Technical Forum

Epigenetic Changes During Mechanically Induced Osteogenic Lineage Commitment

[+] Author and Article Information
Julia C. Chen

Department of Biomedical Engineering,
Columbia University,
New York, NY 10027

Mardonn Chua

Department of Biotechnology,
University of British Columbia,
Vancouver, BC V6T 1Z4, Canada

Raymond B. Bellon

Department of Chemical Engineering,
Columbia University,
New York, NY 10027

Christopher R. Jacobs

Department of Biomedical Engineering,
Columbia University,
New York, NY 10027

1Corresponding author.

Manuscript received December 13, 2014; final manuscript received January 4, 2015; published online January 26, 2015. Editor: Victor H. Barocas.

J Biomech Eng 137(2), 020902 (Feb 01, 2015) (6 pages) Paper No: BIO-14-1628; doi: 10.1115/1.4029551 History: Received December 13, 2014; Revised January 04, 2015; Online January 26, 2015

Osteogenic lineage commitment is often evaluated by analyzing gene expression. However, many genes are transiently expressed during differentiation. The availability of genes for expression is influenced by epigenetic state, which affects the heterochromatin structure. DNA methylation, a form of epigenetic regulation, is stable and heritable. Therefore, analyzing methylation status may be less temporally dependent and more informative for evaluating lineage commitment. Here we analyzed the effect of mechanical stimulation on osteogenic differentiation by applying fluid shear stress for 24 hr to osteocytes and then applying the osteocyte-conditioned medium (CM) to progenitor cells. We analyzed gene expression and changes in DNA methylation after 24 hr of exposure to the CM using quantitative real-time polymerase chain reaction and bisulfite sequencing. With fluid shear stress stimulation, methylation decreased for both adipogenic and osteogenic markers, which typically increases availability of genes for expression. After only 24 hr of exposure to CM, we also observed increases in expression of later osteogenic markers that are typically observed to increase after seven days or more with biochemical induction. However, we observed a decrease or no change in early osteogenic markers and decreases in adipogenic gene expression. Treatment of a demethylating agent produced an increase in all genes. The results indicate that fluid shear stress stimulation rapidly promotes the availability of genes for expression, but also specifically increases gene expression of later osteogenic markers.

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Kato, Y., Windle, J. J., and Koop, B. A., 1997, “Establishment of an Osteocyte-Like Cell Line, MLO-Y4,” J. Bone Miner. Res., 12(12), pp. 2014–2023. [CrossRef] [PubMed]
Antequera, F., 2003, “Structure, Function and Evolution of CpG Island Promoters,” Cell. Mol. Life Sci., 60(8) pp. 1647–1658. [CrossRef] [PubMed]
Ballestar, E., and Wolffe, A. P., 2001, “Methyl-CpG-Binding Proteins. Targeting Specific Gene Repression,” Eur. J. Biochem., 268(1), pp. 1–6. [CrossRef] [PubMed]
El-Osta, A., and Wolffe, A. P., 2000, “DNA Methylation and Histone Deacetylation in the Control of Gene Expression: Basic Biochemistry to Human Development and Disease,” Gene Expression, 9(1–2) pp. 63–75. [PubMed]
Fuks, F., Hurd, P. J., and Wolf, D., 2003, “The Methyl-CpG-Binding Protein MeCP2 Links DNA Methylation to Histone Methylation,” J. Biol. Chem., 278(6) pp. 4035–4040. [CrossRef] [PubMed]
Wolffe, A. P., and Matzke, M. A., 1999, “Epigenetics: Regulation Through Repression,” Science, 286(5439), pp. 481–486. [CrossRef] [PubMed]
Noer, A., Sorensen, A. L., and Boquest, A. C., 2006, “Stable CpG Hypomethylation of Adipogenic Promoters in Freshly Isolated, Cultured, and Differentiated Mesenchymal Stem Cells From Adipose Tissue,” Mol. Biol. Cell, 17(8) pp. 3543–3556. [CrossRef] [PubMed]
Friedl, G., Schmidt, H., and Rehak, I., 2007, “Undifferentiated Human Mesenchymal Stem Cells (hMSCs) are Highly Sensitive to Mechanical Strain: Transcriptionally Controlled Early Osteo-Chondrogenic Response In Vitro,” Osteoarthritis Cartilage, 15(11) pp. 1293–1300. [CrossRef] [PubMed]
Lachner, M., 2002, “Epigenetics: SUPERMAN Dresses Up,” Curr. Biol., 12(12), pp. R434–R436. [CrossRef] [PubMed]
Lachner, M., and Jenuwein, T., 2002, “The Many Faces of Histone Lysine Methylation,” Curr. Opin. Cell Biol., 14(3) pp. 286–298. [CrossRef] [PubMed]
Turner, C. H., Owan, I., and Alvey, T., 1998, “Recruitment and Proliferative Responses of Osteoblasts After Mechanical Loading In Vivo Determined Using Sustained-Release Bromodeoxyuridine,” Bone, 22(5), pp. 463–469. [CrossRef] [PubMed]
David, V., Martin, A., and Lafage-Proust, M. H., 2007, “Mechanical Loading Down-Regulates Peroxisome Proliferator-Activated Receptor Gamma in Bone Marrow Stromal Cells and Favors Osteoblastogenesis at the Expense of Adipogenesis,” Endocrinology, 148(5) pp. 2553–2562. [CrossRef] [PubMed]
McBeath, R., Pirone, D. M., and Nelson, C. M., 2004, “Cell Shape, Cytoskeletal Tension, and RhoA Regulate Stem Cell Lineage Commitment,” Dev. Cell, 6(4), pp. 483–495. [CrossRef] [PubMed]
Estes, B. T., Gimble, J. M., and Guilak, F., 2004, “Mechanical Signals as Regulators of Stem Cell Fate,” Curr. Topics Dev. Biol., 60, pp. 91–126. [CrossRef]
Hoey, D. A., Tormey, S., and Ramcharan, S., 2012, “Primary Cilia-Mediated Mechanotransduction in Human Mesenchymal Stem Cells,” Stem Cells (Dayton, Ohio), 30(11), pp. 2561–2570. [CrossRef] [PubMed]
Li, Y. J., Batra, N. N., and You, L., 2004, “Oscillatory Fluid Flow Affects Human Marrow Stromal Cell Proliferation and Differentiation,” J. Orthop. Res., 22(6), pp. 1283–1289. [CrossRef] [PubMed]
Kreke, M. R., Huckle, W. R., and Goldstein, A. S., 2005, “Fluid Flow Stimulates Expression of Osteopontin and Bone Sialoprotein by Bone Marrow Stromal Cells in a Temporally Dependent Manner,” Bone, 36(6), pp. 1047–1055. [CrossRef] [PubMed]
Kreke, M. R., Sharp, L. A., and Lee, Y. W., 2008, “Effect of Intermittent Shear Stress on Mechanotransductive Signaling and Osteoblastic Differentiation of Bone Marrow Stromal Cells,” Tissue Eng. Part A, 14(4), pp. 529–537. [CrossRef] [PubMed]
Arnsdorf, E. J., Tummala, P., and Castillo, A. B., 2010, “The Epigenetic Mechanism of Mechanically Induced Osteogenic Differentiation,” J. Biomech., 43(15), pp. 2881–2886. [CrossRef] [PubMed]
Tatsumi, S., Ishii, K., and Amizuka, N., 2007, “Targeted Ablation of Osteocytes Induces Osteoporosis With Defective Mechanotransduction,” Cell Metab., 5(6), pp. 464–475. [CrossRef] [PubMed]
Batra, N. N., Li, Y. J., and Yellowley, C. E., 2005, “Effects of Short-Term Recovery Periods on Fluid-Induced Signaling in Osteoblastic Cells,” J. Biomech., 38(9), pp. 1909–1917. [CrossRef] [PubMed]
Malone, A. M., Anderson, C. T., and Tummala, P., 2007, “Primary Cilia Mediate Mechanosensing in Bone Cells by a Calcium-Independent Mechanism,” Proc. Natl. Acad. Sci. USA, 104(33), pp. 13325–13330. [CrossRef]
You, J., Reilly, G. C., and Zhen, X., 2001, “Osteopontin Gene Regulation by Oscillatory Fluid Flow Via Intracellular Calcium Mobilization and Activation of Mitogen-Activated Protein Kinase in MC3T3-E1 Osteoblasts,” J. Biol. Chem., 276(16), pp. 13365–13371. [CrossRef] [PubMed]
You, J., Yellowley, C. E., and Donahue, H. J., 2000, “Substrate Deformation Levels Associated With Routine Physical Activity are Less Stimulatory to Bone Cells Relative to Loading-Induced Oscillatory Fluid Flow,” ASME J. Biomech. Eng., 122(4), pp. 387–393. [CrossRef]
Hoey, D. A., Kelly, D. J., and Jacobs, C. R., 2011, “A Role for the Primary Cilium in Paracrine Signaling Between Mechanically Stimulated Osteocytes and Mesenchymal Stem Cells,” Biochem. Biophys. Res. Commun., 412(1), pp. 182–187. [CrossRef] [PubMed]
Zhou, X., Liu, D., and You, L., 2010, “Quantifying Fluid Shear Stress in a Rocking Culture Dish,” J. Biomech., 43(8), pp. 1598–1602. [CrossRef] [PubMed]
Li, L. C., and Dahiya, R., 2002, “MethPrimer: Designing Primers for Methylation PCRs,” Bioinformatics, 18(11), pp. 1427–1431. [CrossRef] [PubMed]
Jaiswal, N., Haynesworth, S. E., and Caplan, A. I., 1997, “Osteogenic Differentiation of Purified, Culture-Expanded Human Mesenchymal Stem Cells In Vitro,” J. Cell. Biochem., 64(2), pp. 295–312. [CrossRef] [PubMed]
Bourne, S., Polak, J. M., and Hughes, S. P., 2004, “Osteogenic Differentiation of Mouse Embryonic Stem Cells: Differential Gene Expression Analysis by cDNA Microarray and Purification of Osteoblasts by Cadherin-11 Magnetically Activated Cell Sorting,” Tissue Eng., 10(5–6), pp. 796–806. [CrossRef] [PubMed]
Sorensen, A. L., Jacobsen, B. M., and Reiner, A. H., 2010, “Promoter DNA Methylation Patterns of Differentiated Cells are Largely Programmed at the Progenitor Stage,” Mol. Biol. Cell, 21(12), pp. 2066–2077. [CrossRef] [PubMed]
Tontonoz, P., Hu, E., and Spiegelman, B. M., 1994, “Stimulation of Adipogenesis in Fibroblasts by PPAR Gamma 2, a Lipid-Activated Transcription Factor,” Cell, 79(7), pp. 1147–1156. [CrossRef] [PubMed]
Hill, M. R., Young, M. D., and McCurdy, C. M., 1997, “Decreased Expression of Murine PPARgamma in Adipose Tissue During Endotoxemia,” Endocrinology, 138(7), pp. 3073–3076. [PubMed]
Schoonjans, K., Peinado-Onsurbe, J., and Lefebvre, A. M., 1996, “PPARalpha and PPARgamma Activators Direct a Distinct Tissue-Specific Transcriptional Response Via a PPRE in the Lipoprotein Lipase Gene,” EMBO J., 15(19), pp. 5336–5348. [PubMed]
Meissner, A., Mikkelsen, T. S., and Gu, H., 2008, “Genome-Scale DNA Methylation Maps of Pluripotent and Differentiated Cells,” Nature, 454(7205), pp. 766–770. [PubMed]
Wu, H., Whitfield, T. W., and Gordon, J. A., 2014, “Genomic Occupancy of Runx2 With Global Expression Profiling Identifies a Novel Dimension to Control of Osteoblastogenesis,” Genome Biol., 15(3), p. R52. [CrossRef] [PubMed]
Chahrour, M., Jung, S. Y., and Shaw, C., 2008, “MeCP2, a Key Contributor to Neurological Disease, Activates and Represses Transcription,” Science, 320(5880), pp. 1224–1229. [CrossRef] [PubMed]


Grahic Jump Location
Fig. 1

Gene expression changes after treatment with demethylating agent. mRNA levels for (a) Runx2, (b) Dlx5, (c) OSX, (d) OPN, (e) OCN, (f) PPARγ, (g) FABP4, and (h) LPL (bars indicate mean ± SE, n = 4, **p < 0.01, ***p < 0.001).

Grahic Jump Location
Fig. 2

Gene expression changes after treatment with CM from fluid shear stress stimulated osteocytes. mRNA levels for (a) Runx2, (b) Dlx5, (c) OSX, (d) OPN, (e) OCN, (f) PPARγ, (g) FABP4, and (h) LPL (bars indicate mean ± SE, n = 3, *p < 0.05, **p < 0.01, ***p < 0.001).

Grahic Jump Location
Fig. 3

CpG methylation levels in cells treated with CM from static (open circles) or fluid shear stress stimulated (closed circles) osteocytes. Location of CpG site is shown as number of base pairs relative to transcription start site (0). Levels for (a) Runx2, (b) Dlx5, (c) OSX, (d) OPN, (e) OCN, (f) PPARγ, (g) FABP4, and (h) LPL (percentages were calculated from DNA from at least 10 colonies, n = 1).



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