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Originally published In Press as doi:10.1074/jbc.M009846200 on January 26, 2001

J. Biol. Chem., Vol. 276, Issue 16, 13365-13371, April 20, 2001
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Osteopontin Gene Regulation by Oscillatory Fluid Flow via Intracellular Calcium Mobilization and Activation of Mitogen-activated Protein Kinase in MC3T3-E1 Osteoblasts*

Jun YouDagger , Gwendolen C. ReillyDagger , Xuechu Zhen§, Clare E. YellowleyDagger , Qian ChenDagger , Henry J. DonahueDagger , and Christopher R. Jacobs||**

From the Dagger  Musculoskeletal Research Laboratory, Department of Orthopaedics and Rehabilitation, The Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033, § Department of Pharmacology and Physiology, MCP-Hahneman School of Medicine, Drexel University, Philadelphia, Pennsylvania 19129,  Biomechanical Engineering Division, Department of Mechanical Engineering, Stanford University, Stanford, California 94305, and || Rehabilitation Research and Development Center, Palo Alto Health Care System, Department of Veterans Affairs, Palo Alto, California 94304

Recently fluid flow has been shown to be a potent physical stimulus in the regulation of bone cell metabolism. However, most investigators have applied steady or pulsing flow profiles rather than oscillatory fluid flow, which occurs in vivo because of mechanical loading. Here oscillatory fluid flow was demonstrated to be a potentially important physical signal for loading-induced changes in bone cell metabolism. We selected three well known biological response variables including intracellular calcium (Ca2+i), mitogen-activated protein kinase (MAPK) activity, and osteopontin (OPN) mRNA levels to examine the response of MC3T3-E1 osteoblastic cells to oscillatory fluid flow with shear stresses ranging from 2 to -2 Newtons/m2 at 1 Hz, which is in the range expected to occur during routine physical activities. Our results showed that within 1 min, oscillatory flow induced cell Ca2+i mobilization, whereas two MAPKs (ERK and p38) were activated over a 2-h time frame. However, there was no activation of JNK. Furthermore 2 h of oscillatory fluid flow increased steady-state OPN mRNA expression levels by approximately 4-fold, 24 h after exposure to fluid flow. The presence of both ERK and p38 inhibitors and thapsigargin completely abolished the effect of oscillatory flow on steady-state OPN mRNA levels. In addition, experiments using a variety of pharmacological agents suggest that oscillatory flow induces Ca2+i mobilization via the L-type voltage-operated calcium channel and the inositol 1,4,5-trisphosphate pathway.

* This work was supported by National Institutes of Health Grants AR45989, AG13087, AG00811, and AG17021, by the Whitaker Foundation, Arthritis Foundation, and the United States Army Medical Research and Materiel Command Award DAMD 17-98-1-8509.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

** To whom correspondence should be addressed: Biomechanical Engineering Division, Durand 211, Stanford University, Stanford, CA 94305-3030. Tel.: 650-723-3610; Fax: 650-725-1587; E-mail: christopher.jacobs@stanford.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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