RhoA-Mediated Signaling in Mechanotransduction of Osteoblasts

Osteoblasts play a pivotal role in load-driven bone formation by activating Wnt signaling through a signal from osteocytes as a mechanosensor. Osteoblasts are also sensitive to mechanical stimulation, but the role of RhoA, a small GTPase involved in the regulation of cytoskeleton adhesion complexes, in mechanotransduction of osteoblasts is not completely understood. Using MC3T3-E1 osteoblast-like cells under 1 hr flow treatment at 10 dyn/cm2, we examined a hypothesis that RhoA signaling mediates the cellular responses to flow-induced shear stress. To test the hypothesis, we conducted genome-wide pathway analysis and evaluated the role of RhoA in molecular signaling. Activity of RhoA was determined with a RhoA biosensor, which determined the activation state of RhoA based on a fluorescence resonance energy transfer between CFP and YFP fluorophores. A pathway analysis indicated that flow treatment activated phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling as well as a circadian regulatory pathway. Western blot analysis revealed that in response to flow treatment phosphorylation of Akt in PI3K signaling and phosphorylation of p38 and ERK1/2 in MAPK signaling were induced. FRET measurement showed that RhoA was activated by flow treatment, and an inhibitor to a Rho kinase significantly reduced flow-induced phosphorylation of p38, ERK1/2, and Akt as well as flow-driven elevation of the mRNA levels of osteopontin and cyclooxygenase-2. Collectively, the result demonstrates that in response to 1 hr flow treatment to MC3T3-E1 cells at 10 dyn/cm2, RhoA plays a critical role in activating PI3K and MAPK signaling as well as modulating the circadian regulatory pathway.

[1]  H J Donahue,et al.  Substrate deformation levels associated with routine physical activity are less stimulatory to bone cells relative to loading-induced oscillatory fluid flow. , 2000, Journal of biomechanical engineering.

[2]  S. Cowin,et al.  A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses. , 1994, Journal of biomechanics.

[3]  T. Burdo,et al.  Osteopontin prevents monocyte recirculation and apoptosis , 2007, Journal of leukocyte biology.

[4]  D. Burr,et al.  Mechanotransduction in bone: osteoblasts are more responsive to fluid forces than mechanical strain. , 1997, The American journal of physiology.

[5]  L. Bonewald,et al.  The Amazing Osteocyte , 2010, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[6]  E H Burger,et al.  The production of nitric oxide and prostaglandin E(2) by primary bone cells is shear stress dependent. , 2001, Journal of biomechanics.

[7]  Y. Amagai,et al.  In vitro differentiation and calcification in a new clonal osteogenic cell line derived from newborn mouse calvaria , 1983, The Journal of cell biology.

[8]  P. Gong,et al.  Response of osteoblasts to low fluid shear stress is time dependent. , 2011, Tissue & cell.

[9]  F. Reinholt,et al.  Osteopontin--a possible anchor of osteoclasts to bone. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Sunil Wadhwa,et al.  Fluid flow induces COX-2 expression in MC3T3-E1 osteoblasts via a PKA signaling pathway. , 2002, Biochemical and biophysical research communications.

[11]  I D L Bogle,et al.  A model of localised Rac1 activation in endothelial cells due to fluid flow. , 2011, Journal of theoretical biology.

[12]  P. Nijweide,et al.  Pulsating fluid flow increases nitric oxide (NO) synthesis by osteocytes but not periosteal fibroblasts--correlation with prostaglandin upregulation. , 1995, Biochemical and biophysical research communications.

[13]  T J Chambers,et al.  Induction of NO and prostaglandin E2 in osteoblasts by wall-shear stress but not mechanical strain. , 1997, American journal of physiology. Endocrinology and metabolism.

[14]  S. Miyamoto,et al.  Focal Adhesion Kinase as a RhoA-activable Signaling Scaffold Mediating Akt Activation and Cardiomyocyte Protection* , 2008, Journal of Biological Chemistry.

[15]  Stretch augments TGF-beta1 expression through RhoA/ROCK1/2, PTK, and PI3K in airway smooth muscle cells. , 2010, American journal of physiology. Lung cellular and molecular physiology.

[16]  J A Frangos,et al.  Fluid flow stimulates rapid and continuous release of nitric oxide in osteoblasts. , 1996, The American journal of physiology.

[17]  D. Cui,et al.  Synthetic osteogenic growth peptide promotes differentiation of human bone marrow mesenchymal stem cells to osteoblasts via RhoA/ROCK pathway , 2011, Molecular and Cellular Biochemistry.

[18]  M. Matsuda,et al.  Activity of Rho-family GTPases during cell division as visualized with FRET-based probes , 2003, The Journal of cell biology.

[19]  Ping Zhang,et al.  Joint loading-driven bone formation and signaling pathways predicted from genome-wide expression profiles. , 2009, Bone.

[20]  A. Robling,et al.  Mechanical signaling for bone modeling and remodeling. , 2009, Critical reviews in eukaryotic gene expression.

[21]  A. Anwar,et al.  PI3K, Rho, and ROCK play a key role in hypoxia-induced ATP release and ATP-stimulated angiogenic responses in pulmonary artery vasa vasorum endothelial cells. , 2009, American journal of physiology. Lung cellular and molecular physiology.

[22]  E H Burger,et al.  Signal transduction pathways involved in fluid flow-induced PGE2 production by cultured osteocytes. , 1999, American journal of physiology. Endocrinology and metabolism.

[23]  H J Donahue,et al.  Osteopontin Gene Regulation by Oscillatory Fluid Flow via Intracellular Calcium Mobilization and Activation of Mitogen-activated Protein Kinase in MC3T3–E1 Osteoblasts* , 2001, The Journal of Biological Chemistry.

[24]  T. Notomi,et al.  Per‐1 is a specific clock gene regulated by parathyroid hormone (PTH) signaling in osteoblasts and is functional for the transcriptional events induced by PTH , 2011, Journal of cellular biochemistry.

[25]  Mark L. Johnson,et al.  Bisphosphonate-Related Osteonecrosis of the Jaw: Model and Diagnosis with Cone Beam Computerized Tomography , 2008, Cells Tissues Organs.

[26]  A. Franzén,et al.  Possible recruitment of osteoblastic precursor cells from hypertrophic chondrocytes during initial osteogenesis in cartilaginous limbs of young rats. , 1989, Matrix.

[27]  E. Wagner,et al.  The Molecular Clock Mediates Leptin-Regulated Bone Formation , 2005, Cell.

[28]  K. Piekarski,et al.  Transport mechanism operating between blood supply and osteocytes in long bones , 1977, Nature.

[29]  Lutz Claes,et al.  Signal transduction pathways involved in mechanotransduction in bone cells. , 2006, Biochemical and biophysical research communications.

[30]  Shelly R. Peyton,et al.  ECM Compliance Regulates Osteogenesis by Influencing MAPK Signaling Downstream of RhoA and ROCK , 2009, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[31]  N. Obermüller,et al.  Synergistic induction of osteopontin by aldosterone and inflammatory cytokines in mesangial cells , 2008, Journal of cellular biochemistry.

[32]  S. Gay,et al.  Immunohistochemical demonstration of a 44-KD phosphoprotein in developing rat bones. , 1987, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[33]  Mark L. Johnson,et al.  Osteocytes, mechanosensing and Wnt signaling. , 2008, Bone.

[34]  A. C. Stumbo,et al.  Osteopontin expression in coculture of differentiating rat fetal skeletal fibroblasts and myoblasts , 2006, In Vitro Cellular & Developmental Biology - Animal.

[35]  E R Morey,et al.  Inhibition of bone formation during space flight. , 1978, Science.

[36]  J. Kreisberg,et al.  RhoA-dependent murine prostate cancer cell proliferation and apoptosis: role of protein kinase Czeta. , 2002, Cancer research.

[37]  Alexander G Robling,et al.  Biomechanical and molecular regulation of bone remodeling. , 2006, Annual review of biomedical engineering.

[38]  R L Duncan,et al.  Ca(2+) regulates fluid shear-induced cytoskeletal reorganization and gene expression in osteoblasts. , 2000, American journal of physiology. Cell physiology.

[39]  J. Reiners,et al.  Suppression of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-mediated aryl hydrocarbon receptor transformation and CYP1A1 induction by the phosphatidylinositol 3-kinase inhibitor 2-(4-morpholinyl)-8-phenyl-4H-1- benzopyran-4-one (LY294002). , 2000, Biochemical pharmacology.

[40]  P. Khatri,et al.  A systems biology approach for pathway level analysis. , 2007, Genome research.

[41]  A. C. Stumbo,et al.  Osteopontin expression in coculture of differentiating rat fetal skeletal fibroblasts and myoblasts , 2006, In Vitro Cellular & Developmental Biology - Animal.

[42]  David B. Burr,et al.  Osteoblast Responses One Hour After Load-Induced Fluid Flow in a Three-Dimensional Porous Matrix , 2005, Calcified Tissue International.

[43]  Jason W. Triplett,et al.  Osteoblasts and osteocytes respond differently to oscillatory and unidirectional fluid flow profiles , 2007, Journal of cellular biochemistry.

[44]  F. Pavalko,et al.  Focal Adhesion Kinase Is Important for Fluid Shear Stress‐Induced Mechanotransduction in Osteoblasts , 2009, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[45]  Gwo‐Jaw Wang,et al.  Constitutively expressed COX-2 in osteoblasts positively regulates Akt signal transduction via suppression of PTEN activity. , 2011, Bone.

[46]  H. Sun,et al.  CITED2-mediated Regulation of MMP-1 and MMP-13 in Human Chondrocytes under Flow Shear* , 2003, Journal of Biological Chemistry.

[47]  S. Cowin,et al.  A case for bone canaliculi as the anatomical site of strain generated potentials. , 1995, Journal of biomechanics.

[48]  D. Bikle,et al.  Bone response to normal weight bearing after a period of skeletal unloading. , 1989, The American journal of physiology.

[49]  D. Burr,et al.  Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions. , 1998, American journal of physiology. Cell physiology.