Abstract
During physiological activities, osteoblasts experience a variety of mechanical forces that stimulate anabolic responses at the cellular level necessary for the formation of new bone. Previous studies have primarily investigated the osteoblastic response to individual forms of mechanical stimuli. However in this study, we evaluated the response of osteoblasts to two simultaneous, but independently controlled stimuli; fluid flow-induced shear stress (FSS) and static or cyclic hydrostatic pressure (SHP or CHP, respectively). MC3T3-E1 osteoblasts-like cells were subjected to 12 dynes/cm2 FSS along with SHP or CHP of varying magnitudes to determine if pressure enhances the anabolic response of osteoblasts during FSS. For both SHP and CHP, the magnitude of hydraulic pressure that induced the greatest release of ATP during FSS was 15 mmHg. Increasing the hydraulic pressure to 50 mmHg or 100 mmHg during FSS attenuated the ATP release compared to 15 mmHg during FSS. Decreasing the magnitude of pressure during FSS to atmospheric pressure reduced ATP release to that of basal ATP release from static cells and inhibited actin reorganization into stress fibers that normally occurred during FSS with 15 mmHg of pressure. In contrast, translocation of nuclear factor kappa B (NFκB) to the nucleus was independent of the magnitude of hydraulic pressure and was found to be mediated through the activation of phospholipase-C (PLC), but not src kinase. In conclusion, hydraulic pressure during FSS was found to regulate purinergic signaling and actin cytoskeleton reorganization in the osteoblasts in a biphasic manner, while FSS alone appeared to stimulate NFκB translocation. Understanding the effects of hydraulic pressure on the anabolic responses of osteoblasts during FSS may provide much needed insights into the physiologic effects of coupled mechanical stimuli on osteogenesis.
Similar content being viewed by others
References
Bakker, A. D., K. Soejima, J. Klein-Nulend, and E. H. Burger. The production of nitric oxide and prostaglandin E-2 by primary bone cells is shear stress dependent. J. Biomech. 34:671–677, 2001.
Baldwin, Jr., A. S. The NF-kappa B and I kappa B proteins: new discoveries and insights. Annu. Rev. Immunol. 14:649–683, 1996.
Brown, E. J. Integrin-associated proteins. Curr. Opin. Cell Biol. 14:603–607, 2002.
Case, N., B. Sen, J. A. Thomas, M. Styner, Z. Xie, C. R. Jacobs, and J. Rubin. Steady and oscillatory fluid flows produce a similar osteogenic phenotype. Calcif. Tissue Int. 88:189–197, 2011.
Chang, J., Z. Wang, E. Tang, Z. Fan, L. McCauley, R. Franceschi, K. Guan, P. H. Krebsbach, and C. Y. Wang. Inhibition of osteoblastic bone formation by nuclear factor-kappaB. Nat. Med. 15:682–689, 2009.
Charras, G. T., and M. A. Horton. Single cell mechanotransduction and its modulation analyzed by atomic force microscope indentation. Biophys. J. 82:2970–2981, 2002.
Chen, N. X., D. J. Geist, D. C. Genetos, F. M. Pavalko, and R. L. Duncan. Fluid shear-induced NFkappaB translocation in osteoblasts is mediated by intracellular calcium release. Bone 33:399–410, 2003.
Chen, N. X., K. D. Ryder, F. M. Pavalko, C. H. Turner, D. B. Burr, J. Qiu, and R. L. Duncan. Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts. Am. J. Physiol. Cell Physiol. 278:C989–C997, 2000.
Cowin, S. C., G. Gailani, and M. Benalla. Hierarchical poroelasticity: movement of interstitial fluid between porosity levels in bones. Philos. Trans. A 367:3401–3444, 2009.
Cox, B. D., M. Natarajan, M. R. Stettner, and C. L. Gladson. New concepts regarding focal adhesion kinase promotion of cell migration and proliferation. J. Cell. Biochem. 99:35–52, 2006.
Desai, L. P., Y. Wu, R. S. Tepper, and S. J. Gunst. Mechanical stimuli and IL-13 interact at integrin adhesion complexes to regulate expression of smooth muscle myosin heavy chain in airway smooth muscle tissue. Am. J. Physiol. Lung Cell. Mol. Physiol. 301:L275–L284, 2011.
Donahue, T. L., T. R. Haut, C. E. Yellowley, H. J. Donahue, and C. R. Jacobs. Mechanosensitivity of bone cells to oscillating fluid flow induced shear stress may be modulated by chemotransport. J. Biomech. 36:1363–1371, 2003.
Duncan, R. L., K. A. Akanbi, and M. C. Farach-Carson. Calcium signals and calcium channels in osteoblastic cells. Semin. Nephrol. 18:178–190, 1998.
Duncan, R. L., and C. H. Turner. Mechanotransduction and the functional response of bone to mechanical strain. Calcif. Tissue Int. 57:344–358, 1995.
Erb, L., J. Liu, J. Ockerhausen, Q. M. Kong, R. C. Garrad, K. Griffin, C. Neal, B. Krugh, L. I. Santiago-Perez, F. A. Gonzalez, H. D. Gresham, J. T. Turner, and G. A. Weisman. An RGD sequence in the P2Y(2) receptor interacts with alpha(v)beta(3) integrins and is required for G(0)-mediated signal transduction. J. Cell Biol. 153:491–501, 2001.
Evans, E. A., and D. A. Calderwood. Forces and bond dynamics in cell adhesion. Science 316:1148–1153, 2007.
Fan, R. S., R. O. Jacamo, X. H. Jiang, J. Sinnett-Smith, and E. Rozengurt. G protein-coupled receptor activation rapidly stimulates focal adhesion kinase phosphorylation at ser-843-mediation by Ca2+, calmodulin, and Ca2+ calmodulin-dependent kinase II. J. Biol. Chem. 280:24212–24220, 2005.
Forwood, M. R. Inducible cyclo-oxygenase (COX-2) mediates the induction of bone formation by mechanical loading in vivo. J. Bone Miner. Res. 11:1688–1693, 1996.
Frangos, J. A., L. V. Mcintire, and S. G. Eskin. Shear-stress induced stimulation of mammalian-cell metabolism. Biotechnol. Bioeng. 32:1053–1060, 1988.
Gardinier, J., S. Majumdar, R. Duncan, and L. Wang. Cyclic hydraulic pressure and fluid flow differentially modulate cytoskeleton re-organization in MC3t3 osteoblasts. Cell. Mol. Bioeng. 2:133–143, 2009.
Gardinier, J. D., C. W. Townend, K. P. Jen, Q. Wu, R. L. Duncan, and L. Wang. In situ permeability measurement of the mammalian lacunar-canalicular system. Bone 46:1075–1081, 2010.
Genetos, D. C., D. J. Geist, D. Liu, H. J. Donahue, and R. L. Duncan. Fluid shear-induced ATP secretion mediates prostaglandin release in MC3T3-E1 osteoblasts. J. Bone Miner. Res. 20:41–49, 2005.
Genetos, D. C., N. J. Karin, D. J. Geist, H. J. Donahue, and R. L. Duncan. Purinergic signaling is required for fluid shear stress-induced NF-kappa B translocation in osteoblasts. Exp. Cell Res. 317:737–744, 2011.
Glogauer, M., P. Arora, G. Yao, I. Sokholov, J. Ferrier, C. A. McCulloch. Calcium ions and tyrosine phosphorylation interact coordinately with actin to regulate cytoprotective responses to stretching. J. Cell Sci. 110(Pt 1):11–21, 1997.
Hughes, D. E., D. M. Salter, S. Dedhar, and R. Simpson. Integrin expression in human bone. J. Bone Miner. Res. 8:527–533, 1993.
Ingber, D. E. Tensegrity I. Cell structure and hierarchical systems biology. J. Cell Sci. 116:1157–1173, 2003.
Jaasma, M. J., W. M. Jackson, R. Y. Tang, and T. M. Keaveny. Adaptation of cellular mechanical behavior to mechanical loading for osteoblastic cells. J. Biomech. 40:1938–1945, 2007.
Kainulainen, T., A. Pender, M. D’Addario, Y. Feng, P. Lekic, and C. A. McCulloch. Cell death and mechanoprotection by filamin a in connective tissues after challenge by applied tensile forces. J. Biol. Chem. 277:21998–22009, 2002.
Katz, S., R. Boland, and G. Santillan. Modulation of ERK 1/2 and p38 MAPK signaling pathways by ATP in osteoblasts: involvement of mechanical stress-activated calcium influx, PKC and Src activation. Int. J. Biochem. Cell Biol. 38:2082–2091, 2006.
Klein-Nulend, J., A. van der Plas, C. M. Semeins, N. E. Ajubi, J. A. Frangos, P. J. Nijweide, and E. H. Burger. Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J. 9:441–445, 1995.
Lanyon, L. E., and C. T. Rubin. Static vs dynamic loads as an influence on bone remodelling. J. Biomech. 17:897–905, 1984.
Li, W., J. D. Gardinier, C. Price, and L. Wang. Does blood pressure enhance solute transport in the bone lacunar-canalicular system? Bone 47:353–359, 2010.
Liao, Z., C. I. Seye, G. A. Weisman, and L. Erb. The P2Y2 nucleotide receptor requires interaction with alpha v integrins to access and activate G12. J. Cell Sci. 120:1654–1662, 2007.
Liu, D., D. C. Genetos, Y. Shao, D. J. Geist, J. Li, H. Z. Ke, C. H. Turner, and R. L. Duncan. Activation of extracellular-signal regulated kinase (ERK1/2) by fluid shear is Ca(2+)- and ATP-dependent in MC3T3-E1 osteoblasts. Bone 42:644–652, 2008.
Nakamura, I., L. Lipfert, G. A. Rodan, and T. D. Le. Convergence of alpha(v)beta(3) integrin- and macrophage colony stimulating factor-mediated signals on phospholipase Cgamma in prefusion osteoclasts. J. Cell Biol. 152:361–373, 2001.
Orriss, I. R., G. Burnstock, and T. R. Arnett. Purinergic signalling and bone remodelling. Curr. Opin. Pharmacol. 10:322–330, 2010.
Pavalko, F. M., N. X. Chen, C. H. Turner, D. B. Burr, S. Atkinson, Y. F. Hsieh, J. Qiu, and R. L. Duncan. Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions. Am. J. Physiol. 275:C1591–C1601, 1998.
Ponik, S. M., and F. M. Pavalko. Formation of focal adhesions on fibronectin promotes fluid shear stress induction of COX-2 and PGE2 release in MC3T3-E1 osteoblasts. J. Appl. Physiol. 97:135–142, 2004.
Qin, Y. X., and H. Lam. Intramedullary pressure and matrix strain induced by oscillatory skeletal muscle stimulation and its potential in adaptation. J. Biomech. 42:140–145, 2009.
Roelofsen, J., J. Klein-Nulend, and E. H. Burger. Mechanical stimulation by intermittent hydrostatic compression promotes bone-specific gene expression in vitro. J. Biomech. 28:1493–1503, 1995.
Rubin, J., C. Rubin, and C. R. Jacobs. Molecular pathways mediating mechanical signaling in bone. Gene 367:1–16, 2006.
Sah, V. P., T. M. Seasholtz, S. A. Sagi, and J. H. Brown. The role of rho in g protein-coupled receptor signal transduction. Annu. Rev. Pharmacol. Toxicol. 40:459–489, 2000.
Shao, Y., K. J. Czymmek, P. A. Jones, V. P. Fomin, K. Akanbi, R. L. Duncan, and M. C. Farach-Carson. Dynamic interactions between l-type voltage-sensitive calcium channel Cav1.2 subunits and ahnak in osteoblastic cells. Am. J. Physiol. Cell Physiol. 296:C1067–C1078, 2009.
Stevens, H. Y., D. R. Meays, and J. A. Frangos. Pressure gradients and transport in the murine femur upon hindlimb suspension. Bone 39:565–572, 2006.
Takai, E., K. D. Costa, A. Shaheen, C. T. Hung, and X. E. Guo. Osteoblast elastic modulus measured by atomic force microscopy is substrate dependent. Ann. Biomed. Eng. 33:963–971, 2005.
Wadhwa, S., S. L. Godwin, D. R. Peterson, M. A. Epstein, L. G. Raisz, and C. C. Pilbeam. Fluid flow induction of cyclo-oxygenase 2 gene expression in osteoblasts is dependent on an extracellular signal-regulated kinase signaling pathway. J. Bone Miner. Res. 17:266–274, 2002.
Wang, N., K. Naruse, D. Stamenovic, J. J. Fredberg, S. M. Mijailovich, I. M. Tolic-Norrelykke, T. Polte, R. Mannix, and D. E. Ingber. Mechanical behavior in living cells consistent with the tensegrity model. Proc. Natl Acad. Sci. U.S.A. 98:7765–7770, 2001.
Wu, D., P. Ganatos, D. C. Spray, and S. Weinbaum. On the electrophysiological response of bone cells using a stokesian fluid stimulus probe for delivery of quantifiable localized piconewton level forces. J. Biomech. 44:1702–1708, 2011.
Xue, Z., W. Zhang, L. P. Desai, H. Gao, S. J. Gunst, and R. S. Tepper. Increased mechanical strain imposed on murine lungs during ventilation in vivo depresses airway responsiveness and activation of protein kinase Akt. J. Appl. Physiol. 114:1506–1510, 2013.
You, J., C. E. Yellowley, H. J. Donahue, Y. Zhang, Q. Chen, and C. R. Jacobs. Substrate deformation levels associated with routine physical activity are less stimulatory to bone cells relative to loading-induced oscillatory fluid flow. J. Biomech. Eng. 122:387–393, 2000.
Young, S. R., R. Gerard-O’Riley, M. Harrington, and F. M. Pavalko. Activation of NF-kappaB by fluid shear stress, but not TNF-alpha, requires focal adhesion kinase in osteoblasts. Bone 47:74–82, 2010.
Young, S. R., R. Gerard-O’Riley, J. B. Kim, and F. M. Pavalko. Focal adhesion kinase is important for fluid shear stress-induced mechanotransduction in osteoblasts. J. Bone Miner. Res. 24:411–424, 2009.
Zhang, X., A. Chattopadhyay, Q. S. Ji, J. D. Owen, P. J. Ruest, G. Carpenter, and S. K. Hanks. Focal adhesion kinase promotes phospholipase C-gamma1 activity. Proc. Natl Acad. Sci. U.S.A. 96:9021–9026, 1999.
Zhang, J., K. D. Ryder, J. A. Bethel, R. Ramirez, and R. L. Duncan. PTH-induced actin depolymerization increases mechanosensitive channel activity to enhance mechanically stimulated Ca2+ signaling in osteoblasts. J. Bone Miner. Res. 21:1729–1737, 2006.
Acknowledgments
This study was supported by funding from NIH/NIAMS (AR043222, AR051901, AR054385, P30GM103333, and AR064668).
Conflict of interest
The authors Joseph D. Gardinier, Vimal Gangadharan, Liyun Wang, and Randall L. Duncan declare that they have no conflicts of interest.
Ethical Standards
No human studies were carried out by the authors for this article. No animal studies were carried out by the authors for this article.
Author information
Authors and Affiliations
Corresponding author
Additional information
Associate Editor Michael R. King oversaw the review of this article.
Rights and permissions
About this article
Cite this article
Gardinier, J.D., Gangadharan, V., Wang, L. et al. Hydraulic Pressure During Fluid Flow Regulates Purinergic Signaling and Cytoskeleton Organization of Osteoblasts. Cel. Mol. Bioeng. 7, 266–277 (2014). https://doi.org/10.1007/s12195-014-0329-8
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12195-014-0329-8