Fluid shear‐induced ATP secretion mediates prostaglandin release in MC3T3‐E1 osteoblasts

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Genetos, Damian C.¹; Geist, Derik J.²; Liu, Dawei²; Donahue, Henry J.^1,3; Duncan, Randall L.²

Fluid shear‐induced ATP secretion mediates prostaglandin release in MC3T3‐E1 osteoblasts

J Bone Miner Res. January 2005;20(1):41-49

©2005 American Society for Bone and Mineral Research

Affiliations

¹Departments of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA

²Department of Orthopaedic Surgery, Indiana University School of Medicine, Indianapolis, Indiana, USA

³Orthopaedics and Rehabilitation, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USA
Abstract and keywords

ATP is rapidly released from osteoblasts in response to mechanical load. We examined the mechanisms involved in this release and established that shear‐induced ATP release was mediated through vesicular fusion and was dependent on Ca2+ entry into the cell through L‐type voltage‐sensitive Ca2+ channels. Degradation of secreted ATP by apyrase prevented shear‐induced PGE2 release.

Introduction Fluid shear induces a rapid rise in intracellular calcium ([Ca2+]i) in osteoblasts that mediates many of the cellular responses associated with mechanotransduction in bone. A potential mechanism for this increase in [Ca2+]i is the activation of purinergic (P2) receptors resulting from shear‐induced extracellular release of ATP. This study was designed to determine the effects of fluid shear on ATP release and the possible mechanisms associated with this release.

Materials and Methods MC3T3‐E1 preosteoblasts were plated on type I collagen, allowed to proliferate to 90% confluency, and subjected to 12 dynes/cm2 laminar fluid flow using a parallel plate flow chamber. ATP release into the flow media was measured using a luciferin/luciferase assay. Inhibitors of channels, gap junctional intercellular communication (GJIC), and vesicular formation were added before shear and maintained in the flow medium for the duration of the experiment.

Results and Conclusions Fluid shear produced a transient increase in ATP release compared with static MC3T3‐E1 cells (59.8 ± 15.7 versus 6.2 ± 1.8 nM, respectively), peaking within 1 minute of onset. Inhibition of calcium entry through the L‐type voltage‐sensitive Ca2+ channel (L‐VSCC) with nifedipine or verapamil significantly attenuated shear‐induced ATP release. Channel inhibition had no effect on basal ATP release in static cells. Ca2+‐dependent ATP release in response to shear seemed to result from vesicular release and not through gap hemichannels. Vesicle disruption with N‐ethylmaleimide, brefeldin A, or monensin prevented increases in flow‐induced ATP release, whereas inhibition of gap hemichannels with either 18α‐glycyrrhetinic acid or 18β‐glycyrrhetinic acid did not. Degradation of extracellular ATP with apyrase prevented shear‐induced increases in prostaglandin E2 (PGE2) release. These data suggest a time line of mechanotransduction wherein fluid shear activates L‐VSCCs to promote Ca2+ entry that, in turn, stimulates vesicular ATP release. Furthermore, these data suggest that P2 receptor activation by secreted ATP mediates flow‐induced prostaglandin release.

Keywords: ATP release; mechanotransduction; Ca2+ signaling; osteoblasts; fluid shear
Links

DOI: 10.1359/JBMR.041009

PubMed: 15619668

WoS: 000225986900009
History

Received: 2004-03-4

Revised: 2004-07-20

Accepted: 2004-08-11

Online: 2004-10-18

Cited Works (15)

Year	Entry
1997	Owan I, Burr DB, Turner CH, Qiu J, Tu Y, Onyia JE, Duncan RL. Mechanotransduction in bone: osteoblasts are more responsive to fluid forces than mechanical strain. Am J Physiol Cell Physiol. September 1997;273(3):C810-C815.
1996	Rawlinson SCF, Pitsillides AA, Lanyon LE. Involvement of different ion channels in osteoblasts' and osteocytes' early responses to mechanical strain. Bone. December 1996;19(6):609-614.
1993	Reich KM, Frangos JA. Protein kinase C mediates flow-induced prostaglandin E₂ production in osteoblasts. Calcif Tiss Int. January 1993;52(1):62-66.
1997	Klein‐Nulend J, Burger EH, Semeins CM, Raisz LG, Pilbeam CC. Pulsating fluid flow stimulates prostaglandin release and inducible prostaglandin g/h synthase mrna expression in primary mouse bone cells. J Bone Miner Res. January 1997;12(1):45-51.
1999	McAllister TN, Frangos JA. Steady and transient fluid shear stress stimulate NO release in osteoblasts through distinct biochemical pathways. J Bone Miner Res. June 1999;14(6):930-936.
1998	Pavalko FM, Chen NX, Turner CH, Burr DB, Atkinson S, Hsieh Y-F, Qiu J, Duncan RL. Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions. Am J Physiol. December 1998;275(6):C1591-C1601.
1996	Hung CT, Allen FD, Pollack SR, Brighton CT. Intracellular Ca²⁺ stores and extracellular Ca²⁺ are required in the real-time Ca²⁺ response of bone cells experiencing fluid flow. J Biomech. November 1996;29(11):1411-1417.
2000	Yellowley CE, Li Z, Zhou Z, Jacobs CR, Donahue HJ. Functional gap junctions between osteocytic and osteoblastic cells. J Bone Miner Res. February 2000;15(2):209-217.
1996	Forwood MR. Inducible cyclo‐oxygenase (COX‐2) mediates the induction of bone formation by mechanical loading in vivo. J Bone Miner Res. November 1996;11(11):1688-1693.
1991	Reich KM, Frangos JA. Effect of flow on prostaglandin E₂ and inositol trisphosphate levels in osteoblasts. Am J Physiol. September 1991;261(3):C428-C432.
2001	Ryder KD, Duncan RL. Parathyroid hormone enhances fluid shear-induced [Ca²⁺]_i signaling in osteoblastic cells through activation of mechanosensitive and voltage-sensitive Ca²⁺ channels. J Bone Miner Res. February 2001;16(2):240-248.
2002	Burr DB, Robling AG, Turner CH. Effects of biomechanical stress on bones in animals. Bone. May 2002;30(5):781-786.
2001	You J, Reilly GC, Zhen X, Yellowley CE, Chen Q, Donahue HJ, Jacobs CR. 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. April 20, 2001;276(16):13365-13371.
1999	Ajubi NE, Klein-Nulend J, Alblas MJ, Burger EH, Nijweide PJ. Signal transduction pathways involved in fluid flow-induced PGE₂ production by cultured osteocytes. Am J Physiol Endocrinol Metab. January 1999;276(1):E171-E178.
2000	Chen NX, Ryder KD, Pavalko FM, Turner CH, Burr DB, Qiu J, Duncan RL. Ca²⁺ regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts. Am J Physiol Cell Physiol. May 2000;278(5):C989-C997.

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Year	Entry
2013	Hum JM. Signaling Mechanisms That Suppress the Anabolic Response of Osteoblasts and Osteocytes to Fluid Shear Stress [PhD thesis]. Bloomington, IN: Indiana University; September 2013.
2006	Robling AG, Castillo AB, Turner CH. Biomechanical and molecular regulation of bone remodeling. Annu Rev Biomed Eng. 2006;8:455-498.
2010	Kamel MA, Picconi JL, Lara-Castillo N, Johnson ML. Activation of β-catenin signaling in MLO-Y4 osteocytic cells versus 2T3 osteoblastic cells by fluid flow shear stress and PGE2: implications for the study of mechanosensation in bone. Bone. November 2010;47(5):872-881.
2020	Williams KM, Leser JM, Gould NR, Joca HC, Lyons JS, Khairallah RJ, Ward CW, Stains JP. TRPV4 calcium influx controls sclerostin protein loss independent of purinergic calcium oscillations. Bone. July 2020;136:115356.
2013	Dallas SL, Prideaux M, Bonewald LF. The osteocyte: an endocrine cell … and more. Endocr Rev. October 2013;34(4):658-690.
2006	Rubin J, Rubin C, Jacobs CR. Molecular pathways mediating mechanical signaling in bone. Gene. February 15, 2006;367:1-16.
2012	Thompson WR, Rubin CT, Rubin J. Mechanical regulation of signaling pathways in bone. Gene. July 25, 2012;503(2):179-193.
2005	Li J, Liu D, Ke HZ, Duncan RL, Turner CH. The P2X7 nucleotide receptor mediates skeletal mechanotransduction. J Biol Chem. December 30, 2005;280(52):42952-42959.
2006	Sawakami K, Robling AG, Ai M, Pitner ND, Liu D, Warden SJ, Li J, Maye P, Rowe DW, Duncan RL, Warman ML, Turner CH. The Wnt co-receptor LRP5 is essential for skeletal mechanotransduction but not for the anabolic bone response to parathyroid hormone treatment. J Biol Chem. August 18, 2006;281(33):23698-23711.
2011	Bonewald LF. The amazing osteocyte. J Bone Miner Res. 2011;26(2):229-238.
2012	Lu XL, Huo B, Chiang V, Guo XE. Osteocytic network is more responsive in calcium signaling than osteoblastic network under fluid flow. J Bone Miner Res. March 2012;27(3):563-574.
2007	Genetos DC, Kephart CJ, Zhang Y, Yellowley CE, Donahue HJ. Oscillating fluid flow activation of gap junction hemichannels induces ATP release from MLO‐Y4 osteocytes. J Cell Physiol. July 2007;212(1):207-214.
2009	Riddle RC, Donahue HJ. From streaming-potentials to shear stress: 25 years of bone cell mechanotransduction. J Orthop Res. February 2009;27(2):143-149.
2015	Scully N. Differentiation of Osteoblasts to Osteocytes in 3D Type I Collagen Gels: A Novel Tool to Study Osteocyte Responses to Mechanical Loading [PhD thesis]. Cardiff, UK: Cardiff University; April 2015.
2014	Steward AJ. The Mechanotransduction of Hydrostatic Pressure by Mesenchymal Stem Cells [PhD thesis]. University of Notre Dame; April 2014.
2016	Metzger TA. Experimental and Computational Investigation of Bone Marrow Mechanobiology [PhD thesis]. University of Notre Dame; April 2016.
2020	Simfia I. Estrogen Deficiency Alters the Mechanobiological Responses of Osteoblasts and Osteocytes [PhD thesis]. National University of Ireland Galway; April 2020.
2012	Sun X. Mechanically Induced Calcium Efflux from Bone Matrix Stimulates Osteoblasts [PhD thesis]. Purdue University; May 2012.
2018	Yang F. The Synergistic Effect of Bone Graft Substitute Architecture and Mechanical Environment on HMSCs Responses in Vitro [PhD thesis]. Queen Mary University of London; December 2018.
2013	Delaine-Smith RM. Mechanical and Physical Guidance of Osteogenic Differentiation and Matrix Production [PhD thesis]. University of Sheffield; January 2013.
2007	Malone AMD. Mechanotransduction Mechanisms in Bone Cells [PhD thesis]. Stanford University; August 2007.
2008	Kwon RY. Mechanical Behavior and Early Molecular Signaling During Mechanotransduction of Fluid Flow in Bone Cells [PhD thesis]. Stanford University; August 2008.
2009	Litzenberger JB. The Role of Integrin Signaling in Bone Mechanotransduction [PhD thesis]. Stanford University; 2009.
2009	Temiyasathit S. Investigating the Role of Primary Cilia in Mechanotransduction in Bone [PhD thesis]. Stanford University; September 2009.
2010	Thompson WR. Perlecan Modulates the Function of the Osteocyte Lacuno-Canalicular System [PhD thesis]. University of Delaware; 2010.
2011	Boggs ME. Osteocyte-Neuron Communication in Bone: A Mechanism for Transmission of Pain Sensation Potentially Associated With Bone Cancer [PhD thesis]. University of Delaware; 2011.
2011	Jones PA. L-Type Voltage-Sensitive Calcium Channel-Mediated Regulation of Osteoblast Behavior [PhD thesis]. University of Delaware; 2011.
2011	Majumdar S. Cell Surface ATP Synthase: A Novel Mechanism for ATP Signaling in Osteoblasts [PhD thesis]. University of Delaware; Summer, 2011.
2014	Gangadharan V. Delineating the Cellular Mechanism of Osteoblast Desensitization to Mechanical Stimulation [PhD thesis]. University of Delaware; 2014.
2013	Daley E. On the Phenotype of White-Tailed Deer Antlerogenic Progenitor Cells [PhD thesis]. University of Michigan; 2013.
2015	Heo SC. Differentiation Induces Dynamic Alterations in Mesenchymal Stem Cell Nuclear Architecture and Mechanotransduction [PhD thesis]. Philadelphia, PA: University of Pennsylvania; 2015.