Peroxisome Proliferator-Activated Receptor-γ Knockdown Impairs Bone Morphogenetic Protein-2–Induced Critical-Size Bone Defect Repair
2018; Elsevier BV; Volume: 189; Issue: 3 Linguagem: Inglês
10.1016/j.ajpath.2018.11.019
ISSN1525-2191
AutoresChenchao Wang, Justine Tanjaya, Jia Shen, Soonchul Lee, Bharti Bisht, Hsin Chuan Pan, Shen Pang, Yulong Zhang, Emily Berthiaume, Eric Chen, Andrew L. Da Lio, Xinli Zhang, Kang Ting, Shu Guo, Chia Soo,
Tópico(s)Orthopaedic implants and arthroplasty
ResumoThe Food and Drug Administration–approved clinical dose (1.5 mg/mL) of bone morphogenetic protein-2 (BMP2) has been reported to induce significant adverse effects, including cyst-like adipose-infiltrated abnormal bone formation. These undesirable complications occur because of increased adipogenesis, at the expense of osteogenesis, through BMP2-mediated increases in the master regulatory gene for adipogenesis, peroxisome proliferator-activated receptor-γ (PPARγ). Inhibiting PPARγ during osteogenesis has been suggested to drive the differentiation of bone marrow stromal/stem cells toward an osteogenic, rather than an adipogenic, lineage. We demonstrate that knocking down PPARγ while concurrently administering BMP2 can reduce adipogenesis, but we found that it also impairs BMP2-induced osteogenesis and leads to bone nonunion in a mouse femoral segmental defect model. In addition, in vitro studies using the mouse bone marrow stromal cell line M2-10B4 and mouse primary bone marrow stromal cells confirmed that PPARγ knockdown inhibits BMP2-induced adipogenesis; attenuates BMP2-induced cell proliferation, migration, invasion, and osteogenesis; and escalates BMP2-induced cell apoptosis. More important, BMP receptor 2 and 1B expression was also significantly inhibited by the combined BMP2 and PPARγ knockdown treatment. These findings indicate that PPARγ is critical for BMP2-mediated osteogenesis during bone repair. Thus, uncoupling BMP2-mediated osteogenesis and adipogenesis using PPARγ inhibition to combat BMP2's adverse effects may not be feasible. The Food and Drug Administration–approved clinical dose (1.5 mg/mL) of bone morphogenetic protein-2 (BMP2) has been reported to induce significant adverse effects, including cyst-like adipose-infiltrated abnormal bone formation. These undesirable complications occur because of increased adipogenesis, at the expense of osteogenesis, through BMP2-mediated increases in the master regulatory gene for adipogenesis, peroxisome proliferator-activated receptor-γ (PPARγ). Inhibiting PPARγ during osteogenesis has been suggested to drive the differentiation of bone marrow stromal/stem cells toward an osteogenic, rather than an adipogenic, lineage. We demonstrate that knocking down PPARγ while concurrently administering BMP2 can reduce adipogenesis, but we found that it also impairs BMP2-induced osteogenesis and leads to bone nonunion in a mouse femoral segmental defect model. In addition, in vitro studies using the mouse bone marrow stromal cell line M2-10B4 and mouse primary bone marrow stromal cells confirmed that PPARγ knockdown inhibits BMP2-induced adipogenesis; attenuates BMP2-induced cell proliferation, migration, invasion, and osteogenesis; and escalates BMP2-induced cell apoptosis. More important, BMP receptor 2 and 1B expression was also significantly inhibited by the combined BMP2 and PPARγ knockdown treatment. These findings indicate that PPARγ is critical for BMP2-mediated osteogenesis during bone repair. Thus, uncoupling BMP2-mediated osteogenesis and adipogenesis using PPARγ inhibition to combat BMP2's adverse effects may not be feasible. The current gold standard for repairing critical-sized bone defects, which are addressed in >2.2 million surgical cases at costs exceeding $23.9 billion each year, is an autologous bone graft procedure.1Giannoudis P.V. Dinopoulos H. Tsiridis E. Bone substitutes: an update.Injury. 2005; 36 Suppl 3: S20-S27Abstract Full Text Full Text PDF PubMed Scopus (1517) Google Scholar, 2Yelin E.H. Trupin L.S. Sebesta D.S. 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Berthiaume E.A. Zara J.N. Turner A.S. Seim H.B. Kwak J.H. Zhang X.L. Soo C. Cyst-like osteolytic formations in recombinant human bone morphogenetic protein-2 (rhBMP-2) augmented sheep spinal fusion.Am J Pathol. 2017; 187: 1485-1495Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar, 21Fan J. Pi-Anfruns J. Guo M. Im D.C.S. Cui Z.K. Kim S. Wu B.M. Aghaloo T.L. Lee M. Small molecule-mediated tribbles homolog 3 promotes bone formation induced by bone morphogenetic protein-2.Sci Rep. 2017; 7: 7518Crossref PubMed Scopus (14) Google Scholar Of particular importance, it was reported that high BMP2 concentrations (150, 300, and 600 μg/mL) are able to induce bone union in a rat femoral segmental defect model, but form cyst-like bony shells that are filled with adipose tissue.19Zara J.N. Siu R.K. Zhang X. Shen J. Ngo R. Lee M. Li W. Chiang M. Chung J. Kwak J. Wu B.M. Ting K. Soo C. High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo.Tissue Eng Part A. 2011; 17: 1389-1399Crossref PubMed Scopus (443) Google Scholar This finding suggests that large doses of BMP2 may induce excessive adipogenesis during the healing process, which produces newly formed bone of poor quality. As such, the dual effect of BMP2 on the induction of osteogenic and adipogenic signaling must be closely regulated to promote strong and healthy bone growth. PPARγ is a critical signaling molecule involved in the promotion of adipocyte differentiation from mesenchymal stem cell progenitors.22Wahli W. Braissant O. Desvergne B. Peroxisome proliferator activated receptors: transcriptional regulators of adipogenesis, lipid metabolism and more.Chem Biol. 1995; 2: 261-266Abstract Full Text PDF PubMed Scopus (262) Google Scholar In addition, it has been investigated as an anti-inflammatory molecule.23Moraes L.A. Piqueras L. Bishop-Bailey D. Peroxisome proliferator-activated receptors and inflammation.Pharmacol Ther. 2006; 110: 371-385Crossref PubMed Scopus (300) Google Scholar PPARγ has been assumed to have negative impacts on osteoblast differentiation due to the reciprocal relationship between osteoblastic and adipogenic differentiation.24Wahli W. PPAR gamma: ally and foe in bone metabolism.Cell Metab. 2008; 7: 188-190Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 25Ge C. Cawthorn W.P. Li Y. Zhao G. Macdougald O.A. Franceschi R.T. Reciprocal control of osteogenic and adipogenic differentiation by ERK/MAP kinase phosphorylation of Runx2 and PPARgamma transcription factors.J Cell Physiol. 2016; 231: 587-596Crossref PubMed Scopus (92) Google Scholar Indeed, the intramedullary delivery of PPARγ lentiviral shRNA interference (PPARγ shRNA), also known as gene silencing in which gene expression is knocked down in a stable manner, results in increased trabecular bone formation with a significant decrease in lipid accumulation in rat femoral bone marrow.26James A.W. Shen J. Khadarian K. Pang S. Chung G. Goyal R. Asatrian G. Velasco O. Kim J. Zhang X. Ting K. Soo C. Lentiviral delivery of PPARgamma shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture.Tissue Eng Part A. 2014; 20: 2699-2710Crossref PubMed Scopus (13) Google Scholar PPARγ suppression appears to enhance osteogenesis through the promotion of osteoblast formation and differentiation from bone marrow progenitors.27Lee M.J. Chen H.T. Ho M.L. Chen C.H. Chuang S.C. Huang S.C. Fu Y.C. Wang G.J. Kang L. Chang J.K. PPARgamma silencing enhances osteogenic differentiation of human adipose-derived mesenchymal stem cells.J Cell Mol Med. 2013; 17: 1188-1193PubMed Google Scholar, 28Akune T. Ohba S. Kamekura S. Yamaguchi M. Chung U.I. Kubota N. Terauchi Y. Harada Y. Azuma Y. Nakamura K. Kadowaki T. Kawaguchi H. PPAR gamma insufficiency enhances osteogenesis through osteoblast formation from bone marrow progenitors.J Clin Invest. 2004; 113: 846-855Crossref PubMed Scopus (764) Google Scholar, 29Takada I. Suzawa M. Matsumoto K. Kato S. Suppression of PPAR transactivation switches cell fate of bone marrow stem cells from adipocytes into osteoblasts.Ann NY Acad Sci. 2007; 1116: 182-195Crossref PubMed Scopus (114) Google Scholar These studies indicate that the inhibition of PPARγ may be an effective method to tip the balance of bone marrow stromal/stem cell (BMSC) differentiation toward an osteogenic, rather than an adipogenic, lineage. Previous reports have revealed that BMP2, at a regular dose of 50 to 100 ng/mL, can significantly up-regulate osteoblast transcription factors [runt related transcription factor (Runx) 2, distal-less homeobox (Dlx) 5, Osterix, Msh homeobox (Msx) 2, and AJ18] and an adipocyte transcription factor (PPARγ), which induces C26 mesenchymal progenitor differentiation into mature osteoblasts and adipocytes in basal medium.30Kato S. Kawabata N. Suzuki N. Ohmura M. Takagi M. Bone morphogenetic protein-2 induces the differentiation of a mesenchymal progenitor cell line, ROB-C26, into mature osteoblasts and adipocytes.Life Sci. 2009; 84: 302-310Crossref PubMed Scopus (33) Google Scholar Moreover, 300 ng/mL BMP2 has the capacity to induce C3H10T1/2 cell differentiation into adipocytes in basal medium.11Shen J. James A.W. Zhang X. Pang S. Zara J.N. Asatrian G. Chiang M. Lee M. Khadarian K. Nguyen A. Lee K.S. Siu R.K. Tetradis S. Ting K. Soo C. Novel Wnt regulator NEL-like molecule-1 antagonizes adipogenesis and augments osteogenesis induced by bone morphogenetic protein 2.Am J Pathol. 2016; 186: 419-434Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar With large doses of BMP2 seemingly able to activate adipocyte differentiation, inhibiting PPARγ during BMP2 administration was expected to increase osteogenic differentiation and new bone formation without the undesirable induction of adipogenesis. In this study, we determined whether PPARγ inhibition with BMP2 administration could reduce excessive adipogenesis and improve the quality of regenerating bone in a critical-sized mouse femoral bone defect model. Reducing PPARγ expression may assist in unveiling the complex interactions and cross talk between key factors that regulate osteogenesis and adipogenesis in vivo. The possible underlying interactions between PPARγ and BMP2 were also explored to further determine osteogenic and adipogenic regulation. Human BMP2 (rhBMP2; Infuse Bone Graft) was purchased from Medtronic (Minneapolis, MN). The primary antibodies used in the immunohistochemical staining were as follows: anti-PPARγ (sc-7273; Santa Cruz Biotechnology, Dallas, TX), anti-osteocalcin (OCN; sc-18322; Santa Cruz Biotechnology), anti-Runx2 (sc-101145; Santa Cruz Biotechnology), anti–β-catenin (610153; BD Biosciences, East Rutherford, NJ), anti-CD31 (102503; Biolegend, San Diego, CA), anti–vascular endothelial growth factor (sc-57496; Santa Cruz Biotechnology), and anti-BMP receptor 2 (sc-393304; Santa Cruz Biotechnology). All other reagents for immunohistochemical staining were purchased from Dako (Agilent Technologies, Carpinteria, CA) unless otherwise specified. The primary antibodies used in the flow cytometric assay were mouse monoclonal anti–BMP receptor 2 (BMPR2; Ab130206; Abcam, London, UK) with a goat anti-mouse IgG (Ab150119, AF-647; Abcam) secondary antibody, anti-CD44 (560568, allophycocyanin-Cy7; BD Biosciences), anti–CD-105 (562759, phosphatidylethanolamine; BD Biosciences), and antibodies contained in the FITC Annexin V/Dead Cell Apoptosis Kit (V13242; Invitrogen, Paisley, UK). Lentiviral vectors with integrated PPARγ shRNA were used for long-term gene silencing of PPARγ. Scrambled shRNA or green fluorescent protein shRNA was integrated into lentiviral vectors and used as negative control. The lentiviral vectors encoding PPARγ shRNA, scramble shRNA, or green fluorescent protein shRNA were generated by cotransfection of 293T cells with the PPARγ shRNA plasmid vector (SC-29455-SH; Santa Cruz Biotechnology), scramble shRNA plasmid vector (1864 Addgene; Santa Cruz Biotechnology), or FG12 vector (14884 Addgene; Santa Cruz Biotechnology) with the pCMV-dR8.2-vprX helper plasmid and pCMV-VSVG envelope plasmid.26James A.W. Shen J. Khadarian K. Pang S. Chung G. Goyal R. Asatrian G. Velasco O. Kim J. Zhang X. Ting K. Soo C. Lentiviral delivery of PPARgamma shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture.Tissue Eng Part A. 2014; 20: 2699-2710Crossref PubMed Scopus (13) Google Scholar The viral vectors were collected from transfected cell cultures, cell debris was removed by 0.22-mm filtration, and the remaining solution was concentrated by ultracentrifuge at 4°C, 32,310 × g for 60 minutes using Beckman SW32 rotors (Beckman Coulter, Inc., Brea, CA).26James A.W. Shen J. Khadarian K. Pang S. Chung G. Goyal R. Asatrian G. Velasco O. Kim J. Zhang X. Ting K. Soo C. Lentiviral delivery of PPARgamma shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture.Tissue Eng Part A. 2014; 20: 2699-2710Crossref PubMed Scopus (13) Google Scholar The pellets containing the lentiviral vectors were resuspended with Dulbecco's modified Eagle's medium (Gibco, Gaithersburg, MD) to a concentration of 5 × 107 tissue culture infective dosage/mL. The lentiviral vector titer was estimated by measuring the gag p24 protein; 1 pg of the p24 reading was ascribed as the 10 tissue culture infective dosage for freshly isolated lentiviral vectors.26James A.W. Shen J. Khadarian K. Pang S. Chung G. Goyal R. Asatrian G. Velasco O. Kim J. Zhang X. Ting K. Soo C. Lentiviral delivery of PPARgamma shRNA alters the balance of osteogenesis and adipogenesis, improving bone microarchitecture.Tissue Eng Part A. 2014; 20: 2699-2710Crossref PubMed Scopus (13) Google Scholar Real-time PCR and DAPI/green fluorescent protein histologic quantification were used to analyze the efficiency of the shRNA lentiviral activity.31Xu Y. Hammerick K.E. James A.W. Carre A.L. Leucht P. Giaccia A.J. Longaker M.T. Inhibition of histone deacetylase activity in reduced oxygen environment enhances the osteogenesis of mouse adipose-derived stromal cells.Tissue Eng Part A. 2009; 15: 3697-3707Crossref PubMed Scopus (39) Google Scholar Cylindrical poly(lactic-co-glycolic acid) (PLGA) scaffold implants were prepared as previously described.11Shen J. James A.W. Zhang X. Pang S. Zara J.N. Asatrian G. Chiang M. Lee M. Khadarian K. Nguyen A. Lee K.S. Siu R.K. Tetradis S. Ting K. Soo C. Novel Wnt regulator NEL-like molecule-1 antagonizes adipogenesis and augments osteogenesis induced by bone morphogenetic protein 2.Am J Pathol. 2016; 186: 419-434Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 19Zara J.N. Siu R.K. Zhang X. Shen J. Ngo R. Lee M. Li W. Chiang M. Chung J. Kwak J. Wu B.M. Ting K. Soo C. High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo.Tissue Eng Part A. 2011; 17: 1389-1399Crossref PubMed Scopus (443) Google Scholar, 32Chou Y.F. Huang W.B. Dunn J.C.Y. Miller T.A. Wu B.M. The effect of biomimetic apatite structure on osteoblast viability, proliferation, and gene expression.Biomaterials. 2005; 26: 285-295Crossref PubMed Scopus (218) Google Scholar Briefly, the scaffold was fabricated from an 85:15 d, l-PLGA (inherent viscosity, 0.61 dL/g; Absorbable Polymers, Pelham, AL)/chloroform solution, briefly mixed with 200- to 300-μm diameter sucrose, and compressed into a polytetrafluoroethylene-ethylene mold to form a cylindrical shape (2.5-mm length × 1-mm diameter).11Shen J. James A.W. Zhang X. Pang S. Zara J.N. Asatrian G. Chiang M. Lee M. Khadarian K. Nguyen A. Lee K.S. Siu R.K. Tetradis S. Ting K. Soo C. Novel Wnt regulator NEL-like molecule-1 antagonizes adipogenesis and augments osteogenesis induced by bone morphogenetic protein 2.Am J Pathol. 2016; 186: 419-434Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar The scaffolds were freeze dried overnight and were rinsed three times by immersion in distilled water to dissolve the sucrose to obtain a porous structure (94% porosity). All scaffolds were disinfected by immersion in 70% ethanol for 30 minutes, and then rinsed three times with distilled water.11Shen J. James A.W. Zhang X. Pang S. Zara J.N. Asatrian G. Chiang M. Lee M. Khadarian K. Nguyen A. Lee K.S. Siu R.K. Tetradis S. Ting K. Soo C. Novel Wnt regulator NEL-like molecule-1 antagonizes adipogenesis and augments osteogenesis induced by bone morphogenetic protein 2.Am J Pathol. 2016; 186: 419-434Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Next, the scaffolds were coated with hydroxyapatite to increase the biomechanical strength and efficiently load BMP2 and enhance the overall osteoconductivity, as the scaffolds were naturally hydrophobic after fabrication.11Shen J. James A.W. Zhang X. Pang S. Zara J.N. Asatrian G. Chiang M. Lee M. Khadarian K. Nguyen A. Lee K.S. Siu R.K. Tetradis S. Ting K. Soo C. Novel Wnt regulator NEL-like molecule-1 antagonizes adipogenesis and augments osteogenesis induced by bone morphogenetic protein 2.Am J Pathol. 2016; 186: 419-434Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar Briefly, two sterile supersaturated solutions with ion concentrations five times greater than those in human plasma [simulated body fluid (SBF) 1 and 2] were sequentially applied to the scaffolds.11Shen J. James A.W. Zhang X. Pang S. Zara J.N. Asatrian G. Chiang M. Lee M. Khadarian K. Nguyen A. Lee K.S. Siu R.K. Tetradis S. Ting K. Soo C. Novel Wnt regulator NEL-like molecule-1 antagonizes adipogenesis and augments osteogenesis induced by bone morphogenetic protein 2.Am J Pathol. 2016; 186: 419-434Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar The preparation and ionic concentrations of SBF 1 and SBF 2 were similar to the methods and final concentrations used in our previous studies.19Zara J.N. Siu R.K. Zhang X. Shen J. Ngo R. Lee M. Li W. Chiang M. Chung J. Kwak J. Wu B.M. Ting K. Soo C. High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo.Tissue Eng Part A. 2011; 17: 1389-1399Crossref PubMed Scopus (443) Google Scholar, 33Li W. Zara J.N. Siu R.K. Lee M. Aghaloo T. Zhang X. Wu B.M. Gertzman A.A. Ting K. Soo C. Nell-1 enhances bone regeneration in a rat critical-sized femoral segmental defect model.Plast Reconstr Surg. 2011; 127: 580-587Crossref PubMed Scopus (54) Google Scholar To improve wetting and coating uniformity, dried PLGA scaffolds were subjected to glow discharge argon plasma etching (Harrick Scientific, Ossining, NY) immediately before the coating process. Etched scaffolds were then incubated in SBF 1 for 12 hours, and then immersed into SBF 2 for another 12 hours inside of a water-jacketed incubator at 37°C. Coated PLGA scaffolds were rinsed gently with sterile distilled water to wash away excess ions, and then dried in a laminar flow hood. Apatite-coated PLGA scaffolds did not experience a reduction in porosity, and maintained a static pore interconnectivity and pore size ranging between 200 and 300 μm. Shortly before implantation, scaffolds were impregnated with phosphate-buffered saline (PBS), BMP2, or BMP2 in combination with a control lentivirus or PPARγ shRNA lentivirus.11Shen J. James A.W. Zhang X. Pang S. Zara J.N. Asatrian G. Chiang M. Lee M. Khadarian K. Nguyen A. Lee K.S. Siu R.K. Tetradis S. Ting K. Soo C. Novel Wnt regulator NEL-like molecule-1 antagonizes adipogenesis and augments osteogenesis induced by bone morphogenetic protein 2.Am J Pathol. 2016; 186: 419-434Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar The BMP2 dose selected for this study has been previously established and consistently induces adipocytic bone cysts in a femoral segmental defect (FSD) model.19Zara J.N. Siu R.K. Zhang X. Shen J. Ngo R. Lee M. Li W. Chiang M. Chung J. Kwak J. Wu B.M. Ting K. Soo C. High doses of bone morphogenetic protein 2 induce structurally abnormal bone and inflammation in vivo.Tissue Eng Part A. 2011; 17: 1389-1399Crossref PubMed Scopus (443) Google Scholar The BMP2 was diluted to the appropriate concentration in PBS, uniformly added to the scaffolds in a dropwise manner for 20 minutes, and lyophilized in a freeze drier overnight at −20°C.11Shen J. James A.W. Zhang X. Pang S. Zara J.N. Asatrian G. Chiang M. Lee M. Khadarian K. Nguyen A. Lee K.S. Siu R.K. Tetradis S. Ting K. Soo C. Novel Wnt regulator NEL-like molecule-1 antagonizes adipogenesis and augments osteogenesis induced by bone morphogenetic protein 2.Am J Pathol. 2016; 186: 419-434Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar The 3-month–old male Axin2+/− transgenic mice were used as the FSD model for potential Wnt signaling applications.34Yu H.M. Jerchow B. Sheu T.J. Liu B. Costantini F. Puzas J.E. Birchmeier W. Hsu W. 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