized two variants of Msx2, Msx2(P148H) and Msx2(T147A), that exhibit enhanced DNA binding (3-fold improvement in K a) and no DNA binding, respectively (13Newberry E.P. Latifi T. Battaile J.T. Towler D.A. Biochemistry. 1997; 36: 10451-10462Crossref PubMed Scopus (70) Google Scholar). Of note, Msx2(P148H) corresponds to a gain-of-function variant that causes precocious calvarial mineralization in humans. To evaluate the contributions of DNA binding to the pro-osteogenic actions of Msx2, we compared the activity of SFG-Msx2(P148H) and SFG-Msx2(T147A) on osteogenic differentiation in the C3H10T1/2 cell system, assessing mineralization with Alizarin red and differentiation with alkaline phosphatase staining. A time course with and without BMP2 (100 ng/ml supplementation) was undertaken to accentuate any functional differences. As shown in Fig. 5, both Msx2 and Msx2(P148H) enhanced matrix min
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