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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
Author(s) -
Xiao Guozhi,
Gopalakrishnan Rajaram,
Jiang Di,
Reith Elizabeth,
Benson M. Douglas,
Franceschi Renny T.
Publication year - 2002
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.2002.17.1.101
Subject(s) - osteoblast , microbiology and biotechnology , extracellular matrix , signal transduction , protein kinase a , bone morphogenetic protein , mitogen activated protein kinase , biology , gene , bone morphogenetic protein 2 , kinase , chemistry , genetics , in vitro
Osteoblasts secrete a complex extracellular matrix (ECM) containing collagenous and noncollagenous proteins, bone morphogenetic proteins (BMPs), and growth factors. Osteoblast‐specific gene expression requires ascorbic acid (AA)‐dependent assembly of a collagenous ECM. Matrix responsiveness requires an α 2 β 1 integrin‐collagen interaction and mitogen‐activated protein kinase (MAPK) activity, which phosphorylates and activates the osteoblast‐specific transcription factor Cbfa1. This study examines interactions between this integrin/MAPK‐mediated pathway and signals initiated by BMPs contained in the osteoblast matrix. MC3T3‐E1 cells were shown to constitutively express BMP‐2, BMP‐4, and BMP‐7. Noggin, a specific BMP inhibitor, reversibly blocked AA‐induced gene expression, indicating that BMP production by MC3T3‐E1 cells was necessary for differentiation. The ability of exogenously added BMP‐2, BMP‐4, or BMP‐7 to stimulate osteocalcin (OCN) and bone sialoprotein (BSP) mRNAs or OCN promoter activity was synergistically increased in cells that were actively synthesizing an ECM (i.e., were grown in the presence of AA). A minimum of 4 days of ECM accumulation was required for this synergistic response to be observed. Neither BMP‐7, AA, nor a combination of these two treatments had major effects on Cbfa1 messenger RNA (mRNA) or protein levels, as would be expected if regulation was mainly at the posttranscriptional level. U0126, a specific inhibitor of MAPK/extracellular signal‐regulated kinase (MEK), blocked AA‐ or BMP‐7/AA‐dependent gene expression in a time‐ and dose‐dependent manner that was closely correlated with inhibition of extracellular signal‐regulated kinase (ERK) phosphorylation. This work establishes that autocrine BMP production as well as integrin‐mediated cell‐collagen interactions are both required for osteoblast differentiation, and both these pathways require MAP kinase activity.