ECM Compliance Regulates Osteogenesis by Influencing MAPK Signaling Downstream of RhoA and ROCK

The compliance of the extracellular matrix (ECM) regulates osteogenic differentiation by modulating extracellular signal‐regulated kinase (ERK) activity. However, the molecular mechanism linking ECM compliance to the ERK‐mitogen‐activated protein kinase (MAPK) pathway remains unclear. Furthermore, RhoA has been widely implicated in integrin‐mediated signaling and mechanotransduction. We studied the relationship between RhoA and ERK‐MAPK signaling to determine their roles in the regulation of osteogenesis by ECM compliance. Inhibition of RhoA and ROCK in MC3T3‐E1 pre‐osteoblasts cultured on substrates of varying compliance reduced ERK activity, whereas constitutively active RhoA enhanced it. The expression of RUNX2, a potent osteogenic transcription factor, was increased on stiffer matrices and correlated with elevated ERK activity. Inhibition of RhoA, ROCK, or the MAPK pathway diminished RUNX2 activity and delayed the onset of osteogenesis as shown by altered osteocalcin (OCN) and bone sialoprotein (BSP) gene expression, alkaline phosphatase (ALP) activity, and matrix mineralization. These data establish that one possible mechanism by which ECM rigidity regulates osteogenic differentiation involves MAPK activation downstream of the RhoA‐ROCK signaling pathway.

[1]  Renny T. Franceschi,et al.  Critical role of the extracellular signal–regulated kinase–MAPK pathway in osteoblast differentiation and skeletal development , 2007, The Journal of cell biology.

[2]  Richard O. Hynes,et al.  Integrins: Versatility, modulation, and signaling in cell adhesion , 1992, Cell.

[3]  A. Hall,et al.  Rho GTPases in cell biology , 2002, Nature.

[4]  N. Balaban,et al.  Adhesion-dependent cell mechanosensitivity. , 2003, Annual review of cell and developmental biology.

[5]  Richard O. Hynes,et al.  Integrin-mediated Signals Regulated by Members of the Rho Family of GTPases , 1998, The Journal of cell biology.

[6]  A. Hall,et al.  Rho GTPases and the actin cytoskeleton. , 1998, Science.

[7]  D. Baylink,et al.  Skeletal alkaline phosphatase activity as a bone formation index in vitro. , 1986, Metabolism: clinical and experimental.

[8]  P. Ducy.  CBFA1: A molecular switch in osteoblast biology , 2000, Developmental dynamics : an official publication of the American Association of Anatomists.

[9]  Phil G. Campbell,et al.  Phosphophoryn Regulates the Gene Expression and Differentiation of NIH3T3, MC3T3-E1, and Human Mesenchymal Stem Cells via the Integrin/MAPK Signaling Pathway* , 2004, Journal of Biological Chemistry.

[10]  Shelly R. Peyton,et al.  Extracellular matrix rigidity governs smooth muscle cell motility in a biphasic fashion , 2005, Journal of cellular physiology.

[11]  D. Mooney,et al.  External mechanical strain regulates membrane targeting of Rho GTPases by controlling microtubule assembly. , 2003, American journal of physiology. Cell physiology.

[12]  H. Yoshikawa,et al.  Continuous Inhibition of MAPK Signaling Promotes the Early Osteoblastic Differentiation and Mineralization of the Extracellular Matrix , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[13]  J. Sadoshima,et al.  Angiotensin II activates RhoA in cardiac myocytes: a critical role of RhoA in angiotensin II-induced premyofibril formation. , 1998, Circulation research.

[14]  Donald E. Ingber,et al.  Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels , 2006, Journal of Cell Science.

[15]  Joyce Y. Wong,et al.  Directed Movement of Vascular Smooth Muscle Cells on Gradient-Compliant Hydrogels† , 2003 .

[16]  H. van Essen,et al.  Role of the Rhoa/Rho Kinase System in Flow-Related Remodeling of Rat Mesenteric Small Arteries in Vivo , 2004, Journal of Vascular Research.

[17]  Shelly R Peyton,et al.  The effects of matrix stiffness and RhoA on the phenotypic plasticity of smooth muscle cells in a 3-D biosynthetic hydrogel system. , 2008, Biomaterials.

[18]  M. Hurley,et al.  Signal transduction by basic fibroblast growth factor in rat osteoblastic py1a cells , 1996, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[19]  Adam J. Engler,et al.  Myotubes differentiate optimally on substrates with tissue-like stiffness , 2004, The Journal of cell biology.

[20]  L. Chong,et al.  The small GTP-binding protein Rho regulates a phosphatidylinositol 4-phosphate 5-kinase in mammalian cells , 1994, Cell.

[21]  G. Xiao,et al.  Regulation of the osteoblast‐specific transcription factor, Runx2: Responsiveness to multiple signal transduction pathways , 2003, Journal of cellular biochemistry.

[22]  A. Seth,et al.  Ets transcription factors and targets in osteogenesis , 2000, Oncogene.

[23]  T. Peterson,et al.  Fluid shear stress stimulates mitogen-activated protein kinase in endothelial cells. , 1995, Circulation research.

[24]  Ying Zhang,et al.  Mechanical stress increases RhoA activation in airway smooth muscle cells. , 2003, American journal of respiratory cell and molecular biology.

[25]  Karl Kingsley,et al.  Laminin-5 induces osteogenic gene expression in human mesenchymal stem cells through an ERK-dependent pathway. , 2004, Molecular biology of the cell.

[26]  Dimitris Kletsas,et al.  The Bone-specific Transcriptional Regulator Cbfa1 Is a Target of Mechanical Signals in Osteoblastic Cells* , 2002, The Journal of Biological Chemistry.

[27]  Sunil Wadhwa,et al.  Fluid Flow Induction of Cyclo‐Oxygenase 2 Gene Expression in Osteoblasts Is Dependent on an Extracellular Signal‐Regulated Kinase Signaling Pathway , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[28]  Christopher S. Chen,et al.  Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. , 2004, Developmental cell.

[29]  G. Xiao,et al.  Fibroblast Growth Factor 2 Induction of the Osteocalcin Gene Requires MAPK Activity and Phosphorylation of the Osteoblast Transcription Factor, Cbfa1/Runx2* , 2002, The Journal of Biological Chemistry.

[30]  R L Juliano,et al.  Integrin signaling and cell growth control. , 1998, Current opinion in cell biology.

[31]  C. Waterman-Storer,et al.  Cell motility: can Rho GTPases and microtubules point the way? , 2001, Journal of cell science.

[32]  Shu Chien,et al.  Effects of cell tension on the small GTPase Rac , 2002, The Journal of cell biology.

[33]  Shelly R. Peyton,et al.  Intrinsic mechanical properties of the extracellular matrix affect the behavior of pre-osteoblastic MC3T3-E1 cells. , 2006, American journal of physiology. Cell physiology.

[34]  S. Sheen-Chen,et al.  Superoxide Mediates Shock Wave Induction of ERK-dependent Osteogenic Transcription Factor (CBFA1) and Mesenchymal Cell Differentiation toward Osteoprogenitors* , 2002, The Journal of Biological Chemistry.

[35]  Shelly R. Peyton,et al.  The use of poly(ethylene glycol) hydrogels to investigate the impact of ECM chemistry and mechanics on smooth muscle cells. , 2006, Biomaterials.

[36]  John C. Koch,et al.  The laws of bone architecture , 1917 .

[37]  S. Sen,et al.  Matrix Elasticity Directs Stem Cell Lineage Specification , 2006, Cell.

[38]  G. Xiao,et al.  Fibroblast Growth Factor 2 Induction of the Osteocalcin Gene Requires MAPK Activity and Phosphorylation of the Osteoblast Transcription Factor , Cbfa 1 / Runx 2 * , 2002 .

[39]  J. Wolff.  Das Gesetz der Transformation der Knochen , 1893 .

[40]  Y. Wang,et al.  Cell locomotion and focal adhesions are regulated by substrate flexibility. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Shelly R. Peyton,et al.  The regulation of osteogenesis by ECM rigidity in MC3T3‐E1 cells requires MAPK activation , 2007, Journal of cellular physiology.

[42]  D. E. Discher,et al.  Matrix elasticity directs stem cell lineage — Soluble factors that limit osteogenesis , 2009 .

[43]  J. Settleman,et al.  Modulation of Rho GTPase Signaling Regulates a Switch between Adipogenesis and Myogenesis , 2003, Cell.

[44]  G. Xiao,et al.  Bone Morphogenetic Proteins, Extracellular Matrix, and Mitogen‐Activated Protein Kinase Signaling Pathways Are Required for Osteoblast‐Specific Gene Expression and Differentiation in MC3T3‐E1 Cells , 2002, Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research.

[45]  E. Nishida,et al.  Differentiation and Transforming Growth Factor-β Receptor Down-regulation by Collagen-α2β1 Integrin Interaction Is Mediated by Focal Adhesion Kinase and Its Downstream Signals in Murine Osteoblastic Cells* , 1997, The Journal of Biological Chemistry.

[46]  J. Aubin,et al.  Advances in the osteoblast lineage. , 1998, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[47]  C. Nobes,et al.  Rho, Rac, and Cdc42 GTPases regulate the assembly of multimolecular focal complexes associated with actin stress fibers, lamellipodia, and filopodia , 1995, Cell.

[48]  Makoto Sato,et al.  Targeted Disruption of Cbfa1 Results in a Complete Lack of Bone Formation owing to Maturational Arrest of Osteoblasts , 1997, Cell.

[49]  G. Karsenty,et al.  MAPK Pathways Activate and Phosphorylate the Osteoblast-specific Transcription Factor, Cbfa1* , 2000, The Journal of Biological Chemistry.

[50]  E. Nishida,et al.  Differentiation and transforming growth factor-beta receptor down-regulation by collagen-alpha2beta1 integrin interaction is mediated by focal adhesion kinase and its downstream signals in murine osteoblastic cells. , 1997, The Journal of biological chemistry.

[51]  Shelly R. Peyton,et al.  The emergence of ECM mechanics and cytoskeletal tension as important regulators of cell function , 2007, Cell Biochemistry and Biophysics.

[52]  Shu Chien,et al.  Shear stress-induced c-fos activation is mediated by Rho in a calcium-dependent manner. , 2003, Biochemical and biophysical research communications.

[53]  B. Chen,et al.  Distinct roles for the small GTPases Cdc42 and Rho in endothelial responses to shear stress. , 1999, The Journal of clinical investigation.

[54]  N. D’Silva,et al.  Impact of the Mitogen-activated Protein Kinase Pathway on Parathyroid Hormone-related Protein Actions in Osteoblasts* , 2004, Journal of Biological Chemistry.

[55]  W. Kiosses,et al.  Regulation of the small GTP‐binding protein Rho by cell adhesion and the cytoskeleton , 1999, The EMBO journal.

[56]  J. Brugge,et al.  Integrins and signal transduction pathways: the road taken. , 1995, Science.

[57]  Anne J. Ridley,et al.  The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors , 1992, Cell.

[58]  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.

[59]  Y. Zou,et al.  Rho family small G proteins play critical roles in mechanical stress-induced hypertrophic responses in cardiac myocytes. , 1999, Circulation research.

[60]  In‐San Kim,et al.  Expression patterns of bone‐related proteins during osteoblastic differentiation in MC3T3‐E1 cells , 1996, Journal of cellular biochemistry.

[61]  J. Aubin,et al.  Osteoblast and chondroblast differentiation. , 1995, Bone.

[62]  Donald E Ingber,et al.  Extracellular matrix controls myosin light chain phosphorylation and cell contractility through modulation of cell shape and cytoskeletal prestress. , 2004, American journal of physiology. Cell physiology.

[63]  T. Yamakawa,et al.  Mechanotransduction of rat aortic vascular smooth muscle cells requires RhoA and intact actin filaments. , 1999, Circulation research.

[64]  K. Burridge,et al.  Bidirectional signaling between the cytoskeleton and integrins. , 1999, Current opinion in cell biology.

[65]  Dennis Discher,et al.  Substrate compliance versus ligand density in cell on gel responses. , 2004, Biophysical journal.

[66]  R. Franceschi,et al.  The developmental control of osteoblast-specific gene expression: role of specific transcription factors and the extracellular matrix environment. , 1999, Critical reviews in oral biology and medicine : an official publication of the American Association of Oral Biologists.

[67]  M. Pittenger,et al.  Adult Human Mesenchymal Stem Cell Differentiation to the Osteogenic or Adipogenic Lineage Is Regulated by Mitogen-activated Protein Kinase* , 2000, The Journal of Biological Chemistry.

[68]  G. Xiao,et al.  Multiple signaling pathways converge on the Cbfa1/Runx2 transcription factor to regulate osteoblast differentiation. , 2003, Connective tissue research.

[69]  V. Sikavitsas,et al.  Biomaterials and bone mechanotransduction. , 2001, Biomaterials.

[70]  K. Burridge,et al.  Rho-stimulated contractility drives the formation of stress fibers and focal adhesions , 1996, The Journal of cell biology.

[71]  K. Lau,et al.  Fluid flow shear stress stimulates human osteoblast proliferation and differentiation through multiple interacting and competing signal transduction pathways. , 2003, Bone.

[72]  G. Plopper,et al.  ERK Signaling Pathways Regulate the Osteogenic Differentiation of Human Mesenchymal Stem Cells on Collagen I and Vitronectin , 2004, Cell communication & adhesion.

[73]  M. Schwartz.  Integrins, Oncogenes, and Anchorage Independence , 1997, The Journal of cell biology.

[74]  R. Nemenoff,et al.  Activation of JNK/SAPK and ERK by mechanical strain in vascular smooth muscle cells depends on extracellular matrix composition. , 1997, Biochemical and biophysical research communications.